In Vivo Longitudinal Monitoring of Disease Progression in Inflammatory Arthritis Animal Models Using Raman Spectroscopy.

Current techniques for monitoring disease progression and testing drug efficacy in animal models of inflammatory arthritis are either destructive, time-consuming, subjective, or require ionizing radiation. To accommodate this, we have developed a non-invasive and label-free optical system based on Raman spectroscopy for monitoring tissue alterations in rodent models of arthritis at the biomolecular level. To test different sampling geometries, the system was designed to collect both transmission and reflection mode spectra. Mice with collagen antibody-induced arthritis and controls were subject to in vivo Raman spectroscopy at the tibiotarsal joint every 3 days for 14 days. Raman-derived measures of bone content correlated well with micro-computed tomography bone mineral densities. This allowed for time-resolved quantitation of bone densities, which indicated gradual bone erosion in mice with arthritis. Inflammatory pannus formation, bone erosion, and bone marrow inflammation were confirmed by histological analysis. In addition, using library-based spectral decomposition, we quantified the progression of bone and soft tissue components. In general, the tissue components followed significantly different tendencies in mice developing arthritis compared to the control group in line with the histological analysis. In total, this demonstrates Raman spectroscopy as a versatile technique for monitoring alterations to both mineralized and soft tissues simultaneously in rodent models of musculoskeletal disorders. Furthermore, the technique presented herein allows for objective repeated within-animal measurements potentially refining and reducing the use of animals in research while improving the development of novel antiarthritic therapeutics.

Spectral Unmixing for Label-Free, In-Liquid Characterization of Biomass Microstructure and Biopolymer Content by Coherent Raman Imaging.

Characterization of lignocellulosic biomass microstructure with chemical specificity and under physiological conditions could provide invaluable insights to our understanding of plant tissue development, microstructure, origins of recalcitrance, degradation, and solubilization. However, most methods currently available are either destructive, are not compatible with hosting a physiological environment, or introduces exogenous probes, complicating their use for studying changes in microstructure and mechanisms of plant development, recalcitrance, or degradation in situ. To address these challenges, we here present a multi-modal chemically specific imaging technique based on coherent anti-Stokes Raman scattering (CARS) microspectroscopy with simplex maximization and entropy-based spectral unmixing enabling label-free, chemically specific characterization of plant microstructure in liquid. We describe how spatial drift of samples suspended in liquid can introduce artifacts in spectral unmixing procedures for single-frequency CARS and propose a mitigative strategy toward these effects using simultaneously acquired forward-scattered CARS signals and epi-detected autofluorescence. We further apply the technique for chemical and microstructural characterization of untreated and liquid hot water pretreated rapeseed straw by CARS and show how the framework can be extended for 3D imaging with chemical specificity. Finally, we provide examples of the intricate chemical and microstructural details recovered by this hybrid imaging technique, including discerning between primary and secondary cell walls, localization of aqueous components to cell lumina, and the presence of funnel-type pits in samples ofBrassica napus.

