Wednesday, December 30, 2015

Characterization of Membrane Proteins in Isolated Native Cellular Membranes by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy without Purification and Reconstitution


Jacso, T., et al., Characterization of Membrane Proteins in Isolated Native Cellular Membranes by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy without Purification and Reconstitution. Angewandte Chemie, 2012. 124(2): p. 447-450.


Structural information is key for understanding biological processes. Insoluble proteins, like membrane proteins and amyloid fibrils, are a large class of proteins that are underrepresented in the protein data bank (PDB). As of today, only 7% of all entries in the PDB refer to either a membrane protein or an amyloid fibril structure (membrane protein: 4994 entries; amyloid fibril: 67 entries; total number of entries: 70,303; http://www.rcsb.org/pdb/home/home.do). Given the fact that many drugs target membrane proteins, involved in signal transduction, [1] structural information is highly desirable for a better understanding of the underlying biochemical mechanisms.

Monday, December 28, 2015

Metabolic imaging of patients with prostate cancer using hyperpolarized [1-(1)(3)C]pyruvate


Nelson, S.J., et al., Metabolic imaging of patients with prostate cancer using hyperpolarized [1-(1)(3)C]pyruvate. Sci Transl Med, 2013. 5(198): p. 198ra108.


This first-in-man imaging study evaluated the safety and feasibility of hyperpolarized [1-(1)(3)C]pyruvate as an agent for noninvasively characterizing alterations in tumor metabolism for patients with prostate cancer. Imaging living systems with hyperpolarized agents can result in more than 10,000-fold enhancement in signal relative to conventional magnetic resonance (MR) imaging. When combined with the rapid acquisition of in vivo (1)(3)C MR data, it is possible to evaluate the distribution of agents such as [1-(1)(3)C]pyruvate and its metabolic products lactate, alanine, and bicarbonate in a matter of seconds. Preclinical studies in cancer models have detected elevated levels of hyperpolarized [1-(1)(3)C]lactate in tumor, with the ratio of [1-(1)(3)C]lactate/[1-(1)(3)C]pyruvate being increased in high-grade tumors and decreased after successful treatment. Translation of this technology into humans was achieved by modifying the instrument that generates the hyperpolarized agent, constructing specialized radio frequency coils to detect (1)(3)C nuclei, and developing new pulse sequences to efficiently capture the signal. The study population comprised patients with biopsy-proven prostate cancer, with 31 subjects being injected with hyperpolarized [1-(1)(3)C]pyruvate. The median time to deliver the agent was 66 s, and uptake was observed about 20 s after injection. No dose-limiting toxicities were observed, and the highest dose (0.43 ml/kg of 230 mM agent) gave the best signal-to-noise ratio for hyperpolarized [1-(1)(3)C]pyruvate. The results were extremely promising in not only confirming the safety of the agent but also showing elevated [1-(1)(3)C]lactate/[1-(1)(3)C]pyruvate in regions of biopsy-proven cancer. These findings will be valuable for noninvasive cancer diagnosis and treatment monitoring in future clinical trials.

Wednesday, December 23, 2015

Coherent Control of Two Nuclear Spins Using the Anisotropic Hyperfine Interaction


Zhang, Y., C.A. Ryan, R. Laflamme, and J. Baugh, Phys. Rev. Lett., 107, (2011)


We demonstrate coherent control of two nuclear spins mediated by the magnetic resonance of a hyperfine-coupled electron spin. This control is used to create a double-nuclear coherence in one of the two electron spin manifolds, starting from an initial thermal state, in direct analogy to the creation of an entangled (Bell) state from an initially pure unentangled state. We identify challenges and potential solutions to obtaining experimental gate fidelities useful for quantum information processing in this type of system.

Monday, December 21, 2015

Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy


Day, S.E., M.I. Kettunen, F.A. Gallagher, D.-E. Hu, M. Lerche, J. Wolber, K. Golman, J.H. Ardenkjaer-Larsen, and K.M. Brindle, Nat Med, 13, (2007)


Measurements of early tumor responses to therapy have been shown, in some cases, to predict treatment outcome. We show in lymphoma-bearing mice injected intravenously with hyperpolarized [1-13C]pyruvate that the lactate dehydrogenase–catalyzed flux of13C label between the carboxyl groups of pyruvate and lactate in the tumor can be measured using 13C magnetic resonance spectroscopy and spectroscopic imaging, and that this flux is inhibited within 24 h of chemotherapy. The reduction in the measured flux after drug treatment and the induction of tumor cell death can be explained by loss of the coenzyme NAD(H) and decreases in concentrations of lactate and enzyme in the tumors. The technique could provide a new way to assess tumor responses to treatment in the clinic.

Friday, December 18, 2015

Microwave frequency modulation to enhance Dissolution Dynamic Nuclear Polarization


Bornet, A., J. Milani, B. Vuichoud, A.J. Perez Linde, G. Bodenhausen, and S. Jannin, Chem. Phys. Lett., 602, (2014)


Hyperpolarization by Dissolution Dynamic Nuclear Polarization is usually achieved by monochromatic microwave irradiation of the ESR spectrum of free radicals embedded in glasses at 1.2 K and 3.35 T. Hovav et al. (2014) have recently shown that by using frequency-modulated (rather than monochromatic) microwave irradiation one can improve DNP at 3.35 T in the temperature range 10–50 K. We show in this Letter that this is also true under Dissolution-DNP conditions at 1.2 K and 6.7 T. We demonstrate the many virtues of using frequency-modulated microwave irradiation: higher polarizations, faster build-up rates, lower radical concentrations, less paramagnetic broadening, more efficient cross-polarization, and less critical frequency adjustments.

Wednesday, December 16, 2015

Production of hyperpolarized [1,4-13C2]malate from [1,4-13C2]fumarate is a marker of cell necrosis and treatment response in tumors


Gallagher, F.A., M.I. Kettunen, D.E. Hu, P.R. Jensen, R.I. Zandt, M. Karlsson, A. Gisselsson, S.K. Nelson, T.H. Witney, S.E. Bohndiek, G. Hansson, T. Peitersen, M.H. Lerche, and K.M. Brindle, Proc Natl Acad Sci U S A, 106, (2009)


Dynamic nuclear polarization of (13)C-labeled cell substrates has been shown to massively increase their sensitivity to detection in NMR experiments. The sensitivity gain is sufficiently large that if these polarized molecules are injected intravenously, their spatial distribution and subsequent conversion into other cell metabolites can be imaged. We have used this method to image the conversion of fumarate to malate in a murine lymphoma tumor in vivo after i.v. injection of hyperpolarized [1,4-(13)C(2)]fumarate. In isolated lymphoma cells, the rate of labeled malate production was unaffected by coadministration of succinate, which competes with fumarate for transport into the cell. There was, however, a correlation with the percentage of cells that had lost plasma membrane integrity, suggesting that the production of labeled malate from fumarate is a sensitive marker of cellular necrosis. Twenty-four hours after treating implanted lymphoma tumors with etoposide, at which point there were significant levels of tumor cell necrosis, there was a 2.4-fold increase in hyperpolarized [1,4-(13)C(2)]malate production compared with the untreated tumors. Therefore, the formation of hyperpolarized (13)C-labeled malate from [1,4-(13)C(2)]fumarate appears to be a sensitive marker of tumor cell death in vivo and could be used to detect the early response of tumors to treatment. Given that fumarate is an endogenous molecule, this technique has the potential to be used clinically.

Monday, December 14, 2015

Metabolic Imaging by Hyperpolarized 13C Magnetic Resonance Imaging for In vivo Tumor Diagnosis


Golman, K., R.I. Zandt, M. Lerche, R. Pehrson, and J.H. Ardenkjaer-Larsen, Cancer research, 66, (2006)


The "Warburg effect," an elevation in aerobic glycolysis, may be a fundamental property of cancer cells. For cancer diagnosis and treatment, it would be valuable if elevated glycolytic metabolism could be quantified in an image in animals and humans. The pyruvate molecule is at the metabolic crossroad for energy delivery inside the cell, and with a noninvasive measurement of the relative transformation of pyruvate into lactate and alanine within a biologically relevant time frame (seconds), it may be possible to quantify the glycolytic status of the cells. We have examined the metabolism after i.v. injection of hyperpolarized (13)C-pyruvate in rats with implanted P22 tumors. The strongly enhanced nuclear magnetic resonance signal generated by the hyperpolarization techniques allows mapping of pyruvate, lactate, and alanine in a 5 x 5 x 10 mm(3) imaging voxel using a 1.5 T magnetic resonance scanner. The magnetic resonance scanning (chemical shift imaging) was initiated 24 seconds after the pyruvate injection and had a duration of 14 seconds. All implanted tumors showed significantly higher lactate content than the normal tissue. The results indicate that noninvasive quantification of localized Warburg effect may be possible.

