Non‐invasive identification of proteoglycans and chondrocyte differentiation state by Raman microspectroscopy

Proteoglycans (PGs) are crucial extracellular matrix (ECM) components that are present in all tissues and organs. Pathological remodeling of these macromolecules can lead to severe diseases such as osteoarthritis or rheumatoid arthritis. To date, PG‐associated ECM alterations are routinely diagnosed by invasive analytical methods. Here, we employed Raman microspectroscopy, a laser‐based, marker‐free and non‐destructive technique that allows the generation of spectra with peaks originating from molecular vibrations within a sample, to identify specific Raman bands that can be assigned to PGs within human and porcine cartilage samples and chondrocytes. Based on the non‐invasively acquired Raman spectra, we further revealed that a prolonged in vitro culture leads to phenotypic alterations of chondrocytes, resulting in a decreased PG synthesis rate and loss of lipid contents. Our results are the first to demonstrate the applicability of Raman microspectroscopy as an analytical and potential diagnostic tool for non‐invasive cell and tissue state monitoring of cartilage in biomedical research. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Evidence for a krill‐rich diet from non‐destructive analyses of penguin bone

Diet strongly influences the chemistry of vertebrate soft and hard tissues. Bird bone and eggshell mineral preserve reliable records of prey consumption, even beyond the life of the predator, and analyses of hard tissues have usefully reconstructed avian diet. Here, we assess the feasibility of a non‐destructive method for distinguishing krill‐poor from krill‐rich diets in penguins. Krill (Euphausiaceae) are fluoride‐rich, and penguins that consume krill produce fluoride‐rich bones. The chemistry of bone mineral may be elucidated using Raman spectroscopy without recourse to specialised sample preparation. Published data from the diet of six penguin species were compared to a fluoride‐informative spectral band (phosphate symmetric stretch, ν₁‐PO₄^3?) in the Raman spectra of penguin humeri. Penguins that consume abundant krill (e.g. Adélie and emperor) have ν₁‐PO₄^3?‐band positions higher than 963 cm^?1, whereas penguins that primarily eat teleost fish or cephalopods (e.g. Fiordland crested, Humboldt, little blue and yellow‐eyed) have ν₁‐PO₄^3?‐band positions lower than 963 cm^?1. A krill‐rich diet can therefore be determined from the Raman spectra of penguin bones. Raman spectroscopy could be a useful supplement to existing diet analysis techniques.     

Cover Picture: J. Biophotonics 1/2012

Raman spectroscopy has been used in this study to obtain biochemical fingerprint patterns of collagen fibers in native aortic heart valve tissues. Using this non‐contact screening tool, we were able to monitor the increasing damage of collagen fibers due to enzymatic treatment or cryopreservation. (Picture: M. Votteler et al., pp. 47–56 in this issue)     

Laser tissue welding analyzed using fluorescence,Stokes shift spectroscopy,and Huang‐Rhys parameter

Near infrared (NIR) continuous wave laser radiation at the 1,450 nm wavelength was used to weld porcine aorta and skin samples via the absorption of combitional vibrational modes of native water in the tissues. The fluorescence spectra were measured from the key native molecules of welded and non‐welded tissues at specific excitation and emission wavelengths from collagen, elastin, and tryptophan. The changes in the fluorescence intensities and differences in Stokes shift (Δν_ss) of key native fluorophores were measured to differentiate the Huang‐Rhys parameter values (S) of the chromophores. The strength of coupling depends on the local electron‐vibration intra‐tissue molecular environment and the amount of polar solvent water surrounding the net charges on collagen, elastin, and tryptophan. The S values for both non‐welded and welded tissues were almost the same and less than 3, suggesting minimal changes in the local molecular environment as a result of welding. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Extracellular matrix‐associated proteome changes during non‐host resistance in citrus–Xanthomonas interactions

