Structural changes in cartilage and collagen studied by high temperature Raman spectroscopy

Understanding the high temperature behavior of collagen and collagenous tissue is important for surgical procedures and biomaterials processing for the food, pharmaceutical, and cosmetics industries. One primary event for proteins is thermal denaturation that involves unfolding the polypeptide chains while maintaining the primary structure intact. Collagen in the extracellular matrix of cartilage and other connective tissue is a hierarchical material containing bundles of triple-helical fibers associated with water and proteoglycan components. Thermal analysis of dehydrated collagen indicates irreversible denaturation at high temperature between 135°C and 200°C, with another reversible event at ∼60-80°C for hydrated samples. We report high temperature Raman spectra for freeze-dried cartilage samples that show an increase in laser-excited fluorescence interpreted as conformational changes associated with denaturation above 140°C. Spectra for separated collagen and proteoglycan fractions extracted from cartilage indicate the changes are associated with collagen. The Raman data also show appearance of new features indicating peptide bond hydrolysis at high temperature implying that molecular H2O is retained within the freeze-dried tissue. This is confirmed by thermogravimetric analysis that show 5-7 wt% H2O remaining within freeze-dried cartilage that is released progressively upon heating up to 200°C. Spectra obtained after exposure to high temperature and re-hydration following recovery indicate that the capacity of the denatured collagen to re-absorb water is reduced. Our results are important for revealing the presence of bound H2O within the collagen component of connective tissue even after freeze-drying and its role in denaturation that is accompanied by or perhaps preceded by breakdown of the primary polypeptide structure

Exposure of a tendon extracellular matrix to synovial fluid triggers endogenous and engrafted cell death: A mechanism for failed healing of intrathecal tendon injuries

Aim: The purpose of this study was to investigate the effect of normal synovial fluid (SF) on exposed endogenous tendon-derived cells (TDC) and engrafted mesenchymal stem cells (MSCs) within the tendon extracellular matrix. Methods: Explants from equine superficial digital flexor (extra-synovial) and deep digital flexor tendons (DDFT) from the compressed, intra-synovial and the tensile, extra-synovial regions were cultured in allogeneic or autologous SF-media. Human hamstring explants were cultured in allogeneic SF. Explant viability was assessed by staining. Proliferation of equine monolayer MSCs and TDCs in SF-media and co-culture with DDFT explants was determined by alamarblue®. Non-viable Native Tendon matrices (NNTs) were re-populated with MSCs or TDCs and cultured in SF-media. Immunohistochemical staining of tendon sections for the apoptotic proteins caspase-3, -8 and -9 was performed. Results: Contact with autologous or allogeneic SF resulted in rapid death of resident tenocytes in equine and human tendon. SF did not affect the viability of equine epitenon cells, or of MSCs and TDCs in monolayer or indirect explant co-culture. MSCs and TDCs, engrafted into NNTs, died when cultured in SF. Caspase-3, -8 and -9 expression was greatest in SDFT explants exposed to allogeneic SF. Conclusions: The efficacy of cells administered intra-synovially for tendon lesion repair is likely to be limited, since once incorporated into the matrix, cells become vulnerable to the adverse effects of SF. These observations could account for the poor success rate of intra-synovial tendon healing following damage to the epitenon and contact with SF, common with most soft tissue intra-synovial pathologies

A new scheme for sulphur dioxide retrieval from IASI measurements: application to the Eyjafjallajökull eruption of April and May 2010

