13^{13}C and 15^{15}N benchtop NMR detection of metabolites via relayed hyperpolarization

Parahydrogen-based nuclear spin hyperpolarization allows various magnetic-resonance applications, and it is particularly attractive because of its technical simplicity, low cost, and ability to quickly (in seconds) produce large volumes of hyperpolarized material. Although many parahydrogen-based techniques have emerged, some of them remain unexplored due to the lack of careful optimization studies. In this work, we investigate and optimize a novel parahydrogen-induced polarization (PHIP) technique that relies on proton exchange referred to below as PHIP-relay. An INEPT (insensitive nuclei enhanced by polarization transfer) sequence is employed to transfer polarization from hyperpolarized protons to heteronuclei ($^{15}$N and $^{13}$C) and nuclear signals are detected using benchtop NMR spectrometers (1 T and 1.4 T, respectively). We demonstrate the applicability of the PHIP-relay technique for hyperpolarization of a wide range of biochemicals by examining such key metabolites as urea, ammonium, glucose, amino acid glycine, and a drug precursor benzamide. By optimizing chemical and NMR parameters of the PHIP-relay, we achieve a 17,100-fold enhancement of $^{15}$N signal of [$^{13}$C, $^{15}$N$_{2}$]-urea compared to the thermal signal measured at 1 T. We also show that repeated measurements with shorter exposure to parahydrogen provide a higher effective signal-to-noise ratio compared to longer parahydrogen bubbling

Rapid hyperpolarization and purification of the metabolite fumarate in aqueous solution

Hyperpolarized fumarate is a promising biosensor for carbon-13 magnetic resonance metabolic imaging. Such molecular imaging applications require nuclear hyperpolarization to attain sufficient signal strength. Dissolution dynamic nuclear polarization is the current state-of-the-art methodology for hyperpolarizing fumarate, but this is expensive and relatively slow. Alternatively, this important biomolecule can be hyperpolarized in a cheap and convenient manner using parahydrogen-induced polarization. However, this process requires a chemical reaction, and the resulting solutions are contaminated with the catalyst, unreacted reagents, and reaction side-product molecules, and are hence unsuitable for use in vivo. In this work we show that the hyperpolarized fumarate can be purified from these contaminants by acid precipitation as a pure solid, and later redissolved to a desired concentration in a clean aqueous solvent. Significant advances in the reaction conditions and reactor equipment allow for formation of hyperpolarized fumarate at ¹³C polarization levels of 30–45%

The effects of transnational mobility and language on Japanese national identity in three groups: Japanese graduate students studying abroad, kikokushijo, and Nikkei Brazilians

