Characterization of Distinct Chondrogenic Cell Populations of Patients Suffering from Microtia Using Single-Cell Micro-Raman Spectroscopy

Microtia is a congenital condition of abnormal development of the outer ear. Tissue engineering of the ear is an alternative treatment option for microtia patients. However, for this approach, the identification of high regenerative cartilage progenitor cells is of vital importance. Raman analysis provides a novel, non-invasive, label-free diagnostic tool to detect distinctive biochemical features of single cells or tissues. Using micro-Raman spectroscopy, we were able to distinguish and characterize the particular molecular fingerprints of differentiated chondrocytes and perichondrocytes and their respective progenitors isolated from healthy individuals and microtia patients. We found that microtia chondrocytes exhibited lower lipid concentrations in comparison to healthy cells, thus indicating the importance of fat storage. Moreover, we suggest that collagen is a useful biomarker for distinguishing between populations obtained from the cartilage and perichondrium because of the higher spectral contributions of collagen in the chondrocytes compared to perichondrocytes from healthy individuals and microtia patients. Our results represent a contribution to the identification of cell markers that may allow the selection of specific cell populations for cartilage tissue engineering. Moreover, the observed differences between microtia and healthy cells are essential for gaining better knowledge of the cause of microtia. It can be useful for designing novel treatment options based on further investigations of the discovered biochemical substrate alterations

Investigation of magnetic field effects on protein photochemistry using cavity enhanced spectroscopy

Some animals, such as migratory birds, are known to have the ability to sense and use magnetic fields for orientation. At the heart of this remarkable ability is believed to be a photoinduced chemical reaction taking place within a blue-light receptor protein called cryptochrome (Cry). It is hypothesised that magnetic fields can play a decisive role in determining reaction rates (and, hence, yields) by acting on the spin states of spin correlated radical pairs (RPs) formed between a flavin chromophore and a tryptophan (Trp) electron-transfer (ET) chain within Crys. In this work, cavity ring-down spectroscopy (CRDS) has been used for the detection of magnetic field effects (MFEs) via small changes in the differential absorbance (ΔΔA~10−6) of aqueous protein samples, with sub-microsecond time resolution. The photochemistry and effects of magnetic fields on European robin (Er) Cry, fruit fly Cry, and a plant Cry are characterised. In a key result for this field of research, an MFE in the photochemistry of ErCry is revealed for the first time, lending considerable support to its proposed involvement in bird magnetoreception. Using Cry mutants with modified Trp ET chains, the role of individual Trps in the manifestation of MFEs was explored. Replacing the terminal (i.e. fourth) Trp of the ET chain in ErCry with a redox inert amino acid is shown to significantly increase the optically-detected MFE, indicating that the RP formed between the flavin and the third Trp might be the primarily magnetically sensitive species. To better understand the relevant biochemical processes in Cry, de novo designed artificial flavoproteins (maquettes) have been used to model the photochemistry of Cry (Chapter 4). The comparatively simple and adaptable design of these model proteins facilitates detailed MFE studies by reducing the complexity of natural Cry. In these maquettes, photoinduced ET leads to the formation of a RP which exhibits MFEs analogous to those in Crys. The profound effect of the donor-acceptor distance on the MFE is demonstrated using flavomaquettes with varying flavin-Trp distances. The application of cavity enhanced spectroscopy to both natural cryptochromes and artificial flavoproteins, opens up new pathways for the detection and characterisation of MFEs in biologically relevant environments. The enhanced sensitivity of these approaches constitutes a significant step forward in understanding the underlying biochemical processes of animal magnetoreception.</p

Investigation of magnetic field effects on protein photochemistry using cavity enhanced spectroscopy

Some animals, such as migratory birds, are known to have the ability to sense and use magnetic fields for orientation. At the heart of this remarkable ability is believed to be a photoinduced chemical reaction taking place within a blue-light receptor protein called cryptochrome (Cry). It is hypothesised that magnetic fields can play a decisive role in determining reaction rates (and, hence, yields) by acting on the spin states of spin correlated radical pairs (RPs) formed between a flavin chromophore and a tryptophan (Trp) electron-transfer (ET) chain within Crys. In this work, cavity ring-down spectroscopy (CRDS) has been used for the detection of magnetic field effects (MFEs) via small changes in the differential absorbance (ÎÎA~10â6) of aqueous protein samples, with sub-microsecond time resolution. The photochemistry and effects of magnetic fields on European robin (Er) Cry, fruit fly Cry, and a plant Cry are characterised. In a key result for this field of research, an MFE in the photochemistry of ErCry is revealed for the first time, lending considerable support to its proposed involvement in bird magnetoreception. Using Cry mutants with modified Trp ET chains, the role of individual Trps in the manifestation of MFEs was explored. Replacing the terminal (i.e. fourth) Trp of the ET chain in ErCry with a redox inert amino acid is shown to significantly increase the optically-detected MFE, indicating that the RP formed between the flavin and the third Trp might be the primarily magnetically sensitive species. To better understand the relevant biochemical processes in Cry, de novo designed artificial flavoproteins (maquettes) have been used to model the photochemistry of Cry (Chapter 4). The comparatively simple and adaptable design of these model proteins facilitates detailed MFE studies by reducing the complexity of natural Cry. In these maquettes, photoinduced ET leads to the formation of a RP which exhibits MFEs analogous to those in Crys. The profound effect of the donor-acceptor distance on the MFE is demonstrated using flavomaquettes with varying flavin-Trp distances. The application of cavity enhanced spectroscopy to both natural cryptochromes and artificial flavoproteins, opens up new pathways for the detection and characterisation of MFEs in biologically relevant environments. The enhanced sensitivity of these approaches constitutes a significant step forward in understanding the underlying biochemical processes of animal magnetoreception.</p

