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The Henryk Niewodniczański INSTITUTE OF NUCLEAR PHYSICS
Polish Academy of Sciences
152 Radzikowskiego str., 31-342 Kraków, Poland
www.ifj.edu.pl/reports/2014/
Kraków, December 2014
Report No. 2077/AP
XLV Polish Seminar on Nuclear Magnetic Resonance
and Its Applications. Kraków, 1-2 December 2014
ABSTRACTS
Organized by:
The H. Niewodniczański Institute
of Nuclear Physics PAN, Kraków
and
Committee of Physics,
Polish Academy of Sciences
Organizing Committee:
Members:
Ż. Bartel A. Osiak
A. Birczyński A. Osip
J. Blicharski Z. Olejniczak
B. Błasiak W. Rutkowski
K. Byk W. Piędzia
M. Jabłońska T. Skórka
K. Jasiński G. Stoch
K. Kalita U. Tyrankiewicz
Z.T. Lalowicz /Chairman/ W. P. Węglarz /V-Chairman/
M. Noga /Secretary/
Sponsors:
BRUKER-POLSKA Sp. z o.o,
Wydział III Nauk Ścisłych i Nauk o Ziemi POLSKIEJ AKADEMII NAUK
Addresses of the sponsors:
BRUKER POLSKA SP. Z O.O
mgr W. Leszczyński
ul. Budziszyńska 69
60-179 Poznań
tel. (061) 868 90 08
fax. (061) 868 90 96
e-mail: [email protected]
www.bruker.poznan.pl
Wydział III Nauk Ścisłych I Nauk o Ziemi
POLSKIEJ AKADEMII NAUK Pl. Defilad 1
00-901 Warszawa
i
CONTENTS
1. FREEZING RESISTANCE OF CRYPTOBIOTIC POLYPEDILUM
VANDERPLANKI LARVA BY 1H-NMR
Ewelina Baran, Piotr Nowak, Hubert Harańczyk, Stanisław Knutelski, Kazimierz
Strzałka, Takashi Okuda
1
2. MOLECULAR DYNAMICS OF METHANOL CONFINED IN NANOSCALE
CAGES OF FAUJASITES. A DEUTERON NMR INVESTIGATION
Z.T. Lalowicz, A. Birczyński, G. Stoch, K. Góra-Marek, J. Datka
2
3. 1H NMR BASED METABOLOMIC APPROACH TO MONITORING OF THE
HEAD AND NECK CANCER TREATMENT RESPONSE
Łukasz Boguszewicz, Agata Hajduk, Jolanta Mrochem-Kwarciak, Agnieszka
Skorupa, Mateusz Ciszek, Krzysztof Składowski, Maria Sokół
3
4. LIVER CONDITION IN MICE MODEL OF ACUTE HEPATITIS BASED ON
MAGNETIC - RESONANCE IMAGING
Katarzyna Byk, Żaneta Bartel, Krzysztof Jasiński, Bogusław Tomanek, Tomasz
Skórka
4
5. METABOLIC HALLMARKS OF VISUAL CORTEX DEGENERATION IN
MOUSE MODEL OF GLAUCOMA (DBA/2J MICE) REVEALED BY IN
VIVO PROTON MAGNETIC RESONANCE SPECTROSCOPY
Michał Fiedorowicz, Jarosław Orzeł, Bartosz Kossowski,
Marlena Wełniak-Kamińska, Piotr Bogorodzki, Paweł Grieb
5
6. IS HIGH-FAT HIGH-CARBOHYDRATE DIET (HFCD)
NEUROPROTECTIVE? A MAGNETIC RESONANCE IMAGING STUDY IN
WISTAR RATS
Stefan Gaździński, Zuzanna Setkowicz, Joanna Osoba, Karolina Karwowska,
Piotr Majka, Jarosław Orzeł, Bartosz Kossowski, Piotr Bogorodzki, Marlena
Kamińska, Michał Fiedorowicz
6
7. VOXEL-WISE ANALYSES OF HIGH-FAT HIGH-CARBOHYDRATE DIET
ON BRAIN STRUCTURE IN WISTAR RATS
Piotr Majka, Zuzanna Setkowicz, Joanna Osoba, Karolina Karwowska, Jarosław
Orzeł, Bartosz Kossowski, Piotr Bogorodzki, Marlena Kamińska, Michał
Fiedorowicz, Stefan Gaździński
7
8. EFFECTS OF HIGH-FAT HIGH-CARBOHYDRATE DIET ON WHITE
MATTER INTEGRITY: A DIFFUSION TENSOR IMAGING STUDY IN
WISTAR RATS
Stefan Gaździński, Andrzej Gaździński, Zuzanna Setkowicz, Joanna Osoba,
Karolina Karwowska, You Zhang, Jarosław Orzeł, Bartosz Kossowski, Piotr
Bogorodzki
9
9. HR MAS NMR METABOLIC PROFILES OF CARDIOMYOCYTES AFTER
RADIATION EXPOSURE
Michalina Gramatyka, Agnieszka Skorupa, Mateusz Ciszek, Łukasz
10
ii
Boguszewicz, Maria Sokół
10. THEORETICAL CALCULATIONS OF 13C NMR CHEMICAL SHIFTS OF
BROMINE-SUBSTITUTED CARBON ATOMS
Adam Gryff-Keller, Dominika Kubica, Artur Wodyński
11
11. H-NMR IN LIVING ORGANISMS, TISSUES, AND OTHER BIOLOGICAL
SYSTEM RESISTANT TO DRASTIC DEHYDRATION OR TO LOW
TEMPERATURES
Hubert Harańczyk
12
12. REMARKS ON THE FREE-APPROACH MODELS
Łukasz Jaremko, Mariusz Jaremko, Michał Nowakowski, Andrzej Ejchart
14
13. COMPARISON OF MAGNETIC RESONANCE IMAGING OF THE MOUSE
BRAIN IN VIVO USING DIFFERENT TYPES OF RF COILS AT 9.4 T
J. Jasieniak, W. Piedzia, K. Jasinski, W.P. Weglarz
16
14. NMR STUDY OF P(MEO2MA) POLYMER NETWORKS
J. Jenczyk, S. Kadłubowski, M. Olejniczak, M. Kozanecki, S. Jurga
17
15. QUANTITATIVE MRI IN STUDYING WHITE MATTER DAMAGE
FOLLOWING SPINAL CORD INJURY
Piotr Kozłowski
18
16. NEW HIGH DIMENSIONALITY NMR EXPERIMENTS FOR
BIOMOLECULES
Wiktor Koźmiński
20
17. INTRAMOLECULAR INTERACTION OF HYBRID OF URIDINE AND
STILBENE DERIVATIVE
Hanna Krawczyk, Przemysław Szczeciński
21
18. IMAGING METHODS IN RESEARCH AND DEVELOPMENT PROCESS OF
GENERIC MODIFIED RELEASE MATRIX TABLETS
Piotr Kulinowski, Krzysztof Woyna-Orlewicz, Gerd-Martin Rappen, Dorota
Haznar-Garbacz, Władysław P. Węglarz, Przemysław P. Dorożyński
22
19. MOLECULAR MODELING OF SINGLE WALL CARBON NANOTUBE
(SWCNT) CHEMICAL SHIFT DUE TO ADDITION OF DIATOMICS
Teobald Kupka, Marzena Nieradka, Leszek Stobiński, Jakub Kaminský
24
20. MOLECULAR MODELING OF CHEMICAL SHIFT OF ATOMS, SMALL
AND LARGE MOLECULES
Teobald Kupka, Michał Stachów, Marzena Nieradka, Klaudia Radula-Janik,
Roksana Wałęsa, Aneta Buczek, Małgorzata Broda
25
21. BADANIA SPEKTROSKOPOWE PT(II) Z 7,8-BENZOCHINOLINĄ ORAZ 2-
FENOKSYPIRYDYNĄ
Daria Niedzielska, Leszek Pazderski
26
iii
22. FREEZING AND DRYING RESISTANCE OF ANTARCTIC
TURGIDOSCULUM COMPLICATULUM THALLI AS OBSERVED BY 1H-
NMR METHODS
Magdalena Bacior, Piotr Nowak, Paulina Kijak, Ewelina Baran, Hubert
Harańczyk, Maria A. Olech
27
23. THE SOLUTION STRUCTURE OF THE MANEC-TYPE DOMAIN FROM
HEPATOCYTE GROWTH FACTOR INHIBITOR 1 REVEALS AN
UNEXPECTED PAN/APPLE DOMAIN-TYPE FOLD
Michał Nowakowski, Zebin Hong, Chris Spronk, Steen V. Petersen, Jan S.
Pedersen, Wiktor Koźmiński, Frans A.A. Mulder, Jan K. Jensen
28
24. ATLAS-BASED AUTOMATIC BRAIN MORPHOMETRY APPLIED TO
DBA/2J MOUSE MODEL OF GLAUCOMA
Jarosław Orzeł, Michał Fiedorowicz, Bartosz Kossowski,
Marlena Wełniak-Kamińska, Piotr Bogorodzki, Paweł Grieb
29
25. MR TAGGING FOR EVALUATION OF MECHANICAL PROPERTIES OF
FATTY LIVER TISSUE
Anna Osiak, Krzysztof Jasiński, Paweł T. Jochym, Edyta Maślak, Tomasz Skórka
30
26. MRI-BASED METHOD FOR THE IN VIVO ASSESSMENT OF
ENDOTHELIAL STATE IN MURINE MODELS
Anna Osip, Krzysztof Jasiński, Żaneta Bartel, Tomasz Skórka, Stefan Chłopicki
31
27. 23Na NMR STUDY OF Al- AND Ga- NANOPOROUS NATROLITES
Mateusz Paczwa, Marcin Olszewski, Nikolaj Sergeev
Aleksey.A. Sapiga, Aleksey.V. Sapiga
33
28. 2D AND 3D CP-VC AS TOOLS FOR DYNAMICS STUDY
Piotr Paluch, Tomasz Pawlak, Julien Trébosc, Tatyana Polenova, Jean-Paul
Amoureux, Marek J. Potrzebowski
35
29. EVALUATION OF THE RELAXATION AND THE IMAGING PROPERTIES
OF SPIO LOADED NANOCAPSULES AT 9.4 T
P. Piechota, K. Szczepanowicz, P. Warszyński, W. P. Węglarz
36
30. MR IMAGING OF THE MOUSE BRAIN USING CRYO-COIL AT 9.4 T -
HISTOLOGY IN VIVO?
W.Piedzia, N. Bock, K. Jasinski, K. Kalita, G. Stanisz, W.P. Weglarz
37
31. SUPERCONDUCTING DETECTION COIL FOR 0.2 T MRI SYSTEM
Bartosz Proniewski, Henryk Figiel
39
32. PARA HYDROGEN INDUCED POLARIZATION OF PYRIDINE-LABELLED
OLIGOPEPTIDES
Tomasz Ratajczyk
40
33. APPLICATION OF MICRO-MRI TECHNIQUES IN THE EVALUATION OF 41
iv
MUSCLE DEGENERATION AND REPAIR PROCESSES AFTER FEMORAL
ARTERY OCCLUSION IN MICE
Agnieszka Skorupa, Mateusz Ciszek, Łukasz Boguszewicz, Tomasz Cichoń,
Ryszard Smolarczyk, Stanisław Szala, Maria Sokół
34. APPLICATION OF NMR RELAXATION MEASUREMENTS TO THE
STUDY OF OXIDATION PROCESSES IN BIOLOGICAL SYSTEMS
Dorota Wierzuchowska, Lech Skórski, Barbara Blicharska
42
35. CHEMICAL EXCHANGE SATURATION TRANSFER (CEST). FROM AN
AGAR TO A MAN
Greg J. Stanisz
43
36. NMR TOP SYGNALS OF THE 27Al IN SOLID SOLUTIONS BASED ON THE
YAG CRYSTAL
Piotr Stępień, Marcin Olszewski, Nikolaj Sergeev, Bohdan Padlyak
44
37. SPI IMPLEMENTATION FOR 4.7T SYSTEM
Grzegorz Stoch
46
38. DYNAMIC EFFECTS IN SINGLE CRYSTAL OF 9,10-
DIMETHYLTRIPTYCENE-D12 ON BASIS OF PROTON NMR SPECTRA
Piotr Bernatowicz, Tomasz Ratajczyk, Alexander Shkurenko, Bohdan
Kamienski, Sławomir Szymański
47
39. DIVERSE DYNAMICS OF WATER MOLECULES CONFINED IN
FAUJASITES. DEUTERON NMR INVESTIGATION
A. Szymocha, Z.T. Lalowicz, Birczynski, K. Gora-Marek
49
40. CHLORINS - SYNTHESIS AND NMR SPECTROSCOPY STUDIES
Justyna Śniechowska, Piotr Paluch, Marek J. Potrzebowski
50
41. NMR SPECTROSCOPY OF SERUM LIPID EXTRACTS OF SARCOIDOSIS
PATIENTS
Toczylowska Beata, Jastrzebski Dariusz, Zieminska Elzbieta, Zieleznik Karolina,
Zebrowska Aleksandra, Ziora Dariusz, Mierzejewska Aneta, Kozielski Jerzy
51
42. EYE MORPHOLOGY QUANTITATED BY MAGNETIC RESONANCE
IMAGING IN MICE
Marlena Wełniak-Kamińska, Tomasz Chorągiewicz, Michał Fiedorowicz,
Jarosław Orzeł, Piotr Bogorodzki, Paweł Grieb
53
43. THE CYTOSTATIC AGENT AS A CONTRAST AGENT FOR MRI
Beata Wereszczyńska, Tomasz Zalewski, Magdalena Hałupka-Bryl, Marek
Kempka, Stefan Jurga
54
44. USEFULNESS OF MR SEQUENCES: DTI AND 3D ASL IN RARE CHILD
BRAIN TUMOR BASED ON A MR BRAIN CHILD EXAM BEFORE AND
AFTER SURGERY
Magdalena Wicher, Magdalena Machnikowska-Sokołowska, Anna Plechta,
55
v
Katarzyna Zymella, Dominika Wieja-Błach, Marcin Basiak, Marek Konopka
45. EXOGENOUS SILK PROTEIN AND SLES EFFECT ON PROPERTIES OF
HYDRATED HAIR BY 1H-NMR AND SORPTION ISOTHERM
D. Zalitacz, P. Nowak, A. Ciułkowska, K. Pieńkowska, H. Harańczyk
57
46. 4D NMR EXPERIMENT FOR PHOSPHORYLATION STUDIES OF IDPS
Szymon Żerko, Gerald Platzer, Robert Konrat, Wiktor Koźmiński
58
47. THE ZN IONS AS IMPORTANT FACTOR REGULATED UBIQUITIN-
ACTIVATING PROCESS. STRUCTURAL STUDIES OF THE PEPTIDE
DERIVED FROM CYSTEINE CATALYTIC HALF-DOMAIN (SCCH) OF
MOUSE E1 ENZYME
Ilona Marszalek, Arkadiusz Bonna, Wojciech Bal, Igor Zhukov
59
FREEZING RESISTANCE OF CRYPTOBIOTIC Polypedilum vanderplanki LARVA
by 1H-NMR
Ewelina Baran, Piotr Nowak, Hubert Harańczyk, Stanisław Knutelski, Kazimierz Strzałka, Takashi Okuda
Jagiellonian University, Cracow, Poland 1Institute of Physics, 31-059 Cracow, Reymonta St. 4
2Institute of Zoology, Jagiellonian University, Cracow, Poland 3National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan
E-mail: [email protected]
African chironomid Polypedilum vanderplanki is its larval form is the largest multicellular
animal capable to survive anhydrobiosis process [1]. It populates temporary basins In Northern
Nigeria and Uganda. Its maggot may survive deep dehydration during the prolonged dry season
[2].
Dehydrated larvas of the chironomid Polypedilum vanderplanki were grown in a laboratory
conditions [3]. We monitored the rehydration kinetics, the sorption isotherm, and 1H-NMR
spectra. We analysed water content in dehydrated larva, a number and an arrangement of water
binding sites on inner surfaces of its organism, and formation of tightly and of loosely bound
water fractions at different temperatures. 1H-NMR spectra were recorded on a Bruker Avance III spectrometer (Bruker Biospin), operating
at the resonance frequency 300 MHz (B0 = 7 T). The pulse length was π/2 = 2.1 ms.
For low values of the relative humidity, the gaseous phase hycdration courses revealed the
anomalous form, which presumably may be caused by the growths of bacterias in the testins of
specimen. For higher humidities the gaseous phase hydration courses show two phases of bound
water, namely (i) very tightly bound water, and (ii) tightly bound water. The sorption isotherm is
sigmoidal in form, much better fitted using Dent model than BET-approach. The relative mass of
water bound to primary binding sites was ΔM/m0 = 0.046. 1H-NMR spectra show superposition of the solid component well fitted by Gaussian function (ν ≈
44 kHz), coming from protons of dried tissues of P. vanderplanki; and Lorentzian line
component (with ν ≈ 1.6 kHz) coming from water tightly bound on inner and outer surfaces of
solid tissue. This behaviour of bound water resembles that for Coleoptera alytron [4] or in DNA-
CTMA complex [5].
1H-NMR spectra temperature dependence show the gradual immobilization of bound water
fraction without the formation of the ice crystallites, as it was detected in thalli of Antarctic
lichenized fungi experienceed very low temperature, in vivo [6]. The detected by us contribution
of liquid signal in dry form of P. vanderplanki maggot is higher as it was expected.
References M. Sakurai, T. Furuki, K. Akao, D. Tanaka, Y. Nakahara, T. Kikawada, M. Watanabe, T. Okuda,
vol. 105 no. 13 , 5093–5098.
R. Cornette, Y. Kanamori, M. Watanabe, Y. Nakahara, O. Gusev, K. Mitsumasu, K. Kadono-
Okuda, M. Shimomura, K. Mita,T. Kikawada, T. Okuda., J Biol Chem. 2010 Nov
12;285(46):35889-99.
T. Okuda. O. Gusev, 2012, 121-138
H. Harańczyk, P. Nowak, , M. Florek, S. Knutelski, APP, 2011
H. Harańczyk, J. Kobierski, J. Nizioł, E. Hebda, J. Pielichowski, D. Zalitacz, M. Marzec, and A.
El-Ghayoury, Journal of Applied Physics,113, 044702 (2013)
H. Harańczyk, P. Nowak, M. Bacior, Ł. Pater, M.A. Olech, APP, 2011
H. Harańczyk, P. Nowak, M. Bacior, M. Lisowska, M. Marzec, M. Florek, M.A. Olech, Antarctic
Science, Volume 24, Issue 04, August 2012, pp 342-352
The research was carried out with the equipment purchased thanks to the financial support of the
European Regional Development Fund in the framework of the Polish Innovation Economy
Operational Program (POIG.02.01.00-12-023/08). 1
MOLECULAR DYNAMICS OF METHANOL CONFINED IN NANOSCALE
CAGES OF FAUJASITES. A DEUTERON NMR INVESTIGATION.
Z.T. Lalowicza, A. Birczyńskia, G. Stocha, K. Góra-Marekb, J. Datkab
aH. Niewodniczański Institute of Nuclear Physics PAS, Radzikowskiego 152, Kraków, Poland
bFaculty of Chemistry, Jagellonian University. Ingardena 3, 30-060 Kraków, Poland
Nuclear magnetic resonance (NMR) provides means to investigate molecular
dynamics at every state of matter. Molecules confined in nanoscale cages of zeolites represent
a particularly interesting system. Features of molecular dynamics characteristic for the gas
phase, liquid-like layers and immobilized molecules were observed in the temperature range
from 300K down to 20K. Narrow lines were observed at high temperature indicating basically
isotropic reorientation. Spin-lattice relaxation rates provide evidence for a transition from
translational diffusion to isotropic reorientation as the main mechanism of relaxation for
molecules confined in nanoscale zeolite cages for D2, CD4 and CD3OD. Other molecules like
D2O, ND3, and (CD3)2CO are strongly bonded, both mutually and to zeolite framework, and
exhibit a much more restricted diffusion.
