Optimization of TRPV6 Calcium Channel Inhibitors Using a 3D Ligand-Based Virtual Screening Method

Herein, we report the discovery of the first potent and selective inhibitor of TRPV6, a calcium channel overexpressed in breast and prostate cancer, and its use to test the effect of blocking TRPV6-mediated Ca2+-influx on cell growth. The inhibitor was discovered through a computational method, xLOS, a 3D-shape and pharmacophore similarity algorithm, a type of ligand-based virtual screening (LBVS) method described briefly here. Starting with a single weakly active seed molecule, two successive rounds of LBVS followed by optimization by chemical synthesis led to a selective molecule with 0.3 μM inhibition of TRPV6. The ability of xLOS to identify different scaffolds early in LBVS was essential to success. The xLOS method may be generally useful to develop tool compounds for poorly characterized targets.

Optimization of a DNSH passive sampling method to measure airborne carbonyls

Various airborne aldehydes and ketones (i.e., airborne carbonyls) present in outdoor, indoor, and personal air pose a risk to human health at present environmental concentrations. To date, there is no adequate, simple-to-use sampler for monitoring carbonyls at parts per billion concentrations in personal air. The Passive Aldehydes and Ketones Sampler (PAKS) originally developed for this purpose has been found to be unreliable in a number of relatively recent field studies. The PAKS method uses dansylhydrazine, DNSH, as the derivatization agent to produce aldehyde derivatives that are analyzed by HPLC with fluorescence detection. The reasons for the poor performance of the PAKS are not known but it is hypothesized that the chemical derivatization conditions and reaction kinetics combined with a relatively low sampling rate may play a role. This study evaluated the effect of absorption and emission wavelengths, pH of the DNSH coating solution, extraction solvent, and time post-extraction for the yield and stability of formaldehyde, acetaldehyde, and acrolein DNSH derivatives. The results suggest that the optimum conditions for the analysis of DNSHydrazones are the following. The excitation and emission wavelengths for HPLC analysis should be at 250nm and 500nm, respectively. The optimal pH of the coating solution appears to be pH 2 because it improves the formation of di-derivatized acrolein DNSHydrazones without affecting the response of the derivatives of the formaldehyde and acetaldehyde derivatives. Acetonitrile is the preferable extraction solvent while the optimal time to analyze the aldehyde derivatives is 72 hours post-extraction. ^

An evaluation of the Cypress Creek watershed for inorganic chemical load as it flows into Lake Houston

Background. The Cypress Creek is one of the main tributaries of Lake Houston, which provides drinking water to 21.4 million customers. Furthermore, the watershed is being utilized for contact and non-contact recreation, such as canoeing, swimming, hiking trail, and picnics. Water along the creek is impacted by numerous wastewater outfalls from both point and non-point sources. As the creek flows into Lake Houston, it carries both organic and inorganic contaminants that may affect the drinking water quality of this important water source reservoir. Objective. This study was carried out to evaluate the inorganic chemical load of the water in Cypress Creek along its entire length, from the headwaters in Waller County and up to the drainage into Lake Houston. The purpose was to determine whether there are hazardous concentrations of metals in the water and what would be the likely sources. Method. Samples were collected at 29 sites along the creek and analyzed for 29 metals, 17 of which were on the Environmental Protection Agency priority pollution list. Public access sites primarily at bridges were used for sample collection. Samples were transported on ice to the University Of Texas School Of Public Health laboratory, spiked with 2 ml HNO3 kept overnight in the refrigerator, and the following day transported to the EPA laboratory for analysis. Analysis was done by EPA Method 200.7-ICP, Method 200.8ICP/MS and Method 245.1-CVAAS. Results. Metals were present above the detection limits at 65% of sites. Concentrations of aluminum, iron, sodium, potassium, magnesium, and calcium, were particularly high at all sites. Aluminum, sodium, and iron concentrations greatly exceeded the EPA secondary drinking water standards at all sites. ^ Conclusion. The recreational water along Cypress Creek is impacted by wastewater from both permitted and non-permitted outfalls, which deposit inorganic substances into the water. Although a number of inorganic contaminants were present in the water, toxic metals regulated by the EPA were mostly below the recommended limits. However, high concentrations of aluminum, sodium, and iron in the Cypress Creek bring forward the issue of unauthorized discharges of salt water from mining, as well as industrial and domestic wastewater.^