L'explosiu creixement del PIB del Japó a les dècades de 1980 i 1990 va crear les condicions per a una major mobilitat de diversos grups de persones, és a dir, treballadors d'empreses japoneses que es van expandir a l'estranger, treballadors de l'estranger que es necessitaven a les indústries manufactureres i agrícoles japoneses i, la majoria, estudiants de la nova classe mitjana i famílies adinerades que tenien els mitjans econòmics per estudiar a l'estranger. Els treballadors que anaven a l'estranger sovint es quedaven durant anys desplaçant normalment al costat d'ells les seves famílies. Els fills d'aquests treballadors, que finalment van tornar a Japó, es denominen kikokushijo o repatriats. La força laboral massiva que va arribar després dels canvis en la llei d'immigració japonesa el 1990 es diu Nikkei-jin, perquè el Japó només va permetre una gran afluència d'immigrants amb visats especials de treball en el cas de persones amb llaços ancestrals japonesos comprovats. El grup Nikkei més gran que va anar a treballar a Japó va ser el de Brasil. El nombre d'estudiants japonesos a l'estranger també va augmentar dràsticament durant aquest període, amb al voltant de 25.000 estudiants cursant estudis a l'estranger el 1990 a més de 75.000 estudiants el 2005. No obstant això, l'economia del Japó, especialment després de la crisi financera de 2008, ha crescut més lentament. El govern ha emprat diverses mesures per augmentar la seva competitivitat global per tal de revitalitzar la seva economia, inclòs el suport a les escoles de japonès a l'estranger i els exàmens especials d'ingrés a la universitat per kikokushijo, la creació d'objectius per augmentar el nombre de participants en programes d'estudis a l'estranger i noves lleis d'immigració dirigides a Nikkei-jin. Com ha afectat aquest augment de la mobilitat i l'aprenentatge d'idiomes a les identitats nacionals de kikokushijo, Nikkei-jin i els estudiants japonesos a l'estranger? Per això, els meus objectius de recerca van ser estudiar els efectes de la llengua i la mobilitat transnacional sobre la identitat nacional japonesa en aquests tres grups. Cap altre estudi no ha combinat aquests tres grups per aprofundir en aquests temes de recerca. Per fer la meva recerca, vaig entrevistar un total de 20 participants: 4 brasilers Nikkei, 14 kikokushijo i 2 estudiants d'estudis de llarga durada a l'estranger. Em vaig concentrar principalment en les històries d'aprenentatge d'idiomes dels participants i el seu ús actual de l'anglès i el japonès, i com els participants veien les seves identitats nacionals a les nostres entrevistes. L'anàlisi les dades co-construïdes a les nostres entrevistes es basa la teoria del posicionament. Aquesta anàlisi va posar de relleu que algunes de les identitats nacionals japoneses dels meus participants es van veure afectades per l'aprenentatge i l'ús de l'idioma japonès i l'anglès. La mobilitat transnacional també va semblar que tenia efectes variats en les identitats nacionals japoneses dels meus participants. L'anàlisi també inclou una reflexió sobre els esforços del govern japonès per augmentar la mobilitat d’aquests tres grups i les seves estratègies de promoció de l'idioma.El explosivo crecimiento del PIB de Japón en las décadas de 1980 y 1990 creó las condiciones para una mayor movilidad de varios grupos de personas, a saber, trabajadores de empresas japonesas que se expandieron en el extranjero, trabajadores del extranjero que se necesitaban en las industrias manufactureras y agrícolas japonesas y, en su mayoría, estudiantes de la nueva clase media y familias adineradas que tenían los medios económicos para estudiar en el extranjero. Los trabajadores que iban al extranjero a menudo se quedaban durante varios años desplazando frecuentemente junto a ellos a sus familias. Los hijos de estos trabajadores, que finalmente regresaron a Japón, se denominan kikokushijo o repatriados. La fuerza laboral masiva que llegó después de los cambios en la ley de inmigración japonesa en 1990 se llama Nikkei-jin, porque Japón solo permitió una gran afluencia de inmigrantes con visas especiales de trabajo en el caso de personas con lazos ancestrales japoneses comprobados. El grupo Nikkei más grande que fue a trabajar a Japón fue el de Brasil. El número de estudiantes en el extranjero también aumentó drásticamente durante este período, con alrededor de 25.000 estudiantes estudiando en el extranjero en 1990 a más de 75.000 estudiantes en 2005. Sin embargo, la economía de Japón, especialmente después de la crisis financiera de 2008, ha crecido más lentamente. El gobierno ha empleado varias medidas para aumentar su competitividad global con el fin de revitalizar su economía, incluido el apoyo a las escuelas de japonés en el extranjero y los exámenes especiales de ingreso a la universidad para kikokushijo, la creación de objetivos para aumentar el número de participantes en programas de estudios en el extranjero y nuevas leyes de inmigración dirigidas a Nikkei-jin. ¿Cómo ha afectado este aumento de la movilidad y el aprendizaje de idiomas a las identidades nacionales de kikokushijo, Nikkei-jin y los estudiantes japoneses en el extranjero? Por ello, mis objetivos de investigación fueron estudiar los efectos del idioma y la movilidad transnacional sobre la identidad nacional japonesa en estos tres grupos. Ningún otro estudio ha combinado estos tres grupos para profundizar en estos temas de investigación. Para realizar mi investigación, entrevisté a un total de 20 participantes: 4 brasileños Nikkei, 14 kikokushijo y 2 estudiantes de estudios de larga duración en el extranjero. Me concentré principalmente en las historias de aprendizaje de idiomas de los participantes y su uso actual del inglés y japonés, así como en cómo los participantes veían sus identidades nacionales en nuestras entrevistas. El análisis de los datos co-construidos en nuestras entrevistas se basa en la teoría del posicionamiento. Dicho análisis puso de relieve que algunas de las identidades nacionales japonesas de mis participantes se vieron afectadas por el aprendizaje y el uso del idioma japonés e inglés. La movilidad transnacional también pareció tener efectos variados en las identidades nacionales japonesas de mis participantes. El análisis también incluye una reflexión sobre los esfuerzos del gobierno japonés para aumentar la movilidad de estos tres grupos y sus estrategias de promoción del idioma.Japan’s explosive GDP growth in the 1980s and 1990s created conditions for increased mobility of several groups of people, namely workers of Japanese companies that expanded abroad, workers from overseas that were needed in Japanese manufacturing and agricultural industries, and mostly students from newly middle class and wealthy families who had the financial means to study abroad. The workers who went abroad often stayed for several years and brought their families with them. The children of these workers, who eventually returned to Japan, are called kikokushijo, or returnees. The massive workforce that arrived after Japanese immigration law changes in 1990 are called Nikkei-jin, because Japan only allowed a large influx of immigrants with proven Japanese ancestral ties to enter Japan on special work visas. The largest Nikkei group that went to Japan to work was from Brazil. The number of study abroad students also increased dramatically during this time period, with about 25,000 students studying abroad in 1990 to more than 75,000 students in 2005. However, Japan’s economy, especially after the 2008 financial crisis, has grown more slowly. The government has employed several measures to increase its global competitiveness in order to revitalize its economy, including supporting Japanese language schools abroad and special university entrance examinations for kikokushijo, creating goals to increase the number of study abroad participants, and new immigration laws targeting Nikkei-jin. How has this increased mobility and language learning affected the national identities of kikokushijo, Nikkei-jin, and study abroad students? My research goals, therefore, were to study the effects of language and transnational mobility on Japanese national identity in these three groups. No other study has combined these three groups to investigate these research themes. To conduct my research, I interviewed a total of 20 participants: 4 Nikkei Brazilians, 14 kikokushijo, and 2 long-term study abroad students. I focused mainly on the participants’ language learning histories and their current use of English and Japanese as well as how the participants viewed their national identities in our interviews. I used positioning theory to analyze the data that was co-constructed in our interviews. It was found that some of my participants’ Japanese national identities were affected by Japanese and English language learning and usage. Transnational mobility also seemed to have varied effects on my participants’ Japanese national identities. The Japanese government’s efforts to increase mobility of these three groups and its language promotion strategies will also be discussed