Deuteron spin-lattice relaxation was measured for methanol-d4 molecules confined in
zeolite NaX and NaY cages [1]. Experimental evidence was given for the formation of
trimers, their existence was so far proposed only by theory. The conclusion was based on
observation of different relaxation rates for methyl and hydroxyl deuterons undergoing a
common dynamics. A change in the slope of the temperature dependence of both relaxation
rates indicates a transition from the relaxation dominated by translational motion to prevailing
contribution of reorientation at 222K. Trimers undergoing isotropic reorientation disintegrate
and separate methanol molecules become localized on adsorption centers at 169.5K and
153.8K for NaX and NaY, respectively as indicated by extreme broadening of deuteron NMR
spectra. The transition temperature, higher for NaX, indicates the dominating role of the
hydrogen bonding to framework oxygen, contradicting common assumption of preferred
adsorption on sodium cation. NMR spectra at low temperature are consistent with the model
in which molecules are bonded at two positions: horizontal (methanol oxygen bonded to
sodium cation) and vertical (hydrogen bonding of hydroxyl deuteron of methanol to zeolite
framework oxygen). Molecules at vertical position remain localized up to high temperature.
Mobility of single methanol molecules was observed for a lower loading (86 molecules/uc) in
NaX. A direct transition from diffusion to localization was observed at 190K in this case.
The magnetization recovery can be fitted quite accurately by three exponentials in the
low temperature phase of localized molecules. Therefore a new method is introduced for
analyzing deuteron spin-lattice relaxation in molecular systems with different mobility and a
broad distribution of activation energies and correlation times [2]. A Gaussian distribution of
the activation energy was assumed. Three parameters: the mean activation energy, the
distribution width and the pre-exponential factor in the Arrhenius equation were calculated.
The obtained parameters characterize the methyl and hydroxyl mobility of the methanol
molecules at two different locations.
[1] Z. T. Lalowicz, G. Stoch, A. Birczyński, M. Punkkinen, E. E. Ylinen, M. Krzystyniak,
K. Góra-Marek, J. Datka, Solid State Nucl. Magn. Reson. 45-46 (2012) 66-74
[2] G. Stoch, E. E. Ylinen, A. Birczyński, Z. T. Lalowicz, K. Góra-Marek, M. Punkkinen,
Solid State Nucl. Magn. Reson. 49-50 (2013) 33-41
The project was generously supported by the National Science Centre, Grant No. N N202
127939 during 2010-2013.
2
1H NMR BASED METABOLOMIC APPROACH TO MONITORING OF
THE HEAD AND NECK CANCER TREATMENT RESPONSE
Łukasz Boguszewicz1, Agata Hajduk2, Jolanta Mrochem-Kwarciak3, Agnieszka Skorupa1,
Mateusz Ciszek1, Krzysztof Składowski2, Maria Sokół1.
1 Department of Medical Physics, Maria Sklodowska-Curie Memorial Cancer Center and
Institute of Oncology, Gliwice Branch, Poland. 2 I Radiotherapy Clinic, Maria Sklodowska-Curie Memorial Cancer Center and Institute of
Oncology, Gliwice Branch, Poland. 3 Analytics and Clinical Biochemistry Department, Maria Sklodowska-Curie Memorial
Cancer Center and Institute of Oncology, Gliwice Branch, Poland.
Introduction and methodology
The response of organisms to anticancer treatment is reflected in morphological and functional
changes in treated volume as well as systemic and alterations. It may be also assessed by analyzing
alterations in composition and concentrations of metabolites in body fluids, inter alia blood serum.
The studied group consisted of 77 patients (median age 59) treated with radical radiotherapy or
chemoradiotherapy in case of head and neck cancer (HNC). Serum samples were collected before or
during the first days of treatment and after the treatment or within the last three days of treatment.
During the treatment patients underwent weekly medical (laryngological and endoscopic, sonographic
of neck lymphatic system) examinations, laboratory blood tests and acute radiation syndrome (ARS)
evaluation.
Serum samples were measured on 400.13 MHz Bruker Avance III spectrometer using NOESY,
CPMG and diffusion edited sequences with water presaturation. Acquired spectra were referenced to
alanine signal and bucketed over the region 9.0 – 0.5 ppm with a bucket size 0.002 ppm.
Multivariate projection techniques: principal component analysis, non-orthogonal and orthogonal
partial least squares discriminant analysis and regression were exploited in order to find metabolic
patterns reflecting response to treatment as well as to find correlations with available clinical and
laboratory data.
Results and discussion
The directions of the highest variance in the NMR spectra are strongly influenced by the treatment
itself but are independent of the treatment type (radiotherapy or chemoradiotherapy) and duration.
Serum metabolic profiles show significant changes around 45th day after the start of the treatment:
increased 3-hydroxybutyrate (3HBT) levels became significantly lower while signals from lipids and
fatty acids start to increase. 3HBT can be used as an energy source (when blood glucose is low) for
high-energy demanding processes connected to healing acute radiation syndrome and tumor necrosis
cleaning in irradiated place.
3
Liver condition in mice model of acute hepatitis based on magnetic –
resonance imaging Katarzyna Byk1, Żaneta Bartel1, Krzysztof Jasiński1, Bogusław Tomanek1,2, Tomasz Skórka1 1Department of Magnetic Resonance Imaging, Institute of Nuclear Physics, Polish Academy
of Sciences, Krakow, Poland 2Faculty of Medicine & Dentistry, Department of Oncology, Medical Physics Division,
University of Alberta, 3-12 University Terrace, 8303 - 112 Street NW, Edmonton, AB T6G
2T4,Canada
Purpose: The aim of this study was to prepare a mice model of acute hepatitis and assessing
the influence of this disease on liver metabolism.
Materials and methods: 15 BALB/c mice weighting (20.5 ± 1.5)g were divided into 2
groups based on concanavalin A (ConA, Sigma-Aldrich, USA) dose: 1 - control (8 animals)
and 2 - 8mg/kg ConA dose administrated 24hrs before experiment (7 animals). ECG,
temperature and respiratory were monitored (SA Inc., Stony Brook, NY). The experiment was
conducted on Bruker 9.4T magnet (Ettlingen, Germany). Anatomical (TurboRARE
sequence), perfusion (FAIR_Epi sequence) and contrast enhanced dynamic imaging
(IntraGATE sequence) were performed. Gadolinium based contrast (Primovist, Bayer
Schering Pharma AG, Germany) was injected into tail vain. Data were analyzed using
empirical mathematical modelling [1], texture [2] and fractal [3] analyses. Analyses were
performed with the use of ImageJ (NIH, USA), OriginPro (OriginLab Corporation, USA) and
Statistica (StatSoft, USA) software with the p-value equal to 0.05.
Results: Animals from ConA group lost weight from 20.93 g to 19.86 g during 24 hrs while
control animals weight remained stable. Peak of maximal enhancement after contrast injection
appeared later in ConA group (9.7 ± 1.7) min than in control group (5.9 ± 1.1) min. The
elimination half-life of enhancement increased from (32.3 ± 6.9) min in control group to (66 ±
11) min in ConA group. Perfusion in diseased group was equal to (61.9 ± 6.4) ml/min/100g
and was reduced in comparison to healthy group (95.5 ± 2.6) ml/min/100g.
Discussion: The parameters extracted from empirical mathematical modelling are useful for
evaluating acute hepatitis in created mice model. The elimination half-life of enhancement
was significantly increased in diseased group suggesting lesions and weakened metabolism of
liver and the disruption of clearance function. Decrease in mice weight after ConA injection
implied the digestive problems following the damaged livers. Blood perfusion was limited in
diseased group pointing to liver tissue lesions.
Acknowledgements: This work was supported by the European Regional Development Fund
from European Union (grant coordinated by JCET-UJ, No WND-POIG.01.01.02-00-069/09-
00).
References: [1] - Fan, X., M. Medved, J. N. River, et al. “New model for analysis of dynamic
contrast‐enhanced MRI data distinguishes metastatic from nonmetastatic transplanted rodent
prostate tumors”. Magnetic Resonance in Medicine 2004:51(3):487-494.
[2] - Cross, S. S. “Fractals in pathology.” The Journal of Pathology 1999:182(1):1-8.
[3] - Jirak, D., M. Dezortova, P. Taimr and M. Hajek. “Texture analysis of human liver.”
Journal of Magnetic Resonance Imaging 2002:15(1):68-74
4
Metabolic hallmarks of visual cortex degeneration
in mouse model of glaucoma (DBA/2J mice)
revealed by in vivo proton magnetic resonance spectroscopy
Michał Fiedorowicz (1), Jarosław Orzeł (1,2), Bartosz Kossowski (1,2),
Marlena Wełniak - Kamińska (1), Piotr Bogorodzki (1,2), Paweł Grieb (1)
(1) Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw
(2) Faculty of Electronics and Information Technology, Warsaw University of Technology
Objective: Glaucoma, a neurodegenerative disease of optic nerve and retinal ganglion cells
extends beyond retina and affects extra-retinal vision-related brain structures, including visual
cortex. The aim of this study was to indicate the hallmarks of degeneration within visual
cortex in advanced stage mouse model of glaucoma (DBA/2J).
Methods: Aged DBA/2J mice (14 months, n=10) and age matched C57Bl/6 mice (n=10) were
anaesthetized with isoflurane (4% in oxygen – induction, 1.5-2% - maintenance) and placed
in 7T small animal-dedicated magnetic resonance tomograph (BioSpec 70/30USR; Bruker
BioSpin, Ettlingen, Germany). T2-weighted images were obtained in order to precisely
position two distinct voxels of interests (VOIs) corresponding to visual cortex (5x2x1.2 mm3)
and frontal cortex (4x2x1.5mm3). Local shimming procedures (FASTMAP) were repeated for
each voxel followed by manual water suppression procedure (VAPOR). The unsuppressed
water line width was typically maintained at around 10-15 Hz. Spectra were obtained with
PRESS sequence (TR=2000ms, TE=20ms, NA=1024, Scan time=34min). The data were
analyzed with LCModel software (Stephen Provencher Inc, Oakville, Ontario, Canada). Metabolite concentrations are reported as ratios to total creatine (sum of creatine and
phosphocreatine, tCr). Spectra of poor quality (SNR ratio < 15) were excluded from further
analysis. Statistical analysis was performed with U Mann-Whitney test.
Results: Glutamate to tCr ratio was significantly higher (P<0.05) in the visual cortex of
DBA/2J mice than in the control mice, while no significant difference was observed in the
frontal cortex. Glutamine to tCr ratio was lower in DBA/2J than in C57Bl/6 mice in both
VOIs (P<0.05). Taurine to tCr ratio was lower in DBA/2J mice than in controls in the visual
cortex (P<0.001) but not in the frontal cortex. No significant differences were detected for N-
acetylaspartate and total choline.
Conclusion: Aged DBA/2J display complex picture within visual cortex related to the
ongoing degenerative process. This neurodegeneration could be related to excitotoxicity that
was indicated by increased glutamate levels.
The study was supported by National Science Centre grant No. 2012/07/D/NZ4/04199.
5
Is High-Fat High-Carbohydrate Diet (HFCD) Neuroprotective? A Magnetic
Resonance Imaging Study in Wistar Rats.
Stefan Gaździński1, Zuzanna Setkowicz2, Joanna Osoba2, Karolina Karwowska2,
Piotr Majka3, Jarosław Orzeł4, Bartosz Kossowski4, Piotr Bogorodzki4, Marlena Kamińska4,
Michał Fiedorowicz4
1Military Institute of Aviation Medicine, Warsaw, 2Jagiellonian University, Krakow, 3Nencki
Institute of Experimental Biology, 4Mossakowski Medical Research Centre Polish Academy of
Sciences, Warsaw, Poland
Introduction: Obesity was associated with accelerated aging and elevated risk of
neurodegenerative diseases. In animal models, high-fat high-carbohydrate diet (HFCD) is
commonly used to induce obesity. We hypothesized that HFCD will lead to poorer memory,
smaller hippocampi and lower concentrations of brain metabolites in hippocampi, which are
predictors of neurodegenerative diseases both in humans and in laboratory animals.
Methods: Twenty five male Wistar rats were put on HFCD (~35% fat, ~35% carbohydrates)
on their 55th day of life, while 25 control male rats (CON) remained on chow. Both groups
underwent memory tests in 8-arm radial maze at 3rd, 6th, and 9th month. At one year, all
animals underwent MRI to evaluate hippocampal volumes and 1H magnetic resonance
spectroscopy at 7T.
Results: HFCD rats consumed slightly more calories than CON, but less proteins. However,
their protein intake was within recommended amounts. Levels of sugar and ketone bodies
were within healthy norms in both groups; however, numerically they were higher in the
HFCD group.
Contrary to our hypotheses, HFCD rats had better scores of memory than CON throughout the
experiment. At one year, their hippocampi were by 3% larger than in CON (p=0.05), whereas
concentration of N-acetylo-aspartate (NAA, marker of neuronal viability) was 8% higher
(p=0.01).
Conclusions: The results do not support the thesis that HFCD per se leads to degeneration of
the nervous system. On the contrary, they consistently suggest that HFCD enhances memory
and slows aging. More research is needed to pinpoint the mediating factors.
Support: Polish National Science Centre (2011/03/B/NZ4/03771) to Stefan Gazdzinski.
6
Voxel-wise analyses of high-fat high-carbohydrate diet on brain structure
in Wistar rats
Piotr Majka1, Zuzanna Setkowicz2, Joanna Osoba2, Karolina Karwowska2, Jarosław Orzeł3,
Bartosz Kossowski3, Piotr Bogorodzki3, Marlena Kamińska3, Michał Fiedorowicz3,
Stefan Gaździński4
1 Nencki Institute for Experimental Biology, Polish Academy of Sciences, Warsaw, Poland, 2Department of Neuroanatomy, Jagiellonian University, Krakow, Poland, 3Mossakowski
Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland, 4Military Institute of
Aviation Medicine, Warsaw, Poland
Introduction: Smaller hippocampal volumes and extensive body fat accumulaton in midlife
are risk factors for developing Alzheimer’s disease. Our ROI analysis demonstrated that high
fat high carbohydrate diet lead to larger hippocampal volumes in Wistar rats, without
significant changes in cortical volume. Here, we applied voxel-wise analysis to determine
focal changes in brain tissue structure. Furthermore, we compared the effects of template
selection (Valdés-Hernández et. al. [1] template vs. study specific template) on the results.
Methods: Twenty five male Wistar rats were put on HFCD (~40% fat, ~40% carbohydrates,
~7% proteins) on their 55th day of life, while 25 control male rats (CON) remained on chow.
Structural T2-weighted TurboRARE (TR/TE=4700/30ms, RARE factor=4,
resolution=125x125x500μm, no gap, NEX=7, TA=27 min) acquired on Bruker BioSpin
working at 7T, , with a transmit cylindrical radiofrequency coil (15 cm inner diameter) and a
receive-only coil array (2x2 elements) positioned over the animal’s head. Eighteen datasets
acquired for CON, 18 datasets for HFCD selected for analyses. Images resampled to isotropic
resolution of 125μm/vox and processed with N4 algorithm to correct for intensity
inhomogenity. Image of each specimen was registered into the Valdés-Hernández et. al. [1]
template or study-specific template using SyN algorithm [2] resulting in a series of
deformation field. Jacobian determinant of each deformation field was then computed and
modulated with a gray matter probability, blurred with Gaussian filter of 250μm. Significance
of differences between CON and HFCD was determined with two-Sample unpaired T-test.
Threshold-Free Cluster Enhancement permutation method [3] was used to threshold t-maps
(FSL-randomise software). 10,000 permutations were used in tests and p=0.05 was chosen as
a significance threshold.
Results: Hippocampal volume are larger in HFCD-fed rats than in controls, especially in
hippocampal CA1 field, but also in surrounding cortical areas, regardless of used template.
Analysis with study specific template does not show regions of smaller volumes in HFCD fed
group compared to CON.
Conclusions: The results of voxel-wise comparisons not only confirm our ROI findings of
larger hippocmpal volumes in HFCD fed rats, but also point to focal volume increases in
temporal association cortex and ectorhinal cortex. Our results do not support the thesis that
HFCD per se leads to degeneration of the nervous system. Moreover, as CA1 is selectively
7
affected by neurodegenerarative processes in Alzheimer’s disease, our results seem to be of
functional significance.
References: 1. Valdes-Hernandez PA., Frontiers in Neuroinformatics,
November2011|Volume5| Article26, doi: 10.3389/fninf.2011.00026. 2. Avants, BB., et al.,
(2011) NeuroImage, 54(3), 2033–44. 3. Smith, SM., & Nichols, TE. (2009), NeuroImage,
44(1), 83–98.
Support: Polish National Science Centre (2011/03/B/NZ4/03771) to Stefan Gazdzinski.
8
Effects of High-Fat High-Carbohydrate Diet on White Matter Integrity:
A Diffusion Tensor Imaging Study in Wistar Rats
Stefan Gaździński1, Andrzej Gaździński1, Zuzanna Setkowicz2, Joanna Osoba2,
Karolina Karwowska2, You Zhang3, Jarosław Orzeł4, Bartosz Kossowski4, Piotr Bogorodzki4
1Military Institute of Aviation Medicine, Warsaw, Poland, 2Department of Neuroanatomy,
Jagiellonian University, Krakow, Poland, 3 Department of Radiology, Unoversity of
California San Francisco, USA, 4Mossakowski Medical Research Centre, Polish Academy of
Sciences, Warsaw, Poland.
Introduction: Obesity worldwide has reached epidemic proportions, with more than 400
milion people affected. Currently, every third person in the world is overweight. Human DTI
studies have demonstrated lower fractional anisotrophy (FA) and higher mean diffusivity
(MD) in obese humans [1,2]. In animal models, high-fat high-carbohydrate diet (HFCD) is
commonly used to induce obesity. We evaluate hypotheses that long-term HFCD use in male
Wistar rats leads to Lower FA and higher MD than in control group.
Methods: Twenty five male Wistar rats were put on HFCD (~40% fat, ~40% carbohydrates,
~7% proteins) on their 55th day of life, while 25 control male rats (CON) remained on chow;
we obtained DTI data on 21 CON. MR measurements were performed on a 7T wide bore (30
cm) Bruker BioSpec at Mossakowski Medical Research Centre, Warsaw, Poland. Diffusion
tensor was acquired with TE/TR=33/3750ms, along 72 directions, with resolution
0.156x0.172x0.7mm, no gap. Data were skull stripped and eddy-current corrected with FSL.
Than, images were resized to 0.1x0.1x0.1 mm. FA images were than normalized to a study
specific template using DARTEL in SPM8; Study-specific template was created by averaging
all FA images. These transformations were applied to FA, MD, as well as perpendicular and
parallel diffusivities. Images after normalization and smoothing with a smoothing kernel of
0.3mm at FWHM were compared between groups using two-sample t-tests (FWE<0.05,
cluster size >27) with SPM8.
Results: Right cerebral peduncle contains a region of lower MD in in rats fed with HFCD
than in CON (p<0.05, FWE), accompanied by increased FA (p<0.001, uncorrected), contrary
to our hypotheses. MD is elevated in corpus callosum and fimbria of HFCD-fed rats than in
CON, as well as in trigeminal nerve (2b), consistent with our hypothesis. These changes were
not accompanied by significant FA changes. No differences in parallel and radial diffusivities
were noted.
Conclusions: The results partially support the thesis that high fat high carbohydrate diet leads
to worsening of WM integrity. The reason for such behavior is not known.
References: 1. Stanek K. et al., Obesity, 500-4, 2011. 2. Mueller K. et al., PLoS One, Apr
11;6(4):e18544, 2011.
Support: Polish National Science Centre (2011/03/B/NZ4/03771) to Stefan Gazdzinski.
9
HR MAS NMR METABOLIC PROFILES OF CARDIOMYOCYTES
AFTER RADIATION EXPOSURE
Michalina Gramatyka, Agnieszka Skorupa, Mateusz Ciszek, Łukasz Boguszewicz, Maria
Sokół
Department of Medical Physics, Maria Skłodowska-Curie Memorial Cancer Center and
Institute of Oncology, Gliwice, Poland.