Characterization of the East Houston particulate matter <2.5 atmosphere; a novel method for desorbing organic constituents from airborne particulates

I have developed a novel approach to test for toxic organic substances adsorbed onto ultra fine particulate particles present in the ambient air in Northeast Houston, Texas. These particles are predominantly carbon soot with an aerodynamic diameter (AD) of <2.5 μm. If present in the ambient air, many of the organic substances will be absorbed to the surface of the particles (which act just like a charcoal air filter), and may be adducted into the respiratory system. Once imbedded into the lungs these particles may release the adsorbed toxic organic substances with serious health consequences. I used a Airmetrics portable Minivol air sampler time drawing the ambient air through collection filters samples from 6 separate sites in Northeast Houston, an area known for high ambient PM 2.5 released from chemical plants and other sources (e.g. vehicle emissions).(1) In practice, the mass of the collected particles were much less than the mass of the filters. My technique was designed to release the adsorbed organic substances on the fine carbon particles by heating the filter samples that included the PM 2.5 particles prior to identification by gas chromatography/mass spectrometry (GCMS). The results showed negligible amounts of target chemicals from the collection filters. However, the filters alone released organic substances and GCMS could not distinguish between the organic substances released from the soot particles from those released from the heated filter fabric. However, an efficacy tests of my method using two wax burning candles that released soot revealed high levels of benzene. This suggests that my method has the potential to reveal the organic substances adsorbed onto the PM 2.5 for analysis. In order to achieve this goal, I must refine the particle collection process which would be independent of the filters; the filters upon heating also release organic substances obscuring the contribution from the soot particles. To obtain pure soot particles I will have to filter more air so that the soot particles can be shaken off the filters and then analyzed by my new technique. ^

Use of short term test systems for the prediction of the hazard represented by potential chemical carcinogens

It has been hypothesized that results from the short term bioassays will ultimately provide information that will be useful for human health hazard assessment. Although toxicologic test systems have become increasingly refined, to date, no investigator has been able to provide qualitative or quantitative methods which would support the use of short term tests in this capacity.^ Historically, the validity of the short term tests have been assessed using the framework of the epidemiologic/medical screens. In this context, the results of the carcinogen (long term) bioassay is generally used as the standard. However, this approach is widely recognized as being biased and, because it employs qualitative data, cannot be used in the setting of priorities. In contrast, the goal of this research was to address the problem of evaluating the utility of the short term tests for hazard assessment using an alternative method of investigation.^ Chemical carcinogens were selected from the list of carcinogens published by the International Agency for Research on Carcinogens (IARC). Tumorigenicity and mutagenicity data on fifty-two chemicals were obtained from the Registry of Toxic Effects of Chemical Substances (RTECS) and were analyzed using a relative potency approach. The relative potency framework allows for the standardization of data "relative" to a reference compound. To avoid any bias associated with the choice of the reference compound, fourteen different compounds were used.^ The data were evaluated in a format which allowed for a comparison of the ranking of the mutagenic relative potencies of the compounds (as estimated using short term data) vs. the ranking of the tumorigenic relative potencies (as estimated from the chronic bioassays). The results were statistically significant (p $<$.05) for data standardized to thirteen of the fourteen reference compounds. Although this was a preliminary investigation, it offers evidence that the short term test systems may be of utility in ranking the hazards represented by chemicals which may be human carcinogens. ^

Chemical analysis and argon geochronology from dive SAR_1 on Cornacya seamount of the Sardinia Channel

