Towards Chemical Imaging of Living Cells: Design and Application of a Confocal Raman Microscope

Raman microspectroscopy is a technique that can be used to obtain\ud information about the chemical composition of a very small measurement\ud volume (0.5 fl) in a (biological) sample. Molecules present in the sample\ud can be identified based on their scattering characteristics and no special\ud treatment or preparation of the samples is necessary. Therefore, biological\ud samples can be measured under physiological conditions and reactions in\ud living cells can be monitored.\ud We have developed a Confocal Direct Imaging Raman Microscope\ud (CDIRM) which enables the measurement of both Raman microspectra of\ud a small measurement volume and of Raman images which show the\ud distribution of a specific compound over the sample. The CDIRM is the\ud first example in literature of a confocal microscope which is based upon\ud direct imaging. All currently used confocal Raman microscopes work with\ud image reconstruction. Direct imaging has several advantages among which\ud the shorter measurement times that can be used in most applications.\ud In chapter 2 the design of the system has been discussed and its mains\ud characteristics, like resolution and image quality have been described. The\ud resolution of the set up as determined with a 0.282 μm sphere appeared to\ud be 0.37 μm in the lateral direction and 1.2 μm in the axial direction (Full\ud Width at Half Maximum (FWHM)). The resolution for a 275 nm layer was\ud determined to be 1.4 μm in the axial direction. We have demonstrated that\ud high resolution Raman images of biological samples can be made with the\ud CDIRM. Raman images have been measured of the DNA and protein\ud distribution in a polytene chromosome. These images illustrate the\ud capability of our system to make Raman images of a sample with a\ud relatively weak Raman signal: only 0.1 photons/(second×pixel) were\ud detected. Further, we have shown that our system can be used to make 3-\ud dimensional Raman images of biological samples. 3-dimensional images of\ud the distribution of a drug in a living cell and of cholesterol in an eye lens\ud slice have been presented.\ud Raman microspectroscopy is one of the few techniques that enables the\ud monitoring of processes in single living cells, without chemical treatment\ud of the sample which might disturb the cellular system. In chapter 3 Raman\ud measurements on single activated human neutrophilic and eosinophilic\ud granulocytes have been shown. The granulocytes were activated by\ud addition of the soluble activator Phorbol Myristate Acetate or by\ud opsonized particles. Raman spectra were measured in the cytoplasm and\ud the phagosome of activated granulocytes. The resulting spectra were\ud y\ud 123\ud compared with spectra of the native cells and clear differences could be\ud recognized. The results indicated an intracellular reduction of both\ud Myeloperoxidase and cytochrome b558, two heme-proteins which are\ud known to play a role in the human immune system.\ud An important advantage of Raman imaging compared to fluorescence\ud imaging is that no extrinsic labels have to be introduced to distinguish\ud specific molecules. However, in samples with a low concentration of weak\ud Raman scattering molecules it can be advantageous to introduce extrinsic\ud labels. These Raman labels should bind specifically to the molecules of\ud interest and have a relatively large Raman scattering cross section. In\ud certain applications it can be preferable to use such Raman labels instead\ud of fluorescent labels, because of their much narrower bandwidth, which\ud allows the detection of many more different labels in a limited wavelength\ud range and because they do not bleach. In chapter 4 two examples of\ud extrinsic Raman labeling have been demonstrated: the use of the\ud cholesterol specific label filipin for visualizing the cholesterol distribution\ud in an eye lens and the application of antibody coated polystyrene spheres\ud to distinguish different phenotypes of human leukocytes. Further, a\ud discussion is given about which molecules and structures can be used in\ud the development of other suitable Raman labels

Laminitis: risk factors and outcome in a group of Danish horses

Reasons for performing study: Recent systematic reviews have highlighted the lack of quality information with respect to the epidemiology of equine laminitis. Objectives: To identify in Denmark the risk factors for new (i.e. not believed to have suffered from laminitis previously) cases of laminitis (NL) and to look at the outcome and incidence of repeated episodes of laminitis in these animals as well as those which had previously suffered an episode of laminitis (i.e. chronic cases) over the following 12 months. Methods: Information was obtained from 110 veterinary diagnosed cases of laminitis (69 new and 41 chronic) and 80 control animals (the next non-laminitic horse/pony seen by that participating practice). All animals were followed for up to one year. Univariable and multivariable conditional logistic regression was conducted for the NL case control pairs. Variables were retained within the final multivariable models if the likelihood ratio p-value was < 0.05. Results: There was no association between sex or gender and laminitis. A recent change of grass, being on what was considered high quality grass and being a cold-blooded type, <149cm ( i.e. Shetland, Fell, Welsh, or Dartmoor pony, Icelandic horse, Norwegian fjords, or a mix of these breeds) were all significant risk factors for laminitis. Although cresty neck score (CNS), and body condition Score (BCS), were significantly associated with NL at the univariable screening stage, they were found to be confounders of breed and each other during the multivariable model building process. Other factors such as weight, and estimated starch intake were not found to be significant .Thirty three percent of all the laminitis cases had been humanely destroyed within 12 months of diagnosis, mainly for laminitis associated reasons, compared with only 7.5% of the controls (none for laminitis associated reasons). Conclusions: and potential Relevance: This study confirms the importance of grass turn out and breed on laminitis risk. Horses in work at the time of diagnosis as well as those diagnosed in the winter and spring were more likely to be humanely destroyed within the next 12 months than those not in work or diagnosed in the autumn and summer

Influence of the development stage of the plant biomass for protein yield in a green biorefinery.