Clinical data from population of radiotherapy patients show that exposure of the heart
to ionizing radiation during radiotherapy increases the subsequent rate of ischemic heart
disease. Initially heart was considered as relatively radioresistant organ, but since the 1960s
many studies have demonstrated that even low doses of radiation (<4 Gy) can have a negative
impact on the cardiovascular system. The underlying mechanisms of this toxicity are not well
understood. To address this topic we used human cardiomyocytes as a model system, and
studied their metabolic response to radiation using high resolution magic angle spinning
nuclear magnetic resonance techniques (HR MAS NMR).
Cultured in vitro human cardiomyocytes were exposed to ionizing radiation and their
survival was assessed by clonogenic assay. Changes in apoptosis intensity (TUNEL assay)
after irradiation with a dose of 2 Gy was measured as well. Water-presaturated 1H NOESY
and CPMG MAS NMR spectra of cardiomyocytes were acquired at 293K using Bruker
Avance 400 MHz spectrometer at a spinning rate of 3200 Hz. Survival of cardiomyocytes
after NMR experiments was assessed by the Trypan blue exclusion assay.
Exposure of cardiomyocytes to small doses of ionizing radiation (less than 4 Gy) had
no effect on cell proliferation potential and intensity of cell death. However, analysis of HR
MAS NMR metabolic profiles revealed changes in phospholipid and creatine metabolism.
Trypan blue staining showed that after NMR experiments the cells remain viable. Results of
this study show that ionizing radiation affects metabolic profiles of cardiomyocytes even at
low doses, which potentially have no effect on cell viability. The results obtained from this
experiment will be used in further in vivo studies on animal model.
10
THEORETICAL CALCULATIONS OF 13C NMR CHEMICAL SHIFTS
OF BROMINE-SUBSTITUTED CARBON ATOMS
Adam Gryff-Kellera, Dominika Kubicaa and Artur Wodyńskib
aFaculty of Chemistry, Warsaw University of Technology,
Noakowskiego 3, 00-664 Warsaw
bFaculty of Chemistry, University of Warsaw,
Pasteura 1, 02-093 Warsaw
Reliable DFT-based methods of predicting 13C NMR chemical shifts for carbon atoms
bonded exclusively to the atoms of the first-row and even second-row of the periodic table of
elements are now in common use. On the other hand, such predictions, in the case of carbons
bonded to heavier atoms, are much more difficult, since theoretical calculations have to treat
somehow the relativistic effects experienced by electrons in the vicinity of heavy nuclei. Such
heavy atom on light atom (HALA) effects are well visible in the case of carbon atoms
substituted by bromine or bromines. Presently, the DFT so-ZORA method accessible in ADF
commercial program that allows introducing the scalar and spin-orbit relativistic corrections
seems to be the most popular way of including relativity into quantum mechanical
calculations of NMR parameters. In the case of halogens bearing three electron lone pairs,
however, the electron correlation (EC) effects are also important. Actually, the EC and HALA
effects cooperate in the case of bromine and a simple application of a relativistic analogue of
the otherwise efficient non-relativistic method such as DFT/B3LYP/6-311++G(2d,p) yields
results which are not fully satisfying. We have found out that another, less popular functional,
BHandH, collaborates well with ZORA and yields remarkably better results for brominated
carbon atoms than B3LYP functional.
Table 1. Comparison of the experimental and theoretically predicted 13C NMR chemical
shifts [ppm] calculated using the methods which neglect or include relativistic effects.
Compound Method
Expa B3LYPb BHandHb ZORA/B3LYPc ZORA/BHandHc
CBr4 -27.36 155.00 116.22 -10.97 -12.28
2-Br-pyridine 142.23
163.31 155.52 150.35 144.90
a CDCl3, TMS scale. b Basis: 6-311++G(2d,p), solvent: PCM, program: Gaussian. c Basis: TZ2P, solvent: COSMO, program ADF.
11
1H-NMR IN LIVING ORGANISMS, TISSUES, AND OTHER
BIOLOGICAL SYSTEM RESISTANT TO DRASTIC DEHYDRATION
OR TO LOW TEMPERATURES Hubert Harańczyk
Institute of Physics, Jagiellonian University, Cracow, ul. Reymonta 4, 30-059 Cracow e-mail: [email protected]
For extremophilic living organisms, eg. Antarctic lichenized fungi [1-6] or insects [7];
for solid tissues, eg. Arthropode cuticle [8] or hair [9]; or for other biological systems, eg.
DNA [10] or DNA/surfactant complexes [11-13], which may resist the drastic dehydration,
proton NMR signal resembles the one observed for all microheterogeous solid samples at low
hydration level, eg. for solidifying white synthetic cement [14]. Proton signal consists of a
solid signal component, which may be well approximated using Gaussian function or in time
domain by Abragam function [6]. For Antarctic fungi, the solid signal component reveals the
contribution of Pake doublets [15], with the characteristic distance between relaxing protons
as for protons of water molecule. Liquid signal component may be approximated by one or
two Lorentzian lines which come from tightly or loosely bound water fraction. Both bound
water fractions are defined by the proximity to the surfaces of solid matrix of specimen and
sometimes averaged depending on size of pores in the structure.
In extremophilic living organism at recovery after cryptobiotic form or in resistant
tissue at rehydration, water soluble solid fraction dissolves with the increasing hydration
level. It may ocurr on two ways, namely by simple dissolution mainly of sugars and/or
polyols in case of some plant tissues [17], or by by enzyme-induced active bio-polymer
decomposition, which is the way used by lichenized fungi, or at initial phases of seed
imbibition [18]. This manifests in 1H-NMR signal, either in time or in frequency domain, as a
non-linear dependency of liquid signal component expressed in units of solid signal. If the
rational function approximates this change, it enables to yield the saturation concentration of
water soluble solid fraction.
Acknowledgements: The research was carried out with the equipment purchased thanks to the
financial support of the European Regional Development Fund in the framework of the Polish
Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08).
References
1. R. Del-Prado, L.G. Sancho, Flora 195: 51-60 (2000).
2. H. Harańczyk, On water in extremely dry biological systems, WUJ, Kraków (2003).
3. H. Harańczyk, M. Bacior, M.A. Olech, Antarctic Science 20, 527-535 (2008).
4. H. Harańczyk, M. Bacior, P. Jastrzębska, M.A. Olech, Acta Phys. Polon. A115, 516-520
(2009).
5. H. Harańczyk, Ł. Pater, P. Nowak, M. Bacior, M.A. Olech, Acta Phys. Polon. 121, 478-
482, (2012).
6. H. Harańczyk, P. Nowak, M. Bacior, M. Lisowska, M. Marzec, M. Florek and M.A. Olech,
Antarctic Science 24 (4), (2012); 342-352.
7. M. Watanabe, T. Sakashita, A. Fujita, T. Kikawada, D. D. Horikawa, Y. Nakahara, S.
Wada, T. Funayama, N. Hamada, Y. Kobayashi, T. Okuda, Int. J. Radiat. Biol. 82, 587-592
(2006).
12
8. H. Harańczyk, M. Florek, P. Nowak and S. Knutelski, Acta Phys. Polon. 121, 489-494,
(2012).
9. D. Zalitacz, H. Harańczyk, P. Nowak, P. Delong, J. Investigative Dermatology 133, 1424
(2013).
10. H. Harańczyk, J. Czak, P. Nowak, J. Nizioł, Acta Phys. Polon. A117, 257-262 (2010).
11. H.Harańczyk, J.Kobierski, D.Zalitacz, P.Nowak, A.Romanowicz, M.Marzec, J.Nizioł,
Acta Phys. Polon. 121, 483-488, (2012).
12. H. Harańczyk, J. Kobierski, J. Nizioł, E. Hebda, J. Pielichowski, D. Zalitacz, M. Marzec,
A. El-Ghayoury, J. Appl. Phys. 113, (2013) 044702.
13. J. Nizioł, H. Harańczyk, J. Kobierski, E. Hebda, J. Pielichowski, B. Ostachowicz, J. Appl.
Phys. 114, 144701 (2013).
14. R. Rumm, H. Harańczyk, H. Peemoeller, M. M. Pintar, Cement and Concrete Res. 21,
391-393 (1991).
15. W. Derbyshire, M. Van den Bosch, D. Van Dusschoten, W. MacNaughten, I. A. Farhat,
M. A. Hemminga, J.R. Mitchell, J. Magn. Res. 168, 278-283 (2004).
16. J.Hetmańczyk, Ł.Hetmańczyk, A.Migdał-Mikuli, E.Mikuli, M.Florek-Wojciechowska,
H.Harańczyk, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 124,
429-440 (2014).
17. H. Harańczyk, W. P. Węglarz, Z. Sojka, Holzforschung, 53, 299-310 (1999).
18. H. Harańczyk, K. Strzałka, G. Jasiński, K. Mosna-Bojarska, Colloids &Surfaces, A115,
47-54 (1996).
13
REMARKS ON THE FREE-APPROACH MODELS
Łukasz Jaremko1,2, Mariusz Jaremko1, Michał Nowakowski3, Andrzej Ejchart4
1 Max Planck Institute for Biophysical Chemistry, Department for NMR-based Structural
Biology, Am Fassberg 11, 37077 Göttingen, Germany, e-mail: [email protected]
2 Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Am Fassberg 11, 37077
Göttingen, Germany
3 Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw,
Żwirki i Wigury 101, 02-089 Warsaw, Poland, , e-mail:[email protected] 4 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, 02-
106 Warsaw, Poland, e-mail: [email protected]
One of the most successful and widely used approaches to the interpretation of nuclear
magnetic relaxation data for biologically important polymers, mainly proteins, has become
the model-free approach (MFA) requiring no particular physical model of motion(s) and a
small number of parameters [1]. The model is based on two assumptions imposed on the form
of the autocorrelation function: (1) internal motions and overall molecular tumbling are
uncorrelated allowing to factorize the autocorrelation function into two components
describing overall tumbling and internal motion, and (2) the internal correlation function can
be approximated by a single exponential. In the frame of this model local mobility is
described by two parameters, a generalized order parameter, S2, which corresponds to the
spatial freedom of the motion, and an internal correlation time, int, which corresponds to the
rate of this motion in the pico- to nanosecond time scale faster than a single correlation time
describing an isotropic overall molecular tumbling, R. Fourier transformation of
autocorrelation function results in the expression for the spectral density function represented
by two Lorentzian terms. By analysis of the backbone amide nitrogen relaxation, a global
picture of the dynamics of a protein can be revealed.
Soon it was found that model-free approach sometimes poorly reproduced
experimental data in proteins, especially 15N-{1H} NOEs; experimental NOEs tended to be
larger than their calculated values. This was attributed to the additional slow internal motion
outside the extreme narrowing limit pointing at motions being faster than overall tumbling but
slower than those obtained from the genuine MFA model. Therefore, the extended model-free
approach (EMFA) taking into account more complex description of internal motions
characterized by two different time scales (f - fast motion, s - slow motion) was introduced at
the cost of larger number of model parameters [2]. One should bear in mind that NOE
measurements are especially prone to systematic errors resulting from not fully relaxed
spectra and/or saturation transfer due to exchange with water protons. Both these effects can
increase apparent NOEs even beyond the theoretically expected maximum. An attempt to
compensate such artifacts applying the extended model can result in false values of local
parameters.
Comparison of two free-approach models can be analyzed with the use of
target function given by a widely accepted least-squares expression
χ=∑
i=1
N
Γi=∑
i=1
N
∑j=1
M
[ ( Pij ,exp
− Pij,calc
)2 /σij
2 ]
Simulations performed for MFA and EMFA revealed striking differences in target
function dependences between these two models. The MFA displays well determined minima
of quasi-parabolic target functions, (int) and (S2), in the least-squares procedure of the best
motional parameter search as could be expected for well defined numerical problem (Fig. 1).
On the other hand, the EMFA shows strongly diversified behavior of target function graphs
for model parameters describing slow and fast internal motions (Fig. 2).
14
Fig. 1 (above). Dependence of target function
values vs. local MFA model parameters, int
and S2, obtained from the fit to the synthetic
R1, R2 and NOE data.
Fig. 2 (right). Dependence of target function
values vs. local EMFA model parameters
obtained from the fit to the synthetic R1, R2
and NOE data.
Plots (f) and (Sf2) are similar to those obtained for the MFA. On the contrary, the
dependence (s) is strongly asymmetric and nearly flat within a wide range of s values
larger than its input value used in the simulations; opposite is true for the (Ss2) graph. It
usually results in a painfully long and unstable numerical search of global minimum of
function and strongly unsymmetrical confidence boundaries of slow motion parameters s and
Ss2.
One should focus an attention on the relation between a generalized order parameter
and corresponding correlation time. As a rule, the increase of correlation time results in
decrease of corresponding generalized order parameter. In real life, when experimental
relaxation data bear unavoidable inaccuracies, the least squares procedure can deliver very
large, non physically justified s values often exceeding overall correlation time. It is
particularly important for the Ss2(s) dependence since unrealistically large s value
determined in the error sensitive EMFA-based minimization procedure can result in the
assignment of unjustified internal mobility to given amino acid residues. Such behavior was
observed for a number of residues in several proteins for which relaxation data were available
in the literature and data bases.
References
[1] Lipari, G., Szabo, A., Model-free approach to the interpretation of nuclear magnetic
resonance relaxation in macromolecules. J. Am. Chem. Soc. 1982, 104, 4546-4570.
[2] G.M. Clore, A. Szabo, A. Bax, L.E. Kay, P.C. Driscoll, A.M. Gronenborn, Deviations
from the simple two-parameter model-free approach to the interpretation of nitrogen-
15 nuclear magnetic relaxation of proteins. J. Am. Chem. Soc. 1990, 112, 4989-4991.
15
Comparison of magnetic resonance imaging of the mouse brain in vivo using different types of RF coils at 9.4 T
J. Jasieniak, W. Piędzia1, K. Jasiński1, W.P. Węglarz1
1Department of Magnetic Resonance Imaging, Institute of Nuclear Physics, Polish Academy of Sciences, Krakow, Poland
Purpose: The aim of this study was to compare quality of the mice`s brain MR images in vivo obtained with three different types of RF coils: cryo-coil and room temperature surface and volume coils. The radiofrequency field (B1) mapping has also been made.
Materials and methods: Three different RF coils: transmit/receive Bruker CryoProbe [1], mice brain receive coil (together with volume transmit birdcage coil) and 35 mm ID transmit/receive birdcage volume coil were used at 9.4T Bruker Biospec MR scanner for SNR assessment in doped water phantom and mice. ECG, temperature and respiratory were monitored during in vivo measurements. FLASH, MPRAGE and UTE pulse sequences were used for MR imaging. SNR was measured for all three coils. B1 maps were obtained with double angle method. Images were analysed with the use of Fiji software.
Results: The best quality of magnetic resonance imaging of the mouse brain in vivo has been obtained for cryo-coil. The SNR was ~3 to ~12-fold larger for the cryo-coil as compared to the birdcage [2] and ~2.3 to ~3-fold larger for the cryo-coil as compared to the mouse brain surface coil. Different B1 maps have been obtained for the images of phantom of the 2 pulse position-3.5 mm and 5.5 mm.
Discussion: MR imaging of the mouse brain requires good SNR due to the small size of the imaging object. This can be obtained reducing noise from the electronics of coil by lowering the temperature of the coil. Cryo-coil operates at a temperature of 20 K what makes it get the best SNR, which enable to get superior quality images of mouse brain in vivo in reasonable time. However, due to inhomogenous B1 field (and thus spatially dependent flip angle) using of cryo-coil has also some limitations. Specifically, it often requires careful flip angle adjustment for chosen horizontal slice and/or flip angle (B1) mapping.
References: [1] Baltes, C., Radzwill, N. & Bosshard, S. at al. Micro MRI of the mouse brain using a novel 400 MHz cryogenic quadrature RF probe. NMR in Biomedicine, 2009;22(8),:834-842.
[2]-W. Piędzia, K. Jasiński, K.Kalita, B.Tomanek, W.P. Węglarz. “White and gray matter contrast enhancement in MR images of the mouse brain in vivo using IR UTE with a cryo-coil at 9.4 T” - 2014
16
NMR STUDY OF P(MEO2MA) POLYMER NETWORKS
J. Jenczyk, S. Kadłubowski1, M. Olejniczak2, M. Kozanecki2, S. Jurga
NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Poznań, Poland
1Institute of Applied Radiation Chemistry, Technical University of Lodz, Wroblewskiego 15,
93-590 Lodz, Poland 2Department of Molecular Physics, Technical University of Lodz, Zeromskiego 116, 90-324 Lodz,
Poland
Hydrogels are three-dimensional networks, made of amphiphilic polymer chains, able
to swell in water. Thermo-responsive gels have been extensively studied due to their potential
applications as drug delivery systems, regenerative medicine, biosensors, responsive
membranes, molecular machines, nanotemplates for nanoparticles formation, catalysis and
photonic crystals The polymeric networks of 2-(2-methoxyethoxy)ethyl methacrylate
(MEO2MA), 2-hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate
(EGDMA cross-linking agent) (molar ratios: 100:2:1) have been synthesized [1]. These
cross-linked systems are characterized by lower critical solution temperature (LCST) which is
directly related to volume phase transition (VPT). The VPT results in abrupt deswelling
process (network collapse) and water release. The kinetics of this process can be effectively
monitored by Nuclear Magnetic Resonance (NMR) spectroscopy. Moreover, the time-lapse
NMR experiment enables one to assess the relative cross-linking level of studied samples [2].
Obtained results reveal how the irradiation dose and post-irradiation conditioning temperature
influence on the degree of cross-linking. The analysis relies on the time evolution of the
proton NMR spectra above LCST temperature. This evolution can be described by
exponential relation and characterized by time constant τ. The τ value is directly proportional
to cross-linking level due to the fact that water hydrogen bonding in a more densely
crosslinked hydrogel is more resistant to disruption. Independently, wide line separation
WISE experiments were performed for dried polymers in order to estimate mobile and static
fraction of protons from MEO2MA side chains.
1 Time evolution of NMR spectrum (water signal) above LCST temperature.
[1] S. Kadłubowski, M. Matusiak, J. Jenczyk, M. Olejniczak, M. Kozanecki, L. Okrasa Radiation Physics and
Chemistry 100 (2014) 23-31
[2] J. Yoon, Ch. Gayathri, R. R. Gil, T. Kowalewski, K. Matyjaszewski, Macromolecules 43 (2010) 4791–4797
17
QUANTITATIVE MRI IN STUDYING WHITE MATTER DAMAGE FOLLOWING
SPINAL CORD INJURY
Piotr Kozlowski
University of British Columbia, UBC MRI Research Centre, and International Collaboration
On Repair Discoveries (ICORD), Vancouver, BC, Canada
Spinal cord injury (SCI) is a devastating event affecting mostly young, otherwise healthy
population. It often results in severe physical impairment and disability that persists for the
life of the affected individual. Apart from the obvious human tragedy and social costs, the
economic burden on the health care system and society at large is enormous. It is widely
recognized that the functional loss following SCI is largely caused by the damage to the white
matter, thus most of the therapeutic procedures are oriented towards regeneration and
functional restoration of the interrupted nerve fibre tracts. One of the critical aspects of a
successful therapy is the ability to accurately assess the white matter damage prior to
initiating the therapy, and to follow the efficacy of the treatment throughout the therapy. This
requires a non-invasive imaging technique capable of quantitative measurements of white
matter integrity in spinal cord. MRI is currently the most effective radiological method for
assessing SCI. However, exact estimation of myelin content and axonal integrity in spinal
cord is not possible with the standard MRI techniques used clinically. A number of
quantitative MRI techniques have recently been developed with particular focus on white
matter characterization. Among them, Myelin Water Imaging (MWI), Diffusion Tensor
Imaging (DTI) and phase MRI have been particularly promising in SCI applications. This
presentation will review physical bases of these techniques and show their applications in
characterizing white matter damage in several pre-clinical models of SCI.