A new optimal estimation algorithm for the retrieval of sulphur dioxide (SO<sub>2</sub>) has been developed for the Infrared Atmospheric Sounding Interferometer (IASI) using the channels between 1000–1200 and 1300–1410 cm<sup>−1</sup>. These regions include the two SO<sub>2</sub> absorption bands centred at about 8.7 and 7.3 μm (the ν<sub>1</sub> and ν<sub>3</sub> bands respectively). The retrieval assumes a Gaussian SO<sub>2</sub> profile and returns the SO<sub>2</sub> column amount in Dobson units and the altitude of the plume in millibars (mb). Forward modelled spectra (against which the measurements are compared) are based on the Radiative Transfer for TOVS (RTTOV) code. In our implementation RTTOV uses atmospheric profiles from European Centre for Medium-Range Weather Forecasts (ECMWF) meteorological data. The retrieval includes a comprehensive error budget for every pixel derived from an error covariance matrix that is based on the SO<sub>2</sub>-free climatology of the differences between the IASI and forward modelled spectra. The IASI forward model includes the ability to simulate a cloud or ash layer in the atmosphere. This feature is used to illustrate that: (1) the SO<sub>2</sub> retrieval is not affected by underlying cloud but is affected if the SO<sub>2</sub> is within or below a cloud layer; (2) it is possible to discern if ash (or other atmospheric constituents not considered in the error covariance matrix) affects the retrieval using quality control based on the fit of the measured spectrum by the forward modelled spectrum. In this work, the algorithm is applied to follow the behaviour of SO<sub>2</sub> plumes from the Eyjafjallajökull eruption during April and May 2010. From 14 April to 4 May (during Phase I and II of the eruption) the total amount of SO<sub>2</sub> present in the atmosphere, estimated by IASI measurements, is generally below 0.02 Tg. During the last part of the eruption (Phase III) the values are an order of magnitude higher, with a maximum of 0.18 Tg measured on the afternoon of 7 May

Evaluation of three new surface irrigation parameterizations in the WRF-ARW v3.8.1 model: the Po Valley (Italy) case study

Abstract. Irrigation is a method of land management that can affect the local climate. Recent literature shows that it affects mostly the near-surface variables and it is associated with an irrigation cooling effect. However, there is no common parameterization that also accounts for a realistic water amount, and this factor could ascribe one cause to the different impacts found in previous studies. This work aims to introduce three new surface irrigation parameterizations within the WRF-ARW model (v3.8.1) that consider different evaporative processes. The parameterizations are tested on one of the regions where global studies disagree on the signal of irrigation: the Mediterranean area and in particular the Po Valley. Three sets of experiments are performed using the same irrigation water amount of 5.7 mm d−1, derived from Eurostat data. Two complementary validations are performed for July 2015: monthly mean, minimum, and maximum temperature with ground stations and potential evapotranspiration with the MODIS product. All tests show that for both mean and maximum temperature, as well as potential evapotranspiration simulated fields approximate observation-based values better when using the irrigation parameterizations. This study addresses the sensitivity of the results to human-decision assumptions of the parameterizations: start time, length, and frequency. The main impact of irrigation on surface variables such as soil moisture is due to the parameterization choice itself affecting evaporation, rather than the timing. Moreover, on average, the atmosphere and soil variables are not very sensitive to the parameterization assumptions for realistic timing and length

Development of a Cartilage Oligomeric Matrix Protein Neo-Epitope Assay for the Detection of Intra-Thecal Tendon Disease

The diagnosis of tendon injury relies on clinical signs and diagnostic imaging but imaging is subjective and does not always correlate with clinical signs. A molecular marker would potentially offer a sensitive and specific diagnostic tool that could also provide objective assessment of healing for the comparison of different treatments. Cartilage Oligomeric Matrix Protein (COMP) has been used as a molecular marker for osteoarthritis in humans and horses but assays for the protein in tendon sheath synovial fluids have shown overlap between horses affected by tendinopathy and controls. We hypothesized that quantifying a COMP neoepitope would be more discriminatory of injury. COMP fragments were purified from synovial fluids of horses with intra-thecal tendon injuries and media from equine tendon explants, and mass spectrometry of a consistent and abundant fragment revealed a ~100 kDa COMP fragment with a new N-terminus at the 78th amino-acid (NH2-TPRVSVRP) located just outside the junctional region of the protein. A competitive inhibition ELISA based on a polyclonal antibody raised to this sequence yielded more than a 10-fold rise in the mean neoepitope levels for tendinopathy cases compared to controls (5.3 ± 1.3 µg/mL (n = 7) versus 58.8 ± 64.3 µg/mL (n = 13); p = 0.002). However, there was some cross-reactivity of the neoepitope polyclonal antiserum with intact COMP, which could be blocked by a peptide spanning the neoepitope. The modified assay demonstrated a lower concentration but a significant > 500-fold average rise with tendon injury (2.5 ± 2.2 ng/mL (n = 6) versus 1029.8 ± 2188.8 ng/ml (n = 14); p = 0.013). This neo-epitope assay therefore offers a potentially useful marker for clinical use