Friday, December 11, 2015

Rapid-melt Dynamic Nuclear Polarization


Sharma, M., et al., Rapid-melt Dynamic Nuclear Polarization. J Magn Reson, 2015. 258: p. 40-8.


In recent years, Dynamic Nuclear Polarization (DNP) has re-emerged as a means to ameliorate the inherent problem of low sensitivity in nuclear magnetic resonance (NMR). Here, we present a novel approach to DNP enhanced liquid-state NMR based on rapid melting of a solid hyperpolarized sample followed by 'in situ' NMR detection. This method is applicable to small (10nl to 1mul) sized samples in a microfluidic setup. The method combines generic DNP enhancement in the solid state with the high sensitivity of stripline (1)H NMR detection in the liquid state. Fast cycling facilitates options for signal averaging or 2D structural analysis. Preliminary tests show solid-state (1)H enhancement factors of up to 500 for H2O/D2O/d6-glycerol samples doped with TEMPOL radicals. Fast paramagnetic relaxation with nitroxide radicals, In nonpolar solvents such as toluene, we find proton enhancement factors up to 400 with negligible relaxation losses in the liquid state, using commercially available BDPA radicals. A total recycling delay (including sample freezing, DNP polarization and melting) of about 5s can be used. The present setup allows for a fast determination of the hyper-polarization as function of the microwave frequency and power. Even at the relatively low field of 3.4T, the method of rapid melting DNP can facilitate the detection of small quantities of molecules in the picomole regime.

Wednesday, December 9, 2015

PRESTO polarization transfer to quadrupolar nuclei: implications for dynamic nuclear polarization


Perras, F.A., T. Kobayashi, and M. Pruski, PRESTO polarization transfer to quadrupolar nuclei: implications for dynamic nuclear polarization. Phys Chem Chem Phys, 2015. 17(35): p. 22616-22.


We show both experimentally and numerically on a series of model systems that in experiments involving transfer of magnetization from (1)H to the quadrupolar nuclei under magic-angle-spinning (MAS), the PRESTO technique consistently outperforms traditionally used cross polarization (CP), affording more quantitative intensities, improved lineshapes, better overall sensitivity, and straightforward optimization. This advantage derives from the fact that PRESTO circumvents the convoluted and uncooperative spin dynamics during the CP transfer under MAS, by replacing the spin-locking of quadrupolar nuclei with a single central transition selective 90 degrees pulse and using a symmetry-based recoupling sequence in the (1)H channel. This is of particular importance in the context of dynamic nuclear polarization (DNP) NMR of quadrupolar nuclei, where the efficient transfer of enhanced (1)H polarization is desired to obtain the highest sensitivity.

Tuesday, December 8, 2015

[NMR] PhD position / Solid-State NMR / Utrecht / Bio-inspired Nanomaterials

From the Ampere Magnetic Resonance List


PhD position / Solid-State NMR / Utrecht / Bio-inspired Nanomaterials

We invite applications for a 4-years PhD position in the field of biomolecular solid-state NMR at the Bijvoet Center, Utrecht University, The Netherlands. The research deals with the atomic-level characterization of bio-inspired self-assembling nanomaterials for pharmaceutical and medical applications using a combined approach of modern solid-state NMR & MD simulation methods. Further information to the project is available by email (to m.h.weingarth@uu.nl).

The position is offered in the junior research group of by M. Weingarth, embedded in the NMR group of Prof. M. Baldus. We offer access to high-field NMR machines (950, 800, 700, 500, 400 MHz for solid-state NMR // 900 MHz magnet for solution NMR). A 1.2 GHz machine will be installed within the next years. The 950, 800 and 700 MHz machines are equipped with fast-spinning 1.3 mm probes. The 800 and 400 MHz magnetes are equipped for DNP. The candidate will have access to a powerful computational infrastructure and a well-equipped molecular biology lab. Research will also be carried out in collaboration with pharmaceutical groups.

Applicants should have a degree in biochemistry/biophysics or a related discipline. Cross-disciplinary applications are welcome. Strong experience in molecular biology techniques & protein/nucleic acid preparation is desirable. Experience in either MD simulations, NMR or programming is a plus, but not required. The starting date is March/April 2016 or later.

To apply, please send a (brief) cover letter, your CV as well as the names and the contact addresses of two references to:

Dr. Markus Weingarth / Phone: +31 30 253 2875 / Email : m.h.weingarth@uu.nl

See also here for further information: http://www.uu.nl/medewerkers/MHWeingarth

Project-relevant publications:

I.Rad Malekshahi, M., Visscher. K.M., Rodrigues J.P.G.L.M., de Vries, R., Hennink, W.E., Baldus, M., Bonvin A.M.J.J., Mastrobattista E., Weingarth. M. (2015) J. Am. Chem. Soc., 137, 7775, The supramolecular organization of a peptide based nanocarrier at high molecular detail

II. Mance, D., Sinnige., T., Kaplan, M., Daniels, M., Houben, K., Baldus, M., Weingarth, M. (2015) Angew. Chem., in press,A labeling approach to harness backbone and side chain protons in 1H-detected solid-state NMR

III. Sinnige, T., Daniels, M., Baldus, M., Weingarth, M. (2014) J. Am. Chem. Soc., 136, 4452. Proton clouds to measure nonexchangable sidechain protons in solid-state NMR. Cover article

The PhD position is funded by a NWO VIDI grant to M. Weingarth. The candidate is offered a full-time position for 4 years. The salary is supplemented with a holiday bonus of 8.0% and an end-of-year bonus of 8,3% per year. The salary starts at ca. € 2.100 and increases to € 2.700 gross per month in the fourth year.

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[NMR] Postdoctoral opportunity available – Nanometer-scale magnetic resonance

From the Ampere Magnetic Resonance List




The Karlsruhe Institute of Technology (KIT) is seeking an experienced postdoctoral researcher to initiate activities in the area of nanometer-scale magnetic resonance (nano-MR). Such activities may include, but are not limited to, techniques based on magnetic resonance force microscopy (MRFM), or nitrogen vacancy (NV) centers.

The ideal candidate will have a proven track record in nano-MR technology. She/he will be a highly motivated individual, and will be interested in building an independent research group. The candidate will be expected to build up nano-MR activities, with a focus on room temperature applications, within the highly collaborative environment available at KIT.

The candidate will formally join the Institute of Microstructure Technology (IMT), within the Science and Technology of Nanosystems (STN) framework, under the guidance of Prof. Jan G. Korvink. IMT features excellent infrastructure within which nano-MR activities would be expected to flourish. Specifically, IMT has a fully equipped RF characterization lab, a Bruker 500 MHz wide-bore NMR system, a Bruker 1 T ICON MRI system, e-beam lithography, 3D nanolithography, state-of-the art clean room facilities, as well as an in-house AFM competence centre led by Dr. Hendrik Hölscher. Prof. Korvink’s group specializes in micro-NMR detector design and implementation, has activities in hyperpolarisation techniques (para-H2 and DNP), and hence offers an excellent environment within which to build up new and exciting nano-MR techniques. The position is funded for two years. Interested applicants should send a motivation letter, their CV, and a copy of their most relevant publication(s) as a single pdf file to Prof. Jan G. Korvink (jan.korvink@kit.edu). The position will remain open until a suitable candidate is identified. We aim to balance the number of female and male employees at KIT. We therefore expressly encourage female applicants to apply for this job. Applicants with disabilities will be preferentially considered if equally qualified.

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Monday, December 7, 2015

Solid-State Dynamic Nuclear Polarization at 9.4 and 18.8 T from 100 K to Room Temperature


Lelli, M., et al., Solid-State Dynamic Nuclear Polarization at 9.4 and 18.8 T from 100 K to Room Temperature. J Am Chem Soc, 2015. 137(46): p. 14558-61.


Efficient dynamic nuclear polarization (DNP) in solids, which enables very high sensitivity NMR experiments, is currently limited to temperatures of around 100 K and below. Here we show how by choosing an adequate solvent, (1)H cross effect DNP enhancements of over 80 can be obtained at 240 K. To achieve this we use the biradical TEKPol dissolved in a glassy phase of ortho-terphenyl (OTP). We study the solvent DNP enhancement of both TEKPol and BDPA in OTP in the range from 100 to 300 K at 9.4 and 18.8 T. Surprisingly, we find that the DNP enhancement decreases only relatively slowly for temperatures below the glass transition of OTP (Tg = 243 K), and (1)H enhancements around 15-20 at ambient temperature can be observed. We use this to monitor molecular dynamic transitions in the pharmaceutically relevant solids Ambroxol and Ibuprofen.