Myelin Water Imaging:
In a complex system, such as brain or spinal cord, multiple water compartments (e.g. myelin
bilayers, intra-/extra-cellular space, cerebrospinal fluid) result in spin-spin relaxation
becoming a multi-exponential process giving rise to multiple T2 values [1]. Quantitative
analysis of T2 decay curves acquired with a multi-echo Carr-Purcell-Meiboom-Gill (CPMG)
sequence produces continuous distributions of T2 values representing various T2 components
present in the tissue (Figure 1). MWI was successfully applied to measuring myelin content in
the normal and injured rat spinal cords ex vivo [2] and in vivo [3].
Figure 1. Continuous T2 distribution from a rat spinal cord. Multiple T2
components correspond to myelin water, intra-/extra-cellular water and CSF.
Myelin Water Fraction (MWF) is a measure of myelin content in the tissue.
Figure 2. MWI and DTI parametric maps obtained from rat spinal cords
at 3 and 8 weeks following Dorsal Column transection injury. Luxol
Fast Blue is a histological stain of myelin. Scatter plots show strong
correlation between MWF and histological measure of myelin content.
18
Diffusion Tensor Imaging:
DTI provides information about tissue fine structure and
therefore is particularly useful for studying the structure
and integrity of white matter in the control and injured
spinal cord. It has been shown that diffusion anisotropy is
affected both by the state of myelin and axonal structure
[4]. Longitudinal diffusivity (diffusion coefficient along
the axon) is sensitive to the axonal integrity, while
transverse diffusivity (diffusion coefficient perpendicular
to the axon) can provide some information on the myelin
content (Figure 2). Tractography is a technique that allows reconstruction of white matter
tracts from DTI data. It can be used to identify tracts damaged as a result of SCI (Figure 3).
Phase MRI:
The MRI signal is complex in nature. Commonly, the magnitude of this complex number is
used and the phase information is discarded. However, phase may reflect the relative
resonance frequency of spins and can therefore be utilized to investigate the spins’ magnetic
environment, such as the local tissue magnetic susceptibility. Theoretical models predict that
the tissue microstructure has an influence on the phase of MRI signal [5], thus quantitative
phase MRI can potentially be used to characterize white matter damage in an injury model in
rat spinal cord. In a recent study [6], phase MRI has been shown to correlate with histological
measures of myelin content and axonal damage in a Dorsal Column transection injury model
in rat spinal cord (Figure 4).
References:
1. Whittall KP and MacKay AL, J Magn Reson, 84, 134-152 (1989).
2. Kozlowski P, et al., J Neurotrauma, 25, 653-676 (2008).
3. Kozlowski P, et al., Magn Reson Imaging, 32, 250 – 258 (2014).
4. Beaulieu C, NMR Biomed, 15, 435-455 (2002).
5. He et al., PNAS, 106, 13558-563 (2009).
6. Chen IE, et al., Proceedings of 21st Meeting of ISMRM, p. 347 (2013).
Figure 3. Fibre tracts
reconstructed from a
DTI data acquired ex
vivo from a cervical
spinal cord excised
following contusion
injury. The area of
cavity (red arrows) and
axonal damage to the
ventral WM (yellow
arrows) are clearly
visible.
Figure 4. Frequency shift maps and histology sections of myelin (eriochrome, MBP), axonal (NF/Tub III), and degenerated myelin
(degen-MBP) stains from rat spinal cords at baseline and 3 and 8 weeks after Dorsal Column (DC) transection injury. Images show sections 5 mm caudal (left) and 5 mm cranial (right) to injury site. The ascending DC tract (fasciculus gracilis) undergoes Wallerian
degeneration on the cranial side (right) and the retrograde degeneration on the caudal side (left), while the descending DC tract (cortico-
spinal tract) undergoes Wallerian degeneration on the caudal side (left) and the retrograde degeneration on the cranial side (right).
19
New high dimensionality NMR experiments for biomolecules
Wiktor Koźmiński
Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw,
Żwirki i Wigury 101, 02089 Warsaw, Poland
A variety of different methods was proposed to overcome the sampling limitation in
multidimensional NMR spectroscopy. They could be utilized in two different ways, either to
shorten the experiment duration without loss of resolution, or to perform experiments that are
not obtainable conventionally, i.e. with significantly improved resolution and/or of high
dimensionality. Most often first of these two, so called “Fast NMR” approach, is shown as the
example of the utility of these methods, as it saves expensive spectrometer time. However, in
many cases spectra featuring extraordinary resolution and high number of dimensions may be
more interesting from scientific point of view as they reveal effects that are hidden, when
spectral lines are broad, or enable resolving spectral ambiguities when peaks are overlapped.
This second approach we refer to as “Accurate NMR”. Its full potential is manifested when
the overall experiment time is less important than a new information available from spectra of
high dimensionality (4-6D) or of high resolution approaching natural line-width. The new
methods were applied for NMR studies of intrinsically disordered proteins, where the
structural disorder in combination with highly repetitive amino-acid sequences causes severe
peak overlap in the spectra. Recently, several novel 4-6D pulse sequences are proposed. The
new experiments employ non-uniform sampling that enables achieving high resolution in
indirectly detected dimensions. The experiments facilitate resonance assignment of
intrinsically disordered proteins.
[1] K. Kazimierczuk, J. Stanek, A. Zawadzka-Kazimierczuk, W. Koźmiński,
Prog. Nucl. Mag. Res. Sp., 57, 420–434 (2010).
[2] K. Kazimierczuk, M, Misiak, J. Stanek, A. Zawadzka-Kazimierczuk, W. Koźmiński,
Topics in Current Chemistry, 316, 79–124 (2012).
[3] K. Kazimierczuk, J. Stanek, A. Zawadzka-Kazimierczuk, W. Koźmiński,
ChemPhysChem, 14, 3015–3025 (2013)
20
Intramolecular interaction of hybrid of uridine and stilbene derivative Hanna Krawczyk and Przemysław Szczeciński
Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3,
00-664 Warsaw, Poland, [email protected]
Hybrid molecules are defined as chemical entities with two (or more than two) structural
domains having different biological functions. Their dual activity indicates that a hybrid
molecule acts as two distinct pharmacophores. On the other hand chemical modification of
nucleosides and their incorporation into nucleic acid oligomers represent one of the most
successful drug design strategies when considering chemotherapeutic approaches, as is
evidenced by the significant number of analogues in clinical trials for treatment of various
diseases including cancer, inflammation, and viral infections.[1] Numerous modifications to
the sugar ring as well as the heterocyclic nucleobase moieties have been utilized in recent
years to increase chemotherapeutic activity.[2,3] A new class of modified nucleosides by
stilbene derivative has recently been synthesised by us.[4] The purine nucleoside- uridine
(first structural domain)- connected by a single C-C bond with stilbene derivatives (second
structural domain) creates the hybrid molecules. This allows the modified nucleosides to more
readily adopt to the spatial and other requirements of the binding site, while retaining the
majority of the key structural features required for molecular recognition. The synthesized
molecules have specific spectroscopic properties and could be biologically active, just like
nucleosides-uridine- and stilbenes such as combretastatin- OXi4503, (Vascular Disrupting
Agents ; VDAs), a phase I clinical trial for relapsed and refractory acute myeloid leukemia
(AML) and myelodysplastic syndrome (MDS).[5] Our investigation concerns the
determination of stereochemistry of new compounds focusing on various intramolecular
interactions governing conformational equilibria (e.g. the interactions of the stilbene aromatic
ring with nucleobase moieties and sugar scaffold).
References
[1] Papers presented at XV International Roundtable Nucleosides, Nucleotides and Nucleic Acids, Leuven,
Belgium, September 2002. Nucleosides Nucleotides Nucl. Acids. 2003, 22, 489-1736.
[2] Agrawal, S.; Zhao, Q. Y. Curr. Opin. Chem. Biol. 1998, 2, 519-528.
[3] De Clercq, E. Clin. Microbiol. Rev. 2003, 16, 569-596.
[4] K. Kordowska, H. Krawczyk, Nauka i Przemysł metody spektroskopowe w praktyce nowe wyzwania i
możliwości- praca zbiorowa pod redakcją prof. dr hab. Zbigniewa Hubickiego (ISBN 978-83-939465-2-5)
2014, Lublin, 212-216.
[5] A service of the U.S. National Institutes of Healt, http://clinicaltrials.gov/show/NCT01085656.
21
Imaging Methods in Research and Development Process of Generic
Modified Release Matrix Tablets
Piotr Kulinowski a, Krzysztof Woyna-Orlewicz b, Gerd-Martin Rappen c, Dorota Haznar-
Garbacz d, Władysław P. Węglarz e, Przemysław P. Dorożyński b
a Institute of Technology, The Pedagogical University of Cracow, ul. Podchorążych 2, 30-084
Kraków, Poland b Department of Pharmaceutical Technology and Biopharmaceutics, Pharmaceutical Faculty,
Jagiellonian University, ul. Medyczna 9, 30-688 Kraków, Poland c Physiolution GmbH, Walther-Rathenau-Strasse 49a, 17489 Greifswald, Germany d Center of Drug Absorption and Transport (C_DAT), Dept. of Biopharmaceutics and
Pharmaceutical Technology, Felix-Hausdorff-Str. 3, 17487 Greifswald, Germany e Department of Magnetic Resonance Imaging, Institute of Nuclear Physics PAN, ul.
Radzikowskiego 152, 31-342 Kraków, Poland
In vitro phase of R&D process of pharmaceutical product can be performed at relatively low
cost compared to the biological (bioequivalence) studies, but there are no dedicated and
effective methods for development of oral, generic, modified release formulations. The
purpose of the study was to assess in vitro methodology for bioequivalence study risk
minimization.
Presented approach consisted of three independent steps:
1. Quality by Design / Design of Experiment (QbD/DoE) pharmaceutical strategy using
compendial dissolution tests [1];
2. Application of several, selected, imaging/analytical methods;
3. Biorelevant stress dissolution test [2].
At the first step, pharmaceutically equivalent quetiapine fumarate extended release dosage
form of Seroquel XR was developed.
The second step was performed using following methods:
Magnetic Resonance Imaging in USP4 apparatus 4 performed using 4.7T research
system with TMX (NRC, IBD, Canada) console with spin-echo sequence [3,4].
Multi-Echo Magnetic Resonance Imaging using 9.4T BioSpin research system
(Bruker).
Micro-CT imaging performed using Benchtop 160 CT 160 (Nikon Metrology Inc.)
[5].
Texture analysis (penetrometry) with Compact Tabletop, Universal Tester EZ-SX
(Shimadzu).
The last three measurements were performed on samples removed from the dissolution
apparatus at 2h of hydration. Despite pharmaceutical equivalence of the Seroquel XR and
developed formulation developed and original dosage forms differed in micro/meso structure
and consequently in mechanical properties.
These differences were found to cause failure of biorelevant dissolution test using the stress
dissolution apparatus performed at the third step of the study – the test was used as a surrogate
for bioequivalence study.
The work was supported by the Polish Ministry of Science and Higher Education grant
NN518 407438 and German Federal Ministry of Education and Research grant BMBF FKZ
03IPT612C.
[1] R.A. Lionberger, et al. AAPS J. 2008; 10(2): 268–276.
22
[2] G. Garbacz et al. Exp. Opin. Drug Deliv. 7 (2010) 1251-1261.
[3] P. Kulinowski et al. Pharm. Res. 28 (2011) 1065-1073.
[4] P.P. Dorożyński at al. AAPS Pharmscitech 11 (2010) 588-597.
[5] P.R. Laity et al. Eur. J. Pharm. Biopharm. 74 (2010) 109-119.
23
MOLECULAR MODELING OF SINGLE WALL CARBON NANOTUBE
(SWCNT) CHEMICAL SHIFT DUE TO ADDITION OF DIATOMICS
Teobald Kupka1, Marzena Nieradka1, Leszek Stobiński2 i Jakub Kaminský3
1University of Opole, Faculty of Chemistry, 45-052 Opole, Poland
2Institute of Physical Chemistry, Polish Academy of Sciences, 44/52, Kasprzaka, 01-224 Warsaw,
Poland; 3Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Flemingovo nám. 2.,
166 10 Prague, Czech Republic
Density functional theory (DFT) studies on adsorption of several gaseous homo- and
hetero-diatomic molecules (AB) including H2, O2, N2, NO and CO on external
surface of H-capped pristine armchair (5, 5) single-walled carbon nanotube
(SWCNT) were conducted. Structures of C70H10 and the corresponding C70H10–AB
adducts were fully optimized at the B3LYP/6-311G* level of theory. Calculated 13C
NMR chemical shifts were analyzed and critically compared with available
experimental data. Significant changes of carbon NMR atom chemical shifts (up to –
100 ppm) and shielding anisotropies (up to -180 ppm) at sites of addition were
observed.
Fig. 1. The B3LYP/6-31G* optimized model of armchair (5, 5) SWCNT. A1 and A2 adsorption sites
including C3, C4 and C22, C31 carbons are marked in blue. Two types of C-C bonds, tilted and
perpendicular are marked off, too.
[1] M. Nieradka, T. Kupka*, L. Stobiński and J. Kaminský*, DFT studies on armchair (5, 5) SWCNT
functionalization. Modification of selected structural and spectroscopic parameters upon two-atom
molecule attachment, J. Mol. Graphics Model., in press.
http://dx.doi.org/10.1016/j.jmgm.2014.11.006.
24
MOLECULAR MODELING OF CHEMICAL SHIFT OF ATOMS,
SMALL AND LARGE MOLECULES
Teobald Kupka*, Michał Stachów, Marzena Nieradka, Klaudia Radula-Janik, Roksana Wałęsa, Aneta Buczek i Małgorzata Broda
1University of Opole, Faculty of Chemistry, 45-052 Opole, Poland
In this report we demonstrate the need of proper selection of method and basis set
quality in calculations of NMR parameters.
Hartree-Fock (HF), second-order Moller-Plesset (MP2), density functional theory
(DFT) and coupled cluster (CC) studies on calculation of chemical shift of free
noble gas atoms, their dimers and single atoms encapsulated in selected fullerene
cages are reported. Several examples of DFT predicted chemical shifts and indirect
spin-spin coupling constants (SSCC) in medium size molecules are also discussed.
The importance of accounting for relativistic effects in case of typical 13C NMR
chemical shift in case of iodine attached to carbon atom is also demonstrated.
Fig. 1. The optimized model of He@C28
[1] T. Kupka*, M. Nieradka, J. Kaminský and L. Stobiński, Modeling 21Ne NMR
parameters for carbon nanosystems, Magn. Reson. Chem., 51 (2013) 676-681.
[2] T. Kupka*, M. Stachów, E. Chełmecka, K. Pasterny, M. Stobińska, L. Stobiński and J.
Kaminský, Efficient modeling of NMR parameters in carbon nanosystems, J. Chem. Theor.
Comput., 9 (2013) 4275–4286.
[3] R. Wałęsa, T. Ptak, D. Siodłak, T. Kupka and M. A. Broda, Experimental and
theoretical NMR studies of interaction between phenylalanine derivative and egg yolk
lecithin, Magn. Reson. Chem., 52 (2014) 298-305.
[4] K. Radula-Janik, T. Kupka*, K. Ejsmont, Z. Daszkiewicz and S. P. A. Sauer,
Halogen effect on structure and 13C NMR chemical shift of 3,6-disubstituted-N-alkyl
carbazoles, Magn. Reson. Chem. 51 (2013) 630-635.
25
Badania spektroskopowe Pt(II) z 7,8-benzochinoliną
oraz 2-fenoksypirydyną.
Daria Niedzielska, Leszek Pazderski
Zakład Chemii Analitycznej i Spektroskopii Stosowanej,
Wydział Chemii, Uniwersytet Mikołaja Kopernika, Toruń
Slowa kluczowe: kompleksów Pt (II/IV), związki kompleksowe Pt (II), 7,8-benzochinolina, 2-
fenoksypirydyna, 1H NMR, 13C NMR, luminescencji.
7,8-benzochinolina (bzq) oraz 2-fenoksypirydyna są N-donorowymi ligandami heterocyklicznymi
typu azynowego, które koordynują jony Pt(II/IV) w dwojaki sposób: jako ligand jednodonorowy za
pomocą atomu azotu (związki kompleksowe) oraz ligand dwudonorowy za pomocą atomu azotu i
zdeprotonowanego atomu węgla C(2’/10) (związki metaloorganiczne). Pierwszą formę
kompleksowania obserwuje się w związku [Pt(L)2Cl2], natomiast drugą w [Pt(LL*)Cl]2 (Schemat 2).
Schemat 1 Schemat 2
Otrzymane związki te cieszą się dużym zainteresowaniem ze względu na ich właściwości
katalityczne, luminescencyjne oraz cytotoksyczność. Związki Pt(II) z bzq oraz 2-popy są
również stosowane jako prekursory podczas przygotowania innych związków mających
właściwości przeciwnowotworowe, antybakteryjne, przeciwgrzybiczne oraz
przeciwwirusowe.
Celem niniejszych badań była synteza związków Pt(II/IV) z bzq i 2-popy oraz ich pełna
charakterystyka spektroskopowa 1H, 13C i 15N NMR, przy czym wszystkie sygnały zostały
przypisane metodami 1H-13C i 1H-15N HMQC/HMBC. Zostały również wykonane widma
fluorescencyjne w ciele stałym oraz w dichlorometanie. Wykonano również widma w średniej
oraz dalekiej podczerwieni.
Literatura:
[1] Y.Fuchita, H. Leda, A. Kayama, J. Kinoshita-Nagaoka, H.Kawano, S. i M. Mikuriya
Kamedac, J. Chem. Soc., Dalton Trans., 1998, 4095-4100
[2] Jean-Ho Chu, Pi-Shan Lin Ming-Jung Wu, Organometallics, tom. 29, nr 18, 2010
26
FREEZING AND DRYING RESISTANCE OF ANTARCTIC
TURGIDOSCULUM COMPLICATULUM THALLI AS OBSERVED
BY 1H-NMR METHODS
Magdalena Bacior1, Piotr Nowak2, Paulina Kijak2, Ewelina Baran2, Hubert Harańczyk2,
and Maria A. Olech3
1Department of Chemistry and Physics, Faculty of Agriculture and Economics,
University of Agriculture, Cracow 2Institute of Physics and 3Institute of Botany, Jagiellonian University, Cracow
Lichenized fungi can survive freezing and deep dehydration [1-3]. Numerous data
suggest that freezing and dehydration resistance have a common origin and depend on lichen
morphology and habitat [4, 5, 6].
Turgidosculum complicatulum (foliose thallus) samples were collected in the vicinity of
Arctowski Polar Station, King George Island, Maritime Antarctic. 1H-NMR spectra were
collected on Bruker Avance III 300, Bruker Biospin, spectrometer (transmitter power 400 W;
pulse length π/2 = 2.2 μs; bandwidths 300 kHz). Proton Free Induction Decays (FIDs) were
recorded at 30 MHz on a high power relaxometer WNS HB 65, Waterloo NMR Spectrometers
(pulse lenghts /2 = 1.5 s, transmitter power 400 W).
Proton FID consists of a solid signal component fitted well by Gaussian (
2T 25 s)
and two liquid components described by exponential functions coming from a tightly bound
(
2T 120 s), and a loosely bound water fraction (
2T 500 s). Solid signal is fitted well by
Gaussian [6, 7]. 1H-NMR spectra are superpositions of a Gaussian component (G ≈ 40 kHz) coming
from protons of solid matrix of thallus and one averaged Lorentzian component (L ≈3000
Hz) coming from protons of all water fractions in different motional states.
For thalli at low hydration level (m/m0<0.3) the amplitude (in time domain) and line
area (in frequency domain) of liquid signal expressed in solid signal units, L/S, constantly
decreases with decreasin temperature, what suggests non-cooperative immobilization of water
molecules. For highly hydrated samples rapid decreasing of L/S with decreasing temperature
suggests ice nucleation process.
The hydration dependency of total liquid NMR signal component expressed in units of
solid, L/S, both in time or in frequency domain is well described by the rational function
suggesting the dissolving process of the thallus solid fraction at rehydratation.