Revealing the Nature of Chronic Obstructive Pulmonary Disease Using Self-tracking and Analysis of Contact Patterns: Longitudinal Study.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death and is characterized by a progressive loss of pulmonary function over time with intermittent episodes of exacerbations. Rapid and proactive interventions may reduce the burden of the condition for the patients. Telehealth solutions involving self-tracking of vital parameters such as pulmonary function, oxygen saturation, heart rate, and temperature with synchronous communication of health data may become a powerful solution as they enable health care professionals to react with a proactive and adequate response. We have taken this idea to the next level in the Epital Care Model and organized a person-centered technology-assisted ecosystem to provide health services to COPD patients. OBJECTIVE: The objective is to reveal the nature of COPD by combining technology with a person-centered design aimed to benefit from interactions based on patient-reported outcome data and to assess the needed kind of contacts to best treat exacerbations. We wanted to know the following: (1) What are the incidences of mild, moderate, and severe exacerbations in a mixed population of COPD patients? (2) What are the courses of mild, moderate, and severe exacerbations? And (3) How is the activity and pattern of contacts with health professionals related to the participant conditions? METHODS: Participants were recruited by convenience sampling from November 2013 to December 2015. The participants' sex, age, forced expiratory volume during the first second, pulse rate, and oxygen saturation were registered at entry. During the study, we registered number of days, number of exacerbations, and number of contact notes coded into care and treatment notes. Each participant was classified according to GOLD I-IV and risk factor group A-D. Participants reported their clinical status using a tablet by answering 4 questions and sending 3 semiautomated measurements. RESULTS: Of the 87 participants, 11 were in risk factor group A, 24 in B, 13 in C, and 39 in D. The number of observed days was 31,801 days with 12,470 measurements, 1397 care notes, and 1704 treatment notes. A total of 254 exacerbations were treated and only 18 caused hospitalization. Those in risk factor group D had the highest number of hospitalizations (16), exacerbations (151), and contacts (1910). The initial contacts during the first month declined within 3 months to one-third for care contacts and one-half for treatment contacts and reached a plateau after 4 months. CONCLUSIONS: The majority of COPD patients in risk factor group D can be managed virtually, and only 13% of those with severe exacerbations required hospitalization. Contact to the health care professionals decreases markedly within the first months after enrollment. These results provide a new and detailed insight into the course of COPD. We propose a resilience index for virtual clinical management making it easier to compare results across settings.

Vacuum Ultraviolet Excited State Dynamics of the Smallest Ketone: Acetone.

We combined tunable vacuum-ultraviolet time-resolved photoelectron spectroscopy (VUV-TRPES) with high-level quantum dynamics simulations to disentangle multistate Rydberg-valence dynamics in acetone. A femtosecond 8.09 eV pump pulse was tuned to the sharp origin of the A1(n3dyz) band. The ensuing dynamics were tracked with a femtosecond 6.18 eV probe pulse, permitting TRPES of multiple excited Rydberg and valence states. Quantum dynamics simulations reveal coherent multistate Rydberg-valence dynamics, precluding simple kinetic modeling of the TRPES spectrum. Unambiguous assignment of all involved Rydberg states was enabled via the simulation of their photoelectron spectra. The A1(ππ*) state, although strongly participating, is likely undetectable with probe photon energies ≤8 eV and a key intermediate, the A2(nπ*) state, is detected here for the first time. Our dynamics modeling rationalizes the temporal behavior of all photoelectron transients, allowing us to propose a mechanism for VUV-excited dynamics in acetone which confers a key role to the A2(nπ*) state.

Vacuum ultraviolet excited state dynamics of small amides.

Time-resolved photoelectron spectroscopy in combination with ab initio quantum chemistry calculations was used to study ultrafast excited state dynamics in formamide (FOR), N,N-dimethylformamide (DMF), and N,N-dimethylacetamide (DMA) following 160 nm excitation. The particular focus was on internal conversion processes within the excited state Rydberg manifold and on how this behavior in amides compared with previous observations in small amines. All three amides exhibited extremely rapid (<100 fs) evolution from the Franck-Condon region. We argue that this is then followed by dissociation. Our calculations indicate subtle differences in how the excited state dynamics are mediated in DMA/DMF as compared to FOR. We suggest that future studies employing longer pump laser wavelengths will be useful for discerning these differences.

Symmetry controlled excited state dynamics.

Symmetry effects in internal conversion are studied by means of two isomeric cyclic tertiary aliphatic amines in a velocity map imaging (VMI) experiment on the femtosecond timescale. It is demonstrated that there is a delicate structural dependence on when coherence is preserved after the transition between the 3p and 3s Rydberg states. N-Methyl morpholine (NMM) shows unambiguous preserved coherence, consistent with previous work, which is decidedly switched off by the repositioning of oxygen within the ring. From the differences in these dynamics, and an examination of the potential energy surface following the normal modes of vibration, it becomes clear that there is a striking dependence on atom substitution, which manifests itself in the permitted modes of vibration that take the system out of the Franck-Condon region through to the 3s minimum. It is shown that the non Fermi-like behaviour of NMM is due to a conical intersection (CI) between the 3px and 3s states lying directly along the symmetry allowed path of steepest descent out of the Franck-Condon region. NMI, where the symmetry has been changed, is shown to undergo internal conversion in a more Fermi-like manner as the energy spreads through the available modes ergodically.