Lentiviral vector transduction of spermatozoa as a tool for the study of early development

Spermatozoa and lentiviruses are two of nature’s most efficient gene delivery vehicles. Both can be genetically modified and used independently for the generation of transgenic animals or gene transfer/therapy of inherited disorders. Here we show that mature spermatozoa can be directly transduced with various pseudotyped lentiviral vectors and used in in vitro fertilisation studies. Lentiviral vectors encoding Green Fluorescent Protein (GFP) were shown to be efficiently processed and expressed in sperm. When these transduced sperm were used in in vitro fertilisation studies, GFP expression was observed in arising blastocysts. This simple technique of directly transducing spermatozoa has potential to be a powerful tool for the study of early and pre-implantation development and could be used as a technique in transgenic development and vertical viral transmission studies

A new technique for evaluating mesospheric momentum balance utilizing radars and satellite data

Copyright © 2000 European Geosciences UnionA new method for evaluating momentum balance in the mesosphere using radar and satellite data is presented. This method is applied to radar wind data from two medium frequency installations (near Adelaide, Australia and Christchurch, New Zealand) and satellite temperature data from the Improved Stratospheric and Mesospheric Sounder (ISAMS). Because of limitations in data availability and vertical extent, the technique can only be applied to evaluate the momentum balance at 80 km above the radar sites for May 1992. The technique allows the calculation of the residual terms in the momentum balance which are usually attributed to the effects of breaking gravity waves. Although the results are inconclusive above Adelaide, this method produces values of zonal and meridional residual accelerations above Christchurch which are consistent with expectation. In both locations it is apparent that geostrophic balance is a poor approximation of reality. (This result is not dependent on a mismatch between the radar and satellite derived winds, but rather is inherent in the satellite data alone.) Despite significant caveats about data quality the technique appears robust and could be of use with data from future instruments

Inducing pluripotency in the domestic cat (Felis Catus)

Domestic cats suffer from a range of inherited genetic diseases, many of which display similarities with equivalent human conditions. Developing cellular models for these inherited diseases would enable drug discovery, benefiting feline health and welfare as well as enhancing the potential of cats as relevant animal models for translation to human medicine. Advances in our understanding of these diseases at the cellular level have come from the use of induced pluripotent stem cells (iPSCs). iPSCs are capable of differentiating into derivatives of all three germ layers, therefore overcoming the limitations of primary differentiated cells and the ethical concerns of using embryonic stem cells. No studies however report the generation of iPSCs from domestic cats (fiPSCs). Feline adipose derived fibroblasts were infected with amphotropic retrovirus containing the coding sequences for human Oct4, Sox2, Klf4, cMyc and Nanog. Isolated iPSC clones were expanded on mouse inactivated embryonic fibroblasts in the presence of feline leukaemia inhibitory factor (LIF). Retroviral delivery of human pluripotent genes gave rise to putative fiPSC colonies within 5-7 days. These iPS-like cells required foetal bovine serum and feline LIF for maintenance. Colonies were domed with refractile edges, similar to mouse iPSCs. Immunocytochemistry demonstrated positive staining for stem cell markers: alkaline phosphatase, Oct4, Sox2, Nanog and SSEA1. Cells were negative for SSEA4. Expression of endogenous feline Nanog was confirmed by qPCR. The cells were able to differentiate in vitro into cells representative of the three germ layers. These results confirm the generation of the first induced pluripotent cells from domestic cats. These cells will provide valuable models to study genetic diseases and explore novel therapeutic strategies