Acknowledgements: The research was carried out with the equipment purchased thanks to the
financial support of the European Regional Development Fund in the framework of the Polish
Innovation Economy Operational Program (contract no. POIG.02.01.00-12-023/08).
We are thankful to colleagues from Maitri Station and participants of the 23rd Indian Antarctic
Expedition for their help during the field studies.
[1] H. Harańczyk, On water in extremely dry biological systems, Wyd. UJ 2003.
[2] H. Harańczyk, A. Pietrzyk, A. Leja, M. A. Olech, Acta Phys. Polon. 109, 411 (2006).
[3] H. Harańczyk, M. Bacior, P. Jastrzębska, M.A. Olech Acta Phys. Polon. A115, 516-520 (2009).
[4] B. Schroeter, Ch. Scheidegger, New Phytol., 131, 273-285 (1995)
[5] H. Harańczyk, M. Bacior, M.A. Olech Antarctic Science 20, 527-535 (2008).
[6] Harańczyk H., Nowak P., Bacior M., Lisowska M., Marzec M., Florek M., Olech M.A., Antarctic Science, 24(4),
342-352 (2012).
27
The solution structure of the MANEC-type domain from Hepatocyte
Growth Factor Inhibitor 1 reveals an unexpected PAN/apple domain-type
fold
Michał Nowakowski1, Zebin Hong2, Chris, Spronk4, Steen V. Petersen3, Jan S. Pedersen5,
Wiktor Koźmiński1, Frans A.A. Mulder5 and Jan K. Jensen2
1Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw,
Poland. 2Department of Molecular Biology and Genetics, Danish-Chinese Centre for
Proteases and Cancer, Aarhus University, Denmark. 3Department of Biomedicine, Aarhus
University, Denmark. 4Spronk NMR, Lithuania. 5Interdisciplinary Nanoscience Center
(iNANO), Aarhus University, Denmark.
A decade ago, a Motif at N terminus with Eight-Cysteines or in short MANEC was defined as
a new protein domain family. The domains were found exclusively in the N-terminus of > 400
multi-domain membrane proteins from multicellular animals. Despite the large number of
MANEC-containing proteins, only one has been characterized: hepatocyte growth factor
activator inhibitor-1 (HAI-1). HAI-1 is an essential protein shown to regulate the activity of
matriptase, hepsin and hepatocyte growth factor activator, all serine proteases with crucial
roles in epithelial development, cell growth and homeostasis. Misregulation of these systems
has been implicated in severe pathological conditions such as skin diseases and cancer.
Detailed functional understanding of HAI-1 and other MANEC-containing proteins is
hampered by a lack of any structural information on MANEC. Here we present an NMR
solution structure and biophysical characterization of the MANEC domain from HAI-1, as the
first structure of a representative MANEC domain. Although no homologies were predicted
based on sequence, the MANEC structure revealed it as a new subclass of the PAN/apple
domain family. Intriguingly, in silico protein folding resulted in successful structure-based
homology prediction, where sequence-based approaches fail. The MANEC structure
represents a much needed tool for elucidation of function of MANEC-containing proteins as
indicated by the homology to the PAN/apple domains as mediators of protein-protein and
protein-glycan interactions.
28
Atlas-based automatic brain morphometry applied to
DBA/2J mouse model of glaucoma
Jarosław Orzeł (1,2), Michał Fiedorowicz (1), Bartosz Kossowski (1,2),
Marlena Wełniak - Kamińska (1), Piotr Bogorodzki (1,2), Paweł Grieb (1)
(1) Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw
(2) Faculty of Electronics and Information Technology, Warsaw University of Technology
Objective: Glaucoma is a common disease of sight that is associated with degeneration of
retinal ganglion cells (and its axons forming an optic nerve). Recently, it was shown that
neurodegeneration in glaucoma is not limited to retina but it is also present along whole visual
pathway. However, dynamics and mechanisms underlying this process remain unclear.
DBA/2J mice spontaneously develop glaucoma-like pathology. Magnetic Resonance Imaging
(MRI) is a rapid, non-destructive and three-dimensional method that can be used to quantitate
brain structures volumes. In vivo morphometric analysis of vision-related brain structures
would provide a valuable tool for tracking the ongoing neurodegeneration.
Methods: DBA/2J female mice with advanced glaucoma (aged 14 months, n=5) and age
matched controls (C57BL/6J; n=5) were anaesthetized with isoflurane (4% in oxygen –
induction, 1.5-2% - maintenance) and placed in 7T small animal-dedicated magnetic
resonance tomograph (BioSpec 70/30USR; Bruker BioSpin, Ettlingen, Germany). High
resolution structural imaging with TURBORARE T2 sequence (TR=7000ms, TEeff=30ms,
RARE factor=4, spatial resolution=0.86mm x 0.86mm, slice thickness=0.35mm, slices=42,
scan time=23min). For the volumetric analysis we employed C57BL/6J atlas database
published online by National University of Singapore
(http://www.bioeng.nus.edu.sg/cfa/mouse_atlas.html) and C57BL/6J tissue probability maps
(TPM) from the SPMMouse toolbox (http://www.spmmouse.org/). Data were processed using
SPM software (http://www.fil.ion.ucl.ac.uk/spm/) and custom-made MATLAB scripts.
Statistical analysis was performed with U Mann-Whitney test.
Results: Calculated total brain volumes were significantly lower in DBA/2J mice (P<0.01).
Most of the analyzed structures were also smaller in DBA/2J mice (including frontal cortex,
visual cortex and auditory cortex). However, when normalisations were performed to the total
brain volumes, significant differences were noted only for some of the segmented structures,
namely visual cortex (P<0.01) and auditory cortex (P<0.01) but not for frontal cortex.
Conclusion: Changes in relative brain structures volumes indicate an ongoing
neurodegenerative process that particularly affects vision-related brain structures in DBA/2J
model of glaucoma.
The study was supported by National Science Centre grant No. 2012/07/D/NZ4/04199.
29
MR TAGGING FOR EVALUATION OF MECHANICAL PROPERTIES
OF FATTY LIVER TISSUE
Anna Osiak1, Krzysztof Jasiński1, Paweł T. Jochym2, Edyta Maślak3, and Tomasz Skórka1
1Department of Magnetic Resonance Imaging, Institute of Nuclear Physics Polish Academy of Sciences, Kraków,
Poland, 2Department of Materials Research by Computers, Institute of Nuclear Physics Polish Academy of
Sciences, Kraków, Poland, 3Department of Endothelium Experimental Pharmacology, Jagiellonian Center for
Experimental Therapeutics (JCET), Kraków, Poland
Introduction: MR Tagging (MRt) has recently been proved useful for liver stiffness
assessment. With the use of heart beating force deforming the adjacent liver instead of
external pulsing device used in elastography, MRt can be alternative for MRE in studying
liver elasticity. We propose MRt-based liver strain analysis, taking advantage from full strain
tensor properties such as eigenvalues, eigenvectors, trace and absolute strain magnitude,
enriched with parameterization of deformed area range and shape. We hypothesize that these
features may potentially change in abnormal tissue conditions, thus we aimed to assess the
accuracy of such a method for early alterations in non-alcoholic fatty liver disease (NAFLD).
Methods: Two groups of 6-month old C57BL/6J mice: Control – standard AIN-93G diet,
NAFLD – 45% + 60% kcal high-fat diet, were examined using 9.4T BioSpec scanner
(Bruker, Germany): ECG-gated FLASH-cine sequence preceded with SPAMM tagging (TE
1.5ms, TR 8.5ms, 192×192 data matrix, FOV 30×30mm2, 1.0 mm slice thickness, tagging
grid with 0.2 mm tag line, 0.6 mm tag span; LV short-axis heart projection).
The liver deformation was computed from MRt images based on two-dimensional strain
tensor ε (%) as the maps of: ε1 (stretching) and ε2 (compression) principal strains (%) with
their spreading directions, fractional volume changes FVC=ε1+ε2 (%) and absolute strain
magnitude AbsE=ε1×ε2 (%). The maps were quantified using two-dimensional central and
invariant image moments for the deformation area and shape assessment.
Results: In NAFLD, the deformed area within all maps was more irregular in comparison to
healthy organ. The ε1 and ε2 directions showed symmetrical strain development about the
central point of heart stimulation in Controls, while in NAFLD mice the strain penetration
seemed to be limited. In FVC and AbsE maps of fatty livers the major amount of deformation
was enclosed in narrow elliptical area. The central and invariant moments analysis showed
decreased strain area and level within all ε1, ε2, FVC and AbsE maps in NAFLD group, with
noticeably larger deformation shape eccentricity as compared to healthy Controls.
Conclusions: The extended MRt-based analysis of cardiac-induced liver deformation,
benefiting from all strain tensor properties showed restricted deformation area in NAFLD,
what may suggest increased resistance and viscosity of FL tissue. The observations were
compliant with significant differences between groups in quantitative analysis. The results
proved the routine useful for in-vivo measurements of the structural changes within hepatic
tissue in NAFLD. The method may be good alternative for classic MRE, which is often
insensitive for liver elasticity alterations in early stage of the disease.
Aknowledgements: Work supported by European Union (grant coordinated by JCET-UJ, No
POIG.01.01.02-00-069/09) and by The Marian Smoluchowski Krakow Consortium “Matter-
Energy-Future” from the resources of KNOW.
30
MRI-BASED METHOD FOR THE IN VIVO ASSESSMENT OF
ENDOTHELIAL STATE IN MURINE MODELS
Anna Osip1,2, Krzysztof Jasiński1, Żaneta Bartel1, Tomasz Skórka1, Stefan Chłopicki2,3
1Institute of Nuclear Physics, Polish Academy of Sciences, Department of MRI, Crakow 2Jagiellonian University Medical College, Department of Experimental Pharmacology,
Crakow,
3Jagiellonian Centre for Experimental Therapeutics(JCET), Crakow
Introduction
Evaluation of endothelial dysfunction, in clinical conditions is currently based on the
biochemical methods, involving detection of important endothelial markers or mediators, and
physical direct methods, which allow for invasive or non-invasive assessment (1). On the
other hand commonly used non-invasive methods, which are useful especially in clinical
prevention, are limited to the study of peripheral arteries. Magnetic resonance imaging (MRI)
seems to be promising method for assessing endothelial dysfunction in coronary arteries and
also is adequate method to study this issue in mice models (2). The aim of the study was to
implement a comprehensive method, for the reliable assessment of endothelial function in
vivo in mice with the use of MR imaging techniques.
Materials and methods
MRI was performed on Bruker BioSpec 9.4T system (Ettlingen, Germany).
Brachiocephalic artery (BCA) and left carotid artery (LCA) were imaged using 3D
techniques based on the retrospectively gated IntraGate™ FLASH sequence in control mice
(4-months old C57BL/6J mice, ACh: n=4, Galb/Pearm: n=4), in mice fed high-fat diet
(HFD60 - 60 kcal%) (5-months old C57BL/6J mice fed for 4 months a HFD60, ACh: n=4,
Galb/Pearm: n=8) and C57BL/6J mice treated with L-NAME (4-months old mice, ACh: 4).
Assessment included the evaluation of endothelium-dependent vasomotion in response to
acetylcholine (ACh - Sigma-Aldrich: 50 µl, 16,6 mg/kg i.p), or changes of endothelial
permeability with the use of an albumin-binding gadolinium contrast agent (CA: Galbumin,
BioPal, Worcester, MA - 25 mg/ml, 4.5 µl/g i.v.) and 3D IG-FLASH - based VFA - Variable
Flip Angle technique. Blood vessel cross-sections area and volume after ACh administration
were determined. Endothelial permeability was assessed by detection T1 relaxation time
changes around vessel lumen and the number of pixels (Npx50), for which T1 has changed
about 50%, 30 min after contrast agent administration. Data analysis was performed in
ImageJ 1.46r program (NIH, USA) and scripts in Matlab (MathWorks, Natick, USA).
Statistical analysis was performed in STATISTICA 10 (Stat Soft inc., USA).
Results
25 min after ACh administration, the vasodilation of blood vessels in C57BL/6J mice
and its paradoxical vasoconstriction in HFD60 mice was observed. Additionally,
vasoconstriction response was higher in LCA. In mice treated with L-NAME ACh did not
induced vasodilation.
Group C57BL/6J C57/HFD60 C57/L-NAME
BCA Area change 25.12 ± 5.04 % -29.22 ± 3.40 % 1.81±3.32
LCA Area change 24.63 ± 2.57 % -54.37 ± 4.02 % -3.91±4.99
BCA Volume change 12.38 ± 3.19 % -10.57 ± 2.28 %
LCA Volume change 14.09 ± 2.49 % -20.79 ± 3.38 %
31
In HFD60 mice shortening of the T1 around BCA was observed as opposed to C57BL/6J
mice, where shortening of T1 was not significant. Npx50 for HFD60 mice was significant
different from Npx50 for C57BL/6J mice.
Group T1 change Npx50
C57BL/6J -5.41 ± 1.56 % 8 ± 2
C57/HFD60 -23.43 ± 7.70 % 20 ± 4
Conclusion
3D MRI-based technique is suitable to detect systemic endothelial dysfunction in vivo
in mice. Our results suggest that potentially, MRI-based assessment of endothelial
permeability and endothelium-dependent vasomotion in response to acetylcholine may be
useful for monitoring experimental, endothelial-targeted therapy.
Acknowledgments This study was supported by European Union from the resources of the European Regional
Development Fund under the Innovative Economy Pro- gramme (grant coordinated by JCET-
UJ, No POIG.01.01.02-00-069/09).
References 1. Nadar S, Blann AD, Lip GYH. Endothelial dysfunction: methods of assessment and application to hypertension. Curr. Pharm. 2004, Tom 10, 3591–3605. 2. Phinikaridou A, Andia ME, Protti A et al. Noninvasive MRI Evaluation of Endothelial Permeability in Murine Atherosclerosis Using an Albumin-Binding Contrast Agent . Circulation (Baltimore),. 2012, Tom 126, 707-719.
32
23Na NMR STUDY OF Al- AND Ga- NANOPOROUS NATROLITES
Mateusz Paczwa, Marcin Olszewski, Nikolaj Sergeev
Aleksey.A. Sapiga*), Aleksey.V. Sapiga*)
Institute of Physics, University of Szczecin *) Faculty of Physics, V.I.Vernadskii Taurida National University, Simferopol, Crimea
The Al-natrolite ( OHOSiAlNa 210322 2 ) and Ga-natrolite ( OHOSiGaNa 210322 2 ) are
compounds with nanoporous structure. In the small nanochannels in the structure of these
natrolites the water molecules and Na ions are located in the form of zig-zag chains [1,2].
In present report we represent the experimental results on the study of polycrystalline Al- and
Ga-natrolites by 23Na NMR methods.
Fig.1. 23Na MAS NMR spectra of Al-natrolite at
rot = 10 kHz. For Ga-natrolite we obtained the
same spectra. (a) – experimental spectrum at T =
300 K. (b) - theoretical spectrum with NMR
parameters: Q=879,6 kHz; =0,64; CSA=8,19
ppm; Gauss=73,11 Hz
The sodium neighbours in Al- and Ga-
natrolites have a configuration of a distorted
tetrahedron. In the tetrahedron corners there
are two oxygen atoms belonging to a
framework and two oxygen atoms of water
molecules at an average distances of 2.37 Å.
Furthermore there are two oxygen atoms of a
framework at an average distances of 2.5 Å,
four protons at an average distances of 2.8 Å
and atoms of silicon and aluminum at an
average distances of 3.0 Å [1]. The obtained 23Na MAS NMR spectra of Al-natrolite are
shown in fig.1. The shapes of these spectra
are determined only by the second-order
quadrupolar shift of the central transition and
the obtained theoretical values of the
quadrupolar frequency Q and the asymmetry
parameter well coincide with experimental
values obtained in [3].
From the comparison of the 23Na NMR spectra,
shown in fig.2, it appears that the 23Na NMR
spectra have the same shape at T = 300 K and T=
380 K. The NMR shape of 23Na is determined by
magnetic dipolar interactions and by electric
quadrupolar interaction with the electric field
gradient (EFG) on the 23Na sites. The interaction
with magnetic moment of 1H nuclei give the main
contribution to the magnetic dipolar interaction of 23Na nuclei. However the 1H decoupling, which
was used to record the spectra of 23Na NMR
(fig.2), leads to averaging of the dipolar
interactions between the magnetic moments of 1H
Fig.2. 23Na NMR spectra with 1H-
decoupling in Al-natrolite at T = 300 K and
T= 380 K
and 23Na nuclei and so, from fig.2, it follows that in the temperature region T < 380 the
electric field gradient (EFG) at the 23Na sites does not depend on the temperature. From our
theoretical calculation it follows that the contribution of electric dipolar moments of water
molecules to the EFG at the 23Na sites is compare to the contribution from other ions of
lattice. However, according with 1H NMR data of Al-natrolite water molecules at T < 380 K
rotate about their quasi 2-fold axis [4]. The flipping of the water molecule does not change its
33
electric dipole moment and so the contribution of the dipoles of water molecules to the EFG
of a 23Na nuclei must be temperature independent. So our result do not conflict with the
results obtained in [4].
Fig.3. 23Na NMR spectra without 1H-
decoupling in Al-natrolite at T = 300 K and T=
380 K
Fig.3. shows the 23Na NMR spectra obtained
without 1H decoupling at T = 300 K and T=
380 K. The difference between the NMR
spectra represented in fig.2 and fig.3 are
related to the dipolar interactions between the
magnetic moments of 1H and 23Na nuclei.
From fig.3 it follows that increasing of the
sample temperature leads to thermal averaging
of dipolar interactions between the magnetic
moments of 1H and 23Na nuclei. This averaging
is connected with the rotations of water
molecules about their quasi 2-fold axis [4].
These rotations do not change the contribution
of the electric dipoles of water molecules to the
EFG at the 23Na positions but lead to averaging of the local magnetic fields from magnetic
moments of the protons of water molecules at the 23Na sites.
The temperature dependences of the spin-lattice
relaxation times T1 of 23Na in Al- and Ga-
natrolite are shown in fig.4. An activation
energy of about 25 kJ/mol was obtained from
data presented in fig.4 and this value agrees
with the activation energies of the rotations of
water molecules in other hydrates [4-6]. This
result indicates that the rotation of the water
molecules about their quasi 2-fold axis is
responsible for the spin-lattice relaxation
process of 23Na nuclei in Al- and Ga-natrolites.
These rotations do not change the contribution
of the electric dipoles of water molecules to the
EFG at the 23Na positions but lead to the
averaging of the local magnetic fields from
Fig.4. The temperature dependences of the T1
in Al – natrolite (rhombus) and Ga-natrolite
(circles)
magnetic moments of the protons of water molecules at the 23Na sites.
[1] A.V.Sapiga, N.A.Sergeev, NMR investigation of natrolite structure. Cryst. Res. Technol.,
36 (2001) 8-10.
[2] A.A.Sapiga, M.Olszewski, M.Paczwa, A.V.Sapiga, N.A.Sergeev, NMR study of
gallosilicate natrolite. Functional Materials, 21 (2014) 181-185.
[3] H.E.Petch, K.S.Pennington, Nuclear Quadrupole Coupling Tensors for 23Na and 27Al in
natrolite, a Fibrous Zeolite. J.Chem.Phys., 36 (1962) 1261-1221.
[4] R.T.Thompson, R.R.Knispel, H.E.Petch, NMR study of the molecular motion of water in
natrolite. Can. J. Phys., 52 (1974), 2164-2173.
[5] A.V.Sapiga, N.A.Sergeev, V.N.Shcherbakov, S.P.Gabuda, I.A.Belicky, Diffusion of water
molecules in rhombic natrolite. J. Struct. Chem., 27 (1986) 183-184.
[6] J.Haase, K.D.Park, K.Guo, H.K.C.Timken, E.Oldfield, Nuclear Magnetic Resonance
Spectroscopic study of spin-lattice relaxation of quadrupolar nuclei in zeolite. J. Phys. Chem.,
95 (1991) 6996-7002.