Friday, December 4, 2015

Application of Good's buffers to pH imaging using hyperpolarized (13)C MRI


Flavell, R.R., et al., Application of Good's buffers to pH imaging using hyperpolarized (13)C MRI. Chem Commun (Camb), 2015. 51(74): p. 14119-22.


N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), one of Good's buffers, was applied to pH imaging using hyperpolarized (13)C magnetic resonance spectroscopy. Rapid NMR- and MRI-based pH measurements were obtained by exploiting the sensitive pH-dependence of its (13)C chemical shift within the physiologic range.

Wednesday, December 2, 2015

Hyperpolarized NMR of plant and cancer cell extracts at natural abundance


Dumez, J.N., et al., Hyperpolarized NMR of plant and cancer cell extracts at natural abundance. Analyst, 2015. 140(17): p. 5860-3.


Natural abundance (13)C NMR spectra of biological extracts are recorded in a single scan provided that the samples are hyperpolarized by dissolution dynamic nuclear polarization combined with cross polarization. Heteronuclear 2D correlation spectra of hyperpolarized breast cancer cell extracts can also be obtained in a single scan. Hyperpolarized NMR of extracts opens many perspectives for metabolomics.

Monday, November 30, 2015

Single voxel localization for dynamic hyperpolarized 13C MR spectroscopy


Chen, A.P. and C.H. Cunningham, Single voxel localization for dynamic hyperpolarized 13C MR spectroscopy. J. Magn. Reson., 2015. 258: p. 81-85.


The PRESS technique has been widely used to achieve voxel localization for in vivo 1H MRS acquisitions. However, for dynamic hyperpolarized 13C MRS experiments, the transition bands of the refocusing pulses may saturate the pre-polarized substrate spins flowing into the voxel. This limitation may be overcome by designing refocusing pulses that do not perturb the resonance of the hyperpolarized substrate, but selectively refocuses the spins of the metabolic products. In this study, a PRESS pulse sequence incorporating spectral–spatial refocusing pulses that have a stop band (‘notch’) at the substrate resonance is tested in vivo using hyperpolarized [1-13C]pyruvate. Higher metabolite SNR was observed in experiments using the spectral–spatial refocusing pulses as compared to conventional refocusing pulses.

Sunday, November 29, 2015

[NMR] PhD Position - Uni Leipzig - Solid-state NMR on photoreceptor protein

From the Ampere Magnetic Resonance List



PhD Position - Uni Leipzig - Solid-state NMR on photoreceptor protein


The Institute of Analytical Chemistry at the University of Leipzig invites applications for a PhD position in the field of solid-state NMR characterization of photoreceptors. To be appointed from 01.02.16 or later. The position is limited to 3 years.

The NMR research group of Jörg Matysik works on the development of hyperpolarization and optical NMR techniques for studying photo/spin-chemically relevant systems. The successful candidate will work on an interdisciplinary research project combining biochemical work and solid-state NMR spectroscopy. This DFG financed project aims for understanding of the electronic orbital structure of the chromophore in photoreceptors of the phytochrome family.

The candidate should hold a MSc degree and be highly motivated with a strong interest in protein chemistry and biophysics. Previous experience biochemistry of proteins and is desirable. Experience in the field of magnetic resonance spectroscopy is of advantage.

For further information, please contact Jörg Matysik,

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[NMR] Course on "Dissolution Dynamic Nuclear Polarization" December 9-11, 2015 at EPFL, Switzerland

From the Ampere Magnetic Resonance List



We would like to announce a three-day course for PhD students and Post-Docs on

"Dissolution Dynamic Nuclear Polarization"

The course will start at 1pm on Wednesday December the 9th and will end at 5pm on Friday the 11th, and will be held at the Ecole Polytechnique Fédérale de Lausanne (EPFL), Switzerland, at the Laboratory of Biomolecular Magnetic Resonance (LRMB).

* Registration
please register at the following adress:
subscription fees: 100 CHF

* Information
sami.jannin (at) epfl.ch
geoffrey.bodenhausen (at) epfl.ch

* Objectives
Dissolution Dynamic Nuclear Polarization (D-DNP) provides a way to enhance NMR signals in liquids by more than 4 orders of magnitude. We present the current state-of-the-art and most recent advances of this technique, and we propose experimental demonstrations with hands-on participation. 

* Content
Lectures and seminars: 11 Hours
Hands-on: 7 Hours

Day 1:  Lectures, 1 pm - 5 pm: Theoretical aspects of DNP

- Introduction to DNP-enhanced NMR
- Principles of Dissolution-DNP
- Low temperature DNP mechanisms
- Cross Polarization techniques
- Applications to imaging and chemistry

Day 2:  Lectures, 9 am - 12 am: Experimental aspects of DNP

- Hardware for DNP
- Hardware for Cross Polarization 
- Hardware for Dissolution

Experiments, 1 pm - 5 pm

- Sample Preparation
- Preparation of a dissolution DNP experiment

Day 3: Experiments, 9 am - 12 am: Practical DNP at the Laboratory of Biomolecular Magnetic Resonance (LRMB)

- Low temperature DNP
- Cross Polarization with DNP
- Dissolution DNP


Seminars, 1 pm - 5 pm

 - All participants are invited to give a short presentation, possibly on DNP and/or related to their own research subjects.

*Required prior knowledge
Basic understanding of NMR

Dr. Sami Jannin

EPFL
Av Forel
Batochime BCH 1534
CH-1015 Lausanne 
Switzerland

Phone +41 21 693 97 24
Mobile +41 77 406 61 23
Skype samiboulot
Fax +41 21 693 94 35


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[NMR] Post Doc position in Hyperpolarized Magnetic Resonance

From the Ampere Magnetic Resonance List

Center for Hyperpolarization in Magnetic Resonance at the Technical University of Denmark is looking for a PhD in Magnetic Resonance who wants to work on "HyperPET/MR: a new concept of hybrid molecular imaging in cancer using simultaneous PET and 13C-hyperpolarized MRSI" for a two-year postdoc position. Please share if you know a candidate! "HyperPET/MR" is a collaboration with Copenhagen University, Copenhagen University Hospital, Cambridge University, Stanford University and GE Healthcare. The project is funded by Innovation Fund DK (then Strategic Research Council).


Jan Henrik Ardenkjær-Larsen 
Professor, Center Leader 
Technical University of Denmark 
Department of Electrical Engineering 
Ørsted Plads, bldg 349, office 126 
DK - 2800 Kgs. Lyngby 
Phone +45 40272775 


Monday, November 23, 2015

Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with (13)C hyperpolarised NMR


Breukels, V., et al., Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with (13)C hyperpolarised NMR. NMR Biomed, 2015. 28(8): p. 1040-8.


Hyperpolarised (HP) (13)C NMR allows enzymatic activity to be probed in real time in live biological systems. The use of in vitro models gives excellent control of the cellular environment, crucial in the understanding of enzyme kinetics. The increased conversion of pyruvate to lactate in cancer cells has been well studied with HP (13)C NMR. Unfortunately, the equally important metabolic step of lactate transport out of the cell remains undetected, because intracellular and extracellular lactate are measured as a single resonance. Furthermore, typical experiments must be performed using tens of millions of cells, a large amount which can lead to a costly and sometimes highly challenging growing procedure. We present a relatively simple set-up that requires as little as two million cells with the spectral resolution to separate the intracellular and extracellular lactate resonances. The set-up is tested with suspensions of prostate cancer carcinoma cells (PC3) in combination with HP [1-(13)C]pyruvate. We obtained reproducible pyruvate to lactate label fluxes of 1.2 and 1.7 nmol/s per million cells at 2.5 and 5.0 mM pyruvate concentrations. The existence of a 3-Hz chemical shift difference between intracellular and extracellular lactate enabled us to determine the lactate transport rates in PC3. We deduced a lactate export rate of 0.3 s(-1) and observed a decrease in lactate transport on addition of the lactate transport inhibitor alpha-cyano-4-hydroxycinnamic acid.

Wednesday, November 18, 2015

Dynamic nuclear polarization in solid samples by electrical-discharge-induced radicals


Katz, I. and A. Blank, Dynamic nuclear polarization in solid samples by electrical-discharge-induced radicals. J Magn Reson, 2015. 261: p. 95-100.


