Rapid and Live-Cell Detection of Senescence in Mesenchymal Stem Cells by Micro Magnetic Resonance Relaxometry.

Detection of cellular senescence is important quality analytics of cell therapy products, including mesenchymal stromal cells (MSCs). However, its detection is critically limited by the lack of specific markers and the destructive assays used to read out these markers. Here, we establish a rapid, live-cell assay for detecting senescent cells in heterogeneous mesenchymal stromal cell (MSC) cultures. We report that the T2 relaxation time measured by microscale Magnetic Resonance Relaxometry, which is related to intracellular iron accumulation, correlates strongly with senescence markers in MSC cultures under diverse conditions, including different passages and donors, size-sorted MSCs by inertial spiral microfluidic device, and drug-induced senescence. In addition, the live-cell and non-destructive method presented here has general applicability to other cells and tissues and can critically advance our understanding of cellular senescence.

Enhancing the sensitivity of micro magnetic resonance relaxometry detection of low parasitemia Plasmodium falciparum in human blood.

Upon Plasmodium falciparum infection of the red blood cells (RBCs), the parasite replicates and consumes haemoglobin resulting in the release of free heme which is rapidly converted to hemozoin crystallites. The bulk magnetic susceptibility of infected RBCs (iRBCs) is changed due to ferric (Fe3+) paramagnetic state in hemozoin crystallites which induce a measurable change in spin-spin relaxation (transverse relaxation) rate in proton nuclear magnetic resonance (NMR) of iRBCs. Earlier, our group reported that this transverse relaxation rate (R2) can be measured by an inexpensive, portable 0.5 Tesla bench top magnetic resonance relaxometry (MRR) system with minimum sample preparation and is able to detect very low levels of parasitemia in both blood cultures as well as animal models. However, it was challenging to diagnose malaria in human blood using MRR, mainly due to the inherent variation of R2 values of clinical blood samples, caused by many physiological and genotypic differences not related to the parasite infection. To resolve the problem of baseline R2 rates, we have developed an improved lysis protocol for removing confounding molecular and cellular background for MRR detection. With this new protocol and by processing larger volume of blood (>1 ml), we are able to reliably detect very low level of parasitemia (representing early stage of infection, ~0.0001%) with a stable baseline and improved sensitivity using the current MRR system.

The solid-state photo-CIDNP effect and its analytical application : photo-CIDNP MAS NMR to study radical pairs.

Photochemically induced dynamic nuclear polarization (photo-CIDNP) is an effect that produces non-Boltzmann nuclear spin polarization which can be observed as modification of signal intensity in NMR spectroscopy. The effect is well known in liquid-state NMR where it is explained most generally by the classical radical pair mechanism (RPM). In the solid-state, other mechanisms are operative in the spin-dynamics of radical pairs such as three-spin mixing (TSM) and differential decay (DD). Initially the solid-state photo-CIDNP effect has been solely observed on natural photosynthetic reaction centers (RCs). Therefore the analytical capacity of the method has been explored in experiments on reaction centers (RCs) of the purple bacterium of Rhodobacter (R.) sphaeroides. Here we will provide an account on phenomenology, theory, and analytical capacity of the solid-state photo-CIDNP effect.

Whole cell nuclear magnetic resonance characterization of two photochemically active states of the photosynthetic reaction center in heliobacteria.

Applying photo-CIDNP (photochemically induced dynamic nuclear polarization) MAS (magic-angle spinning) nuclear magnetic resonance to whole cells of Heliobacillus (Hb.) mobilis, we demonstrate that heliobacterial reaction centers are operational in two different states as indicated by the occurrence of a light-induced spin-correlated radical pair. A culture maintained anaerobically is called "Braunstoff" (German for "brown substance"). After exposure to oxygen, Braunstoff is converted to "Grünstoff" ("green substance") as indicated by a color change due to the conversion of BChl g to Chl a(F). It is shown that electron transfer occurs symmetrically via both branches of cofactors in both forms. The donor and acceptor cofactors remain identical and unchanged upon conversion, while the intermediate accessory cofactors are transformed from BChl g to Chl a(F). The donor triplet state in Braunstoff is localized on the special pair donor and lives for 100 µs, demonstrating the absence of nearby carotenoids. In Grünstoff, the donor triplet becomes mobile and appears to be formed on an accessory cofactor.

Electron spin density distribution in the special pair triplet of Rhodobacter sphaeroides R26 revealed by magnetic field dependence of the solid-state photo-CIDNP effect.

Photo-CIDNP (photochemically induced dynamic nuclear polarization) can be observed in frozen and quinone-blocked photosynthetic reaction centers (RCs) as modification of magic-angle spinning (MAS) NMR signal intensity under illumination. Studying the carotenoidless mutant strain R26 of Rhodobacter sphaeroides, we demonstrate by experiment and theory that contributions to the nuclear spin polarization from the three-spin mixing and differential decay mechanism can be separated from polarization generated by the radical pair mechanism, which is partially maintained due to differential relaxation (DR) in the singlet and triplet branch. At a magnetic field of 1.4 T, the latter contribution leads to dramatic signal enhancement of about 80,000 and dominates over the two other mechanisms. The DR mechanism encodes information on the spin density distribution in the donor triplet state. Relative peak intensities in the photo-CIDNP spectra provide a critical test for triplet spin densities computed for different model chemistries and conformations. The unpaired electrons are distributed almost evenly over the two moieties of the special pair of bacteriochlorophylls, with only slight excess in the L branch.

Solid-state photo-CIDNP effect observed in phototropin LOV1-C57S by (13)C magic-angle spinning NMR spectroscopy.

Until now, the solid-state photo-CIDNP effect, discovered in 1994 by Zysmilich and McDermott, has been observed selectively in photosynthetic systems. Here we present the first observation of this effect in a nonphotosynthetic system, the blue-light photoreceptor phototropin LOV1-C57S using (13)C magic-angle spinning (MAS) NMR.