34
2D AND 3D CP-VC AS TOOLS FOR DYNAMICS STUDY
Piotr Palucha*, Tomasz Pawlaka, Julien Tréboscb, Tatyana Polenovac, Jean-Paul Amoureuxb,d and
Marek J. Potrzebowskia.
a Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies, Sienkiewicza 112,
PL-90-363 Lodz, Poland b Unit of Catalysis and Chemistry of Solids (UCCS), CNRS-8181, University Lille North of France,
59652 Villeneuve d’Ascq, France
c Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware, USA d Physics Department & Shanghai Key Laboratory of Magnetic Resonance, East China Normal
University, Shanghai 200062, China
One of the biggest achievements of modern solid state NMR spectroscopy is its ability to
determine accurate inter-nuclear distances, which can be afterward used as structural restraints for
reconstruction of three-dimensional structures of the condensed matter . The most common strategy
is based on the analysis of homo- and/or hetero-nuclear dipolar couplings, which are both inverse
proportional to the cube of inter-nuclear distances. Among different interactions, C–H and N–H
one-bond contacts are of great interest in the context of characterizing inter-molecular arrangement
via hydrogen bonding as well as backbone and side-chain dynamics in biological molecules. Indeed,
the partial averaging of C–H and/or N–H dipolar couplings gives information about the geometry
and amplitude of the motional processes in the solid state. During last decades, different
methodological approaches, both for static samples and samples under magic angle spinning (MAS),
have been introduced in order to improve the quality and reliability of obtained data. The big
achievement in the field of measurements of X–1H dipolar couplings was the introduction of
PISEMA technique , and its different variants which allowed determining dipolar interactions under
MAS eg. PILGRIM. One years ago we demonstrated that a very simple experiment,
Cross-Polarization with Variable Contact-time (CP-VC), is very efficient at ultra-fast MAS (vRO=60
kHz) to measure accurately the C–H and N–H distances, and to analyze the dynamics of
bio-molecules. Very recently we developed new multidimensional solid-state NMR methodology
which permits, in simple and accurate way, the analysis of the 1H-13C dipolar splittings and further
scrutiny of the molecular motions in side chains in nanocrystalline proteins. The power of the
technique is demonstrated in 3D NMR CPVC-RFDR correlation experiments in two proteins, GB1
and DLC8. This presented methodology is general and can be extended to other systems.
Fig. 2. F1/F2 planes extracted from 3D CPVC-RFDR spectrum of GB1 for the following residues:
Tyr (a, b), Phe (c) and Trp (d). The F3 values for each plane are indicated in the figure.
In my presentation I will briefly present some methodology and possibilities of advanced solid state
NMR and latter I will discuss on probing dynamics of aromatic residues: phenylalanine, tyrosine
and tryptophan using our new methodological approaches.
35
Evaluation of the relaxation and the imaging properties of SPIO loaded
nanocapsules at 9.4 T
P. Piechota1, K. Szczepanowicz2, P. Warszyński2, W. P. Węglarz1
1Henryk Niewodniczański Institute of Nuclear Physics PAN, Krakow, Poland 2Jerzy Haber Institute of Catalysis and Surface Chemistry PAN, Krakow, Poland
Purpose: The aim of this work was to evaluate relaxation and visualization properties of
nanocapsules with iron oxide nanoparticles (SPIO) incorporated into their shell, at 9.4 T.
Materials and methods: Four different samples: SPIO, nanocapsules without SPIO, nanocapsules
with single layer of SPIO in the shell and nanocapsules with double layer of SPIO in the shell
were investigated using 9.4T Bruker Biospec MR scanner. CPMG and IR pulse sequences were used
for T2 and T1 relaxation times measurements, respectively. RARE pulse sequence was used for MR
imaging. Dependence of relaxivities on relative SPIO concentrations were obtained with the use of
OriginLab software.
Results: The best contrast effect was obtained for nanocapsules with double layer of iron
oxides in the shell. The T1 relaxation time was 6 to 7 – fold and the T2 relaxation time was
7.5 to 14 – fold shorter as compared to three other samples. The best relaxivity was obtained
for the highest concentration of iron oxides in the sample.
Discussion: The higher the difference between relaxation times of two neighbouring tissues, the better
contrast effect is obtained in MR images. Superparamagnetic iron oxides have ability to shorten
relaxation times of tissues in which they are accumulated. Nanocapsules with double layer of iron
oxides in the shell gave good contrast effect which indicate the possibility of imaging their
distribution in the living organisms using MR imaging.
36
MR Imaging of the Mouse Brain using Cryo-coil at 9.4 T - Histology in vivo ?
W.Piędzia1, N. Bock2, K. Jasiński1, K. Kalita1, G. Stanisz3, W.P. Węglarz1 1Institute of Nuclear Physics, Polish Academy of Sciences, Kraków, Poland
2Medical Physics and Applied Radiation Sciences, McMaster University, Hamilton, Ontario, Canada 3Sunnybrook Health Sciences Centre, Toronto, Ontario, Kanada
Purpose White matter (WM) degeneration is caused by many disorders including Multiple Sclerosis. Among many constituents of WM, myelin damage is the most pronounced and affects brain function. Myelin changes can be evaluated by magnetic resonance imaging (MRI) through T2 and T1 relaxation times [1-5]. The inversion recovery ultra short echo time pulse sequence (IR-UTE) [3] has been shown to be well suited for T1 quantification measurements in vivo. However imaging of the whole brain is challenging using this techniques due to long total acquisition time. Recently, Bock et al [5] suggested MP-RAGE pulse sequence for high resolution and time efficient imaging of mouse brain that yields high quality myelin maps including cortical white matter. The purpose of our study was to assess feasibility of using segmented MP-RAGE pulse sequence and cryo-coil for contrasting WM/GM and quantification of T1 in mouse brain in vivo, as compared to room temperature brain surface coil. Methods A healthy C57BL/6J mice were scanned using a 9.4T/21cm horizontal bore Bruker Biospec MRI system. A dedicated mouse brain surface coil and a cryo-coil were used for obtaining a two sets of corresponding full 3D (multislice) images of the brain. Two mice were scanned with each coil using the same experimental parameters: TR/TE = 15/4.5 ms, Segment Repetition Time (SRT) = 6000 ms, FA = 12º , 22 horizontal slices with 0.3 mm thickness covering the whole brain volume, together with FOV = 1.5 x 2 cm and MTX = 256x256 were used resulting with in-plane resolution of 59x78 µm. Two averages and the following Inversion Times (TI): 500, 900, 1000, 1100, 1200, 1300, 1400, 1600 and 2500 ms were used. Pixel by pixel T1-maps were generated from the data using custom written Matlab based script. For 3 selected inversion times (1000, 1100 and 1200 ms for surface coil while 900, 1000 and 1100 ms for cryo-coil) images with NA = 6 were measured for another animal. Additionally, images without transmitting RF power (i.e. with pulse power attenuation of 150 dB) were generated in order to properly assess noise level for SNR comparison between room temperature surface brain coil and cryo-coil. Results Calculations show 2.3 – 3.0 times higher SNR for cryo-coil as compared to room temperature brain coil. The exact figure depends on which horizontal slice level the flip angle was adjusted for cryo-coil. Fig. 1 shows the comparison of selected horizontal slices obtained at two different TI’s, illustrating switching of contrast between GM and WM, due to nulling of signal from WM at TI=900 ms, while GM signal is nulled at TI=1000ms. The corresponding TI values for room temperature coil were about 100 ms higher. The difference stem from the inhomogenous spatial distribution of flip angle in case of transmit/receive cryo-coil in contrary to room temperature setup where spatially homogenous flip angle is defined by volume transmit coil, while surface coil is used only for signal receiving. Fig. 2. shows calculated T1 maps from cryo-coil and for comparison from room temperature setup. Effect of FA inhomogeneity was not taken into account in calculation. Significantly more fine details is visible in cryo-coil based map, while already mentioned decrease in T1 values is observed.
Fig.1 MP-RAGE images (TI = 1100 ms and 900 ms) and the T1 map of the mouse brain obtained from cryo-coil Conclusions We have shown that MP-RAGE sequence used with cryo-coil at 9.4T, due to significant 2-3 times larger SNR than available from the dedicated room temperature brain coil, allowed for obtaining high resolution images of the whole mouse brain in vivo, with easily adjustable WM/GM contrast trough choice of appropriate inversion time, within the experimental time very attractive for in vivo experiments. Proposed MP-RAGE/cryo-coil setup
37
is very promising for quantitative assessment of myelination in mouse models and potentially for in-vivo mouse brain histology. References [1] Wilhelm M.J. et al: PNAS; 109 (24): 9605–9610 (2012) [2] Larson P.E.Z et al: Magn Reson Med; 56 (1): 94–103 (2006) [3] Piędzia W. et al: Journal of Neuroscience Methods; 232: 30–35 (2014) [4] Chavez S et al..: NMR Biomed.; 25(9): 1043–1055 (2012) [5] Bock N. et al.: NeuroImage 65 1-12 (2013)
38
SUPERCONDUCTING DETECTION COIL FOR 0.2 T MRI
SYSTEM
Bartosz Proniewski1, Henryk Figiel1
1 AGH University of Science and Technology, Faculty of Physics and Applied
Computer Science, Al. Mickiewicza 30, 30-059 Krakow, Poland
In magnetic resonance imaging, the detection coil plays an important role,
being responsible for both, the excitation of the resonance and the detection of the
MR signal. The design of the detection coil is crucial in obtaining high quality
images, as their sensitivity is directly translatable into signal to noise ratio (SNR),
a standard approach in quantification of imaging quality. Quality factor Q of the
radiofrequency (RF) is one electrical parameter that directly impacts the SNR and
therefore influences the imaging quality of an MRI system. Its value can be measured
on the bench and directly compared between coil designs. Using low electrical noise
materials enables higher Q values of the coils, hence providing better image quality.
In this work we have underlined key aspects of utilizing commercially
available high temperature superconducting tapes for manufacturing a surface RF
coil. Numerical calculations were carried out in order to determine the influence of
external magnetic field on current flow in the superconductor with varying orientation
of the tape. Based on the optimum orientation of the tape and the orientation of the
external magnetic field in the system the coil was designed for, a coil type was
selected to be in the form of a Figure of 8 and the tape layout was optimized using
electromagnetic simulations. A cryostat built from multilayered wall was then
designed to keep the room temperature for sufficient amount of time required to
perform imaging experiments. Once all the key factors have been assessed, the final
design was established and a prototype was built.
Three identical coils were built – one from a 1st generation and one from the
2nd generation HTS tapes supplied by American Superconductors Company and a
third reference coil made out of copper wire. All cols have been tuned and matched
inductively to the 8.86 MHz frequency and characterized on the bench, by
measurements of their Q factors. Cryostat design was evaluated by measuring
temperature on the outside, where the imaged object was to be placed. Performing
imaging experiments using a simple phantom carried out experimental validation of
the use of HTS tapes in RF coil design. Images were used to calculate the SNR in
various planes for the three coils working in room temperature and in cryogenic
conditions (77 K). Results show that HTS coils operating in cryogenic temperature
can indeed provide higher SNR relative increase, compared to the copper coil. In fact
the copper coil improved by 28-47% while the HTS coil by 81-127% when immersed
in liquid nitrogen, compared to room temperature operation.
39
PARA HYDROGEN INDUCED POLARIZATION OF
PYRIDINE-LABELLED OLIGOPEPTIDES Tomasz Ratajczyk
Institute of Physical Chemistry Polish Academy of Sciences, Kasprzaka 44/52,
01-224 Warszawa, Poland
The application of Magnetic Resonance techniques is strongly limited by
inherently low sensitivity. This problem can be solved by so-called hyperpolarization
techniques. One of these techniques is the Para Hydrogen Induced Polarization (PHIP)
[1]. In the PHIP variant called SABRE, the NMR signal is enhanced by the interaction
of para-hydrogen with an appropriate molecular system via a catalyst in a complex
arising in a reversible reaction [2]. When the labile complex is formed, high spin
polarization is transferred from para-hydrogen to the substrate. Afterwards, the labile
complex splits up into hydrogen molecule, the catalyst and the SABRE-
hyperpolarized product. Biofunctional SABRE-hyperpolarized molecular systems
would be of central importance for widening the scope of possible MRI applications.
Thus, the design of appropriate SABRE-active molecular systems is crucial.
Fig. 1 SABRE Fig . 2 SABRE activation
Here this problem is addressed. We propose a guideline for designing of
SABRE-active molecules. We show that SABRE-inactive molecular systems can by
SABRE-activated using labelling with pyridine. In particularly, we report SABRE of
simple pyridine-labelled oligopeptides. We reveal that these oligopeptides interact with
catalyst and hydrogen molecule. When the 3-components labile complex is formed,
high spin polarization from para-hydrogen is transferred only to the pyridine unit. For
all studied compounds 1H NMR signal enhancement factors were evaluated.
[1] C.R. Bowers, D.P. Weitekamp, J. Am. Chem. Soc. 109 (1987) 5541; M.G. Pravica,
D.P. Weitekamp, Chem. Phys. Lett. 145 (1988) 255; T.C. Eisenschmid, R.U. Kirss,
P.P. Deutsch, S.I. Hommeltoft, R. Eisenberg, J. Bargon, R.G. Lawler, A.L. Balch,
J. Am. Chem. Soc. 109 (1987) 8089.
[2] R. W. Adams, J. A. Aguilar, K. D. Atkinson, M. J. Cowley, P. I. P. Elliott, S. B.
Duckett, G G. Green, I. G. Khazal, J. Lopez-Serrano and D. C. Williamson,
Science, 323 (2009), 1708;
This work has been supported by the Polish National Science Centre (NCN) under
Contract No.: SONATA-2011/03/D/ST4/02345.
40
APPLICATION OF MICRO-MRI TECHNIQUES IN THE
EVALUATION OF MUSCLE DEGENERATION AND REPAIR
PROCESSES AFTER FEMORAL ARTERY OCCLUSION IN MICE
Agnieszka Skorupa1, Mateusz Ciszek1, Łukasz Boguszewicz1, Tomasz Cichoń2, Ryszard
Smolarczyk2, Stanisław Szala2, Maria Sokół1
1Department of Medical Physics, 2Center for Translational Research and Molecular Biology
of Cancer, Maria Skłodowska – Curie Memorial Cancer Center and Institute of Oncology,
Gliwice, Poland.
Introduction: Development of non-invasive techniques enabling evaluation of muscle
damage and repair processes would provide a valuable insight into the effects of therapeutic
interventions (for example injection mesenchymal stem cells into the injured muscles). The
purpose of this work was to evaluate the kinetics of MRI-derived parameters characterizing
muscle function after femoral artery ligation in mice.
Materials and methods: MRI experiments were performed immediately, 3, 7, 14 and 21
days after femoral artery ligation on a 9.4 T vertical 89-mm-bore Bruker magnet equipped
with a Bruker Micro2.5 gradient system of 1T/m and a transmit/receive birdcage radio
frequency coil with an inner diameter of 30 mm. During data acquisition animals were
anesthetized with sevoflurane. Body temperature and respiration rate were monitored using
ECG/respiratory unit. 2D Time of flight (TOF) angiography was used to visualize collateral
vessel formation after artery ligation. Multi-slice multi-echo (MSME) sequence and diffusion
tensor imaging (DTI) with fat suppression were used to characterize muscle tissue in terms of
transverse relaxation time (T2), fractional anisotropy (FA), apparent diffusion coefficient
(ADC), radial (RD) and axial (AD) diffusities.
Results and discussion: Longitudinal MRI monitoring of mice revealed decreased FA and
increased T2, ADC and RD after experimental injury and progressive normalization of these
parameters with time. The observed changes are presumably due to initial degeneration and
subsequent rapid repair of the injured tissue. Though, the presence of intramuscular oedema
(increased high T2 signal) on MRI carries an extremely broad differential, oedema-like
changes are characteristic for acute phase of muscle injury, while abnormal DTI-derived
parameters reflect disrupted muscle architecture.
Conclusion: Multi-parametric MRI used in this study revealed dynamic response of T2,
ADC, FA and RD parameters after ischemic muscle injury and provides information useful in
monitoring of therapeutic effects of cellular therapies tested in animal models.
41
APPLICATION OF NMR RELAXATION MEASUREMENTS TO THE STUDY OF
OXIDATION PROCESSES IN BIOLOGICAL SYSTEMS
DOROTA WIERZUCHOWSKA*, LECH SKÓRSKI, BARBARA BLICHARSKA
*Institute of Physics, Pedagogical University, ul. Podchorążych 2, 30-084 Kraków, Poland
Institute of Physics, Jagiellonian University, ul. Reymonta 4, 30-059 Kraków, Poland
So far only EPR and CI DNP (Chemically Induced Dynamic Nuclear Polarization)
methods have been used to observe the action of free radicals in biological systems [1,2]. In
this communication we present some new results obtained by NMR relaxation method,
namely the time evolution measurements of relaxation time T1 in protein aqueous solutions
such as: egg white and bovine serum albumin and rabbit blood serum, after initiation of
oxidation process by addition of H2O2.
Hydrogen peroxide is one of the strongest reactive oxygen spiecies and added to
aqueous solutions causes changes of the water proton relaxation times [3,4]. Just after
addition of H2O2/H2O (3%) to albumin solution samples the relaxation time T1 starts to short
and after approximately 25 minutes stabilizes. The kinetics of this process depends on the
structure and concentration of the protein and amount of added hydrogen peroxide. Measured
time evolution curves may be well approximated by exponential decay.
In rabbit serum, instead of stabilization, after reaching the minimum value the
relaxation time T1 starts to regrow. Similar kinetics is observed in experiments with protein
solutions containing a small amount of added antioxidants like ascorbic acid (vitamin C).
Moreover, the supplementary addition of vitamin C to rabbit blood serum enhances the T1
behavior. It means that regrow of T1 might be a consequence of the action of antioxidants,
which are known to be present in blood serum.
We hope that in the future NMR relaxation measurements may be useful not only in
investigation but also in diagnosis of some diseases, especially with free radical background.
[1] D.A. Svistunenko, Biophys. Biochim. Acta 1546 (2001) 365-378
[2] L.T. Kuhn, J. Bargon, Top.Curr.Chem. 276 (2007) 125-154
[3] L.W. Skórski, B. Solnica, B. Blicharska, Journal of Laboratory Diagnostics 47(1) (2011) 85-89
[4] H.C. Bertram, M. Kristensen, H. Osdal, C.P. Baron, J.F. Young, H.J. Andersen, J.Agric.Food
Chem. 55 (2007) 2342-2348
42
CHEMICAL EXCHANGE SATURATION TRANSFER (CEST). FROM AN AGAR TO A MAN.
Greg J Stanisz
Physical Sciences, Sunnybrook Research Institute
Departmental of Medical Biophysics, University of Toronto
Toronto, Ontario, Canada
This work describes the origins of the contrast mechanism chemical exchange saturation transfer (CEST) and methods for proton magnetic resonance imaging (MRI), with application in cancer. It focuses on endogenous techniques, which do not require the injection of a contrast agent, and the experimental and analytic techniques which allow quantitative metrics to be obtained. The theory behind CEST is presented, and it is modelled using a three pool system of Bloch equations which are a function of magnetization relaxation parameters, CEST pool concentration, and hydrogen exchange rate constant. A platform is created for optimization and parameter fitting, which is used to obtain parameter values and for pulse sequence experimental design. The contribution of semi-solid macromolecules to the CEST spectrum is determined, and evidence given that the CEST parameters (in particular, the exchange rate constant) can be obtained independently of this additional pool. It is shown that there is a linear relationship between the log of the exchange rate constant and pH. CEST spectra are obtained in cancer xenografts in mice, exhibiting features from amide, amine and aliphatic protons as well as from magnetization transfer. Semi-quantitative CEST parameter maps are derived, showing the distribution of CEST features in tumours and in contrast with the surrounding normal-appearing muscle. Several exploratory experiments are performed in protein-containing phantoms and cell pellets in order to validate the origins of CEST spectra and their behaviour in conditions of changing pH and temperature. Cell pellet experiments support the hypothesis that CEST is sensitive to primarily intracellular conditions, and furthermore that the cell nucleus is a concentrated source of CEST-contributing proteins. Ultimately, the work contained herein supports the hypothesis that the endogenous CEST experiments provide new information not obtainable from other MRI experiments, leading to quantitative absolute intracellular pH mapping. This has potential for predicting the effectiveness of treatment regimens based upon the relationship between intracellular pH and drug uptake, establishing regions of tumour which are actively proliferating or which may be resistant to therapy.