Sarcya 1 dive explored a previously unknown 12 My old submerged volcano, labelled Cornacya. A well developed fracturation is characterised by the following directions: N 170 to N-S, N 20 to N 40, N 90 to N 120, N 50 to N 70, which corresponds to the fracturation pattern of the Sardinian margin. The sampled lavas exhibit features of shoshonitic suites of intermediate composition and include amphibole-and mica-bearing lamprophyric xenoliths which are geochemically similar to Ti-poor lamproites. Mica compositions reflect chemical exchanges between the lamprophyre and its shoshonitic host rock suggesting their simultaneous emplacement. Nd compositions of the Cornacya K-rich suite indicate that continental crust was largely involved in the genesis of these rocks. The spatial association of the lamprophyre with the shoshonitic rocks is geochemically similar to K-rich and TiO2-poor igneous suites, emplaced in post-collisional settings. Among shoshonitic rocks, sample SAR 1-01 has been dated at 12.6±0.3 My using the 40Ar/39Ar method with a laser microprobe on single grains. The age of the Cornacya shoshonitic suite is similar to that of the Sisco lamprophyre from Corsica, which similarly is located on the western margin of the Tyrrhenian Sea. Thus, the Cornacya shoshonitic rocks and their lamprophyric xenolith and the Sisco lamprophyre could represent post-collisional suites emplaced during the lithospheric extension of the Corsica-Sardinia block, just after its rotation and before the Tyrrhenian sea opening. Drilling on the Sardinia margin (ODP Leg 107) shows that the upper levels of the present day margin (Hole 654) suffered tectonic subsidence before the lower part (Hole 652). The structure of this lower part is interpreted as the result of an eastward migration of the extension during Late Miocene and Early Pliocene times. Data of Cornacya volcano are in good agreement with this model and provide good chronological constraints for the beginning of the phenomenon.

Chemical characterization and quantification of the brown algal storage compound laminarin

In search of a meaningful stress indicator for Fucus vesiculosus we found that the often used quantitative determination procedures for the polysaccharide laminarin (beta-1,3-glucan) result in different kind of problems, uncertainties and limitations. This chemical long-term storage form of carbon enables perennial brown algae in seasonally fluctuating ecosystems to uncouple growth from photosynthesis. Because of this high ecological relevance a reliable and precise method for determination and quantification of laminarin is needed. Therefore, a simple, cold water extraction method coupled to a new quantitative liquid chromatography-mass spectrometrical method (LC-MS) was developed. Laminarin was determined in nine out of twelve brown algal species, and its expected typical molar mass distribution of 2000-7000 Da was confirmed. Furthermore, laminarin consisted of a complex mixture of different chemical forms, since fifteen chemical laminarin species with distinct molecular weights were measured in nine species of brown algae. Laminarin concentrations in the algal tissues ranged from 0.03 to 0.86% dry weight (DW). The direct chemical characterization and quantification of laminarin by LC-MS represents a powerful method to verify the biochemical and ecological importance of laminarin for brown algae. Single individuals of Laminaria hyperborea, L. digitata, Saccharina latissima, F. serratus, F. vesiculosus, F. spiralis, Himanthalia elongata, Cystoseira tamariscifolia, Pelvetia canaliculata, Ascophyllum nodosum, Halidrys siliquosa and Dictyota dichotoma were collected in fall (18.11.2013) during spring low tide from the shore of Finavarra, Co. Clare, west coast of Ireland (53° 09' 25'' N, 09° 06' 58'' W). After sampling, the different algae were immediately transported to the lab, lyophilized and sent to the University of Rostock. Laminarin was extracted with cold ultrapure water from the algal samples. Before extraction they were ground to < 1 mm grain size with an analytical mill (Ika MF 10 Basic). The algal material (approx. 1.5 g DW) was extracted in ultrapure water (8 mL) on a shaker (250 rpm) for 5 h. After the addition of surplus ultrapure water (4 mL) and shaking manually, 1 mL of the sample was filter centrifuged (45 µm) at 14,000 rpm (Hettich Mikro 22 R). The slightly viscous supernatant was free of suspended material and converted into a microvial (300 µL) for further analysis. The extracts were analyzed using liquid chromatography-mass spectrometry (LC-MS) analysis (LTQ Velos Pro ion trap spectrometer with Accela HPLC, Thermo Scientific). Laminarin species were separated on a KinetexTM column (2.6 µm C18, 150 x 3 mm). The mobile phase was 90 % ultrapure water and 10 % acetonitrile, run isocratically at a flow rate of 0.2 mL min-1. MS was working in ESI negative ion mode in a mass range of 100 - 4000 amu. Glucose contents were determined after extraction using high-performance liquid chromatography (HPLC). Extracted samples were analyzed in an HPLC (SmartLine, Knauer GmbH) equipped with a SUPELCOGELTM Ca column (30 x 7,8 mm without preColumn) and RI-detector (S2300 PDA S2800). Water was used as eluent at a flow rate of 0.8 mL min-1 at 75 °C. Glucose was quantified by comparison of the retention time and peak area with standard solutions using ChromGate software. Mannitol was extracted from three subsamples of 10-20 mg powdered alga material (L. hyperborea, L. digitata, S. latissima, F. serratus, F. vesiculosus, F. spiralis, H. elongata, P. canaliculata, A. nodosum, H. siliquosa) and quantified, following the HPLC method described by Karsten et al. (1991). For analyzing carbon and nitrogen contents, dried algal material was ground to powder and three subsamples of 2 mg from each alga thalli were loaded and packed into tin cartridges (6×6×12 mm). The packages were combusted at 950 °C and the absolute contents of C and N were automatically quantified in an elemental analyzer (Elementar Vario EL III, Germany) using acetanilide as standard according to Verardo et al. (1990).

