High Resolution Proton NMR Spectroscopy of Multiple Sclerosis Lesions

Abstract: Tissue from postmortem multiple sclerosis and normal control brains was extracted with perchloric acid and analysed using proton NMR spectroscopy. The content of N -acetyl-derived groups (the sum of N -acetylaspartate, acetate, and N -acetylaspartylglutamate) was decreased in multiple sclerosis plaques compared with normal control white matter (mean, 4.36 vs. 6.64 µmol/g wet weight). In normal appearing white matter adjacent to plaques a corresponding decrease was seen, with no change in white matter distant from plaques. A decrease in the content of total creatine was observed in multiple sclerosis plaques in comparison with normal control white matter (mean, 4.64 vs. 6.56 µmol/g wet weight), which correlated strongly with the decrease in N -acetyl-derived groups. No changes in other metabolites such as total choline or myo -inositol were seen. The decreases in content of N -acetyl-derived groups are in agreement with observations from in vivo proton NMR spectroscopy in multiple sclerosis patients. The decrease in total creatine is in contrast to most of the observations made in vivo where total creatine is assumed to be unchanged and metabolite levels are often expressed as a total creatine ratio. The use of a total creatine ratio in vivo could lead to an underestimation of reductions in N -acetylaspartate and an apparent increase in other metabolites in the multiple sclerosis lesion.     

Retinal Attachment Instability Is Diversified among Mammalian Melanopsins

Melanopsins play a key role in non-visual photoreception in mammals. Their close phylogenetic relationship to the photopigments in invertebrate visual cells suggests they have evolved to acquire molecular characteristics that are more suited for their non-visual functions. Here we set out to identify such characteristics by comparing the molecular properties of mammalian melanopsin to those of invertebrate melanopsin and visual pigment. Our data show that the Schiff base linking the chromophore retinal to the protein is more susceptive to spontaneous cleavage in mammalian melanopsins. We also find this stability is highly diversified between mammalian species, being particularly unstable for human melanopsin. Through mutagenesis analyses, we find that this diversified stability is mainly due to parallel amino acid substitutions in extracellular regions. We propose that the different stability of the retinal attachment in melanopsins may contribute to functional tuning of non-visual photoreception in mammals.     

Force spectroscopy reveals the presence of structurally modified dimers in transthyretin amyloid annular oligomers

Toxicity in amyloidogenic protein misfolding disorders is thought to involve intermediate states of aggregation associated with the formation of amyloid fibrils. Despite their relevance, the heterogeneity and transience of these oligomers have placed great barriers in our understanding of their structural properties. Among amyloid intermediates, annular oligomers or annular protofibrils have raised considerable interest because they may contribute to a mechanism of cellular toxicity via membrane permeation. Here we investigated, by using AFM force spectroscopy, the structural detail of amyloid annular oligomers from transthyretin (TTR), a protein involved in systemic and neurodegenerative amyloidogenic disorders. Manipulation was performed in situ , in the absence of molecular handles and using persistence length‐fit values to select relevant curves. Force curves reveal the presence of dimers in TTR annular oligomers that unfold via a series of structural intermediates. This is in contrast with the manipulation of native TTR that was more often manipulated over length scales compatible with a TTR monomer and without unfolding intermediates. Imaging and force spectroscopy data suggest that dimers are formed by the assembly of monomers in a head‐to‐head orientation with a nonnative interface along their β‐strands. Furthermore, these dimers stack through nonnative contacts that may enhance the stability of the misfolded structure.     

Relationship of Aerial Broad Band Reflectance to Meloidogyne incognita Density in Cotton

Aerial images were obtained on 22 July 1999 and 4 August 2000 from five cotton sites infested with Meloidogyne incognita. Images contained three broad bands representing the green (500-600 nm), red (600-700 nm), and near-infrared (700-900 nm) spectrum. Soil samples were collected and assayed for nematodes in the fall at these sites. Sampling locations were identified from images, by locating the coordinates of a wide range of light intensity (measured as a digital number) for each single band, and combinations of bands. There was no single band or band combination in which reflectance consistently predicted M. incognita density. In all 10 site-year combinations, the minimum number of samples necessary to estimate M. incognita density within 25% of the population mean was greater when sampling by reflectance-based classes (3 to 4 per site) than sampling based on the entire site as one unit. Two sites were sampled at multiple times during the growing season. At these sites, there was no single time during the growing season optimal to take images for nematode sampling. Aerial infrared photography conducted during the growing season could not be used to accurately determine fall population densities of M. incognita.     