Dynamic nuclear polarization (DNP) is a method for enhancing nuclear magnetic resonance (NMR) signals that has many potential applications in chemistry and medicine. Traditionally, DNP signal enhancement is achieved through the use of exogenous radicals mixed in a solution with the molecules of interest. Here we show that proton DNP signal enhancements can be obtained for solid samples without the use of solvent and exogenous radicals. Radicals are generated primarily on the surface of a solid sample using electrical discharges. These radicals are found suitable for DNP. They are stable under moderate vacuum conditions, yet readily annihilate upon compound dissolution or air exposure. This feature makes them attractive for use in medical applications, where the current variety of radicals used for DNP faces regulatory problems. In addition, this solvent-free method may be found useful for analytical NMR of solid samples which cannot tolerate solvents, such as certain pharmaceutical products.

Tuesday, November 17, 2015

Monday, November 16, 2015

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR


Voinov, M.A., et al., Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR. J Phys Chem B, 2015. 119(32): p. 10180-90.


Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with ~17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.

Friday, November 13, 2015

Implementation and characterization of flow injection in dissolution dynamic nuclear polarization NMR spectroscopy


Chen, H.Y. and C. Hilty, Implementation and characterization of flow injection in dissolution dynamic nuclear polarization NMR spectroscopy. ChemPhysChem, 2015. 16(12): p. 2646-52.


The use of dissolution dynamic nuclear polarization (D-DNP) offers substantially increased signals in liquid-state NMR spectroscopy. A challenge in realizing this potential lies in the transfer of the hyperpolarized sample to the NMR detector without loss of hyperpolarization. Here, the use of a flow injection method using high-pressure liquid leads to improved performance compared to the more common gas-driven injection, by suppressing residual fluid motions during the NMR experiment while still achieving a short injection time. Apparent diffusion coefficients are determined from pulsed field gradient echo measurements, and are shown to fall below 1.5 times the value of a static sample within 0.8 s. Due to the single-scan nature of D-DNP, pulsed field gradients are often the only choice for coherence selection or encoding, but their application requires stationary fluid. Sample delivery driven by a high-pressure liquid will improve the applicability of these types of D-DNP advanced experiments.

Wednesday, November 11, 2015

Open Position at Bridge12: Scientist - Magnetic Resonance Spectroscopy

Bridge12 has currently an opening for a Scientist - Magnetic Resonance Spectroscopy. Details can be found here:

http://www.bridge12.com/content/scientist-instrument-development-magnetic-resonance-spectroscopy


Spin Noise Detection of Nuclear Hyperpolarization at 1.2 K


Poschko, M.T., et al., Spin Noise Detection of Nuclear Hyperpolarization at 1.2 K. ChemPhysChem, 2015: p. n/a-n/a.


We report proton spin noise spectra of a hyperpolarized solid sample of commonly used "DNP juice" containing TEMPOL and irradiated by a microwave field at a temperature of 1.2 K in a magnetic field of 6.7 T. The line shapes of the spin noise power spectra are sensitive to the variation of the microwave irradiation frequency and change from dip to bump, when the electron Larmor frequency is crossed, which is shown to be in good accordance with theory by simulations. Small but significant deviations from these predictions are observed, which can be related to spin noise and radiation damping phenomena that have been reported in thermally polarized systems. The non-linear dependence of the spin noise integral on nuclear polarization provides a means to monitor hyperpolarization semi-quantitatively without any perturbation of the spin system by radio frequency irradiation.

Monday, November 9, 2015

Structure of Colloidal Quantum Dots from Dynamic Nuclear Polarization Surface Enhanced NMR Spectroscopy


Piveteau, L., et al., Structure of Colloidal Quantum Dots from Dynamic Nuclear Polarization Surface Enhanced NMR Spectroscopy. J Am Chem Soc, 2015. 137(43): p. 13964-71.


Understanding the chemistry of colloidal quantum dots (QDs) is primarily hampered by the lack of analytical methods to selectively and discriminately probe the QD core, QD surface and capping ligands. Here, we present a general concept for studying a broad range of QDs such as CdSe, CdTe, InP, PbSe, PbTe, CsPbBr3, etc., capped with both organic and inorganic surface capping ligands, through dynamic nuclear polarization (DNP) surface enhanced NMR spectroscopy. DNP can enhance NMR signals by factors of 10-100, thereby reducing the measurement times by 2-4 orders of magnitude. 1D DNP enhanced spectra acquired in this way are shown to clearly distinguish QD surface atoms from those of the QD core, and environmental effects such as oxidation. Furthermore, 2D NMR correlation experiments, which were previously inconceivable for QD surfaces, are demonstrated to be readily performed with DNP and provide the bonding motifs between the QD surfaces and the capping ligands.

Friday, November 6, 2015

Robust hyperpolarized (13)C metabolic imaging with selective non-excitation of pyruvate (SNEP)


Chen, W.C., et al., Robust hyperpolarized (13)C metabolic imaging with selective non-excitation of pyruvate (SNEP). NMR Biomed, 2015. 28(8): p. 1021-30.


In vivo metabolic imaging using hyperpolarized [1-(13)C]pyruvate provides localized biochemical information and is particularly useful in detecting early disease changes, as well as monitoring disease progression and treatment response. However, a major limitation of hyperpolarized magnetization is its unrecoverable decay, due not only to T1 relaxation but also to radio-frequency (RF) excitation. RF excitation schemes used in metabolic imaging must therefore be able to utilize available hyperpolarized magnetization efficiently and robustly for the optimal detection of substrate and metabolite activities. In this work, a novel RF excitation scheme called selective non-excitation of pyruvate (SNEP) is presented. This excitation scheme involves the use of a spectral selective RF pulse to specifically exclude the excitation of [1-(13)C]pyruvate, while uniformly exciting the key metabolites of interest (namely [1-(13)C]lactate and [1-(13)C]alanine) and [1-(13)C]pyruvate-hydrate. By eliminating the loss of hyperpolarized [1-(13)C]pyruvate magnetization due to RF excitation, the signal from downstream metabolite pools is increased together with enhanced dynamic range. Simulation results, together with phantom measurements and in vivo experiments, demonstrated the improvement in signal-to-noise ratio (SNR) and the extension of the lifetime of the [1-(13)C]lactate and [1-(13)C]alanine pools when compared with conventional non-spectral selective (NS) excitation. SNEP has also been shown to perform comparably well with multi-band (MB) excitation, yet SNEP possesses distinct advantages, including ease of implementation, less stringent demands on gradient performance, increased robustness to frequency drifts and B0 inhomogeneity as well as easier quantification involving the use of [1-(13)C]pyruvate-hydrate as a proxy for the actual [1-(13)C] pyruvate signal. SNEP is therefore a promising alternative for robust hyperpolarized [1-(13)C]pyruvate metabolic imaging with high fidelity.

[NMR] job opening in New York

From the Ampere Magenetic Resonance List



Staff scientist / Postdoctoral fellow

The Meriles group at CUNY - City College of New York invites applications for a position at the level of research scientist or postdoctoral associate. The successful candidate will have a strong research background but also the drive and motivation to work in an entrepreneurial setting. Applicants must have a PhD in Physics, Chemistry or a closely related field, be a US citizen or permanent resident, and demonstrate experience in one or more of the following areas:

  • electron or nuclear magnetic resonance, particularly, in connection with schemes of nuclear spin hyperpolarization (e.g., DNP);
  • design and application of microfluidics in liquid state NMR
  • familiarity with nitrogen-vacancy centers in diamond and optically-detected magnetic resonance.

Work will be carried out in close connection with researchers at UC Berkeley and an industrial partner. Records of creative research demonstrated by publications in peer-reviewed journals, excellent oral and written communication skills, and the ability to work collaboratively in a team environment are necessary. Prior entrepreneurial experience or employment within industry is viewed positively. Salary will be commensurate with the applicant’s experience. Interested candidates should submit a CV with a publication list, a brief biosketch, and the contact information of three references to Prof. Carlos Meriles (cmeriles@sci.ccny.cuny.edu).

All qualified applicants will receive consideration for employment without regard to race, color, religion, sex, or disability.


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Wednesday, November 4, 2015

Design and characterization of a W-band system for modulated DNP experiments


Guy, M.L., L. Zhu, and C. Ramanathan, Design and characterization of a W-band system for modulated DNP experiments. J. Magn. Reson., 2015. 261: p. 11-18.