Non‐host resistance (NHR) is a most durable broad‐spectrum resistance employed by the plants to restrict majority of pathogens. Plant extracellular matrix (ECM) is a critical defense barrier. Understanding ECM responses during interaction with non‐host pathogen will provide insights into molecular events of NHR. In this study, the ECM‐associated proteome was compared during interaction of citrus with pathogen Xanthomonas axonopodis pv. citri (Xac) and non‐host pathogen Xanthomonas oryzae pv. oryzae (Xoo) at 8, 16, 24 and 48 h post inoculation. Comprehensive analysis of ECM‐associated proteins was performed by extracting wall‐bound and soluble ECM components using both destructive and non‐destructive procedures. A total of 53 proteins was differentially expressed in citrus–Xanthomonas host and non‐host interaction, out of which 44 were identified by mass spectrometry. The differentially expressed proteins were related to (1) defense‐response (5 pathogenesis‐related proteins, 3 miraculin‐like proteins (MIR, MIR1 and MIR2) and 2 proteases); (2) enzymes of reactive oxygen species (ROS) metabolism [Cu/Zn superoxide dismutase (SOD), Fe‐SOD, ascorbate peroxidase and 2‐cysteine‐peroxiredoxin]; (3) signaling (lectin, curculin‐like lectin and concanavalin A‐like lectin kinase); and (4) cell‐wall modification (α‐xylosidase, glucan 1, 3 β‐glucosidase, xyloglucan endotransglucosylase/hydrolase). The decrease in ascorbate peroxidase and cysteine‐peroxiredoxin could be involved in maintenance of ROS levels. Increase in defense, cell‐wall remodeling and signaling proteins in citrus–Xoo interaction suggests an active involvement of ECM in execution of NHR. Partially compromised NHR in citrus against Xoo, upon Brefeldin A pre‐treatment supported the role of non‐classical secretory proteins in this phenomenon.     

Raman spectroscopy as a non‐invasive technique for the quantification of melanins in feathers and hairs

The quantification of melanins is a complex task due to the chemical heterogeneity of the pigments and the difficulty of their isolation. The best accepted procedure currently consists in the chemical cleavage of melanins and the subsequent detection of degradation products by HPLC, which implies the destruction of samples. Here, we show that Raman spectroscopy is a non‐invasive technique that can be used to quantify melanins. We made parallel analyses of the characteristics of pheomelanin and eumelanin Raman spectra as measured by confocal Raman microscopy and of degradation products of pheomelanin (4‐amino‐3‐hydroxyphenylalanine, 4‐AHP) and eumelanin (pyrrole‐2,3,5‐tricarboxylic acid, PTCA) as measured by HPLC in feathers of red‐legged partridges and hairs of wild boars and humans. We found strong correlations between the spectral Raman characteristics and 4‐AHP and PTCA levels, which indicates that the Raman spectra of melanins can be used to determine their content.     

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.^1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.⁶ As every chemical vibration is assigned to a specific Raman band (wavenumber in cm^-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.^7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.¹ Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).¹⁰Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 °C, 5% CO₂) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 °C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.     

Climate warming increases biological control agent impact on a non‐target species

Climate change may shift interactions of invasive plants, herbivorous insects and native plants, potentially affecting biological control efficacy and non‐target effects on native species. Here, we show how climate warming affects impacts of a multivoltine introduced biocontrol beetle on the non‐target native plant Alternanthera sessilis in China. In field surveys across a latitudinal gradient covering their full distributions, we found beetle damage on A. sessilis increased with rising temperature and plant life history changed from perennial to annual. Experiments showed that elevated temperature changed plant life history and increased insect overwintering, damage and impacts on seedling recruitment. These results suggest that warming can shift phenologies, increase non‐target effect magnitude and increase non‐target effect occurrence by beetle range expansion to additional areas where A. sessilis occurs. This study highlights the importance of understanding how climate change affects species interactions for future biological control of invasive species and conservation of native species.     

