Stabilization of troponin I for applications in clinical chemistry and forensic medicine. Cardiac troponin I: A time of death marker

Cardiac troponin I (cTnI) is one of the most useful serum marker test for the determination of myocardial infarction (MI). The first commercial assay of cTnI was released for medical use in the United States and Europe in 1995. It is useful in determining if the source of chest pains, whose etiology may be unknown, is cardiac related. Cardiac TnI is released into the bloodstream following myocardial necrosis (cardiac cell death) as a result of an infarct (heart attack). In this research project the utility of cardiac troponin I as a potential marker for the determination of time of death is investigated. The approach of this research is not to investigate cTnI degradation in serum/plasma, but to investigate the proteolytic breakdown of this protein in heart tissue postmortem. If our hypothesis is correct, cTnI might show a distinctive temporal degradation profile after death. This temporal profile may have potential as a time of death marker in forensic medicine. The field of time of death markers has lagged behind the great advances in technology since the late 1850's. Today medical examiners are using rudimentary time of death markers that offer limited reliability in the medico-legal arena. Cardiac TnI must be stabilized in order to avoid further degradation by proteases in the extraction process. Chemically derivatized magnetic microparticles were covalently linked to anti-cTnI monoclonal antibodies. A charge capture approach was also used to eliminate the antibody from the magnetic microparticles given the negative charge on the microparticles. The magnetic microparticles were used to extract cTnI from heart tissue homogenate for further bio-analysis. Cardiac TnI was eluted from the beads with a buffer and analyzed. This technique exploits banding pattern on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a western blot transfer to polyvinylidene fluoride (PVDF) paper for probing with anti-cTnI monoclonal antibodies. Bovine hearts were used as a model to establish the relationship of time of death and concentration/band-pattern given its homology to human cardiac TnI. The final concept feasibility was tested with human heart samples from cadavers with known time of death. ^

Synthesis of multisubstituted halo -olefins via Pd-catalyzed cross -coupling reactions. Applications in nucleoside chemistry

The enzyme S-adenosyl-L-homocysteine (AdoHcy) hydrolase effects hydrolytic cleavage of AdoHcy to adenosine (Ado) and L-homocysteine (Hcy). The cellular levels of AdoHcy and Hcy are critical because AdoHcy is a potent feedback inhibitor of crucial transmethylation enzymes. Also, elevated plasma levels of Hcy in humans have been shown to be a risk factor in coronary artery disease. ^ On the basis of the previous finding that AdoHcy hydrolase is able to add the enzyme-sequestered water molecule across the 5',6'-double bond of (halo or dihalohomovinyl)-adenosines causing covalent binding inhibition, we designed and synthesized AdoHcy analogues with the 5',6'-olefin motif incorporated in place of the carbon-5' and sulfur atoms. From the available synthetic methods we chose two independent approaches: the first approach was based on the construction of a new C5'-C6' double bond via metathesis reactions, and the second approach was based on the formation of a new C6'-C7' single bond via Pd-catalyzed cross-couplings. Cross-metathesis of the suitably protected 5'-deoxy-5'-methyleneadenosine with racemic 2-amino-5-hexenoate in the presence of Hoveyda-Grubb's catalyst followed by standard deprotection afforded the desired analogue as 5' E isomer of the inseparable mixture of 9'R/S diastereomers. Metathesis of chiral homoallylglycine [(2S)-amino-5-hexenoate] produced AdoHcy analogue with established stereochemistry E at C5'atom and S at C9' atom. The 5'-bromovinyl analogue was synthesized using the bromination-dehydrobromination strategy with pyridinium tribromide and DBU. ^ Since literature reports on the Pd-catalyzed monoalkylation of dihaloalkenes (Csp2-Csp3 coupling) were scarce, we were prompted to undertake model studies on Pd-catalyzed coupling between vinyl dihalides and alkyl organometallics. The 1-fluoro-1-haloalkenes were found to undergo Negishi couplings with alkylzinc bromides to give multisubstituted fluoroalkenes. The alkylation was trans-selective affording pure Z-fluoroalkenes. The highest yields were obtained with PdCl 2(dppb) catalyst, but the best stereochemical outcome was obtained with less reactive Pd(PPh3)4. Couplings of 1,1-dichloro-and 1,1-dibromoalkenes with organozinc reagents resulted in the formation of monocoupled 1-halovinyl product. ^