Figure 1: A visual comparison of the maps generated from each MRI contrast mechanism examined in this study, for a single scan time-point in a single Lewis Lung Carcinoma tumour xenograft in a mouse. Also shown, are the reference spin echo image and the TUNEL and H&E histology images for apoptosis.
43
NMR TOP SYGNALS OF THE 27Al IN SOLID SOLUTIONS BASED
ON THE YAG CRYSTAL
Piotr Stępień, Marcin Olszewski, Nikolaj Sergeev, Bohdan Padlyak*)
Division of Solis State Physics, Institute of Physics, University of Szczecin *) Spectroscopy Sector, Institute of Physical Optics, 23 Dragomanov Str., 79-005 Lviv, Ukraine
and Division of Spectroscopy of Functional Materials, Institute of Physics,
University of Zielona Góra, 4a Szafrana Str., 65-516 Zielona Góra
The Two-dimensional One Pulse (TOP) experiment is the simplest 2D – experiment [1-4].
In this experiment 2D signal s(t1,t2) of the sample, which rotates with frequency R is obtained
from identical 1D signals - free induction decays (FID), separated by tR = 2/R in both t1 and t2
dimensions. The main strength of TOP method lies in its rapid interpretation of MAS signals of
half-integer quadrupolar nuclei [3]. In [1-3] affirms that TOP spectroscopy leads to a better
resolution of information disguised in conventional 1D MAS spectra and it is an ideal method for
study of satellite transition of quadrupolar nuclei. In this communication we represent the
application of TOP method to study of 27Al 2D – spectra of nominally pure and Cr-doped
yttrium-aluminium garnet YAG (Y3Al5O12 and Y3Al5O12:Cr) crystals.
In Fig. 1 and Fig. 2 are presented the 27Al MAS NMR spectra obtained for powdered
crystalline samples Y3Al5O12 and Y3Al5O12:Cr. All 27Al MAS NMR spectra, which are observed
in YAG, contain two peaks corresponding to the tetragonal (AlO4) and octahedral (AlO6)
structural atomic groups. The simulation of the experimental 27Al MAS NMR spectra of the
Y3Al5O12 and Y3Al5O12:Cr give the fractions of the AlO6 and AlO4 groups: N(AlVI) / N(AlIV)
0.67 for YAG and N(AlVI) / N(AlIV) 0.85 for YAG:Cr. So the doping by Cr of the Y3Al5O12
crystals leads to variation of the occupation by Al atoms both octahedrally- and tetrahedrally-
coordinated sites of the garnet lattice.
Fig. 1. The theoretical (a) and experimental (b)
curves for 27Al MAS NMR spectrum of the
nominally-pure polycrystalline Y3Al5O12
sample.
Fig. 2. The theoretical (a) and experimental (b)
curves for 27Al MAS NMR spectrum of the
Y3Al5O12:Cr polycrystalline sample.
The isotropic chemical shifts (iso), quadrupolar coupling constants (CQ) of the 27Al nuclei
in the AlO4 and AlO6 structural groups obtained by Dmfit program [5] are presented in Table 1.
44
Table 1. Quadrupole coupling constants (CQ), isotropic chemical shifts (iso), and the broadening
parameters (L) of the Lorentzian function, for AlVI and AlIV in the un-doped and Cr-doped
YAG crystals. The asymmetry parameter = 0.
The Al sites in
YAG crystal
Un-doped YAG Cr-doped YAG
CQ,(MHz) iso,(ppm) L ,(kHz) CQ,(MHz) iso,(ppm) L,(kHz)
Tetrahedral 6.1 82 0.15 6.07 80.26 0.54
Octahedral 0.6 5.38 0.9 0.607 5.31 0.9
In Fig. 3 and Fig. 4 are presented the 27Al TOP NMR spectra obtained for powdered
crystalline samples Y3Al5O12 and Y3Al5O12:Cr.
Fig. 3. The 27Al TOP spectrum of YAG with
different projections of 2D-spectrum.
Fig. 4. The 27Al TOP spectrum of YAG:Cr with
different projections of 2D-spectrum.
From comparison of Fig. 3 and Fig. 4 we conclude that the TOP MAS NMR spectroscopy
is a sensitive and powerful method for investigating the local structure of main structural units in
ordered and disordered solids and the redistribution of atoms between different sites caused by
doping impurities.
[1] B. Blumich, P. Blumler, J. Jansen, Presentation of sideband envelopes by two-dimensional
one-pulse (TOP) spectroscopy, Solid State NMR, 1 (1992) 111-113.
[2] P. Blumler, B. Blumich, J. Jamsen, Two-dimensional one-pulse rotational echo spectra, Solid
State NMR, 3 (1994) 237-240.
[3] D. Massiot, J. Hiet, N. Pellerin, F. Fayon, M. Deschamps, S. Sreuernagen, P.J. Grandinetti,
Two dimensional one pulse MAS of half-integer quadrupolar nuclei, J. Magn. Res., 181 (2006)
310-315.
[4] Ph.J. Grandinetti, J.T. Ash, N.M. Trease, Symmetry pathways in solid-state NMR, Progress in
NMR spectroscopy, 59 (2011) 121-196.
[5] D. Massiot, F. Fayon, M. Capron, I. King, S.Le Calve, B. Alonso, J.-O. Durand, B. Bujoli, Z.
Gan, G. Hoatson, (2002) Modelling one- and two-dimentional solid-state NMR spectra. Magnetic
Resonance in Chemistry, 40 (2002) 70-76.
45
SPI Implementation for 4.7T System
Grzegorz Stoch
H. Niewodniczański Institute of Nuclear Physics of PAN, 31–342 Kraków, Poland
SPI is an imaging technique free from artifacts arising from B0 inhomogeneity,
susceptibility variations, and chemical shift [1]. It is capable to deliver data for systems with
very short T2 relaxation times. These capabilities are key points for extending MRI research
on the field of solid state imaging - which has been not the case so far.
Original approach to SPI implementation for our 4.7T system is presented (Oxford
Instruments MARAN DRX console).
References:
[1]. B.J. Balcom, SPRITE imaging of short relaxation time nuclei, in: P. Bleumler, B. Bleumich, R. Botto, E.
Fukushima (Eds.), Spatially Resolved Magnetic Resonance, Wiley-VCH, Toronto, 1998, pp. 75–86.
46
DYNAMIC EFFECTS IN SINGLE CRYSTAL OF 9,10-
DIMETHYLTRIPTYCENE-D12 ON BASIS OF PROTON NMR
SPECTRA
Piotr Bernatowicz,1 Tomasz Ratajczyk,1 Alexander Shkurenko,1 Bohdan Kamienski2,
Sławomir Szymański2
1Institute of Physical Chemistry, Polish Academy of Sciences, 2Institute of Organic
Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warszawa, Poland
Single-crystal and powder X-ray diffraction studies of 9,10-dimethyltriptycene (1)
and its d12-isotopomer deuterated in the aromatic positions (1a), made lately by us and
other authors, consistently delivered undistorted R-3c structure of the material.1 In this
structure, all methyl groups in the crystal occur in closely spaced pairs in a staggered
conformation, with different pairs being both structurally and magnetically equivalent
(see Scheme 1). On the other hand, the once obtained 1H
wide line NMR spectra of polycrystalline 1a were
inconsistent with the above structure.1 The presently
reported 1H spectra of a single crystal of 1a also reveal
severe inconsistency with the R-3c structure determined for
the same crystal from X-ray diffraction data. These NMR and
X-ray data were both collected below 135 K where the methyl group dynamics are
completely frozen on the NMR timescale. Apart from that shown in Scheme 1, the
NMR spectra reveal another sharply defined environment of the methyl groups,
involving as much as about 20 per cent of the latter. In this environment, the methyl
groups apparently have the same orientations as in the pairs but suffer no dipole-dipole
couplings to outer protons. The spectrum at 131 K is shown in Fig. 1, together with the
theoretical fit. The theoretical component spectra corresponding to the two
environments are also shown. The observed effects can tentatively be explained as
follows: (i) in crystals of 1a point vacancies cause their nearest surroundings to form
mesoscopic domains only slightly differing from the ordered R-3c phase; (ii) as such,
these domains are practically invisible in the X-ray diffraction patterns; (iii) within the
domains, the vacancies undergo rapid diffusion by purely translational jumps of the
molecules of 1; (iv) in this way, the dipolar spin-spin couplings between the paired
methyl groups are lost in a similar way as is the loss of J-couplings between rapidly
dissociating and recombining fragments of
molecules in solution. A precise definition of
the domains mentioned under (i) is still to be
elaborated. The dynamic disorder of this type is
unusual because of its persistence down to
relatively low temperatures.
Fig. 1. 1H NMR spectrum of single crystal of 1a at 131 K
(black), with the methyl groups' axes parallel to the
magnetic field. The theoretical best fit spectrum and the
component theoretical spectra of the ordered and
dynamically disordered domains are depicted in red, blue,
and green, respectively. The ratio of the intensities of the
green and blue spectra is 0.220.
0.33 nm
Scheme 1.
47
Actually, below 90 K the spectra do start to broaden and change shape but in a way
that cannot be unambiguously interpreted in terms of gradual freezing of the considered
dynamic disorder.
A distinctive feature of the proposed mechanism of the disorder is that the two types of
methyl groups should experience essentially the same environment on the average. In one
environment, the two given groups remain permanently in contact while in the other such
close contacts are being constantly disrupted to be immediately restored, but with one
partner changed.
Above 160 K, effects of thermally activated dynamics of the methyl groups in 1a are
evidenced in the spectra. A series of variable temperature spectra of 1a documenting
these effects were measured for the orientation where the threefold axes of the methyl
groups are directed at right angle to the external field. The spectra are shown in Fig. 2.
They could be perfectly fitted with the damped quantum rotation (DQR) model2 with
the additional assumption that the dynamic properties of the isolated methyl groups and
those coming in coupled pairs are identical. The conventional fits shown in the right
panel of Fig. 2 are evidently defective in the region of slow and moderate exchange.
Fig. 2. Variable temperature spectra of oriented single crystal of 1a (black lines) superposed with
theoretical best fit spectra calculated with the DQR model (red lines, left panel) and the conventional
random jump model (blue lines, right panel). The green line depicts the best fit spectrum to the
experimental pattern measured at temperature where the methyl group dynamics are frozen. The
threefold axes of the methyl groups are oriented at right angle to the external field. All calculations were
performed under assumption that the ratio of the fractions of the isolated and pairwise coupled methyl
groups is 0.220, i.e., the same as determined at 131 K.
The perfect character of the DQR fits obtained for experimental spectra from an
extended temperature range provides one more corroboration of the postulated
mechanism of the dynamic disorder. If the methyl groups resided in two basically
different environments, it would be unlikely for them to share the same dynamic
parameters controlling their hindered rotation.
1P. Bernatowicz, T. Ratajczyk , P. Kalicki, and S. Szymanski, Solid State NMR, 59-60, 34–44 (2014). 2S. Szymański, J. Chem. Phys. 111, 288-299 (1999).
48
Diverse dynamics of water molecules confined in faujasites.
Deuteron NMR investigation. A. Szymochaa, Z.T. Lalowiczb, Birczynskib, K. Gora-Marekc
a H. Kollataj Academy of Agriculture, Mickiewicza 21, 31-120, Krakow, Poland bH. Niewodniczanski Institute of Nuclear Physics PAS, Radzikowskiego 152, Krakow, Poland cFaculty of Chemistry, Jagellonian University, ul. Ingardena 3, 30-060 Krakow, Poland
Study of dynamic behavior of water molecules in zeolites is a part of investigations
aiming to elucidate also catalytic properties at molecular level. In a detailed microscopic
model one expects features related to various interactions, such as electrostatic water-sodium
cation, hydrogen bonding of water to framework oxygens, and water-water bonding, as well
as their dependence on the loading and Si/Al ratio. There are two narrow components of
different width in the spectra above 220K for all samples considered. Their relative weights
change with temperature. We may conclude at this point that there are two phases and
negligible exchange between them. Contribution of the narrow component undergoes
thermally activated temperature dependence and can be attributed to water molecules forming
clusters freely mowing in space, with O-D performing internal tetrahedral jumps. Other water
molecules perform chaotic rotational jumps, and belong to bottom adsorption layers at sodium
cations. Their contribution increases on decreasing temperature. All molecules become
localized below 220K, as indicated by extreme broadening of the spectra, which in turn
provide evidence for the symmetry of deuteron mobility. Three main components can be
pointed out. Pake doublets, with the separation related to the quadrupole coupling constant,
are attributed to immobile deuterons. The value of the quadrupole coupling constant, there are
four similar values measured in all cases, allows to specify location of a deuteron on four
chemically distinguishable oxygens in the zeolite framework. Twofold exchange of deuteron
positions leads to the characteristic spectral shape. Gaussian spectral components, with the
width decreasing on increasing temperature, represent chaotic reorientations leading to narrow
spectra at high temperature. Contribution of these components depends on Si/Al ratio and
loading. Pake doublets dominate at temperature below 70K, while twofold exchange was
observed at the intermediate range.
The project was generously supported by the National Science Centre, Grant No. N
N202 127939 during 2010-2013.
49
CHLORINS – SYNTHESIS AND NMR SPECTROSCOPY STUDIES
Justyna Śniechowska, Piotr Paluch, Marek J. Potrzebowski
Centre of Molecular and Macromolecular Studies Polish Academy of Science,
Department of Structural Studies, Lodz, Poland
Chlorins belong the group of heterocyclic compounds consisting, at the core, of
three pyrroles and one pyrroline that are connected by bridging carbon atoms into a
macrocyclic structure. Due to unique physical and chemical properties, they can be used
as an excellent photosensitizers in photodynamic diagnosis and therapy as well as
anticancer and antibacterial drugs. Knowledge about their chemistry and structural
complexity is strongly desired.
In this work we present method of the synthesis of several chlorins (Fig. 1) and
their advanced structural study employing NMR spectroscopy in the liquid and solid
state. Using high-resolution NMR techniques such as 1H-13C HSQC/HMBC, 19F-13C
HSQC, 1H-15N HSQC, 19F-19F COSY and 19F-1H HOESY the chemical shift assignment
of signals in the spectra for the individual atoms of the molecule in the liquid state was
done. The structure of major tautomer was established. Furthermore, the chlorins were
found to be a very interesting and unique model for NMR studies, because of
differentiation of hydrogen or fluorine atoms in side groups. It is probably caused by
disturb of planarity system and restricted rotation of rings in meso-positions.
In the solid state we employed both advanced two-dimensional (2D) NMR
techniques and theoretical calculations that provide in-depth information about the
structure and intermolecular interactions within host-guest (H-G) complexes.
N
N
NH
NH
R
RR
RX
N
N
NH
NH
R
RR
R XN
Fig. 1. General structure of obtained chlorins
50
NMR SPECTROSCOPY OF SERUM LIPID EXTRACTS OF
SARCOIDOSIS PATIENTS
Toczylowska Beata1,2, Jastrzebski Dariusz3, Zieminska Elzbieta4, Zieleznik Karolina3,
Zebrowska Aleksandra5, Ziora Dariusz3, Mierzejewska Aneta*, Kozielski Jerzy3
1. Nalecz Institute of Biocybernetics and Biomedical Engineering, Polish Academy of
Sciences, Warsaw, Trojdena 4
2. Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw,
Pawinskiego 5A
3. School of Medicine with the Division of Dentistry, Chair and Department of Lung
Diseases and Tuberculosis, Medical University of Silesia, Zabrze, Koziolka 1
4. Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw,
Pawinskiego 5
5. Department of Physiological and Medical Sciences, Academy of Physical Education,
Katowice, Mikołowska 72a
Student of Warsaw University, work done in Institute of Biochemistry and Biophysics,
Polish Acadamy of Sciences, Warsaw, Pawinskiego 5A
The aim of this study was to determine the use of the lipid profile of patients with
sarcoidosis and compare it with healthy subjects. We assume that lipid profile of serum in
sarcoidosis differs from the lipid profile of control subjects.
Serum was collected from 9 patients with II stage of sarcoidosis and 5 control subjects
(healthy volunteers). Lipids were extracted from serum before analysis using modified Blight
and Dyer method and dissolved in deuterated chloroform (Fig1). Proton NMR spectroscopy
combined with discriminant analyses, PCA (principal component analysis) and PLS-DA
(partial least squares projections to latent structures discriminant analysis), was used. NMR
spectra were collected using 400MHz spectrometer and standard one pulse sequence. Thirty
four NMR signals of lipid compounds were selected using home prepared software.
Quantities of metabolites were expressed as relative intensities of spectral peak magnitudes as
compared to the internal standard – CHCl3 rest signal. Targeted profiling will be applied to
each NMR spectrum as the data reduction technique. Partial least square discriminat analysis
(PLS-DA) with Pareto scaling were used to analyze lipid profile.
Fig1. NMR spectrum of lipid extract
Univariate t-test analysis show significant differences in NMR signals of in 14 out of 34
of NMR signal levels from compounds: total cholesterol, both esterified and free cholesterol,
phosphatidylcholine, triglycerides, fatty acids and unassigned compounds (4.94 ppm,
6 5 4 3 2 1 0
ppm
PL/TG
EC
PC
TGPC/SM
PUFA
FA
TC
TC
EC/FC FA/TC
FC/EC
51
5.14 ppm, 4.05 ppm and 1.84 ppm) (p<0.05) and are presented on (Fig 2.) For analyzing lipid
profile discriminant analysis was applied.
Fig.2 Compound selected as significant different for control and sarcoidosis patients in PLS-
DA analysis and t-test analysis. * - indicated significant differences in t-test (p<0.05)
PLS-DA model consisted of three components and very good explain the data and also
predict the data. Discriminant analysis correctly classified patients according to their groups
for 100% of sarcoidose and 100% of control (Fig.3). Lipidomics indicated significant
differences in phosphatidylcholine, triglycerides, fatty acids, sphigomyelin three unassigned
compounds (4.05ppm, 4.94ppm, 5.14ppm) levels.
Fig 3. Score The scores plot of the two-component PLS-DA model of the first two principal
components t[1] and t[2]; t[1] represents the greatest amount of correlated variation in the
data set, whereas t[2] represents the second greatest amount of correlated variation.
From multivariate discriminant analysis we obtain a list of potential biomarkers which are
statistically significant and which separate one class from another. These biomarkers are
statistically significant, but not necessarily biochemically significant. They may have
biochemical significance and they may be the biomarkers we are interested in, however, this
must be established through extensive testing. Presented method allow to distinguish between
healthy subject and sarcoidosis patients.
52
Eye morphology quantitated by magnetic resonance imaging in mice
Marlena Wełniak - Kamińska (1), Tomasz Chorągiewicz (2) Michał Fiedorowicz (1), Jarosław
Orzeł (1, 3), Piotr Bogorodzki (1, 3), Paweł Grieb (1)
(1) Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw
(2) 1st Eye Hospital, Medical University of Lublin, Lublin, Poland
(3) Faculty of Electronics and Information Technology, Warsaw University of Technology
Objective: Evaluating eye dimensions is crucial in studies of animal models of eye diseases.
However, performing these measurements in alive mice is challenging. The aim of this study
was to develop a method for quantitating eye morphology using planar surface MRI coil.
Methods: Aged (18 months) C57Bl/6 female mice (n=5) were anaesthetized with isoflurane
(4% in oxygen – induction, 1.5-2% - maintenance) and placed in 7T small animal-dedicated
magnetic resonance tomograph (BioSpec 70/30USR; Bruker BioSpin, Ettlingen, Germany).