The influence of relativistic effects on nuclear magnetic resonance spin-spin coupling constant polarizabilities of H2 O2 , H2 S2 , H2 Se2 , and H2 Te2.

Relativistic and nonrelativistic calculations have been performed on hydrogen peroxide, dihydrogen disulfide, dihydrogen diselenide, and dihydrogen ditelluride, H2 X2 (X = O, S, Se, Te), to investigate the consequences of relativistic effects on their structures as well as their nuclear magnetic resonance (NMR) spin-spin coupling constants and spin-spin coupling constant polarizabilites. The study has been performed using both one-component nonrelativistic and four-component relativistic calculations at the density functional theory (DFT) level with the B3LYP exchange-correlation functional. The calculation of nuclear spin-spin coupling constant polarizabilities has been performed by evaluating the components of the third order tensor, nuclear spin-spin coupling polarizability, using quadratic response theory. From this, the pseudoscalar associated with this tensor has also been calculated. The results show that relativistic corrections become very important for H2 Se2 and H2 Te2 and hint that a new chiral discrimination technique via NMR spectroscopy might be possible for molecules containing elements like Se or Te. © 2018 Wiley Periodicals, Inc.

Vacuum ultraviolet excited state dynamics of the smallest ring, cyclopropane. II. Time-resolved photoelectron spectroscopy and ab initio dynamics.

The vacuum-ultraviolet photoinduced dynamics of cyclopropane (C3H6) were studied using time-resolved photoelectron spectroscopy (TRPES) in conjunction with ab initio quantum dynamics simulations. Following excitation at 160.8 nm, and subsequent probing via photoionization at 266.45 nm, the initially prepared wave packet is found to exhibit a fast decay (<100 fs) that is attributed to the rapid dissociation of C3H6 to ethylene (C2H4) and methylene (CH2). The photodissociation process proceeds via concerted ring opening and C-C bond cleavage in the excited state. Ab initio multiple spawning simulations indicate that ring-opening occurs prior to dissociation. The dynamics simulations were subsequently employed to simulate a TRPES spectrum, which was found to be in excellent agreement with the experimental result. On the basis of this agreement, the fitted time constants of 35 ± 20 and 57 ± 35 fs were assigned to prompt (i) dissociation on the lowest-lying excited state, prepared directly by the pump pulse, and (ii) non-adiabatic relaxation from higher-lying excited states that lead to delayed dissociation, respectively.

Putting the Disulfide Bridge at Risk: How UV-C Radiation Leads to Ultrafast Rupture of the S-S Bond.

We investigate the ultrafast photoinduced dynamics of the cyclic disulfide 1,2-dithiane upon 200 nm excitation by time-resolved photoelectron spectroscopy and show that the S-S bond breaks on an ultrafast time scale. This stands in stark contrast to excitation at longer wavelengths where the initially excited S1 state evolves as the wavepacket is guided towards a conical intersection with S0 by a torsional motion involving a partially broken bond between the sulfur atoms. This process at lower excitation energy allows for efficient (re-)population of S0 , rendering dithiane intact. At 200 nm, in contrast, the excitation leads to a manifold of higher excited states, Sn , that are primarily of Rydberg character. We are able to follow the gradual transition from the initially excited state to the dissociative receiver state in real time. The Rydberg states are intersected by a repulsive valence state that mediates a transition to the repulsive S2 surface. Therefore, we propose that the resulting diradical will eventually break apart on a longer timescale. The findings imply that upon going from UV-B to UV-C light the structural integrity of the disulfide moiety is compromised and proteins irradiated in this range will not be able to reform the initial tertiary structure, leading to loss of function.