Synthesis of multisubstituted halo-olefins via Pd-catalyzed cross-coupling reactions : applications in nucleoside chemistry

The enzyme S-adenosyl-L-homocysteine (AdoHey) hydrolase effects hydrolytic cleavage of AdoHcy to adenosine (Ado) and L-homocysteine (Hcy). The cellular levels of AdoHcy and Hcy are critical because AdoHcy is a potent feedback inhibitor of crucial transmethylation enzymes. Also, elevated plasma levels of Hcy in humans have been shown to be a risk factor in coronary artery disease. On the basis of the previous finding that AdoHcy hydrolase is able to add the enzyme-sequestered water molecule across the 5',6'-double bond of (halo or dihalohomovinyl)-adenosines causing covalent binding inhibition, we designed and synthesized AdoHcy analogues with the 5',6'-olefin motif incorporated in place of the carbon-5' and sulfur atoms. From the available synthetic methods we chose two independent approaches: the first approach was based on the construction of a new C5'- C6' double bond via metathesis reactions, and the second approach was based on the formation of a new C6'-C7' single bond via Pd-catalyzed cross-couplings. Cross-metathesis of the suitably protected 5'-deoxy-5'-methyleneadenosine with racemic 2-amino-5-hexenoate in the presence of Hoveyda-Grubb's catalyst followed by standard deprotection afforded the desired analogue as 5'E isomer of the inseparable mixture of 9'RIS diastereomers. Metathesis of chiral homoallylglycine [(2S)-amino-5-hexenoate] produced AdoHcy analogue with established stereochemistry E at C5'atom and S at C9' atom. The 5'-bromovinyl analogue was synthesized using the brominationdehydrobromination strategy with pyridinium tribromide and DBU. Since literature reports on the Pd-catalyzed monoalkylation of dihaloalkenes (Csp2-Csp3 coupling) were scarce, we were prompted to undertake model studies on Pdcatalyzed coupling between vinyl dihalides and alkyl organometallics. The 1-fluoro-1- haloalkenes were found to undergo Negishi couplings with alkylzinc bromides to give multisubstituted fluoroalkenes. The alkylation was trans-selective affording pure Zfluoroalkenes. The highest yields were obtained with PdCl 2(dppb) catalyst, but the best stereochemical outcome was obtained with less reactive Pd(PPh3)4 . Couplings of 1,1- dichloro-and 1,1-dibromoalkenes with organozinc reagents resulted in the formation of monocoupled 1-halovinyl product.

Discharge measurements and storm event chemistry at the San Francisco River station (Planta) in 2007-2008

To improve our knowledge of the influence of land-use on solute behaviour and export rates in neotropical montane catchments we investigated total organic carbon (TOC), Ca, Mg, Na, K, NO3 and SO4 concentrations during April 2007-May 2008 at different flow conditions and over time in six forested and pasture-dominated headwaters (0.7-76 km2) in Ecuador. NO3 and SO4 concentrations decreased during the study period, with a continual decrease in NO3 and an abrupt decrease in February 2008 for SO4. We attribute this to changing weather regimes connected to a weakening La Niña event. Stream Na concentration decreased in all catchments, and Mg and Ca concentration decreased in all but the forested catchments during storm flow. Under all land-uses TOC increased at high flows. The differences in solute behaviour during storm flow might be attributed to largely shallow subsurface and surface flow paths in pasture streams on the one hand, and a predominant origin of storm flow from the organic layer in the forested streams on the other hand. Nutrient export rates in the forested streams were comparable to the values found in literature for tropical streams. They amounted to 6-8 kg/ha/y for Ca, 7-8 kg/ha/y for K, 4-5 kg/ha/y for Mg, 11-14 kg/ha/y for Na, 19-22 kg/ha/y for NO3 (i.e. 4.3-5.0 kg/ha/y NO3-N) and 17 kg/ha/y for SO4. Our data contradict the assumption that nutrient export increases with the loss of forest cover. For NO3 we observed a positive correlation of export value and percentage forest cover.