13C and 15N NMR Detection of Metabolites via Relayed Hyperpolarization at 1 T and 1.4 T

Nuclear-spin hyperpolarization allows various magnetic-resonance applications in chemistry and medicine that are unattainable by standard methods. For such applications, parahydrogen-based hyperpolarization approaches are particularly attractive because of their technical simplicity, low cost, and ability to quickly (in seconds) produce large volumes of hyperpolarized material. Although many parahydrogen-based techniques have emerged, some of them remain unexplored due to the lack of careful optimization studies. In this work, we investigate and optimize a novel parahydrogen-induced polarization (PHIP) technique that relies on proton exchange referred to below as PHIP-relay. An INEPT (insensitive nuclei enhanced by polarization transfer) sequence is employed to transfer polarization from hyperpolarized protons to heteronuclei (15N and 13C) and nuclear signals are detected using benchtop NMR spectrometers (1 T and 1.4 T, respectively). We demonstrate the applicability of the PHIP-relay technique for hyperpolarization of a wide range of biochemicals by examining such key metabolites as urea, ammonium, glucose, amino acid glycine, and a drug precursor benzamide. By optimizing chemical and NMR parameters of the PHIP-relay, we achieve a 17,100-fold enhancement of 15N signal of [13C, 15N2]-urea compared to the thermal signal measured at 1 T. We also show that repeated measurements with shorter exposure to parahydrogen provide a higher effective signal-to-noise ratio compared to longer parahydrogen bubbling

Collagen/kerateine multi-protein hydrogels as a thermally stable extracellular matrix for 3D in vitro models

Objective: To determine whether the addition of kerateine (reduced keratin) in rat tail collagen type I hydrogels increases thermal stability and changes material properties and supports cell growth for use in cellular hyperthermia studies for tumor treatment. Methods: Collagen type I extracted from rat tail tendon was combined with kerateine extracted from human hair fibers. Thermal, mechanical, and biocompatibility properties and cell behavior was assessed and compared to 100% collagen type I hydrogels to demonstrate their utility as a tissue model for 3D in vitro testing. Results: A combination (i.e., containing both collagen ‘C/KNT’) hydrogel was more thermally stable than pure collagen hydrogels and resisted thermal degradation when incubated at a hyperthermic temperature of 47°C for heating durations up to 60 min with a higher melting temperature measured by DSC. An increase in the storage modulus was only observed with an increased collagen concentration rather than an increased KTN concentration; however, a change in ECM structure was observed with greater fiber alignment and width with an increase in KTN concentration. The C/KTN hydrogels, specifically 50/50 C/KTN hydrogels, also supported the growth and of fibroblasts and MDA-MB-231 breast cancer cells similar to those seeded in 100% collagen hydrogels. Conclusion: This multi-protein C/KTN hydrogel shows promise for future studies involving thermal stress studies without compromising the 3D ECM environment or cell growth

