Genetic manipulation of Anopheles stephensi immunity to increase plasmodium falciparum salivary gland sporozoite infection levels

Human malaria is responsible for over 700,000 deaths a year. To stay abreast of the threat posed by the parasite, a constant stream of new drugs and vector control methods are required. This study focuses on a vaccine that has the potential to protect against parasite infection, but has been hindered by developmental challenges. In malaria prevention, live, attenuated, aseptic, Plasmodium falciparum sporozoites (PfSPZ) can be administered as a highly protective vaccine. PfSPZ are produced using adult female Anopheles stephensi mosquitoes as bioreactors. Production volume and cost of a PfSPZ vaccine for malaria are expected to be directly correlated with Plasmodium falciparum infection intensity in the salivary glands. The sporogonic development of Plasmodium falciparum in A. stephensi to fully infected salivary gland stage sporozoites is dictated by the activities of several known components of the mosquito’s innate immune system. Here I report on the use of genetic technologies that have been rarely, if ever, used in Anopheles stephensi Sda500 to increase the yield of sporozoites per mosquito and enhance vaccine production. By combining the Gal4/UAS bipartite system with in vivo expression of shRNA gene silencing, activity of the IMD signaling pathway downstream effector LRIM1, an antagonist to Plasmodium development, was reduced in the midgut, fat body, and salivary glands of A. stephensi. In infection studies using P. berghei and P. falciparum these transgenic mosquitoes consistently produced significantly more salivary gland stage sporozoites than wildtype controls, with increases in P. falciparum ranging from 2.5 to 10 fold. Using Plasmodium infection assays and qRT-PCR, two novel findings were identified. First, it was shown that 14 days post Plasmodium infection, transcript abundance of the IMD immune effector genes LRIM1, TEP1 and APL1c are elevated, in the salivary glands of A. stephensi, suggesting the salivary glands may play a role in post midgut defense against the parasite. Second, a non-pathogenic IMD signaling pathway response was observed which could suggest an alternative pathway for IMD activation. The information gained from these studies has significantly increased our knowledge of Plasmodium defense in A. stephensi and moreover could significantly improve vaccine production.

Analysis of radiation induced DNA damage by LC-MS/MS in isolated and cellular DNA

Abstract: It is well established that ionizing radiation induces a variety of damage in DNA by direct effects that are mediated by one-electron oxidation and indirect effects that are mediated by the reaction of water radiolysis products, e.g., hydroxyl radicals (•OH). In cellular DNA, direct and indirect effects appear to have about an equal effect toward DNA damage. We have shown that ϒ-(gamma) ray irradiation of aqueous solutions of DNA, during which •OH is the major damaging ROS can lead to the formation several lesions. On the other hand, the methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the gene regulation. The C5 position of cytosine in CG dinucleotides is frequently methylated by DNA methyl transferees (DNMTs) and constitutes 4-5% of the total cytosine. Here, my PhD research work focuses on the analysis of oxidative base modifications of model compounds of methylated and non methylated oligonucleotides, isolated DNA (calf-thymus DNA) and F98 cultured cell by gamma radiation. In addition, we identified a series of modifications of the 2-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. We also studied one electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation, which produces cross-links between guanine and thymine bases (G*-T*). To achieve these goals, we developed several methods based on mass spectrometry to analyze base modifications in isolated DNA and cellular DNA.

Effects of supplemental osteopontin on intestinal development and serum antibody responses to rotavirus vaccination in piglets