Magnetic-field and microwave-frequency modulated DNP experiments have been shown to yield improved enhancements over conventional DNP techniques, and even to shorten polarization build-up times. The resulting increase in signal-to-noise ratios can lead to significantly shorter acquisition times in signal-limited multi-dimensional NMR experiments and pave the way to the study of even smaller sample volumes. In this paper we describe the design and performance of a broadband system for microwave frequency- and amplitude-modulated DNP that has been engineered to minimize both microwave and thermal losses during operation at liquid helium temperatures. The system incorporates a flexible source that can generate arbitrary waveforms at 94 GHz with a bandwidth greater than 1 GHz, as well as a probe that efficiently transmits the millimeter waves from room temperature outside the magnet to a cryogenic environment inside the magnet. Using a thin-walled brass tube as an overmoded waveguide to transmit a hybrid HE11 mode, it is possible to limit the losses to 1 dB across a 2 GHz bandwidth. The loss is dominated by the presence of a quartz window used to isolate the waveguide pipe. This performance is comparable to systems with corrugated waveguide or quasi-optical components. The overall excitation bandwidth of the probe is seen to be primarily determined by the final antenna or resonator used to excite the sample and its coupling to the NMR RF coil. Understanding the instrumental limitations imposed on any modulation scheme is key to understanding the observed DNP results and potentially identifying the underlying mechanisms. We demonstrate the utility of our design with a set of triangular frequency-modulated DNP experiments.

Monday, November 2, 2015

Frequency swept microwaves for hyperfine decoupling and time domain dynamic nuclear polarization


Hoff, D.E., et al., Frequency swept microwaves for hyperfine decoupling and time domain dynamic nuclear polarization. Solid State Nucl Magn Reson, 2015.


Hyperfine decoupling and pulsed dynamic nuclear polarization (DNP) are promising techniques to improve high field DNP NMR. We explore experimental and theoretical considerations to implement them with magic angle spinning (MAS). Microwave field simulations using the high frequency structural simulator (HFSS) software suite are performed to characterize the inhomogeneous phase independent microwave field throughout a 198GHz MAS DNP probe. Our calculations show that a microwave power input of 17W is required to generate an average EPR nutation frequency of 0.84MHz. We also present a detailed calculation of microwave heating from the HFSS parameters and find that 7.1% of the incident microwave power contributes to dielectric sample heating. Voltage tunable gyrotron oscillators are proposed as a class of frequency agile microwave sources to generate microwave frequency sweeps required for the frequency modulated cross effect, electron spin inversions, and hyperfine decoupling. Electron spin inversions of stable organic radicals are simulated with SPINEVOLUTION using the inhomogeneous microwave fields calculated by HFSS. We calculate an electron spin inversion efficiency of 56% at a spinning frequency of 5kHz. Finally, we demonstrate gyrotron acceleration potentials required to generate swept microwave frequency profiles for the frequency modulated cross effect and electron spin inversions.

Friday, October 30, 2015

Rational design of dinitroxide biradicals for efficient cross-effect dynamic nuclear polarization


Kubicki, D.J., et al., Rational design of dinitroxide biradicals for efficient cross-effect dynamic nuclear polarization. Chem. Sci., 2015.


A series of 37 dinitroxide biradicals have been prepared and their performance studied as polarizing agents in cross effect DNP NMR experiments at 9.4 T and 100 K in 1,1,2,2-tetrachloroethane (TCE). We observe that in this regime the DNP performance is strongly correlated with the substituents on the polarizing agents, and electron and nuclear spin relaxation times, with longer relaxation times leading to better enhancements. We also observe that deuteration of the radicals generally leads to better DNP enhancement but with longer buildup time. One of the new radicals introduced here provides the best performance obtained so far under these conditions.

Thursday, October 29, 2015

[NMR] 2016 Winter School on Biomolecular Solid-State NMR: Application deadline is Oct 30, 2015

From the Ampere Magnetic Resonance List
Announcement
The 4th U.S.-Canada Winter School on Biomolecular Solid-State NMR
Stowe, Vermont
January 10-15, 2016

Organizers: Mei Hong (MIT), Chris Jaroniec (Ohio State) and Bob Griffin (MIT)

Dear Colleagues,

We invite you to encourage your students, postdocs, and senior associates to attend the 4th Winter School on Biomolecular Solid-State NMR, which will be held on January 10-15, 2016, in Stowe, Vermont. Similar to the three previous highly successful Winter Schools, this pedagogical meeting is aimed at students and postdocs in solid-state NMR as well as more senior scientists in related fields who are interested in entering this vibrant field. Our goals are to provide a focused week of teaching of the core concepts and practices in the increasingly multifaceted and complex field of biological solid-state NMR spectroscopy, and to encourage information sharing among different laboratories. Topics to be covered in the 4th Winter School include:

  • Basics of solid-state NMR: orientation-dependent NMR frequencies, MAS, tensors and rotations, density operator and its time evolution, dipolar recoupling, and average Hamiltonian theory.
  • Multidimensional correlation spectroscopy, resonance assignment and protein structure determination 
  • Theory of dipolar decoupling and polarization transfer
  • Pushing the sensitivity envelope: dynamic nuclear polarization and 1H detection
  • Effective "sensitivity enhancement" by non-uniform sampling
  • Solid-state NMR techniques for measuring molecular motion
  • Solid-state NMR techniques for measuring membrane protein orientation 
  • Beyond spin 1/2: NMR of quadrupolar nuclei 
  • Beating the 800-pound gorilla: NMR of membrane proteins 
  • XPLOR-NIH for structure calculation 
In addition to lectures, problem sets and their discussion sessions will be given at the meeting.

Speakers: The following people have agreed to give lectures:
Tim Cross (Florida State)
Philip Grandinetti (Ohio State)
Bob Griffin (MIT)
Mei Hong (MIT)
Chris Jaroniec (Ohio State)
Vladimir Ladizhansky (Guelph)
Len Mueller (UC Riverside)
Stanley Opella (UC San Diego)
Bernd Reif (Tech Univ Munich)
David Roynyak (Bucknell)
Charles Schwieters (NIH)
Robert Tycko (NIH)

Venue and transportation: The meeting will be held at the beautiful and historical Trapp Family Lodge http://www.trappfamily.com/ in Stowe, Vermont. Stowe is accessible from airports in Burlington, VT, Manchester, NH, and Boston, MA. A block of rooms has been reserved at the TFL. We anticipate space for ~70 attendees.

Cost: Room and board will be free for attendees. The registration fee is $425 for academic attendees and $600 for industrial attendees. 

Application: Interested students and postdocs should send the following application material as a single PDF file to ws2016@mit.edu. The application material includes 1) a CV, 2) publication list, and 3) a 1-page description of your current research and your statement of interest in attending the Winter School. Please indicate your gender in the CV for the purpose of hotel room assignment. Please name this application file as ProfessorLastName_YourLastName_WS2016app.pdf. For example, the filename can be  “Mueller_ Smith_WS2016app.pdf”.

Application deadline: Friday, Oct 30, 2015. Given the space limitation, applications received after the above date may not be possible to be accommodated. 

Please distribute this announcement to members of your research group as well as to colleagues who may be interested in attending or sending their group members.

With kind regards,
Mei and Chris

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Wednesday, October 28, 2015

Polymorphs of Theophylline Characterized by DNP Enhanced Solid-State NMR


Pinon, A.C., et al., Polymorphs of Theophylline Characterized by DNP Enhanced Solid-State NMR. Mol Pharm, 2015.


We show how dynamic nuclear polarization (DNP) enhanced solid-state NMR spectroscopy can be used to characterize polymorphs and solvates of organic solids. We applied DNP to three polymorphs and one hydrated form of the asthma drug molecule theophylline. For some forms of theophylline, sample grinding and impregnation with the radical-containing solution, which are necessary to prepare the samples for DNP, were found to induce polymorphic transitions or desolvation between some forms. We present protocols for sample preparation for solid-state magic-angle spinning (MAS) DNP experiments that avoid the polymorphic phase transitions in theophylline. These protocols include cryogrinding, grinding under inert atmosphere, and the appropriate choice of the impregnating liquid. By applying these procedures, we subsequently demonstrate that two-dimensional correlation experiments, such as 1H-13C and 1H-15N HETCOR or 13C-13C INADEQUATE, can be obtained at natural isotopic abundance in reasonable times, thus enabling more advanced structural characterization of polymorphs.

Monday, October 26, 2015

The effect of Gd on trityl-based dynamic nuclear polarisation in solids


Ravera, E., et al., The effect of Gd on trityl-based dynamic nuclear polarisation in solids. Phys Chem Chem Phys, 2015. 17(40): p. 26969-78.