Relayed Hyperpolarization for Zero-Field Nuclear Magnetic Resonance

Zero- to ultralow-field nuclear magnetic resonance (ZULF NMR) is a rapidly developing form of spectroscopy that drastically reduces the size and expense of portable devices with NMR capabilities. However, signal acquisition still requires a mechanism for orienting nuclear spins (e.g., generating a bulk magnetic moment for detection), and the currently employed methods only apply to a limited pool of chemicals or come at prohibitively high cost. Here, we demonstrate that the parahydrogen-based SABRE-relay method (SABRE = Signal Amplification by Reversible Exchange) can be used as a more general means of generating hyperpolarized analytes for ZULF NMR. This method is applicable to a wide range of small molecules possessing exchangeable protons, as we demonstrate here by observing zero-field J-spectra of [13C]-methanol, [1-13C]-ethanol, and [2-13C]-ethanol. We also explore the magnetic-field dependence of the proton hyperpolarization efficiency in SABRE-relay, and show the existence of a second, previously unexplored maximum at 19 mT. We further demonstrate that water does not significantly diminish SABRE-relay performance using benzylamine as polarization-transfer agent and use this to hyperpolarize ethanol extracted from a store-bought sample of vodka (1H polarization of ~0.1%). Applications for detecting trace chemical impurities and measuring J-spectra from natural extracts are also discussed

Periodontal Disease Augments Cardiovascular Disease Risk Biomarkers in Rheumatoid Arthritis

OBJECTIVES: Periodontal disease (PD) and rheumatoid arthritis (RA) are known chronic conditions with sustained inflammation leading to osteolysis. Cardiovascular diseases (CVD) are frequent comorbidities that may arise from sustained inflammation associated with both PD and RA. In order to determine CVD risk, alterations at the molecular level need to be identified. The objective of this study, therefore, was to assess the relationship of CVD associated biomarkers in RA patients and how it is influenced by PD. METHODS: The study consisted of patient (26 RA with PD, 21 RA without PD, 51 patients with PD only) and systemically and periodontally healthy control (n = 20) groups. Periodontal parameters bleeding on probing, probing pocket depth, and marginal bone loss were determined to characterize the patient groups. Proteomic analysis of 92 CVD-related protein biomarkers was performed using a multiplex proximity extension assay. Biomarkers were clustered using the search tool for retrieval of interacting genes (STRING) to determine protein-protein interaction (PPI) networks. RESULTS: RA patients with PD had higher detection levels for 47% of the measured markers (ANGPT1, BOC, CCL17, CCL3, CD4, CD84, CTRC, FGF-21, FGF-23, GLO1, HAOX1, HB-EGF, hOSCAR, HSP 27, IL16, IL-17D, IL18, IL-27, IL6, LEP, LPL, MERTK, MMP12, MMP7, NEMO, PAPPA, PAR-1, PARP-1, PD-L2, PGF, PIgR, PRELP, RAGE, SCF, SLAMF7, SRC, THBS2, THPO, TNFRSF13B, TRAIL-R2, VEGFD, VSIG2, and XCL1) as compared to RA without PD. Furthermore, a strong biological network was identified amongst these proteins (clustering coefficient = 0.52, PPI enrichment p-value < 0.0001). Coefficients for protein clusters involved in CVD (0.59), metabolic (0.53), and skeletal (0.51) diseases were strongest in the PD group. CONCLUSION: Periodontal disease augments CVD-related biomarkers in RA through shared pathological clusters, concurrently enhancing metabolic and skeletal disease protein interactions, independent of autoimmune status

Relayed hyperpolarization for zero-field nuclear magnetic resonance.

Zero- to ultralow-field nuclear magnetic resonance (ZULF NMR) is a rapidly developing form of spectroscopy that provides rich spectroscopic information in the absence of large magnetic fields. However, signal acquisition still requires a mechanism for generating a bulk magnetic moment for detection, and the currently used methods only apply to a limited pool of chemicals or come at prohibitively high cost. We demonstrate that the parahydrogen-based SABRE (signal amplification by reversible exchange)–Relay method can be used as a more general means of generating hyperpolarized analytes for ZULF NMR by observing zero-field J-spectra of [(13)C]-methanol, [1-(13)C]-ethanol, and [2-(13)C]-ethanol in both (13)C-isotopically enriched and natural abundance samples. We explore the magnetic field dependence of the SABRE-Relay efficiency and show the existence of a second maximum at 19.0 ± 0.3 mT. Despite presence of water, SABRE-Relay is used to hyperpolarize ethanol extracted from a store-bought sample of vodka (%P(H) ~ 0.1%)