Current knowledge on non‐native freshwater fish introductions

This review provides a contemporary account of knowledge on aspects of introductions of non‐native fish species and includes issues associated with introduction pathways, ecological and economic impacts, risk assessments, management options and impact of climate change. It offers guidance to reconcile the increasing demands of certain stakeholders to diversify their activities using non‐native fishes with the long‐term sustainability of native aquatic biodiversity. The rate at which non‐native freshwater fishes have been introduced worldwide has doubled in the space of 30 years, with the principal motives being aquaculture (39%) and improvement of wild stocks (17%). Economic activity is the principal driver of human‐mediated non‐native fish introductions, including the globalization of fish culture, whereby the production of the African cichlid tilapia is seven times higher in Asia than in most areas of Africa, and Chile is responsible for c. 30% of the world's farmed salmon, all based on introduced species. Consequently, these economic benefits need balancing against the detrimental environmental, social and economic effects of introduced non‐native fishes. There are several major ecological effects associated with non‐native fish introductions, including predation, habitat degradation, increased competition for resources, hybridization and disease transmission. Consideration of these aspects in isolation, however, is rarely sufficient to adequately characterize the overall ecological effect of an introduced species. Regarding the management of introduced non‐native fish, pre‐introduction screening tools, such as the fish invasiveness scoring kit (FISK), can be used to ensure that species are not introduced, which may develop invasive populations. Following the introduction of non‐native fish that do develop invasive populations, management responses are typified by either a remediation or a mitigation response, although these are often difficult and expensive to implement, and may have limited effectiveness.     

Comparison of invertebrate herbivores on native and non‐native Senecio species: Implications for the enemy release hypothesis

The enemy release hypothesis posits that non‐native plant species may gain a competitive advantage over their native counterparts because they are liberated from co‐evolved natural enemies from their native area. The phylogenetic relationship between a non‐native plant and the native community may be important for understanding the success of some non‐native plants, because host switching by insect herbivores is more likely to occur between closely related species. We tested the enemy release hypothesis by comparing leaf damage and herbivorous insect assemblages on the invasive species Senecio madagascariensis Poir. to that on nine congeneric species, of which five are native to the study area, and four are non‐native but considered non‐invasive. Non‐native species had less leaf damage than natives overall, but we found no significant differences in the abundance, richness and Shannon diversity of herbivores between native and non‐native Senecio L. species. The herbivore assemblage and percentage abundance of herbivore guilds differed among all Senecio species, but patterns were not related to whether the species was native or not. Species‐level differences indicate that S. madagascariensis may have a greater proportion of generalist insect damage (represented by phytophagous leaf chewers) than the other Senecio species. Within a plant genus, escape from natural enemies may not be a sufficient explanation for why some non‐native species become more invasive than others.     

Fourier transform infrared and Raman‐based biochemical profiling of different grades of pure foetal‐type hepatoblastoma

The biomolecular events resulting from the progression of hepatoblastoma remain to be elucidated. Fourier‐transform infrared (FTIR) and Raman spectroscopies are capable of noninvasively and accurately capturing the biochemical properties of biological tissue from its pathological status. Our aim was to probe critial biomolecular changes of liver accompanying the progression of pure foetal hepatoblastoma (PFH) by FTIR and Raman spectroscopies. Herein, biochemical alterations were both evident in the FTIR spectra (regions of 3100‐2800 cm^?1 and 1800‐900 cm^?1) and the Raman spectra (region of 1800‐400 cm^?1) among normal, borderline and malignant liver tissues. Compared with normal tissues, the ratios of protein‐to‐lipid, α‐helix‐to‐β‐sheet, RNA‐to‐DNA, CH3 methyl‐to‐CH2 methylene, glucose‐to‐phospholipids, and unsaturated‐to‐saturated lipids intensities were significantly higher in malignant tissues, while the ratios of RNA‐to‐Amide II, DNA‐to‐Amide II, glycogen‐to‐cholesterol and Amide I‐to‐Amide II intensities were remarkably lower. These biochemical alterations in the transition from normal to malignant have profound implications not only for cyto‐pathological classification but also for molecular understanding of PFH progression. The successive changes of the spectral characteristics have been shown to be consistent with the development of PFH, indicating that FTIR and Raman spectroscopies are excellent tools to interrogate the biochemical features of different grades of PFH.      