Seawater carbonate chemistry and biometry and dissolution features of the benthic foraminifer Ammonia aomoriensis in a laboratory experiment

Culturing experiments were performed with the benthic foraminifer Ammonia aomoriensis from Flensburg Fjord, western Baltic Sea. The experiments simulated a projected rise in atmospheric CO2 concentrations. We exposed specimens to 5 seawater pCO2 levels ranging from 618 µatm (pH 7.9) to 3130 µatm (pH 7.2) for 6 wk. Growth rates and mortality differed significantly among pCO2 treatments. The highest increase of mean test diameter (19%) was observed at 618 µatm. At partial pressures >1829 µatm, the mean test diameter was observed to decrease, by up to 22% at 3130 µatm. At pCO2 levels of 618 and 751 µatm, A. aomoriensis tests were found intact after the experiment. The outer chambers of specimens incubated at 929 and 1829 µatm were severely damaged by corrosion. Visual inspection of specimens incubated at 3130 µatm revealed wall dissolution of all outer chambers, only their inner organic lining stayed intact. Our results demonstrate that pCO2 values of >=929 µatm in Baltic Sea waters cause reduced growth of A. aomoriensis and lead to shell dissolution. The bottom waters in Flensburg Fjord and adjacent areas regularly experience pCO2 levels in this range during summer and fall. Increasing atmospheric CO2 concentrations are likely to extend and intensify these periods of undersaturation. This may eventually slow down calcification in A. aomoriensis to the extent that net carbonate precipitation terminates. The possible disappearance of this species from the Baltic Sea and other areas prone to seasonal undersaturation would likely cause significant shifts in shallow-water benthic ecosystems in the near future.

Seawater carbonate chemistry and growth rate of Emiliania huxleyi in lab experiment

Predicting the impacts of environmental change on marine organisms, food webs, and biogeochemical cycles presently relies almost exclusively on short-term physiological studies, while the possibility of adaptive evolution is often ignored. Here, we assess adaptive evolution in the coccolithophore Emiliania huxleyi, a well-established model species in biological oceanography, in response to ocean acidification. We previously demonstrated that this globally important marine phytoplankton species adapts within 500 generations to elevated CO2. After 750 and 1000 generations, no further fitness increase occurred, and we observed phenotypic convergence between replicate populations. We then exposed adapted populations to two novel environments to investigate whether or not the underlying basis for high CO2-adaptation involves functional genetic divergence, assuming that different novel mutations become apparent via divergent pleiotropic effects. The novel environment "high light" did not reveal such genetic divergence whereas growth in a low-salinity environment revealed strong pleiotropic effects in high CO2 adapted populations, indicating divergent genetic bases for adaptation to high CO2. This suggests that pleiotropy plays an important role in adaptation of natural E. huxleyi populations to ocean acidification. Our study highlights the potential mutual benefits for oceanography and evolutionary biology of using ecologically important marine phytoplankton for microbial evolution experiments.

Seawater carbonate chemistry and skeletal development, size, weight, total lipid and symbiont density of coral Favia fragum in a laboratory experiment

Ocean acidification (OA) threatens the existence of coral reefs by slowing the rate of calcium carbonate (CaCO3) production of framework-building corals thus reducing the amount of CaCO3 the reef can produce to counteract natural dissolution. Some evidence exists to suggest that elevated levels of dissolved inorganic nutrients can reduce the impact of OA on coral calcification. Here, we investigated the potential for enhanced energetic status of juvenile corals, achieved via heterotrophic feeding, to modulate the negative impact of OA on calcification. Larvae of the common Atlantic golf ball coral, Favia fragum, were collected and reared for 3 weeks under ambient (421 µatm) or significantly elevated (1,311 µatm) CO2 conditions. The metamorphosed, zooxanthellate spat were either fed brine shrimp (i.e., received nutrition from photosynthesis plus heterotrophy) or not fed (i.e., primarily autotrophic). Regardless of CO2 condition, the skeletons of fed corals exhibited accelerated development of septal cycles and were larger than those of unfed corals. At each CO2 level, fed corals accreted more CaCO3 than unfed corals, and fed corals reared under 1,311 µatm CO2 accreted as much CaCO3 as unfed corals reared under ambient CO2. However, feeding did not alter the sensitivity of calcification to increased CO2; Delta calcification/Delta Omega was comparable for fed and unfed corals. Our results suggest that calcification rates of nutritionally replete juvenile corals will decline as OA intensifies over the course of this century. Critically, however, such corals could maintain higher rates of skeletal growth and CaCO3 production under OA than those in nutritionally limited environments.