An engineered, quantifiable in vitro model for analysing the effect of proteostasis-targeting drugs on tissue physical properties.

Cellular function depends on the maintenance of protein homeostasis (proteostasis) by regulated protein degradation. Chronic dysregulation of proteostasis is associated with neurodegenerative and age-related diseases, and drugs targeting components of the protein degradation apparatus are increasingly used in cancer therapies. However, as chronic imbalances rather than loss of function mediate their pathogenesis, research models that allow for the study of the complex effects of drugs on tissue properties in proteostasis-associated diseases are almost completely lacking. Here, to determine the functional effects of impaired proteostatic fine-tuning, we applied a combination of materials science characterisation techniques to a cell-derived, in vitro model of bone-like tissue formation in which we pharmacologically perturbed protein degradation. We show that low-level inhibition of VCP/p97 and the proteasome, two major components of the degradation machinery, have remarkably different effects on the bone-like material that human bone-marrow derived mesenchymal stromal cells (hMSC) form in vitro. Specifically, whilst proteasome inhibition mildly enhances tissue formation, Raman spectroscopic, atomic force microscopy-based indentation, and electron microscopy imaging reveal that VCP/p97 inhibition induces the formation of bone-like tissue that is softer, contains less protein, appears to have more crystalline mineral, and may involve aberrant micro- and ultra-structural tissue organisation. These observations contrast with findings from conventional osteogenic assays that failed to identify any effect on mineralisation. Taken together, these data suggest that mild proteostatic impairment in hMSC alters the bone-like material they form in ways that could explain some pathologies associated with VCP/p97-related diseases. They also demonstrate the utility of quantitative materials science approaches for tackling long-standing questions in biology and medicine, and could form the basis for preclinical drug testing platforms to develop therapies for diseases stemming from perturbed proteostasis or for cancer therapies targeting protein degradation. Our findings may also have important implications for the field of tissue engineering, as the manufacture of cell-derived biomaterial scaffolds may need to consider proteostasis to effectively replicate native tissues.

Conformationally controlled ultrafast intersystem crossing in bithiophene systems.

Bithiophenes serve as model systems for larger polythiophenes used in solar cell applications and molecular electronics. We report a study of ultrafast dynamics of two bithiophene systems measured with femtosecond time-resolved photoelectron spectroscopy, and show that their intersystem crossing takes place within the first few picoseconds after excitation, in line with previous studies. We show that the intersystem crossing rate can be explained in terms of arguments based on symmetry of the S1 minimum energy geometry, which depends on the specific conformation of bithiophene. Furthermore, this work shows that the minor cis-conformer contributes to an even higher intersystem crossing rate than the major trans conformer. The work presented here can provide guiding principles towards the design of solar cell components with even faster formation of long-lived excited states for solar energy harvesting.

Readying Community Water Fluoridation Advocates through Training, Surveillance, and Empowerment.

This paper describes the Community Water Fluoridation Advocacy Training Project that was designed to develop networks of community water fluoridation advocates in rural communities. The South Carolina (SC) Department of Health and Environmental Control Division of Oral Health staff and the SC Dental Association were responsible for developing and facilitating the training sessions for key policy influencers, which included medical and dental providers, early childhood educators, and water system operators and managers. Findings from the post-training survey indicate that participants increased their knowledge and skills to discuss the impact of water fluoridation on the dental health of community residents. Participants identified a need for online access to water fluoridation education and advocacy materials. Dental public health competencies illustrated: communication and collaboration with groups and individuals, and advocate, implement and evaluate public health policy, legislation and regulations.

Simple additive manufacturing of an osteoconductive ceramic using suspension melt extrusion.