Milk contains numerous bioactive substances including immunoglobulins, cytokines, growth factors and components that exert antibiotic and prebiotic activity (Field, 2005). Little is known about the biological effects of individual milk bioactives, despite the fact that natural milk improves intestinal development and immune system functions in neonates (Donovan et al., 1994; Field, 2005) relative to milk formula. Characterization of the biological effects of such components is important for optimal production of infant milk formulas to be used when mother’s milk is not available. Milk components with preliminary evidence of positive effects on the intestinal growth and mucosal immunity include osteopontin (OPN). Osteopontin is a phosphorylated acidic glycoprotein expressed by a number of different immune and non-immune cells and tissues (Sodek et al., 2000). It is also present in body fluids including blood, bile and milk (Sodek et al., 2000). Osteopontin is a multifunctional protein that is implicated in a wide number of biological processes including cell survival, bone remodeling, and immune modulatory functions (Sodek et al., 2000). Furthermore, Schack and colleagues (2009) demonstrated that the concentration of OPN in human milk is considerably higher than in bovine milk and infant formulas. Taken together, it is likely that OPN plays a role in the early development of gastrointestinal tract and mucosal immune responses in infants. Since the neonatal pig shares anatomical, physiological, immunological, and metabolic similarities with the human infants (Moughan, et al., 1992), they were selected as the animal model in our studies. Our first aim was to investigate the effects of OPN on piglet intestinal development. Newborn, colostrum-deprived piglets (n=27) were randomized to receive three treatments: formula with bovine OPN (OPN; 140 mg/L); formula alone (FF); or sow reared (SR) for 21 days. Body weight, intestinal weight and length, mucosal protein and DNA content, disaccharidase activity, villus morphology, and crypt cell proliferation were measured. Statistical significance was assigned at P<0.05. No significant effects of OPN were observed for body weight, intestinal weight and length. Mucosal protein content of SR piglets was lower than FF and OPN piglets in the duodenum, but higher than FF and OPN piglets in the ileum. No significant effects of diet in mucosal DNA content were detected for the three regions of the small intestine. Lactase and sucrase activities of SR piglets were higher than the two formula-fed groups in the duodenum, lower in the ileum. No significant effects of diet on lactase and sucrase activities were noted between two formula-fed groups in the duodenum and ileum. Jejunal lactase activity of FF piglets was higher than SR piglets, whereas no significant effect of diet was observed in jejunal sucrase activity among the three groups. Duodenal and ileal villus height and villus area of SR piglets were lower than two formula-fed groups, while OPN piglets did not differ from FF piglets. There was a significant effect of diet (P<0.0001) on jejunal crypt cell proliferation, with proliferation in OPN piglets being intermediate between that of FF and SR. In summary, supplemental OPN increased jejunal crypt cell proliferation, independent of evident morphological growth, and had a minor impact on disaccharidase activity in the small intestine of neonatal piglets. Rotavirus (RV) is the most common viral cause of severe gastroenteritis in infants and young children worldwide (Parashar et al., 2006). Maeno et al. (2009) reported that OPN knockout (OPN-KO) suckling mice were more susceptible to RV infection compared to wild-type (WT) suckling mice. To detect the role of OPN in intestinal immune responses of neonates, the goal of the second study was to evaluate whether supplemental OPN influenced the serum antibody responses to RV vaccination in neonatal piglets. Newborn, colostrum-deprived piglets were randomized into two dietary groups: formula with bovine OPN (OPN; 140 mg/L) and formula alone (FF) for 35 days. On d7, piglets in each dietary group were further randomized to receive rotavirus (RV) vaccination (Rotarix®) (FF+RV and OPN+RV) or remained non-vaccinated (FF+NV and OPN+NV). Booster vaccination was provided on d14. Blood samples were collected on d7, 14, 21, 28 and 35. RV-specific serum immunoglobulin (Ig) G, IgA, IgM and total serum IgG, IgA, IgM were measured by ELISA. Statistical significance was assigned at P<0.05, with trends reported as P<0.10. Body weight gain was unaffected by diet and/or vaccination. No significant effect of oral OPN supplementation was observed for RV-specific antibody responses and total Igs levels. After the combination of dietary groups, RV piglets had significantly higher RV-specific IgM concentrations compared to NV piglets. Although there were higher means of RV-specific IgG and RV-specific IgA concentrations in RV group than their counterparts in NV group, the difference did not reach statistical significance. RV-specific IgM reached a peak at d7 post booster vaccination (PBV), whereas the RV-specific IgG and IgA peaked later at PBV 14 or 21. Total Igs were unaffected by RV vaccination but were significantly increased over time, following similar pattern as RV-specific Igs. In summary, neonatal piglets generated weak antibody responses to RV vaccination. Supplemental OPN did not enhance RV-specific serum antibody responses and total serum Igs levels in neonatal piglets with or without RV vaccination. In conclusion, we observed normal developmental changes in the small intestine and serum Igs levels in neonatal piglets over time. Oral OPN supplementation showed minimal impacts on intestinal development and no effect on serum Igs levels. The role of supplemental OPN on the growth and development of infants is still inconclusive. Future studies should measure other physiological and immunological parameters by using different models of vaccination or infection.