Mineralogy, mineral chemistry, and genesis of precious metal-bearing volcanogenic massive sulfide deposits in the Newfoundland Appalachians, Canada: the Ming deposit as example

The Ming deposit, Newfoundland Appalachians, is a metamorphosed (upper greenschist to lower amphibolite facies), Cambro-Ordovician, bimodalmafic volcanogenic massive sulfide (VMS) deposit that consists of several, spatially-associated, elongated orebodies composed of stratabound semimassive to massive sulfides and/or discordant sulfide stringers in a rhyodacitic footwall. Copper is the main commodity; however, the deposit contains precious metal-bearing zones with elevated Au grades. In this study, field observations, microscopy, and micro-analytical tools including electron microprobe, laser ablation inductively coupled plasma mass spectrometry, and secondary ion mass spectrometry were used to constrain the relative timing of precious metal emplacement, the physico-chemical conditions of hydrothermal fluid precipitation, and the sources of sulfur, precious metals, semi-metals and metals. The ore mineral assemblage is complex and indicates an intermediate sulfidation state. Pyrite and chalcopyrite are the dominant ore minerals with minor sphalerite and pyrrhotite, and trace galena, arsenopyrite and cubanite. Additional trace phases include tellurides, NiSb phases, sulfosalts, electrum, AgHg±Au alloys, and oxides. Silver phases and precious metals occur predominantly in semi-massive and massive sulfides as free grains, and as grains spatially associated with arsenopyrite and/or sulfosalts. Precious metal phases occurring between recrystallized pyrite and within cataclastic pyrite are rare. Hence, the complex ore assemblage and textures strongly suggest syngenetic precious metal emplacement, whereas metamorphism and deformation only internally and locally remobilized precious metal phases. The ore assemblage formed from reduced, acidic hydrothermal fluids over a range of temperatures (≈350 to below 260ºC). The abundance of telluride and Ag-bearing tetrahedrite, however, varies strongly between the different orebodies indicating variable ƒTe₂, ƒSe₂, mBi, and mSb within the hydrothermal fluids. The variations in the concentrations of semi-metals and metals (As, Bi, Hg, Sb, Se, Te), as well as Au and Ag, were due to variations in temperature but also to a likely contribution of magmatic fluids into the VMS hydrothermal system from presumably different geothermal reservoirs. Sulfur isotope studies indicate at least two sulfur sources: sulfur from thermochemically-reduced seawater sulfate and igneous sulfur. The source of igneous sulfur is the igneous footwall, direct magmatic fluid/volatiles, or both. Upper greenschist to lower amphibolite metamorphic conditions and deformation had no significant effect on the sulfur isotope composition of the sulfides at the Ming deposit.

Academic Experience and Personal Abilities in Analytical Chemistry Education

Report published in the Proceedings of the National Conference on "Education and Research in the Information Society", Plovdiv, May, 2016

Time series of air chemistry measurements at Georg-von-Neumayer station, Dronning Maud Land, Antarctica in the years 1982 to 1991