The importance of factors controlling species abundance and distribution varies in native and non‐native ranges

How variation in factors controlling species abundance and distribution between native and non‐native ranges compares to that within ranges remains poorly understood. We used a globally distributed ruderal, Centaurea solstitialis (Centaurea), to explore the possibility that the importance of those factors exhibits great variation between and within ranges. To test our hypothesis, we established seed addition experiments with soil disturbance (turnover and control) and biocide (fungicides, insecticide, and control) treatments in two regions within native (the Caucasus and south‐western Turkey) and non‐native (the western United States – US – and central Argentina) distributions. Also, we estimated the rate of vegetation recovery after disturbance (resilience) and related it to Centaurea density in experimental plots. Disturbance strongly increased Centaurea density in all regions. Density was similar between the native Caucasus and non‐native Argentina and much greater in those regions than in the native Turkey and non‐native US in biocide‐free plots. Fungicides had positive effects on density in the US and negative ones in the Caucasus and Argentina, resulting in no differences between those three regions and greater density in the US than Turkey. Insecticide applications promoted Centaurea density in Turkey and Argentina, but inter‐regional comparisons of density in treated plots were comparable to those in biocide‐free plots. Overall, plants were smaller and less fecund in Turkey than the other regions, except the US. The greatest fungal attack was documented in Turkey, and herbivory was stronger there and in Argentina than in the Caucasus and US. The resilience of the local community explained a large proportion of variation in Centaurea density. These results support our hypothesis, and reveal that the speed at which competition is re‐gained after disturbance may influence global variation in Centaurea abundance. Because many ruderals exhibit native and non‐native distributions, our results are likely to be generalized to other systems.     

Allelopathy and its coevolutionary implications between native and non‐native neighbors of invasive Cynara cardunculus L.

Invasive plants apply new selection pressures on neighbor plant species by different means including allelopathy. Recent evidence shows allelopathy functions as remarkably influential mediator for invaders to be successful in their invaded range. However, few studies have determined whether native and non‐native species co‐occurring with invaders have evolved tolerance to allelopathy. In this study, we conducted germination and growth experiments to evaluate whether co‐occurring native Juncus pallidus and non‐native Lolium rigidum species may evolve tolerance to the allelochemicals induced by Cyanara cardunculus in Australian agricultural fields. The test species were germinated and grown in pots filled with collected invaded and uninvaded rhizosphere soil of C. cardunculus with and without activated carbon (AC). Additionally, a separate experiment was done to differentiate the direct effects of AC on the test species. The soil properties showed invaded rhizosphere soils had higher total phenolic and lower pH compared with uninvaded soils. We found significant reduction of germination percentage and seedling growth in terms of above‐ and belowground biomass, and maximum plant height and root length of native in the invaded rhizosphere soil of C. cardunculus, but little effect on non‐native grass species. Even soil manipulated with AC showed no significant differences in the measured parameters of non‐native except aboveground biomass. Taken together, the results indicate allelochemicals induced by C. cardunculus exert more suppressive effects on native than non‐native linking the coevolved tolerance of those.     

The presence of non‐native helical structure in the unfolding of a beta‐sheet protein MPT63

MPT63, a major secreted protein from Mycobacterium tuberculosis, has been shown to have immunogenic properties and has been implicated in virulence. MPT63 is a β‐sandwich protein containing 11 β strands and a very short stretch of 3₁₀ helix. The detailed experimental and computational study reported here investigates the equilibrium unfolding transition of MPT63. It is shown that in spite of being a complete β‐sheet protein, MPT63 has a strong propensity toward helix structures in its early intermediates. Far UV‐CD and FTIR spectra clearly suggest that the low‐pH intermediate of MTP63 has enhanced helical content, while fluorescence correlation spectroscopy suggests a significant contraction. Molecular dynamics simulation complements the experimental results indicating that the unfolded state of MPT63 traverses through intermediate forms with increased helical characteristics. It is found that this early intermediate contains exposed hydrophobic surface, and is aggregation prone. Although MPT63 is a complete β‐sheet protein in its native form, the present findings suggest that the secondary structure preferences of the local interactions in early folding pathway may not always follow the native conformation. Furthermore, the Gly25Ala mutant supports the proposed hypothesis by increasing the non‐native helical propensity of the protein structure.     