From the field to the table: ionizing radiation as a feasible postharvest treatment for fresh and dried plant foods

Food irradiation is a treatment that involves subjecting in-bulk or packaged food to a controlled dose of ionizing radiation, with a clearly defined goal. It has been used for disinfestation and sanitization of food commodities and to retard postharvest ripening and senescence processes, being a sustainable alternative to chemical agents 1 . Doses up to 10 kGy are approved by several international authorities for not offering negative effects to food from a nutrition and toxicology point of view 2 . However, the adoption of this technology for food applications has been a slow process due to some misunderstandings by the consumer who often chooses non-irradiated foods. In this study, the effects of the ionizing radiation treatment on physical, chemical and bioactive properties of dried herbs and its suitability for preserving quality attributes of fresh vegetables during cold storage were evaluated. The studied herbs, perennial spotted rockrose (Tuberaria lignosa (Sweet) Samp.) and common mallow (Malva neglecta Wallr.) were freeze-dried and then irradiated up to 10 kGy in a Cobalt-60 chamber. The selected vegetables, watercress (Nasturtium officinale R. Br.) and buckler sorrel (Rumex induratus Boiss. Reut.) were rinsed in tap water, packaged in polyethylene bags, submitted to irradiation doses up to 6 kGy and then were stored at 4 C for a period of up to 12 days. Physical, chemical and bioactive parameters of irradiated and non-irradiated samples were evaluated using different methodologies the colour was measured with a colorimeter, individual chemical compounds were analyzed by chromatographic techniques, antioxidant properties were evaluated using in vitro assays based on different reaction mechanisms, and other quality analyses were performed following official methods of analysis. The irradiation treatment did not significantly affect the colour of the perennial spotted rockrose samples, or its phenolic composition and antioxidant activity 3 . Medium doses preserved the colour of common mallow and a low dose did not induce any adverse effect in the organic acids profile. The green colour of the irradiated vegetables was maintained during cold storage but the treatment had pros and cons in other quality attributes. The 2 kGy dose preserved free sugars and favoured polyunsaturated fatty acids (PUFA) while the 5 kGy dose favoured tocopherols and preserved the antioxidant properties in watercress samples. The 6 kGy dose was a suitable option for preserving PUFA and the ω-6 ω-3 fatty acids ratio in buckler sorrel samples. This comprehensive experimental work allowed selecting appropriate processing doses for the studied plant foods in order to preserve its quality attributes and edibility.

Effects of gamma and electron beam irradiation in the antioxidante potential of methanolic extracts and infusions of Arenaria Montana L

Irradiation is a methodology qualified for dry ingredients preservation or decontamination and can be performed using various radiation sources and energy levels in accordance with the objectives to be achieved [1]. Electron beam irradiation is used mainly for food products with low density, while gamma irradiation is mainly used for large volumes [2]. Arenaria Montana L. has a high antioxidant potential and richness in bioactive phytochemicals. It is used in Portuguese traditional medicine, acting therapeutically as an anti-inflammatory and diuretic plant [3]. The aim of this work was to evaluate the effects of gamma and electron beam irradiation at different doses (I and 10 kGy) in the antioxidant activity of A. montana. Free radicals scavenging activity, reducing power and lipid peroxidation inhibition properties of its methanolic extracts and infusions were evaluated. Through a global analysis, it was concluded that the antioxidant activity proved to be higher in methanolic extracts in comparison with the infusions, where it decreased with increasing irradiation dose regardless of the technology used (gamma or electron beam). For methanolic extracts, electron beam resulted in increased antioxidant activity while gamma irradiation caused a decrease in these extracts. Thus, the antioxidant potential is variable depending not only on the type of radiation and the dose applied, but also on the solvent used in the preparation of the extracts (methanol or water).