Small planar surface receive coil (internal diameter=10 mm, Bruker BioSpin, Ettlingen,
Germany) was placed over left eye of the imaged animal. High resolution structural imaging
with TURBORARE T2 sequence (TR=2700ms, TEeff=30ms, RARE factor=8, NA=12, spatial
resolution=0.062mm x 0.062mm, slice thickness=0.3mm, slices=7, scan time=16min) was
performed. Basic ocular dimensions and anterior chamber angle were measured manually
using OsiriX software (Pixmeo, SARL, Bernex, Switzerland).
Results: Mean anterior chamber depth in C57Bl/6 mice was 0.464 mm (SD ±0.040) and
anterior chamber angle (ACA) 24.98° (SD ±5.599), vitreous chamber depth (VCD) 0.465 mm
(SD ±0.033), axial length (AL) 3.586 mm (SD ±0.079) and horizontal length (HL) 3.394 mm
(SD ±0.061) of the eyeball, lens thickness (LT) 2.146 mm (SD ±0.059) and optic nerve
diameter (ON) 0.250 mm (SD ±0.0520), retinal thickness (RT) 0.206 mm (SD ±0.018).
Conclusion: High resolution magnetic resonance imaging of the eye with use of small planar
coil provide reproducible and consistent measures of key dimensions of eyeball. These results
are consistent with results obtained with optical coherence tomography in C57Bl/6 mice
[Chou et al. 2011, IOVS 52:3604-3612].
The study was supported by National Science Centre grant No. 2012/07/D/NZ4/04199.
53
THE CYTOSTATIC AGENT AS A CONTRAST AGENT FOR MRI
Beata Wereszczyńskaa, Tomasz Zalewskib, Magdalena Hałupka-Brylb,
Marek Kempkaa,b, Stefan Jurgaa,b
a Department of Macromolecular Physics, Adam Mickiewicz University, Umultowska 85,
61-614 Poznań b NanoBioMedical Centre, Adam Mickiewicz University, Umultowska 85, 61-614 Poznań
Superparamagnetic iron oxide nanoparticles (SPIO) are widely used in medicine especially
as drug delivery systems and MRI contrast agents. The idea of combining those two
applications resulted in verifying the possibility of use an existing medicine as MRI
contrast agent. The object of the study were magnetic iron oxide nanoparticles loaded with
doxorubicin (SPIO/DOX) synthesized for targeted drug delivery. Their magnetic
properties and ability to accumulate in the tumor area makes them well suited to use in
monitoring of tumor treatment by magnetic resonance imaging.
In order to evaluate contrasting properties of SPIO/DOX studies of proton relaxation at
three magnetic fields intensities (0.4T, 4.7T and 9.4T) have been performed. Relaxation
times T1 and T2 have been measured (using Inversion-recovery and CPMG pulse
sequences, respectively) for samples of different concentration (mM) of investigated
nanoparticles in water and physiological saline and relaxivities r1 and r2 were calculated.
Obtained results allow to conclude that investigated nanocomplex can be classified as
effective T2 contrast agent in wide range of magnetic fields commonly used in human
diagnosis. SPIO/DOX can also be used as T1 contrast agent up to magnetic fields of 1T.
Series of T2-weighted MRI images of solution of SPIO/DOX injected into agarose gel
phantom were obtained at 9.4T using Fast Spin Echo sequence. The successive images
illustrate the expansion of the of contrast area in function of time after injection. In order to
calculate contrast areas image profiles were performed. Threshold pixel value was
determined and all the pixels, in regions of interest, below that value were counted. Results
confirmed that investigated particles are efficient T2 contrast agent and have ability to
easily spread by diffusion in environment with tissue-like density.
Acknowledgements
Financial support from the National Centre for Research and Development under research
grant “Nanomaterials and their application to biomedicine”, contract number
PBS1/A9/13/2012, is gratefully acknowledged.
54
USEFULNESS OF MR SEQUENCES: DTI AND 3D ASL IN RARE
CHILD BRAIN TUMOR BASED ON A MR BRAIN CHILD EXAM
BEFORE AND AFTER SURGERY
Magdalena Wicher1, Magdalena Machnikowska – Sokołowska1, Anna Plechta1, Katarzyna
Zymella1, Dominika Wieja – Błach1, Marcin Basiak1, Marek Konopka1
1 SCANX Medical Imaging Sp. z o.o., ul. PCK 10, 40 – 057 Katowice
Purpose:
To assess the usefulness of MR sequences: DTI (diffusion tensor imaging) and 3D ASL (3D
Arterial Spin Labeling-non contrast, whole brain quantitative perfusion assessment) in MR
imaging of rare child brain tumors.
Materials and methods:
Two MR brain examinations of a 9 years old boy before and after brain tumor surgery were
analyzed. Exams were performed on GE MR Optima 450w 1,5T GEM with protocol: T1
and T2 SE (spin-echo), T2 FLAIR, 3D BRAVO. The protocol was supplemented with 3D
ASL and DTI. 3D ASL uses magnetic properties of inflowing blood signed by energy with
radio frequency (RF) pulses to invert the water spins of inflowing arterial blood. This
technique uses water in arterial blood as an endogenous contrast media to help visualize
tissue perfusion and provide quantitative assessment of cerebral blood flow (CBF) in
ml/100g/min. 3D ASL image was acquired with following parameters: field of view 24 cm,
echo time (TE) 10,7 ms, repetition time (TR) 4683 ms and 1 NEX (number of excitations).
Arterial spin labeling was achieved on carotid arteries level (can be performed a few
centimeters below FOV). Calculations of cerebral flow in ASL technique depends on time
of arrival of arterial blood to imaging voxels, local tissue and blood time relaxation and
magnetic balance of blood [1],[2],[3]. Pioneered pulsed-continuous arterial spin labeling
method helps amplify signal and improve contrast. 3D ASL with spiral readout helps
increase signal to noise (SNR), while minimizing motion and reducing susceptibility
artifacts, compared to conventional gradient-echo based techniques.
Rys.1. 3D ASL technique [4].
Another technique we used was diffusion tensor imaging (DTI). Diffusion of water
molecules in the brain tissue is characterized as anisotropic, restricted by different barriers,
such as myelin or cell membranes. Multidirectional diffusion differential requires more
complex description with use of diffusion tensor. In case of diffusion tensor calculations it
is necessary to make measurements of diffusion at least in six different directions. To show
degree of anisotropy of examined structure fractional anisotropy (FA) (reaching values
between 0 – 1) is used. 0 value corresponds with isotropic structure, whereas 1 value with
structure, in which diffusion is possible only in one direction. There are many ways of
diffusion tensor imaging (DTI). Most common are MD and FA images in grey scale, FA
images coded by colour and tractography. In parametric FA maps coded by colour, tensor is
defined by direction of maximum component of diffusion tensor (red: right – left; green:
front – back; blue: up – down), and intensity of colour depends on FA quantity [5].
55
Results:
Brain MR examination showed tumor originating in right lateral ventricle.
Non-contrast perfusion (3D ASL) and diffusion tensor (DTI) in 6 directions were used.
To minimize examination time instead of 25 directions we used only 6, which was enough
for good fiber tracks visualization. Sequences were analyzed in Readyview (GE) software,
with quantification of cerebral brain flow (CBF) for tumor and visualization of fiber tracks.
Fiber tracks imaging helped spare neuronal fibres during surgery and was very useful in
their observation in next patient MR exams. Based on 3D ASL, CBF value was calculated,
which in first exam (before surgery) for a lesion deforming and thinning the corpus
callosum CBF was: 86,44; in a second exam (after first surgery) despite bigger mass effect
CBF was: 236,6.
Conclusion:
DTI (diffusion tensor imaging) and 3D ASL in MRI of brain children tumors are very
useful during planning surgery and control examinations, help to spare neuronal fibres and
plan neurosurgery (DTI) and specify total and residual tumor mass. 3D ASL is a
noninvasive technique without a need for an exogenic injection, so it can be repeated as
many times as necessary and enables to monitor perfusion in time. Both sequences have a
vast usefulness in MRI of pediatric patients.
References:
[1] G. Jędrzejewski: Pol. J. Radiol. 2006, 71, 52.
[2] J. Wang, D.C. Alsop, L. Lin: Magn. Reson. Med. 2002, 48, 242.
[3] R. L. Wolf, D. C. Alsop, M. L. McGarvey: J Neuroimaging, 2003; 13, 17–27.
[4] A. Urbanik, K. Sprężak, Przegląd Lekarski 2013/70/5, strony:1-9
[5] R. Brecheisen, B. Platel, A. Vilanova, B. ter Haar Romeny: IEEE Trans.Vis. Comput.
Graph 2009, 15 (6),1444 – 1448.
56
EXOGENOUS SILK PROTEIN AND SLES EFFECT ON PROPERTIES
OF HYDRATED HAIR BY 1H-NMR AND SORPTION ISOTHERM
D. Zalitacz, P. Nowak, A. Ciułkowska, K. Pieńkowska, H. Harańczyk,
Institute of Physics, Jagiellonian University, Cracow,
Hydrolyzed silk protein is a main constituent of silk applied for improvement of hair
properties [1], whereas sodium lauryl sulfate (SLES) is applied for hair purification.
Hydration changes the properties of biological systems [2], eg. biopolymers [3]. The
hydration process of human hair can be effectively approximated by the multilayer sorption of
bond water saturating primary and secondary water binding sites [4].
Using hydration kinetics, sorption isotherm,1H-NMR spectroscopy we analysed the
effect of silk proteins on a human terminal hair of the female individual. The kinetics and the
saturation of the hydration process, and also the formation of tightly and loosely bound water
fractions at different steps of hydration process were analyzed.
We used terminal human hair of 35 years old Caucasian female. Prior to experiments,
the sample was purified with a 25% water solution of SLES, and then rinsed with water [5].
Next on the hair surface the hydrolyzed silk protein was imposed. The results were compared
with the control sample which was solely purified with SLES solution.
The hydration kinetics shows three fractions of bound water, namely (i) a very tightly
bound water fraction, (ii) a tightly bound water, and (iii) a loosely bound water.
The sorption isotherm is sigmoidal in form reasonably well fitted using Dent model
[6]. The relative mass of water saturating primary binding sites is average equal to
ΔM/m0=0.050 for hair washed with a SLES and ΔM/m0 = 0.065 for hair with hydrolyzed silk.
The performed for air dry hair at room temperature 1H-NMR spectra are the
superpositions of a Gaussian component (G≈45kHz), coming from s solid matrix of hair,
and one Lorentz component, coming from residual water bound in a hair structure. The
halfwidth of the NMR liquid component was for SLES washed hair equal to L ≈ 2400 Hz,
whereas L ≈ 2900 Hz) for hair doped with hydrolyzed silk.
References
[1]G.Secchi, Clinics in Dermatology, 26, 321–325 (2008)
[2]H.Harańczyk, On water in extremely dry biological systems, WUJ, Kraków (2003).
[3]H.Harańczyk, J.Kobierski, D.Zalitacz, P.Nowak, A.Romanowicz, M.Marzec, J.Nizioł, Acta
Phys. Polon. A121, 483-488 (2012).
[4]Zalitacz, D.; Haranczyk, H.; Nowak, P, Delong, P, 2013 "Mild hydration effect on bound-
water dynamics in human hair monitored by H-1-NMR" Journal of investigative dermatology
Vol. 133, 1424-1424, (2013)
[5] G.Zhang, L.Senak. D. Moore, Journal of Biomedical Optics, 16(5), 056009, 2011
[6] R.Dent, Textile Research J., 47, 145-152 (1977).
57
4D NMR EXPERIMENT FOR PHOSPHORYLATION
STUDIES OF IDPS
Szymon Żerko*1, Gerald Platzer2, Robert Konrat2, Wiktor Koźmiński1
1Faculty of Chemistry, Biological and Chemical Research Centre, University of Warsaw,
Żwirki I Wigury 101, 02-089, Warsaw, Poland
2 Max F. Perutz Laboratories, University of Vienna, Campus Vienna Biocenter 5, 1030,
Vienna, Austria
The phosphorylation of a specific amino acid residue using NMR is usually detected
basing on a chemical shift change between phosphorylated and not phosphorylated state of
amino acid1,2. Unfortunately, because of the severe signal crowding on spectra of disordered
proteins of big sizes unambiguous identification of modified residue using 2D experiment
such as 2D NHSQC, especially in case of existence of several phosphorylation sites, can be
very difficult.
Presented approach relay on a direct detection of the phosphorylation state. Thanks to the
employment of a phosphorus filtering within the pulse sequence the direct detection of the
phosphorylated residues is possible. To provide a good signal dispersion a three dimensional
experiment was used. The result is a triple resonance experiment HNCO(P) with a
phosphorus filter and a quadruple resonance experiment HNCOP. 3D HNCO(P) allows to an
unambiguous identification of the phosphorylated and not phosphorylated amino acid
residues. Moreover, 4D HNCOP allows to obtain an additional phosphorus chemical shift for
each phosphorylated residue.
Presented experiments were tested on a numerously phosphorylated human osteopontin,
an intrinsically disordered protein containing 302 amino acid residues. Agilent 600MHz
spectrometer equipped with a room temperature Penta probe was used.
The study was carried out at the Biological and Chemical Research Centre, University of Warsaw,
established within the project co-financed by European Union from the European Regional Development Fund
under the Operational Programme Innovative Economy, 2007 ‒ 2013.
[1] . Landrieu, L. Lacosse, A. Leroy, J. Wieruszeski, X. Trivelli, A. Sillen, N. Sibille, H. Schwalbe, K. Saxena, T.
Langer, G. Lippens. JACS, 128(11):3575–3583, 2006.
[2] S. Liokatis, A. Dose, D. Schwarzer, P. Selenko. JACS, 132(42):14704–14705, 2010.
58
THE ZN IONS AS IMPORTANT FACTOR REGULATED UBIQUITIN-
ACTIVATING PROCESS. STRUCTURAL STUDIES OF THE PEPTIDE
DERIVED FROM CYSTEINE CATALYTIC HALF-DOMAIN (SCCH) OF
MOUSE E1 ENZYME.
Ilona Marszalek,1 Arkadiusz Bonna,1 Wojciech Bal,1 and Igor Zhukov1,2
1Institute of Biochemistry and Biophysics, Polish Academy of Sciences
ul. Pawińskiego 5a, 02-106 Warsaw, Poland
2NanoBioMedical Centre, Adam Mickiewicz University
ul. Umultowska 85, 61-614 Poznań, Poland
Post-translational ubiquitination signal regulate the mechanism of protein degradation. It
regulate a wide variety of cellular activities in eukaryotic organisms. The ubiquitilation process
activated by an E1 enzyme by catalytic adenylation of the C-terminal -COOH group in ubiquitin
(UBI) followed by linking via thioester bond UBI to the catalytic cysteine in catalytic domain of
E1. The ubiquitin-activation signal triggering a cascade consisting of three classes of enzymes –
activation (E1), conjugation (E2), and ligation (E3). The ubiquitin-activating E1 enzyme
presented as a one multidomain protein contained two adenylation (AAD – active and IAD –
inactive) domains, four helix bundle domain (4HB), ubiquitin-like folded domain (UFD), and
catalytic domain [1]. The later catalytic domain could be divided on two autonomously folded
subunits – FCCH (first catalytic cysteine half-domain) and SCCH (second catalytic cysteine half-
domain) [2]. The both sub-domains are located close in space, but sequentially interspersed with
adenylation domain [2,3]. There are two other small proteins – SUMO and NEDD8 – which
demonstrated similar activities, and referred as ubiquitin-like proteins (UBL). They are share the
similar fold with UBI, but constitutes different set of E1, E2 and E3 enzymes.
The structural and dynamic aspects of activation of ubiquitination and sumoilation activity
were studied in details by several groups [4,5]. Nevertheless the structural studies were focused
on yeasts (S. cerevisiae, S. pombe). From the other hand, the alignment performed for E1
enzymes reveal a specific sequence, which is highly conserved through all mammalian
organisms, not observed in invertebrates and in plants (Figure 1). The selected sequence
corresponded to 703WGDCVTWACHHWHTEYC719 segment comprise three cysteines, three
histidines and three tryptophanes. This motif,
which is very unusual for proteins, in crystal
structure of SCCH it reveal an α-helical fold (pdb
1Z7L). We speculate that selected segment
constitute the half of Zn(II)-binding interface,
which is facilitated dimerization phenomena of
ubiquitin-activating E1 enzyme. Recently, the
Zn(II) binding motif was detected in the SUMO
E1 enzyme with unclear function [6]. In fact, only
structures of catalytic domain from mouse is
available in pdb [2,3], but consequences of Zn(II)
binding on SCCH dimerization and protein degradation cascade were not studied before.
The NMR experiments were performed on NMR sample obtained by solution of 3 mM
nonlabeled peptide in 90%/10% H2O/D2O buffer contained 20 mM TRIS. pH was stabilized at
7.2. The measurements were conducted on Agilent DDR2 800 NMR spectrometer equipped with
Figu
re 1. Sequence alignments of ubiquitin-activating E1
enzymes from different organisms.
59
four channels, z-gradient unit and 1H/13C/15N triple probehead with inverse detection. Acquired
homonuclear NMR data included 2D TOCSY (mixing time 80 ms) and NOESY (mixing time 150
ms) experiments. Dimerization process was controlled by measuring coefficient of translation
diffusion before and after Zn(II) addition. These values were extracted from DPFGDSTE (Double
Polar Field Gradient Double Stimulated Echo) experiment [7] performed with 15 gradient steps,
accumulated with 128 transients and processed with Agilent VnmrJ 4.0 software. The
heteronuclear 1H-13C and 1H-15N HSQC NMR experiments were acquired on natural
abundance of 13C and 15N nuclei. Structure calculations of peptide in both, Zn(II)-free and
Zn(II)-loaded forms, were performed with the XPLOR-NIH program [8] using distance
constraints yielded from analysis of 1H-1H NOESY spectra. Additionally, backbone torsion angle
restraints were extracted from chemical shifts with the TALOS-N approach [9].
Our studies exhibited possible role of Zn(II) ions in ubiquitin-activation process in
mammalians organisms. The ubiquitin-activation together with UBI transfer to E2 conjugation
enzyme could be regulated with Zn(II) through E1 dimerization process. The interface of
interaction comprised residues 703-719 of mouse E1 enzyme. The detail analysis of impact of
Zn(II) binding on initial stage of protein degradation cascade is in progress in our group.
Acknowledgments This is financially supported by Polish Ministry of Higher Education grant N N301
318539 (for IZ). Financial support from the Polish National Centre for Research and
Development under research grant number 178479 (contract number PBS1/A9/ 13/2012) is
gratefully acknowledged. The used equipment was sponsored in part by the Centre for Preclinical
Research and Technology (CePT), a project co-sponsored by European Regional Development
Fund and Innovative Economy, The National Cohesion Strategy of Poland.
References: 1. Lois, L.M., and Lima, C.D. (2005) EMBO J., 24, 439–451.
2. Szczepanowski, R.H., Filipek, R., and Bochtler, M. (2005) J. Biol. Chem., 280, 22006–22011.
3. Jaremko, M., Jaremko, L., Nowakowski, M., Wojciechowski, M., Szczepanowski, R.H.,
Panecka, R., Zhukov, I., Bochtler, M., and Ejchart, A. (2014) J. Struct. Biol., 185, 69–78.
4. Olsen, S.K., and Lima, C.D. (2013) Molec. Cell, 49, 884–896.
5. Schafer, A., Kuhn, M., and Schindelin, H. (2014) Acta Cryst. D., 70, 1311–1320.
6. Wang, J., and Chen, Y. (2010) J. Biol. Chem., 285, 23732–23738.
7. Nilsson, M., Gil, A.M., Delgadillo, I., and Morris, G.A. (2004) Anal. Chem., 76, 5418–5422.
8. Schwieters, C.D., Kuszewski, J.J., and Clore, G.M. (2006) Progr. NMR Spectroscopy, 48, 47–
62.
9. Shen, Y., and Bax, A. (2013) J. Biomol. NMR, 56, 227–241.
60