In dynamic nuclear polarisation (DNP) experiments performed under static conditions at 1.4 K we show that the presence of 1 mM Gd(iii)-DOTAREM increases the (13)C polarisation and decreases the (13)C polarisation buildup time of (13)C-urea dissolved in samples containing water/DMSO mixtures with trityl radical (OX063) concentrations of 10 mM or higher. To account for these observations further measurements were carried out at 6.5 K, using a combined EPR and NMR spectrometer. At this temperature, frequency swept DNP spectra of samples with 5 or 10 mM OX063 were measured, with and without 1 mM Gd-DOTA, and again a (13)C enhancement gain was observed due to the presence of Gd-DOTA. These measurements were complemented by electron-electron double resonance (ELDOR) measurements to quantitate the effect of electron spectral diffusion (eSD) on the DNP enhancements and lineshapes. Simulations of the ELDOR spectra were done using the following parameters: (i) a parameter defining the rate of the eSD process, (ii) an "effective electron-proton anisotropic hyperfine interaction parameter", and (iii) the transverse electron spin relaxation time of OX063. These parameters, together with the longitudinal electron spin relaxation time, measured by EPR, were used to calculate the frequency profile of electron polarisation. This, in turn, was used to calculate two basic solid effect (SE) and indirect cross effect (iCE) DNP spectra. A properly weighted combination of these two normalized DNP spectra provided a very good fit of the experimental DNP spectra. The best fit simulation parameters reveal that the addition of Gd(iii)-DOTA causes an increase in both the SE and the iCE contributions by similar amounts, and that the increase in the overall DNP enhancements is a result of narrowing of the ELDOR spectra (increased electron polarisation gradient across the EPR line). These changes in the electron depolarisation profile are a combined result of shortening of the longitudinal and transverse electron spin relaxation times, as well as an increase in the eSD rate and in the effective electron-proton anisotropic hyperfine interaction parameter.

Friday, October 23, 2015

High-resolution NMR of hydrogen in organic solids by DNP enhanced natural abundance deuterium spectroscopy


Rossini, A.J., et al., High-resolution NMR of hydrogen in organic solids by DNP enhanced natural abundance deuterium spectroscopy. J. Magn. Reson., 2015. 259: p. 192-198.


We demonstrate that high field (9.4 T) dynamic nuclear polarization (DNP) at cryogenic (∼100 K) sample temperatures enables the rapid acquisition of natural abundance 1H–2H cross-polarization magic angle spinning (CPMAS) solid-state NMR spectra of organic solids. Spectra were obtained by impregnating substrates with a solution of the stable DNP polarizing agent TEKPol in tetrachloroethane. Tetrachloroethane is a non-solvent for the solids, and the unmodified substrates are then polarized through spin diffusion. High quality natural abundance 2H CPMAS spectra of histidine hydrochloride monohydrate, glycylglycine and theophylline were acquired in less than 2 h, providing direct access to hydrogen chemical shifts and quadrupolar couplings. The spectral resolution of the 2H solid-state NMR spectra is comparable to that of 1H spectra obtained with state of the art homonuclear decoupling techniques.

Wednesday, October 21, 2015

Hyperpolarized choline as an MR imaging molecular probe: feasibility of in vivo imaging in a rat model


Friesen-Waldner, L.J., et al., Hyperpolarized choline as an MR imaging molecular probe: feasibility of in vivo imaging in a rat model. J Magn Reson Imaging, 2015. 41(4): p. 917-23.


PURPOSE: To assess the feasibility of choline MRI using a new choline molecular probe for dynamic nuclear polarization (DNP) hyperpolarized MRI. MATERIALS AND METHODS: Male Sprague-Dawley rats with an average weight of 400 +/- 20 g (n = 5), were anesthetized and injection tubing was placed in the tail vein. [1,1,2,2-D4 , 1-(13) C]choline chloride (CMP1) was hyperpolarized by DNP and injected into rats at doses ranging from 12.6 to 50.0 mg/kg. Coronal projection (13) C imaging was performed on a 3 Tesla clinical MRI scanner (bore size 60 cm) using a variable flip angle gradient echo sequence. Images were acquired 15 to 45 s after the start of bolus injection. Signal intensities in regions of interest were determined at each time point and compared. RESULTS: (13) C MRI images of hyperpolarized CMP1 at a 50 mg/kg dose showed time-dependent organ distribution patterns. At 15 s, high intensities were observed in the inferior vena cava, heart, aorta, and kidneys. At 30 s, most of the signal intensity was localized to the kidneys. These distribution patterns were reproduced using 12.6 and 25 mg/kg doses. At 45 s, only signal in the kidneys was detected. CONCLUSION: Hyperpolarized choline imaging with MRI is feasible using a stable-isotope labeled choline analog (CMP1). Nonradioactive imaging of choline accumulation may provide a new investigatory dimension for kidney physiology. J. Magn. Reson. Imaging 2015;41:917-923. (c) 2014 Wiley Periodicals, Inc.

Friday, October 16, 2015

Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with (13)C hyperpolarised NMR


Breukels, V., et al., Direct dynamic measurement of intracellular and extracellular lactate in small-volume cell suspensions with (13)C hyperpolarised NMR. NMR Biomed, 2015. 28(8): p. 1040-8.


Hyperpolarised (HP) (13)C NMR allows enzymatic activity to be probed in real time in live biological systems. The use of in vitro models gives excellent control of the cellular environment, crucial in the understanding of enzyme kinetics. The increased conversion of pyruvate to lactate in cancer cells has been well studied with HP (13)C NMR. Unfortunately, the equally important metabolic step of lactate transport out of the cell remains undetected, because intracellular and extracellular lactate are measured as a single resonance. Furthermore, typical experiments must be performed using tens of millions of cells, a large amount which can lead to a costly and sometimes highly challenging growing procedure. We present a relatively simple set-up that requires as little as two million cells with the spectral resolution to separate the intracellular and extracellular lactate resonances. The set-up is tested with suspensions of prostate cancer carcinoma cells (PC3) in combination with HP [1-(13)C]pyruvate. We obtained reproducible pyruvate to lactate label fluxes of 1.2 and 1.7 nmol/s per million cells at 2.5 and 5.0 mM pyruvate concentrations. The existence of a 3-Hz chemical shift difference between intracellular and extracellular lactate enabled us to determine the lactate transport rates in PC3. We deduced a lactate export rate of 0.3 s(-1) and observed a decrease in lactate transport on addition of the lactate transport inhibitor alpha-cyano-4-hydroxycinnamic acid.

Wednesday, October 14, 2015

Pushing NMR sensitivity limits using dynamic nuclear polarization with closed-loop cryogenic helium sample spinning


Bouleau, E., et al., Pushing NMR sensitivity limits using dynamic nuclear polarization with closed-loop cryogenic helium sample spinning. Chemical Science, 2015.


We report a strategy to push the limits of solid-state NMR sensitivity far beyond its current state-of-the-art. The approach relies on the use of dynamic nuclear polarization and demonstrates unprecedented DNP enhancement factors for experiments performed at sample temperatures much lower than 100 K, and can translate into 6 orders of magnitude of experimental time-savings. This leap-forward was made possible thanks to the employment of cryogenic helium as the gas to power magic angle sample spinning (MAS) for dynamic nuclear polarization (DNP) enhanced NMR experiments. These experimental conditions far exceed what is currently possible and allows currently reaching sample temperatures down to 30 K while conducting experiments with improved resolution (thanks to faster spinning frequencies, up to 25 kHz) and highly polarized nuclear spins. The impressive associated gains were used to hyperpolarize the surface of an industrial catalyst as well as to hyperpolarize organic nano-assemblies (self-assembling peptides in our case), for whom structures cannot be solved using diffraction techniques. Sustainable cryogenic helium sample spinning significantly enlarges the realm and possibilities of the MAS-DNP technique and is the route to transform NMR into a versatile but also sensitive atomic-level characterization tool.

Friday, October 9, 2015

Metabolic response of prostate cancer to nicotinamide phophoribosyltransferase inhibition in a hyperpolarized MR/PET compatible bioreactor


Keshari, K.R., et al., Metabolic response of prostate cancer to nicotinamide phophoribosyltransferase inhibition in a hyperpolarized MR/PET compatible bioreactor. Prostate, 2015. 75(14): p. 1601-9.