Gamma radiation preserves chemical and bioactive properties of Boletus edulis wild mushrooms

Boletus edulis Bull: Fr. is an edible mushroom quite appreciated for its organoleptic and nutritional properties. However, the seasonality and perishability cause some difficulties in its distribution and marketing in fresh form; losses associated with this type of food during marketing can reach 40% [1]. Irradiation is recognized as a safe and effective method for food preservation, being used worldwide to increase shelf life of fresh and dehydrated products (e.g. fruits, vegetables and spices) [2]. In particular, gamma irradiation has already been applied to cultivated mushrooms (especially Agaricus, Lentinula and Pleurotus Genus) and proved to be an interesting conservation technology [3]. However, the studies with added-value wild species are scarce. In this work, the effects of gamma irradiation on chemical and antioxidant properties of wild B. edulis, were evaluated. Fruiting bodies were obtained in Trás-os-Montes, in the Northeast of Portugal, in November 2012. The irradiation was performed in experimental equipment with 60Co sources at 1 and 2 kGy. All the results were compared with nonirradiated samples (control). Macronutrients and energy value were determined following official procedures of food analysis; fatty acids were analyzed by gas-chromatography coupled to flame ionization detection (GC-FID), while sugars and tocopherols were determined by high performance liquid chromatography (HPLC) coupled to refraction index (RI) and fluorescence detectors, respectively. Antioxidant activity was evaluated in the methanolic extracts by in vitro assays measuring DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity, reducing power, inhibition of β- carotene bleaching and inhibition of lipid peroxidation using thiobarbituric acid reactive substances (TBARS) assay. Total phenolics were also determined by the Folin-Ciocalteu assay. The nutritional profiles were not affected in high extension. Fatty acids and sugars were slightly affected, decreasing with the increasing doses. The performed assays for antioxidant activity, indicate that irradiated samples tended to have lower scavenging activity and reducing power, but higher lipid peroxidation inhibition. Despite the detected differences in individual compounds, the results of nutritional parameters, the most relevant in terms of mushroom acceptability by consumers, were less affected, indicating an interesting potential of gamma-irradiation to be used as an effective conservation technology for the studied mushrooms.

Effects of gamma and electron-beam irradiation on the individual phenolics of Viola tricolor L. edible flowers.

Edible flowers are being used in culinary preparations to improve the sensorial and nutritional qualities of food, besides improving human health due to the profusion in bioactive compounds [1]. Nevertheless, edible flowers are highly perishable and must be free of insects, which is difficult because they are usually cultivated without using pesticides [2]. Food irradiation is an economically viable technology to extend shelf life of foods, improving their hygiene and quality, while disinfesting insects [3]. The efficiency and safety of radiation processing (using Co-60 or electronaccelerators) have been approved by legal authorities (FDA, USDA, WHO, FAO), as also by the scientific community, based on extensive research [4]. Viola tricolor L. (heartseases), from Violaceae family, is one of the most popular edible flowers. Apart from being used as food, it has also been applied for its medicinal properties, mainly due to their biological activity and phenolic composition [5]. Herein, the phenolic compounds were analyzed by HPLC-DAD-ESI/MS and linear discriminant analysis (LDA) was performed to compare the results from flowers submitted to different irradiation doses and technologies (Co-60 and electron-beam). Quercetin-3-O-(6-O-rhamnosylglucoside)-7-O-rhamnoside (Figure 1) was the most abundant compound, followed by quercetin-3-O-rutinoside and acetyl-quercetin-3-O (6-O-rhamnosylglucoside)-7-O-rhamnoside. In general, irradiated samples (mostly with 1 kGy) showed the highest phenolic compounds content. The LDA outcomes indicated that differences among phenolic compounds effectively discriminate the assayed doses and technologies, defining which variables contributed mostly to that separation. This information might be useful to define which dose and/or technology optimizes the content in a specific phenolic compound. Overall, irradiation did not negatively affect the levels of phenolic compounds, providing the possibility of its application to expand the shelf life of V. tricolor and highlighting new commercial solutions for this functional food.