Community‐level plant–soil feedbacks explain landscape distribution of native and non‐native plants

Plant–soil feedbacks (PSFs) have gained attention for their potential role in explaining plant growth and invasion. While promising, most PSF research has measured plant monoculture growth on different soils in short‐term, greenhouse experiments. Here, five soil types were conditioned by growing one native species, three non‐native species, or a mixed plant community in different plots in a common‐garden experiment. After 4 years, plants were removed and one native and one non‐native plant community were planted into replicate plots of each soil type. After three additional years, the percentage cover of each of the three target species in each community was measured. These data were used to parameterize a plant community growth model. Model predictions were compared to native and non‐native abundance on the landscape. Native community cover was lowest on soil conditioned by the dominant non‐native, Centaurea diffusa, and non‐native community cover was lowest on soil cultivated by the dominant native, Pseudoroegneria spicata. Consistent with plant growth on the landscape, the plant growth model predicted that the positive PSFs observed in the common‐garden experiment would result in two distinct communities on the landscape: a native plant community on native soils and a non‐native plant community on non‐native soils. In contrast, when PSF effects were removed, the model predicted that non‐native plants would dominate all soils, which was not consistent with plant growth on the landscape. Results provide an example where PSF effects were large enough to change the rank‐order abundance of native and non‐native plant communities and to explain plant distributions on the landscape. The positive PSFs that contributed to this effect reflected the ability of the two dominant plant species to suppress each other's growth. Results suggest that plant dominance, at least in this system, reflects the ability of a species to suppress the growth of dominant competitors through soil‐mediated effects.     

A novel environmental DNA approach to quantify the cryptic invasion of non‐native genotypes

The invasion of non‐native species that are closely related to native species can lead to competitive elimination of the native species and/or genomic extinction through hybridization. Such invasions often become serious before they are detected, posing unprecedented threats to biodiversity. A Japanese native strain of common carp (Cyprinus carpio) has become endangered owing to the invasion of non‐native strains introduced from the Eurasian continent. Here, we propose a rapid environmental DNA‐based approach to quantitatively monitor the invasion of non‐native genotypes. Using this system, we developed a method to quantify the relative proportion of native and non‐native DNA based on a single‐nucleotide polymorphism using cycling probe technology in real‐time PCR. The efficiency of this method was confirmed in aquarium experiments, where the quantified proportion of native and non‐native DNA in the water was well correlated to the biomass ratio of native and non‐native genotypes. This method provided quantitative estimates for the proportion of native and non‐native DNA in natural rivers and reservoirs, which allowed us to estimate the degree of invasion of non‐native genotypes without catching and analysing individual fish. Our approach would dramatically facilitate the process of quantitatively monitoring the invasion of non‐native conspecifics in aquatic ecosystems, thus revealing a promising method for risk assessment and management in biodiversity conservation.     

Diversity and distribution of genetic variation in gammarids: Comparing patterns between invasive and non‐invasive species

Biological invasions are worldwide phenomena that have reached alarming levels among aquatic species. There are key challenges to understand the factors behind invasion propensity of non‐native populations in invasion biology. Interestingly, interpretations cannot be expanded to higher taxonomic levels due to the fact that in the same genus, there are species that are notorious invaders and those that never spread outside their native range. Such variation in invasion propensity offers the possibility to explore, at fine‐scale taxonomic level, the existence of specific characteristics that might predict the variability in invasion success. In this work, we explored this possibility from a molecular perspective. The objective was to provide a better understanding of the genetic diversity distribution in the native range of species that exhibit contrasting invasive propensities. For this purpose, we used a total of 784 sequences of the cytochrome c oxidase subunit I of mitochondrial DNA (mtDNA‐COI) collected from seven Gammaroidea, a superfamily of Amphipoda that includes species that are both successful invaders (Gammarus tigrinus, Pontogammarus maeoticus, and Obesogammarus crassus) and strictly restricted to their native regions (Gammarus locusta, Gammarus salinus, Gammarus zaddachi, and Gammarus oceanicus). Despite that genetic diversity did not differ between invasive and non‐invasive species, we observed that populations of non‐invasive species showed a higher degree of genetic differentiation. Furthermore, we found that both geographic and evolutionary distances might explain genetic differentiation in both non‐native and native ranges. This suggests that the lack of population genetic structure may facilitate the distribution of mutations that despite arising in the native range may be beneficial in invasive ranges. The fact that evolutionary distances explained genetic differentiation more often than geographic distances points toward that deep lineage divergence holds an important role in the distribution of neutral genetic diversity.     