Investigation and comparison of the binding between tolvaptan and pepsin and trypsin: Multi‐spectroscopic approaches and molecular docking

Tolvaptan (TF), a selective arginine vasopressin V2 receptor antagonist, was approved by the Food and Drug Administration in 2009. This study mainly investigated the differences between the binding of TF with pepsin and trypsin by using a series of spectroscopy and molecular modeling methods. Thermodynamic parameters and molecular docking results suggested that the binding of TF to pepsin and trypsin were both spontaneous but driven by different forces. For pepsin, the binding was driven by hydrogen bonds and van der Waals forces; but for trypsin, it was driven by electrostatic forces and hydrophobic forces. The quenching mechanism between TF and pepsin and trypsin was investigated by fluorescence experiments and time‐resolved fluorescence spectroscopy. Synchronous fluorescence and 3‐dimensional fluorescence were used to investigate the micro‐environmental and conformational changes of pepsin and trypsin after the insertion of TF. In addition, activity‐measurement results showed that both the pepsin and trypsin activities increased with increasing TF concentration, which may help to understand the possible effect of TF on the digestion and absorption of nutrients in vivo.     

Focus: A Multifaceted Battle Against Cancer: Extracting Knowledge from Chemical Imaging Data Using Computational Algorithms for Digital Cancer Diagnosis

Fourier transform infrared (FTIR) spectroscopic imaging is an emerging microscopy modality for clinical histopathologic diagnoses as well as for biomedical research. Spectral data recorded in this modality are indicative of the underlying, spatially resolved biochemical composition but need computerized algorithms to digitally recognize and transform this information to a diagnostic tool to identify cancer or other physiologic conditions. Statistical pattern recognition forms the backbone of these recognition protocols and can be used for highly accurate results. Aided by biochemical correlations with normal and diseased states and the power of modern computer-aided pattern recognition, this approach is capable of combating many standing questions of traditional histology-based diagnosis models. For example, a simple diagnostic test can be developed to determine cell types in tissue. As a more advanced application, IR spectral data can be integrated with patient information to predict risk of cancer, providing a potential road to precision medicine and personalized care in cancer treatment. The IR imaging approach can be implemented to complement conventional diagnoses, as the samples remain unperturbed and are not destroyed. Despite high potential and utility of this approach, clinical implementation has not yet been achieved due to practical hurdles like speed of data acquisition and lack of optimized computational procedures for extracting clinically actionable information rapidly. The latter problem has been addressed by developing highly efficient ways to process IR imaging data but remains one that has considerable scope for progress. Here, we summarize the major issues and provide practical considerations in implementing a modified Bayesian classification protocol for digital molecular pathology. We hope to familiarize readers with analysis methods in IR imaging data and enable researchers to develop methods that can lead to the use of this promising technique for digital diagnosis of cancer.     

The effect of calcium and vitamin D supplementation on osteoporotic rabbit bones studied by vibrational spectroscopy

Fourier transform infrared spectroscopy is utilized to examine the effects of increased calcium, vitamin D, and combined calcium-vitamin D supplementation on osteoporotic rabbit bones with induced inflammation. The study includes different bone sites (femur, tibia, humerus, vertebral rib) in an effort to explore possible differences among the sites. We evaluate the following parameters: mineral-to-matrix ratio, carbonate content, and non-apatitic species (labile acid phosphate and labile carbonate) contribution to bone mineral. Results show that a relatively high dose of calcium or calcium with vitamin D supplementation increases the bone mineralization index significantly. On the other hand, vitamin D alone is not as effective in promoting mineralization even with high intake. Mature B-type apatite was detected for the group with calcium supplementation similar to that of aged bone. High vitamin D intake led to increased labile species concentration revealing bone formation. This is directly associated with the suppression of pro-inflammatory cytokines linked to induced inflammation. The latter is known to adversely alter bone metabolism, contributing to the aetiopathogenesis of osteoporosis. Thus, a high intake of vitamin D under inflammation-induced osteoporosis does not promote mineralization but suppresses bone resorption and restores metabolic balance.     