OBJECTIVE: Craniofacial bone trauma is a leading reason for surgery at most hospitals. Large pieces of destroyed or resected bone are often replaced with non-resorbable and stock implants, and these are associated with a variety of problems. This paper explores the use of a novel fatty acid/calcium phosphate suspension melt for simple additive manufacturing of ceramic tricalcium phosphate implants. METHODS: A wide variety of non-aqueous liquids were tested to determine the formulation of a storable 3D printable tricalcium phosphate suspension ink, and only fatty acid-based inks were found to work. A heated stearic acid-tricalcium phosphate suspension melt was then 3D printed, carbonized and sintered, yielding implants with controllable macroporosities. Their microstructure, compressive strength and chemical purity were analyzed with electron microscopy, mechanical testing and Raman spectroscopy, respectively. Mesenchymal stem cell culture was used to assess their osteoconductivity as defined by collagen deposition, alkaline phosphatase secretion and de-novo mineralization. RESULTS: After a rapid sintering process, the implants retained their pre-sintering shape with open pores. They possessed clinically relevant mechanical strength and were chemically pure. They supported adhesion of mesenchymal stem cells, and these were able to deposit collagen onto the implants, secrete alkaline phosphatase and further mineralize the ceramic. SIGNIFICANCE: The tricalcium phosphate/fatty acid ink described here and its 3D printing may be sufficiently simple and effective to enable rapid, on-demand and in-hospital fabrication of individualized ceramic implants that allow clinicians to use them for treatment of bone trauma.

Spatially resolved spectroscopic differentiation of hydrophilic and hydrophobic domains on individual insulin amyloid fibrils.

The formation of insoluble ß-sheet-rich protein structures known as amyloid fibrils is associated with numerous neurodegenerative diseases, such as Alzheimer's and Parkinson's disease. A detailed understanding of the molecular structure of the fibril surface is of interest as the first contact with the physiological environment in vivo and plays a decisive role in biological activity and associated toxicity. Recent studies reveal that the inherent sensitivity and specificity of tip-enhanced Raman scattering (TERS) renders this technique a compelling method for fibril surface analysis at the single-particle level. Here, the reproducibility of TERS is demonstrated, indicating its relevance for detecting molecular variations. Consequently, individual fibrils are systematically investigated at nanometer spatial resolution. Spectral parameters were obtained by band-fitting, particularly focusing on the identification of the secondary structure via the amide III band and the differentiation of hydrophobic and hydrophilic domains on the surface. In addition multivariate data analysis, specifically the N-FINDR procedure, was employed to generate structure-specific maps. The ability of TERS to localize specific structural domains on fibril surfaces shows promise to the development of new fibril dissection strategies and can be generally applied to any (bio)chemical surface when structural variations at the nanometer level are of interest.

Quantitative multi-image analysis for biomedical Raman spectroscopic imaging.

Imaging by Raman spectroscopy enables unparalleled label-free insights into cell and tissue composition at the molecular level. With established approaches limited to single image analysis, there are currently no general guidelines or consensus on how to quantify biochemical components across multiple Raman images. Here, we describe a broadly applicable methodology for the combination of multiple Raman images into a single image for analysis. This is achieved by removing image specific background interference, unfolding the series of Raman images into a single dataset, and normalisation of each Raman spectrum to render comparable Raman images. Multivariate image analysis is finally applied to derive the contributing 'pure' biochemical spectra for relative quantification. We present our methodology using four independently measured Raman images of control cells and four images of cells treated with strontium ions from substituted bioactive glass. We show that the relative biochemical distribution per area of the cells can be quantified. In addition, using k-means clustering, we are able to discriminate between the two cell types over multiple Raman images. This study shows a streamlined quantitative multi-image analysis tool for improving cell/tissue characterisation and opens new avenues in biomedical Raman spectroscopic imaging.

Transient IR Spectroscopic Observation of Singlet and Triplet States of 2-Nitrofluorene: Revisiting the Photophysics of Nitroaromatics.