Smad‐induced alterations of matrix metabolism by a myristoyl tetra peptide

Regulation of extracellular matrix (ECM) components is essential for tissue homeostasis and function. We screened a small peptide that induces ECM protein synthesis for its usefulness in protecting keratinocytes. In this report, we demonstrate that myristoyl tetrapeptide Ala‐Ala‐Pro‐Val (mAAPV) stimulates the expression of ECM proteins and inhibits the expression of metalloproteinases (MMPs) that degrade ECM proteins in Hs68 human fibroblast cells. In order to elucidate the underlying molecular mechanisms for the effects of mAAVP, we investigated the changes in gene expression in the presence of mAAPV using a cDNA microarray. Treatment with mAAPV resulted in decreased expression of MMP‐related genes such as MMP1, MMP3, TIMP1 and TIMP3 and increased expression of collagen genes, including COL1A1, COL1A2, COL3A1, COL5A1 and COL6A3. The pattern of gene expression regulated by mAAPV was very similar to that of gene expression induced by transforming growth factor (TGF)‐β, indicating that the TGF‐β signaling pathway is crucial for simultaneous activation of several ECM‐related genes by mAAPV. We examined whether the activation of SMAD, a downstream protein of TGF‐β receptor, is involved in the signal transduction pathway induced by mAAPV. The results demonstrate that mAAVP directly activates SMAD2 and induces SMAD3 to bind to DNA. In conclusion, our results demonstrate that mAAPV both enhances the expression of collagen and inhibits its degradation via production of protease inhibitors that prevent enzymatic breakdown of the ECM. The results suggest that mAAPV would be a useful ECM‐protecting agent. Copyright © 2014 John Wiley & Sons, Ltd.     

Lipid‐polymorphism of plant thylakoid membranes. Enhanced non‐bilayer lipid phases associated with increased membrane permeability

Earlier experiments, using ³¹P‐NMR and time‐resolved merocyanine fluorescence spectroscopy, have shown that isolated intact, fully functional plant thylakoid membranes, in addition to the bilayer phase, contain three non‐bilayer (or non‐lamellar) lipid phases. It has also been shown that the lipid polymorphism of thylakoid membranes can be characterized by remarkable plasticity, i.e. by significant variations in ³¹P‐NMR signatures. However, changes in the lipid‐phase behaviour of thylakoids could not be assigned to changes in the overall membrane organization and the photosynthetic activity, as tested by circular dichroism and 77 K fluorescence emission spectroscopy and the magnitude of the variable fluorescence of photosystem II, which all showed only marginal variations. In this work, we investigated in more detail the temporal stability of the different lipid phases by recording ³¹P‐NMR spectra on isolated thylakoid membranes that were suspended in sorbitol‐ or NaCl‐based media. We observed, at 5°C during 8 h in the dark, substantial gradual enhancement of the isotropic lipid phases and diminishment of the bilayer phase in the sorbitol‐based medium. These changes compared well with the gradually increasing membrane permeability, as testified by the gradual acceleration of the decay of flash‐induced electrochromic absorption changes and characteristic changes in the kinetics of fast chlorophyll a‐fluorescence transients; all variations were much less pronounced in the NaCl‐based medium. These observations suggest that non‐bilayer lipids and non‐lamellar lipid phases play significant roles in the structural dynamics and functional plasticity of thylakoid membranes.     

No evidence for local adaptation and an epigenetic underpinning in native and non‐native ruderal plant species in Germany

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