BACKGROUND: Metabolic shifts in disease are of great interest for the development of novel therapeutics. In cancer treatment, these therapies exploit the metabolic phenotype associated with oncogenesis and cancer progression. One recent strategy involves the depletion of the cofactors needed to maintain the high rate of glycolysis seen with the Warburg effect. Specifically, blocking nicotinamide adenine dinucleotide (NAD) biosynthesis via nicotinamide phosphoribosyltransferase (NAMPT) inhibition depletes cancer cells of the NAD needed for glycolysis. To characterize this metabolic phenotype in vivo and describe changes in flux with treatment, non-invasive biomarkers are necessary. One such biomarker is hyperpolarized (HP) [1-(13) C] pyruvate, a clinically translatable probe that allows real-time assessment of metabolism. METHODS: We therefore developed a cell perfusion system compatible with HP magnetic resonance (MR) and positron emission tomography (PET) to develop translatable biomarkers of response to NAMPT inhibition in reduced volume cell cultures. RESULTS: Using this platform, we observed a reduction in pyruvate flux through lactate dehydrogenase with NAMPT inhibition in prostate cancer cells, and showed that both HP lactate and 2-[(18) F] fluoro-2-deoxy-D-glucose (FDG) can be used as biomarkers for treatment response of such targeted agents. Moreover, we observed dynamic flux changes whereby HP pyruvate was re-routed to alanine, providing both positive and negative indicators of treatment response. CONCLUSIONS: This study demonstrated the feasibility of a MR/PET compatible bioreactor approach to efficiently explore cell and tissue metabolism, the understanding of which is critical for developing clinically translatable biomarkers of disease states and responses to therapeutics. Prostate 75:1601-1609, 2015. (c) 2015 Wiley Periodicals, Inc.

Wednesday, October 7, 2015

Affinity screening using competitive binding with fluorine-19 hyperpolarized ligands


Kim, Y. and C. Hilty, Affinity screening using competitive binding with fluorine-19 hyperpolarized ligands. Angew Chem Int Ed Engl, 2015. 54(16): p. 4941-4.


Fluorine-19 NMR and hyperpolarization form a powerful combination for drug screening. Under a competitive equilibrium with a selected fluorinated reporter ligand, the dissociation constant (K(D)) of other ligands of interest is measurable using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment, without the need for a titration. This method is demonstrated by characterizing the binding of three ligands with different affinities for the serine protease trypsin. Monte Carlo simulations show that the highest accuracy is obtained when about one-half of the bound reporter ligand is displaced in the binding competition. Such conditions can be achieved over a wide range of affinities, allowing for rapid screening of non-fluorinated compounds when a single fluorinated ligand for the binding pocket of interest is known.

Monday, October 5, 2015

Polarization Transfer from Ligands Hyperpolarized by Dissolution Dynamic Nuclear Polarization for Screening in Drug Discovery


Min, H., G. Sekar, and C. Hilty, Polarization Transfer from Ligands Hyperpolarized by Dissolution Dynamic Nuclear Polarization for Screening in Drug Discovery. ChemMedChem, 2015. 10(9): p. 1559-63.


Nuclear magnetic resonance (NMR) spectroscopy is a valuable technique for ligand screening, because it exhibits high specificity toward chemical structure and interactions. Dissolution dynamic nuclear polarization (DNP) is a recent advance in NMR methodology that enables the creation of non-equilibrium spin states, which can dramatically increase NMR sensitivity. Here, the transfer of such spin polarization from hyperpolarized ligand to protein is observed. Mixing hyperpolarized benzamidine with the serine protease trypsin, a "fingerprint" of enhanced protein signals is observed, which shows a different intensity profile than the equilibrium NMR spectrum of the protein, but coincides closely to the frequency profile of a saturation transfer difference (STD) NMR experiment. The DNP experiment benefits from hyperpolarization and enables observation of all frequencies in a single, rapid experiment. Based on these merits, it is an interesting alternative to the widely used STD experiment for identification of protein-ligand interactions.

Friday, October 2, 2015

Quantitative Structural Constraints for Organic Powders at Natural Isotopic Abundance Using Dynamic Nuclear Polarization Solid-State NMR Spectroscopy


Mollica, G., et al., Quantitative Structural Constraints for Organic Powders at Natural Isotopic Abundance Using Dynamic Nuclear Polarization Solid-State NMR Spectroscopy. Angewandte Chemie, 2015. 127(20): p. 6126-6129.


A straightforward method is reported to quantitatively relate structural constraints based on 13C–13C double-quantum build-up curves obtained by dynamic nuclear polarization (DNP) solid-state NMR to the crystal structure of organic powders at natural isotopic abundance. This method relies on the significant gain in NMR sensitivity provided by DNP (approximately 50-fold, lowering the experimental time from a few years to a few days), and is sensitive to the molecular conformation and crystal packing of the studied powder sample (in this case theophylline). This method allows trial crystal structures to be rapidly and effectively discriminated, and paves the way to three-dimensional structure elucidation of powders through combination with powder X-ray diffraction, crystal-structure prediction, and density functional theory computation of NMR chemical shifts.

Wednesday, September 30, 2015

X-Band DNP Hyperpolarization of Viscous Liquids and Polymer Melts


Neudert, O., et al., X-Band DNP Hyperpolarization of Viscous Liquids and Polymer Melts. Macromol Rapid Commun, 2015. 36(10): p. 885-9.


NMR studies of synthetic polymers and biomacromolecules, which provide insight into the conformation and dynamics of these materials, can benefit strongly from the increased sensitivity offered by dynamic nuclear polarization (DNP) and other hyperpolarizing methods. In this study (1) H DNP nuclear spin hyperpolarization of two polybutadiene samples, representing a supercooled liquid and an entangled polymer melt, is demonstrated at 0.35 T magnetic field strength and at temperatures between -80 and +50 degrees C. Electron spin polarization transfer from the alpha,gamma-bisdiphenylene-beta-phenylallyl radical to the sample nuclei is achieved by the Overhauser and solid effect. DNP signal enhancements are studied, varying the electron spin resonance offset, microwave power, and sample temperature. The influence of spin relaxation times, line widths, and molecular dynamics are discussed. The results show promising, up to 15-fold NMR signal enhancements using noncryogenic temperatures and an inexpensive setup that is less technically demanding than current high-field DNP setups.

Monday, September 28, 2015

Natural Abundance (17)O DNP Two-Dimensional and Surface-Enhanced NMR Spectroscopy


Perras, F.A., T. Kobayashi, and M. Pruski, Natural Abundance (17)O DNP Two-Dimensional and Surface-Enhanced NMR Spectroscopy. J Am Chem Soc, 2015. 137(26): p. 8336-9.


Due to its extremely low natural abundance and quadrupolar nature, the (17)O nuclide is very rarely used for spectroscopic investigation of solids by NMR without isotope enrichment. Additionally, the applicability of dynamic nuclear polarization (DNP), which leads to sensitivity enhancements of 2 orders of magnitude, to (17)O is wrought with challenges due to the lack of spin diffusion and low polarization transfer efficiency from (1)H. Here, we demonstrate new DNP-based measurements that extend (17)O solid-state NMR beyond its current capabilities. The use of the PRESTO technique instead of conventional (1)H-(17)O cross-polarization greatly improves the sensitivity and enables the facile measurement of undistorted line shapes and two-dimensional (1)H-(17)O HETCOR NMR spectra as well as accurate internuclear distance measurements at natural abundance. This was applied for distinguishing hydrogen-bonded and lone (17)O sites on the surface of silica gel; the one-dimensional spectrum of which could not be used to extract such detail. Lastly, this greatly enhanced sensitivity has enabled, for the first time, the detection of surface hydroxyl sites on mesoporous silica at natural abundance, thereby extending the concept of DNP surface-enhanced NMR spectroscopy to the (17)O nuclide.

Friday, September 25, 2015

Feasibility of multianimal hyperpolarized (13) C MRS


Ramirez, M.S., et al., Feasibility of multianimal hyperpolarized (13) C MRS. Magn Reson Med, 2015. 73(5): p. 1726-32.


PURPOSE: There is great potential for real-time investigation of metabolism with MRS and hyperpolarized (HP) (13) C agents. Unfortunately, HP technology has high associated costs and efficiency limitations that may constrain in vivo studies involving many animals. To improve the throughput of preclinical investigations, we evaluate the feasibility of performing HP MRS on multiple animals simultaneously. METHODS: Simulations helped assess the viability of a dual-coil strategy for spatially localized multivolume MRS. A dual-mouse system was assembled and characterized with bench- and scanner-based experiments. Enzyme phantoms mixed with HP [1-(13) C] pyruvate emulated real-time metabolism and offered a controlled mechanism for evaluating system performance. Finally, a normal mouse and a mouse bearing a subcutaneous xenograft of colon cancer were simultaneously scanned in vivo using an agent containing HP [1-(13) C] pyruvate. RESULTS: Geometric separation/rotation, active decoupling, and use of low input impedance preamplifiers permitted an encode-by-channel approach for spatially localized MRS. A precalibrated shim allowed straightforward metabolite differentiation in enzyme phantom and in vivo experiments at 7 Tesla, with performance similar to conventional acquisitions. CONCLUSION: The initial feasibility of multi-animal HP (13) C MRS was established. Throughput scales with the number of simultaneously scanned animals, demonstrating the potential for significant improvements in study efficiency.