The first Air Chemistry Observatory at the German Antarctic station Georg von Neumayer (GvN) was operated for 10 years from 1982 to 1991. The focus of the established observational programme was on characterizing the physical properties and chemical composition of the aerosol, as well as on monitoring the changing trace gas composition of the background atmosphere, especially concerning greenhouse gases. The observatory was designed by the Institut für Umweltphysik, University of Heidelberg (UHEIIUP). The experiments were installed inside the bivouac lodge, mounted on a sledge and put upon a snow hill to prevent snow accumulation during blizzards. All experiments were under daily control and daily performance protocols were documented. A ventilated stainless steel inlet stack (total height about 3-4 m above the snow surface) with a 50% aerodynamic cut-off diameter around 7-10 µm at wind velocities between 4-10 m/s supplied all experiments with ambient air. Contamination free sampling was realized by several means: (i) The Air Chemistry Observatory was situated in a clean air area about 1500 m south of GvN. Due to the fact that northern wind directions are very rare, contamination from the base can be excluded for most of the time. (ii) The power supply (20 kW) is provided by a cable from the main station, thus no fuel-driven generator is operated in the very vicinity. (iii) Contamination-free sampling is controlled by the permanently recorded wind velocity, wind direction and by condensation particle concentration. Contamination was indicated if one of the following criteria were given: Wind direction within a 330°-30° sector, wind velocity <2.2 m/s or >17.5 m/s, or condensation particle concentrations >2500/cm**3 during summer, >800/cm**3 during spring/autumn and >400/cm**3 during winter. If one or a definable combination of these criteria were given, high volume aerosol sampling and part of the trace gas sampling were interrupted. Starting at 1982 through 1991-01-14 surface ozone was measured with an electrochemical concentration cell (ECC). Surface ozone mixing ratio are given in ppbv = parts per 10**9 by volume. The averaging time corresponds to the given time intervals in the data sheet. The accuracy of the values are better than ±1 ppbv and the detection limit is around 1.0 ppbv. Aerosols were sampled on two Whatman 541 cellulose filters in series and analyzed by ion chromatography at the UHEI-IUP. Generally, the sampling period was seven days but could be up to two weeks on occasion. The air flow was around 100 m**3/h and typically 10000-20000 m**3 of ambient air was forced through the filters for one sample. Concentration values are given in nanogram (ng) per 1 m**3 air at standard pressure and temperature (1013 mbar, 273.16 K). Uncertainties of the values were approximately ±10% to ±15% for the main components MSA, chloride, nitrate, sulfate and sodium, and between ±20% and ±30% for the minor species bromide, ammonium, potassium, magnesium and calcium.

Seawater carbonate chemistry and calcification in the tropical urchin Echinometra viridis in a laboratory experiment

Atmospheric carbon dioxide (pCO2) has risen from approximately 280 to 400 ppm since the Industrial Revolution, due mainly to the combustion of fossil fuels, deforestation, and cement production. It is predicted to reach as high as 900 ppm by the end of this century. Ocean acidification resulting from the release of anthropogenic CO2 has been shown to impair the ability of some marine calcifiers to build their shells and skeletons. Here, we present the results of ocean acidification experiments designed to assess the effects of an increase in atmospheric pCO2 from ca. 448 to 827 ppm on calcification rates of the tropical urchin Echinometra viridis. Experiments were conducted under the urchin's winter (20 °C) and summer (30 °C) water temperatures in order to identify seasonal differences in the urchin's response to ocean acidification. The experiments reveal that calcification rates decreased for urchins reared under elevated pCO2, with the decline being more pronounced under wintertime temperatures than under summertime temperatures. These results indicate that the urchin E. viridis will be negatively impacted by CO2-induced ocean acidification that is predicted to occur by the end of this century. These results also suggest that impact of CO2-induced ocean acidification on urchin calcification will be more severe in the winter and in cooler waters.

Seawater carbonate chemistry and growth, carbon acquisition, and species interaction of Antarctic phytoplankton species in a laboratory experiment

Despite the fact that ocean acidification is considered to be especially pronounced in the Southern Ocean, little is known about CO2-dependent physiological processes and the interactions of Antarctic phytoplankton key species. We therefore studied the effects of CO2 partial pressure (PCO2) (16.2, 39.5, and 101.3 Pa) on growth and photosynthetic carbon acquisition in the bloom-forming species Chaetoceros debilis, Pseudo-nitzschia subcurvata, Fragilariopsis kerguelensis, and Phaeocystis antarctica. Using membrane-inlet mass spectrometry, photosynthetic O2 evolution and inorganic carbon (Ci) fluxes were determined as a function of CO2 concentration. Only the growth of C. debilis was enhanced under high PCO2. Analysis of the carbon concentrating mechanism (CCM) revealed the operation of very efficient CCMs (i.e., high Ci affinities) in all species, but there were species-specific differences in CO2-dependent regulation of individual CCM components (i.e., CO2 and uptake kinetics, carbonic anhydrase activities). Gross CO2 uptake rates appear to increase with the cell surface area to volume ratios. Species competition experiments with C. debilis and P. subcurvata under different PCO2 levels confirmed the CO2-stimulated growth of C. debilis observed in monospecific incubations, also in the presence of P. subcurvata. Independent of PCO2, high initial cell abundances of P. subcurvata led to reduced growth rates of C. debilis. For a better understanding of future changes in phytoplankton communities, CO2-sensitive physiological processes need to be identified, but also species interactions must be taken into account because their interplay determines the success of a species.