Characterization of Distinct Chondrogenic Cell Populations of Patients Suffering from Microtia Using Single-Cell Micro-Raman Spectroscopy

Microtia is a congenital condition of abnormal development of the outer ear. Tissue engineering of the ear is an alternative treatment option for microtia patients. However, for this approach, the identification of high regenerative cartilage progenitor cells is of vital importance. Raman analysis provides a novel, non-invasive, label-free diagnostic tool to detect distinctive biochemical features of single cells or tissues. Using micro-Raman spectroscopy, we were able to distinguish and characterize the particular molecular fingerprints of differentiated chondrocytes and perichondrocytes and their respective progenitors isolated from healthy individuals and microtia patients. We found that microtia chondrocytes exhibited lower lipid concentrations in comparison to healthy cells, thus indicating the importance of fat storage. Moreover, we suggest that collagen is a useful biomarker for distinguishing between populations obtained from the cartilage and perichondrium because of the higher spectral contributions of collagen in the chondrocytes compared to perichondrocytes from healthy individuals and microtia patients. Our results represent a contribution to the identification of cell markers that may allow the selection of specific cell populations for cartilage tissue engineering. Moreover, the observed differences between microtia and healthy cells are essential for gaining better knowledge of the cause of microtia. It can be useful for designing novel treatment options based on further investigations of the discovered biochemical substrate alterations

Engineering an Artificial Flavoprotein Magnetosensor

Migratory birds use the Earth’s magnetic field as a source of navigational information. This light-dependent magnetic compass is thought to be mediated by cryptochrome proteins in the retina. Upon light activation, electron transfer between the flavin adenine dinucleotide cofactor and tryptophan residues leads to the formation of a spin-correlated radical pair, whose subsequent fate is sensitive to external magnetic fields. To learn more about the functional requirements of this complex chemical compass, we have created a family of simplified, adaptable proteins—maquettes—that contain a single tryptophan residue at different distances from a covalently bound flavin. Despite the complete absence of structural resemblance to the native cryptochrome fold or sequence, the maquettes exhibit a strong magnetic field effect that rivals those observed in the natural proteins in vitro. These novel maquette designs offer unprecedented flexibility to explore the basic requirements for magnetic sensing in a protein environment

Magnetically Sensitive Radical Photochemistry of Non-natural Flavoproteins

It is a remarkable fact that ∼50 μT magnetic fields can alter the rates and yields of certain free-radical reactions and that such effects might be the basis of the light-dependent ability of migratory birds to sense the direction of the Earth’s magnetic field. The most likely sensory molecule at the heart of this chemical compass is cryptochrome, a flavin-containing protein that undergoes intramolecular, blue-light-induced electron transfer to produce magnetically sensitive radical pairs. To learn more about the factors that control the magnetic sensitivity of cryptochromes, we have used a set of <i>de novo</i> designed protein maquettes that self-assemble as four-α-helical proteins incorporating a single tryptophan residue as an electron donor placed approximately 0.6, 1.1, or 1.7 nm away from a covalently attached riboflavin as chromophore and electron acceptor. Using a specifically developed form of cavity ring-down spectroscopy, we have characterized the photochemistry of these designed flavoprotein maquettes to determine the identities and kinetics of the transient radicals responsible for the magnetic field effects. Given the gross structural and dynamic differences from the natural proteins, it is remarkable that the maquettes show magnetic field effects that are so similar to those observed for cryptochromes

Magnetic sensitivity of cryptochrome 4 from a migratory songbird

Night-migratory songbirds are remarkably proficient navigators. Flying alone and often over great distances, they use various directional cues including, crucially, a light-dependent magnetic compass. The mechanism of this compass has been suggested to rely on the quantum spin dynamics of photoinduced radical pairs in cryptochrome flavoproteins located in the retinas of the birds. Here we show that the photochemistry of cryptochrome 4 (CRY4) from the night-migratory European robin (Erithacus rubecula) is magnetically sensitive in vitro, and more so than CRY4 from two non-migratory bird species, chicken (Gallus gallus) and pigeon (Columba livia). Site-specific mutations of ErCRY4 reveal the roles of four successive flavin–tryptophan radical pairs in generating magnetic field effects and in stabilizing potential signalling states in a way that could enable sensing and signalling functions to be independently optimized in night-migratory birds