Thursday, September 24, 2015

Nanometer-scale water- and proton-diffusion heterogeneities across water channels in polymer electrolyte membranes


Song, J., O.H. Han, and S. Han, Nanometer-scale water- and proton-diffusion heterogeneities across water channels in polymer electrolyte membranes. Angew Chem Int Ed Engl, 2015. 54(12): p. 3615-20.


Nafion, the most widely used polymer for electrolyte membranes (PEMs) in fuel cells, consists of a fluorocarbon backbone and acidic groups that, upon hydration, swell to form percolated channels through which water and ions diffuse. Although the effects of the channel structures and the acidic groups on water/ion transport have been studied before, the surface chemistry or the spatially heterogeneous diffusivity across water channels has never been shown to directly influence water/ion transport. By the use of molecular spin probes that are selectively partitioned into heterogeneous regions of the PEM and Overhauser dynamic nuclear polarization relaxometry, this study reveals that both water and proton diffusivity are significantly faster near the fluorocarbon and the acidic groups lining the water channels than within the water channels. The concept that surface chemistry at the (sub)nanometer scale dictates water and proton diffusivity invokes a new design principle for PEMs.

Monday, September 21, 2015

Characterization and optimization of the visualization performance of continuous flow overhauser DNP hyperpolarized water MRI: Inversion recovery approach


Terekhov, M., et al., Characterization and optimization of the visualization performance of continuous flow overhauser DNP hyperpolarized water MRI: Inversion recovery approach. Magn Reson Med, 2015: p. n/a-n/a.


PURPOSE: Overhauser dynamic nuclear polarization (DNP) allows the production of liquid hyperpolarized substrate inside the MRI magnet bore as well as its administration in continuous flow mode to acquire MR images with enhanced signal-to-noise ratio. We implemented inversion recovery preparation in order to improve contrast-to-noise ratio and to quantify the overall imaging performance of Overhauser DNP-enhanced MRI. METHOD: The negative enhancement created by DNP in combination with inversion recovery (IR) preparation allows canceling selectively the signal originated from Boltzmann magnetization and visualizing only hyperpolarized fluid. The theoretical model describing gain of MR image intensity produced by steady-state continuous flow DNP hyperpolarized magnetization was established and proved experimentally. RESULTS: A precise quantification of signal originated purely from DNP hyperpolarization was achieved. A temperature effect on longitudinal relaxation had to be taken into account to fit experimental results with numerical prediction. CONCLUSION: Using properly adjusted IR preparation, the complete zeroing of thermal background magnetization was achieved, providing an essential increase of contrast-to-noise ratio of DNP-hyperpolarized water images. To quantify and optimize the steady-state conditions for MRI with continuous flow DNP, an approach similar to that incorporating transient-state thermal magnetization equilibrium in spoiled fast field echo imaging sequences can be used. Magn Reson Med, 2015. (c) 2015 Wiley Periodicals, Inc.

Friday, September 18, 2015

Hyperpolarized [ C]ketobutyrate, a molecular analog of pyruvate with modified specificity for LDH isoforms


von Morze, C., et al., Hyperpolarized [ C]ketobutyrate, a molecular analog of pyruvate with modified specificity for LDH isoforms. Magn Reson Med, 2015: p. n/a-n/a.


PURPOSE: The purpose of this study was to investigate 13 C hyperpolarization of alpha-ketobutyrate (alphaKB), an endogenous molecular analog of pyruvate, and its in vivo enzymatic conversion via lactate dehydrogenase (LDH) using localized MR spectroscopy. METHODS: Hyperpolarized (HP) 13 C MR experiments were conducted using [13 C]alphaKB with rats in vivo and with isolated LDH enzyme in vitro, along with comparative experiments using [13 C]pyruvate. Based on differences in the kinetics of its reaction with individual LDH isoforms, HP [13 C]alphaKB was investigated as a novel MR probe, with added specificity for activity of LDHB-expressed H ("heart"-type) subunits of LDH (e.g., constituents of LDH-1 isoform). RESULTS: Comparable T1 and polarization values to pyruvate were attained (T1 = 52 s at 3 tesla [T], polarization = 10%, at C1 ). MR experiments showed rapid enzymatic conversion with substantially increased specificity. Formation of product HP [13 C]alpha-hydroxybutyrate (alphaHB) from alphaKB in vivo was increased 2.7-fold in cardiac slabs relative to liver and kidney slabs. In vitro studies resulted in 5.0-fold higher product production from alphaKB with bovine heart LDH-1, as compared with pyruvate. CONCLUSIONS: HP [13 C]alphaKB may be a useful MR probe of cardiac metabolism and other applications where the role of H subunits of LDH is significant (e.g., renal cortex and brain). Magn Reson Med, 2015. (c) 2015 Wiley Periodicals, Inc.

Wednesday, September 16, 2015

Dynamic nuclear polarization of nucleic acid with endogenously bound manganese


Wenk, P., et al., Dynamic nuclear polarization of nucleic acid with endogenously bound manganese. J Biomol NMR, 2015: p. 1-13.


We report the direct dynamic nuclear polarization (DNP) of 13C nuclei of a uniformly [13C,15N]-labeled, paramagnetic full-length hammerhead ribozyme (HHRz) complex with Mn2+ where the enhanced polarization is fully provided by the endogenously bound metal ion and no exogenous polarizing agent is added. A 13C enhancement factor of epsilon = 8 was observed by intra-complex DNP at 9.4 T. In contrast, "conventional" indirect and direct DNP experiments were performed using AMUPol as polarizing agent where we obtained a 1H enhancement factor of epsilon approximately 250. Comparison with the diamagnetic (Mg2+) HHRz complex shows that the presence of Mn2+ only marginally influences the (DNP-enhanced) NMR properties of the RNA. Furthermore two-dimensional correlation spectra (15N-13C and 13C-13C) reveal structural inhomogeneity in the frozen, amorphous state indicating the coexistence of several conformational states. These demonstrations of intra-complex DNP using an endogenous metal ion as well as DNP-enhanced MAS NMR of RNA in general yield important information for the development of new methods in structural biology.

Monday, September 14, 2015

Successive Stages of Amyloid-beta Self-Assembly Characterized by Solid-State Nuclear Magnetic Resonance with Dynamic Nuclear Polarization


Potapov, A., et al., Successive Stages of Amyloid-beta Self-Assembly Characterized by Solid-State Nuclear Magnetic Resonance with Dynamic Nuclear Polarization. J Am Chem Soc, 2015. 137(25): p. 8294-307.


Self-assembly of amyloid-beta (Abeta) peptides in human brain tissue leads to neurodegeneration in Alzheimer's disease (AD). Amyloid fibrils, whose structures have been extensively characterized by solid state nuclear magnetic resonance (ssNMR) and other methods, are the thermodynamic end point of Abeta self-assembly. Oligomeric and protofibrillar assemblies, whose structures are less well-understood, are also observed as intermediates in the assembly process in vitro and have been implicated as important neurotoxic species in AD. We report experiments in which the structural evolution of 40-residue Abeta (Abeta40) is monitored by ssNMR measurements on frozen solutions prepared at four successive stages of the self-assembly process. Measurements on transient intermediates are enabled by ssNMR signal enhancements from dynamic nuclear polarization (DNP) at temperatures below 30 K. DNP-enhanced ssNMR data reveal a monotonic increase in conformational order from an initial state comprised primarily of monomers and small oligomers in solution at high pH, to larger oligomers near neutral pH, to metastable protofibrils, and finally to fibrils. Surprisingly, the predominant molecular conformation, indicated by (13)C NMR chemical shifts and by side chain contacts between F19 and L34 residues, is qualitatively similar at all stages. However, the in-register parallel beta-sheet supramolecular structure, indicated by intermolecular (13)C spin polarization transfers, does not develop before the fibril stage. This work represents the first application of DNP-enhanced ssNMR to the characterization of peptide or protein self-assembly intermediates.