Structural transition of kidney cystatin in dimethylnitrosamine-induced renal cancer in rats: identification as a novel biomarker for kidney cancer and prognosis

In our study, renal cancer is induced in rats making use of dimethylnitrosamine (DMN). G1 – Group 1 were control rats and G2 – Group 2 rats were given a single intra-peritoneal injection of DMN of 50 mg/kg body weight resulting in 100% incidences of renal tumors after 12 months. SEM and histopathology confirmed the presence of renal cancer in the DMN-treated rats. Making use of ammonium sulfate precipitation and gel filtration chromatography on Sephacryl S-100HR column, a thiol protease inhibitor was isolated from kidney of control rats known as Rat kidney Cystatin (RKC) as well as from kidney of cancerous rat called as Cancerous Rat Kidney Cystatin (CRKC). Both these inhibitors were characterized, and interestingly, it was found that CRKC showed greater anti-papain activity and also it was stable in a broad pH and temperature range thus implying that CRKC is more stable as compared to RKC. UV and fluorescence spectroscopy point out in structural difference between RKC and CRKC which was further confirmed by Circular dichroism (CD) and FTIR spectroscopy. Our study clearly showed that kidney cystatin is structurally modified in the case of renal cancer and performs its role in a more efficacious manner.     

Reversible coupling of individual phycobiliprotein isoforms during state transitions in the cyanobacterium Trichodesmium analysed by single-cell fluorescence kinetic measurements

In the non-heterocyst, marine cyanobacterium Trichodesmium nitrogen fixation is confined to the photoperiod and occurs coevally with oxygenic photosynthesis although nitrogenase is irreversibly inactivated by oxygen. In previous studies it was found that regulation of photosynthesis for nitrogen fixation involves Mehler reaction and various activity states with reversible coupling of photosynthetic components. We now investigated these activity states in more detail. Spectrally resolved fluorescence kinetic measurements of single cells revealed that they were related to alternate uncoupling and coupling of phycobilisomes from and to the photosystems, changing the effective cross-section of PSII. Therefore, we isolated and purified the phycobiliproteins of Trichodesmium via ion exchange chromatography and recorded their UV/VIS absorption, fluorescence excitation and fluorescence emission spectra. After describing these spectra by mathematical equations via the Gauss-Peak-Spectra method, we used them to deconvolute the in vivo fluorescence spectra of Trichodesmium cells. This revealed that the contribution of different parts of the phycobilisome antenna to fluorescence quenching changed during the daily activity cycle, and that individual phycobiliproteins can be reversibly coupled to the photosystems, while the expression levels of these proteins did not change much during the daily activity cycle. Thus we propose that variable phycobilisome coupling plays a key role in the regulation of photosynthesis for nitrogen fixation in Trichodesmium.     

Trehalose and dry dipalmitoylphosphatidylcholine revisited

Dry mixtures of sonicated vesicles of DPPC and trehalose which contained a maximum of 0.2 mol water/mol lipid were examined by differential scanning calorimetry, Fourier transform infrared spectroscopy and freeze-fracture electron microscopy. Samples of dry DPPC and trehalose prepared from aqueous solution had a minimum T_m of 24°C for the gel to liquid-crystalline transition provided that the vesicles were dried with trehalose while the lipid was in liquid-crystalline phase. This low transition is compared to a transition of 105–112°C for dry pure DPPC and of 42°C for hydrated pure DPPC. The present work is an extension of earlier work from this laboratory using both other lipids and other methods of preparation.