Changes in the annual harmful algal blooms of Alexandrium minutum : effects of environmental conditions and drainage basin inputs in the Rance estuary (Brittany, France)

Time series of physico-chemical data and concentrations (cell L-1) of the toxic dinoflagellate Alexandrium minutum collected in the Rance macrotidal estuary (Brittany, France) were analyzed to understand the physico-chemical processes of the estuary and their relation to changes in bloom development from 1996 to 2009. The construction of the tidal power plant in the north and the presence of a lock in the south have greatly altered hydrodynamics, blocking the zone of maximum turbidity upstream, in the narrowest part of the estuary. Alexandrium minutum occurs in the middle part of the estuary. Most physical and chemical parameters of the Rance estuary are similar to those observed elsewhere in Brittany with water temperatures between 15–18 °C, slightly lowered salinities (31.8–33.1 PSU), low river flow rates upstream and significant solar radiation (8 h day-1). A notable exception is phosphate input from the drainage basin which seems to limit bloom development: in recent years, bloom decline can be significantly correlated with the decrease in phosphate input. On the other hand, the chemical processes occurring in the freshwater-saltwater interface do not seem to have an influence on these occurrences. The other hypotheses for bloom declines are discussed, including the prevalence of parasitism, but remain to be verified in further studies.

Photo Degradation of Cotnaminants of Emerging concern (CECs) under Simulated Solar Radiation: Implications for their Environmental Fate

Contaminants of emerging concern (CECs) are continuously being released into the environment mainly because of their incomplete removal in the sewage treatment plants (STPs). The CECs selected for the study include antibiotics (macrolides, sulfonamides and ciprofloxacin), sucralose (an artificial sweetener) and dioctyl sulfosuccinate (DOSS, chemical dispersant used in the Deepwater Horizon oil spill). After being discharged into waterways from STPs, photo degradation is a key factor in dictating the environmental fate of antibiotics and sucralose. Photodegradation efficiency depends on many factors such as pH of the matrix, matrix composition, light source and structure of the molecule. These factors exert either synergistic or antagonistic effects in the environment and thus experiments with isolated factors may not yield the same results as the natural environmental processes. Hence in the current study photodegradation of 13 CECs (antibiotics, sucralose and dicotyl sulfosuccinate) were evaluated using natural water matrices with varying composition (deionized water, fresh water and salt water) as well as radiation of different wavelengths (254 nm, 350 nm and simulated solar radiation) in order to mimic natural processes. As expected the contribution of each factor on the overall rate of photodegradation is contaminant specific, for example under similar conditions, the rate in natural waters compared to pure water was enhanced for antibiotics (2-11 fold), significantly reduced for sucralose (no degradation seen in natural waters) and similar in both media for DOSS. In general, it was observed that the studied compounds degraded faster at 254 nm, while when using a simulated sunlight radiation the rate of photolysis of DOSS increased and the rates for antibiotics decreased in comparison to the 350 nm radiation. The photo stability of the studied CECs followed the order sucralose > DOSS > macrolides > sulfonamides > ciprofloxacin and a positive relationship was observed between photo stability and their ubiquitous presence in natural aquatic matrices. An online LC-MS/MS method was developed and validated for sucralose and further applied to reclaimed waters (n =56) and drinking waters (n = 43) from South Florida. Sucralose was detected in reclaimed waters with concentrations reaching up to 18 µg/L. High frequency of detection (> 80%) in drinking waters indicate contamination of ground waters in South Florida by anthropogenic activity.

Effects in ultrafast laser micromachining PMMA-based optical fibre grating

Ultrafast laser owns extreme small beam size and high pulse intensity which enable spatial localised modification either on the surface or in the bulk of materials. Therefore, ultrafast laser has been widely used to micromachine optical fibres to alter optical structures. In order to do the precise control of the micromachining process to achieve the desired structure and modification, investigations on laser parameters control should be carried out to make better understanding of the effects in the laser micromachining process. These responses are important to laser machining, most of which are usually unknown during the process. In this work, we report the real time monitored results of the reflection of PMMA based optical fibre Bragg gratings (POFBGs) during excimer ultraviolet laser micromachining process. Photochemical and thermal effects have been observed during the process. The UV radiation was absorbed by the PMMA material, which consequently induced the modifications in both spatial structure and material properties of the POFBG. The POFBG showed a significant wavelength blue shift during laser micromachining. Part of it attributed to UV absorption converted thermal energy whilst the other did not disappear after POFBG cooling off, which attributed to UV induced photodegradation in POF.