The dynamics of 2-nitrofluorene (2-NF) in deuterated acetonitrile is studied using UV pump, IR probe femtosecond transient absorption spectroscopy. Upon excitation to the vibrationally excited S1 state, the excited-state population of 2-NF branches into two different relaxation pathways. One route leads to intersystem crossing (ISC) to the triplet manifold within a few hundred femtoseconds and the other to internal conversion (IC) to the ground state. The experiments indicate that after relaxation to the energetic minimum on S1, 2-NF undergoes internal conversion to the ground state in about 15 ps. IC within the triplet manifold is also observed as the initially populated triplet state relaxes to T1 in about 6 ps. Rotational anisotropy measurements corroborate the assignment of the transient IR frequencies and indicate a rotational diffusion time of 2-NF in the solvent of about 14 ps. The combined set of results provides a unified picture of the dynamics in photoexcited 2-NF. This to our knowledge is the first example using femtosecond vibrational spectroscopy for the study of the fundamental photoinduced processes in nitroaromatic compounds.

Sparse feature selection methods identify unexpected global cellular response to strontium-containing materials.

Despite the increasing sophistication of biomaterials design and functional characterization studies, little is known regarding cells' global response to biomaterials. Here, we combined nontargeted holistic biological and physical science techniques to evaluate how simple strontium ion incorporation within the well-described biomaterial 45S5 bioactive glass (BG) influences the global response of human mesenchymal stem cells. Our objective analyses of whole gene-expression profiles, confirmed by standard molecular biology techniques, revealed that strontium-substituted BG up-regulated the isoprenoid pathway, suggesting an influence on both sterol metabolite synthesis and protein prenylation processes. This up-regulation was accompanied by increases in cellular and membrane cholesterol and lipid raft contents as determined by Raman spectroscopy mapping and total internal reflection fluorescence microscopy analyses and by an increase in cellular content of phosphorylated myosin II light chain. Our unexpected findings of this strong metabolic pathway regulation as a response to biomaterial composition highlight the benefits of discovery-driven nonreductionist approaches to gain a deeper understanding of global cell-material interactions and suggest alternative research routes for evaluating biomaterials to improve their design.

High resolution Raman spectroscopy mapping of stem cell micropatterns.

We report on the use of high resolution Raman spectroscopy mapping combined with a micro-engineered stem cell platform. This technique obtains quantitative information about the concentration of individual intracellular molecules such as proteins, lipids, and other metabolites, while tightly controlling cell shape and adhesion. This new quantitative analysis will prove highly relevant for in vitro drug screening applications and regenerative medicine.

Model based variable selection as a tool to highlight biological differences in Raman spectra of cells.

In vitro Raman spectroscopy used for non-invasive, non-destructive characterization of single cells and tissues has proven to be a powerful tool for understanding the complex biochemical processes within these biological systems. Additionally it enables the comparison of a wide range of in vitro model systems by discriminating them based on their biomolecular differences. However, one persistent challenge in Raman spectroscopy has been the highly complex structure of cell and tissue spectra, which comprise signals from lipids, proteins, carbohydrates and nucleic acids, which may overlap significantly. This leads to difficulty in discerning which molecular components are responsible for the changes seen between experimental groups. To address this problem, we introduce a technique to highlight the significant biochemical changes between sample groups by applying a novel approach using Partial Least Squares - Discriminant Analysis (PLS-DA) Variable Importance Projection (VIP) scores normally used for variable selection as heat maps combined with group difference spectra to highlight significant differences in Raman band shapes and position. To illustrate this method we analyzed single HeLa cells in their live, fixed, fixed and ethanol dehydrated, to the fixed, dehydrated and then rehydrated states respectively. Fixation, ethanol dehydration and rehydration are known to induce molecular changes in the lipids and proteins within each cell.

Raman spectroscopic evidence of tissue restructuring in heat-induced tissue fusion.

Heat-induced tissue fusion via radio-frequency (RF) energy has gained wide acceptance clinically and here we present the first optical-Raman-spectroscopy study on tissue fusion samples in vitro. This study provides direct insights into tissue constituent and structural changes on the molecular level, exposing spectroscopic evidence for the loss of distinct collagen fibre rich tissue layers as well as the denaturing and restructuring of collagen crosslinks post RF fusion. These findings open the door for more advanced optical feedback-control methods and characterization during heat-induced tissue fusion, which will lead to new clinical applications of this promising technology.