The scarce availability of organic animal feed for monogastric animals is hindering the development of organic farming in many regions. A green biorefinery platform called OrganoFinery aims at producing organic protein-rich animal feed based on organically grown green biomass. The freshly harvested green biomass is separated into a solid fraction (press cake) and a liquid fraction (green juice). Subsequently, the green juice is inoculated with specific lactic acid bacteria which ferment the soluble sugars into lactic acid triggering a pH drop and thus, the precipitation of proteins. Last, proteins are extracted by centrifugation and the organic protein concentrate is suitable for feeding monogastric animals. Green perennial crops can be harvested several times during a season. In the present study, the impact of the development stage of the plant biomass, exemplified by red clover, at harvesting time for the protein extraction yield was assessed in order to develop a harvesting strategy. Red clover was harvested at different developmental stages i.e. early stem elongation, late stem elongation and heading. Right after harvest, the fresh biomass was processed to produce a protein concentrate as described above. The green juices presented higher concentration of soluble proteins and soluble sugars with increasing maturity. After 10 hours fermentation, the pH dropped from 5.9-6.0 to 3.9-4.0 in all green juice samples and the lactic acid concentration reached up to 20.6 g/L. The highest lactic acid concentration (20.6 g/L) was achieved for the late maturity stage and was 1.5 higher than in the early maturity stage. Differences due to the development stage were also observed in the recovery of proteins from the green juice into the protein concentrate. Indeed, the recovery of proteins from the green juice was between 70-80% of the content in the green juice and with the highest values obtained for the late maturity stage. The relevance of this study relies on the fact that the harvesting strategy is crucial in order to maximize the protein yield from green biomass and, to develop an economically viable green biorefinery platform to produce organic protein-rich feed for monogastric animals

Mapping Greenhouse Plastic Wastes in the West Region of Portugal

Plastic materials are used in many applications in agriculture, as greenhouse and tunnel covering films, direct coverings, shading and protective nets, irrigation pipes, fertilizers bags, pots and others. In Mediterranean countries, plastic film is the most common greenhouse covering material. Due to the direct exposure to solar radiation and wind, greenhouse plastic cover needs to be replaced every 6 to 45 months, depending on the material. The huge quantities of plastic waste produced need a proper management in order to avoid environmental and economic impacts. The plastic waste is usually recycled by special companies that collect and treat this waste material. The aim of this study was to apply Geographical Information Systems and photointerpretation techniques to evaluate the amount of plastic waste produced in one of the most important regions of greenhouse production in Portugal, the West Region specifically the Torres Vedras municipality. The methodology has integrated the characterization of the type of plastic covering material and the cartography of the greenhouses land units. It was used the Land Use Map 2007, Bing Map images and administrative boundaries to determine the geographic position and the types of greenhouses. For each administrative unit it was calculated the frequency and total greenhouses area. The identification of the type of plastic material was made by photointerpretation and direct interviews in one Company that sells material for greenhouses in Torres Vedras. The database created allowed to determine the amount of plastic used as greenhouse cover materials and to identify the needs for recycling in this area. The geodatabase produced is a first step to monitoring plastic waste production in time and space

Fatty acid suppression of glial activation prevents central neuropathic pain after spinal cord injury

Supplemental digital content associated with this article can be found online at http://links.lww.com/PAIN/A853Peer reviewedPostprin

Extracellular microRNAs in Relation to Weight Loss—A Systematic Review and Meta-Analysis

Obesity is an important risk factor for cardiovascular disease and type 2 diabetes mellitus. Even a modest weight loss of 5–15% improves metabolic health, but circulating markers to indicate weight loss efficiency are lacking. MicroRNAs, small non-coding post-transcriptional regulators of gene expression, are secreted from tissues into the circulation and may be potential biomarkers for metabolic health. However, it is not known which specific microRNA species are reproducibly changed in levels by weight loss. In this study, we performed a systematic review and meta-analysis to investigate the microRNAs associated with weight loss by comparing baseline to follow-up levels following intervention-driven weight loss. This systematic review was performed according to the PRISMA guidelines with searches in PubMed and SCOPUS. The primary search resulted in a total of 697 articles, which were screened according to the prior established inclusion and exclusion criteria. Following the screening of articles, the review was based on the inclusion of 27 full-text articles, which were evaluated for quality and the risk of bias. We performed systematic data extraction, whereafter the relative values for miRNAs were calculated. A meta-analysis was performed for the miRNA species investigated in three or more studies: miR-26a, miR-126, and miR-223 were overall significantly increased following weight loss, while miR-142 was significantly decreased after weight loss. miR-221, miR-140, miR-122, and miR-146 were not significantly changed by intervention-driven weight loss. These results indicate that few miRNAs are significantly changed during weight loss.</p