Infección por virus sincitial respiratorio y su relación con valores de IGG en niños críticos

Antecedente: La infección por el virus sincitial respiratorio (VSR) representa una elevada morbimortalidad, y en algunos casos necesidad de manejo en unidades de cuidado intensivo pediátrico (UCIP). La respuesta inmunológica influye de manera directa en la expresión de la severidad y pronóstico de los pacientes con infección respiratoria. Metodología: Estudio de una cohorte retrospectiva de pacientes con infección respiratoria grave secundaria a VSR, sin historia de inmunodeficiencia, atendidos en la UCIP del Hospital Universitario Clínica San Rafael. Se realizó análisis descriptivoglobaly de acuerdo a la categorización de las prueba de IgG. Resultados: De 188 pacientes que ingresaron a la UCIP, 13% presentaron infección por VSR (24), con una edad promedio de 7,3 (DE=3,6) meses. Pertenecían al sexo masculino79,83%. Se encontró que 12,5% tenían un valor de IgGbajo para su edad, 58,33% tenían valores en límite inferior y el 29,17% dentro de rangos normales para su edad. En los pacientes con IgG baja, fue mayor la presentación de choque séptico que no responde a líquidos (100 vs 92 vs 86%), la mediana de días de ventilación mecánica fue mayor (8 vs 6 vs 5 respectivamente), así como la mortalidad (67 vs 7,1 vs 0%). Conclusión: Nuestra serie encontró que aquellos pacientes con niveles bajos o valores en el límite inferior de IgG sérica tuvieron mayor compromiso sistémico, mayor duración de ventilación mecánica y mayor mortalidad. Se necesitan estudios prospectivos que relaciones niveles bajos de IgG con severidad y pronostico en estos pacientes con infección grave por VSR.

Solar and thermal radiative effects during the 2011 extreme desert dust episode over Portugal

This paper analyses the influence of the extreme Saharan desert dust (DD) event on shortwave (SW) and longwave (LW) radiation at the EARLINET/AERONET Évora station (Southern Portugal) from 4 up to 7 April 2011. There was also some cloud occurrence in the period. In this context, it is essential to quantify the effect of cloud presence on aerosol radiative forcing. A radiative transfer model was initialized with aerosol optical properties, cloud vertical properties and meteorological atmospheric vertical profiles. The intercomparison between the instantaneous TOA shortwave and longwave fluxes derived using CERES and those calculated using SBDART, which was fed with aerosol extinction coefficients derived from the CALIPSO and lidar-PAOLI observations, varying OPAC dataset parameters, was reasonably acceptable within the standard deviations. The dust aerosol type that yields the best fit was found to be the mineral accumulation mode. Therefore, SBDART model constrained with the CERES observations can be used to reliably determine aerosol radiative forcing and heating rates. Aerosol radiative forcings and heating rates were derived in the SW (ARFSw, AHRSw) and LW (ARFLw, AHRLw) spectral ranges, considering a cloud-aerosol free reference atmosphere. We found that AOD at 440 nm increased by a factor of 5 on 6 April with respect to the lower dust load on 4 April. It was responsible by a strong cooling radiative effect pointed out by the ARFSw value (−99 W/m2 for a solar zenith angle of 60°) offset by a warming radiative effect according to ARFLw value (+21.9 W/m2) at the surface. Overall, about 24% and 12% of the dust solar radiative cooling effect is compensated by its longwave warming effect at the surface and at the top of the atmosphere, respectively. Hence, larger aerosol loads could enhance the response between the absorption and re-emission processes increasing the ARFLw with respect to those associated with moderate and low aerosol loads. The unprecedented results derived from this work complement the findings in other regions on the modifications of radiative energy budget by the dust aerosols, which could have relevant influences on the regional climate and will be topics for future investigations.

In Situ Observation of the Thermal Decomposition of Weddelite by Heating Stage Environmental Scanning Electron Microscopy

The morphological and chemical changes occurring during the thermal decomposition of weddelite, CaC2O4·2H2O, have been followed in real time in a heating stage attached to an Environmental Scanning Electron Microscope operating at a pressure of 2 Torr, with a heating rate of 10 °C/min and an equilibration time of approximately 10 min. The dehydration step around 120 °C and the loss of CO around 425 °C do not involve changes in morphology, but changes in the composition were observed. The final reaction of CaCO3 to CaO while evolving CO2 around 600 °C involved the formation of chains of very small oxide particles pseudomorphic to the original oxalate crystals. The change in chemical composition could only be observed after cooling the sample to 350 °C because of the effects of thermal radiation.