Ion concentration and deduced chemical compounds of the GRIP and Dome Fuji ice cores, Greenland and Antarctica

We have proposed a method of deducing the chemical compounds found in deep polar ice cores by analyzing the balance between six major ions (Cl-, NO3-, SO4**2-, Na+, Mg2+, and Ca2+). The method is demonstrated for the Holocene and last glacial maximum regions of the Dome Fuji and GRIP ice cores. The dominant compounds depend only on the ion balance and the sequence of chemical reactions. In priority order, the principle salts are calcium sulfate, other sulfates, nitrate, chloride, and carbonate. The chemical abundances deduced by this method agree well with the results of Raman spectroscopy on individual salt inclusions. The abundances in the ice cores are shown to reflect differences in climatic periods (the acidic environment of the Holocene versus the reductive environment of the last glacial maximum) and regional conditions (the marine environment of Antarctica versus the continental environment of Greenland).

Chemical composition, characteristics and age of glauconites at DSDP Leg 66 Holes

During the analysis of "glaucony" recovered during Leg 66, off Mexico, we reviewed the data on previously studied glaucony layers in active margin areas. We found the depth of Leg 66 glaucony sediments to be significantly greater than conventionally assumed appropriate to their genesis (100-500 m). Accordingly, we hypothesize their occurrence at unusual depth to be due to (1) transport of shallow sediments and redeposition at greater depths, (2) margin subsidence, or (3) genesis at greater depth than is generally assumed. For the area off Mexico, we reject (1). (2) has already been verified in Japan and is possible as an explanation for the present phenomenon without excluding (3), which we investigate in this chapter.

Chemical composition of Ninetyeast Ridge basalts

On Leg 121 of the Ocean Drilling Program, we recovered basaltic rocks from a total of three basement sites in the southern, central, and northern regions of Ninetyeast Ridge. These new sites complement the previous four basement holes drilled during Legs 22 and 26 of the Deep Sea Drilling Project, and confirm the predominantly tholeiitic, light rare earth element-enriched character of the basalts that cap the ridge. The basalts show marked iron enrichment; ferrobasalts occur at Sites 214 and 216 and oceanic andesites at Site 253. All of the basalts recovered during Leg 121 are altered, and range from aphyric olivine tholeiites (Site 756), to strongly plagioclase-phyric basalts (Site 757). Basalts from Site 758, which were clearly erupted in a submarine environment (pillow basalts are present in the section), are sparsely to strongly plagioclase-phyric. The basalts recovered at any one hole are isotopically homogeneous (except for the basalts from Site 758, which show a range of Pb isotopes), and it is possible to relate the magmas at any one site by high-level fractionation processes. However, there are significant variations in isotope ratios and highly incompatible element ratios between sites, which suggest that the mantle source for the ridge basalts was compositionally variable. Such variation, in view of the large volume of magmatic products that form the ridge system, is not surprising. There is not, however, a systematic variation in basalt composition along the ridge. We agree with previous models that relate Ninetyeast Ridge to a mantle plume in the southern Indian Ocean. The tholeiitic, iron-enriched, and voluminous character of the ridge basalts is typical of oceanic islands associated with plumes on or near a mid-ocean ridge (e.g., Iceland, Galapagos Islands, and St. Paul/Amsterdam islands). The absence of recovered alkalic suites is inconsistent with an intraplate setting, such as the Hawaiian Islands or Kerguelen Island. Thus, the major element data, like the gravity data, strongly suggest that the ridge was erupted on or very close to an active spreading center. Isotopically, the most likely plume that created the excess magmatism on the Ridge is the Kerguelen-Heard plume system, but the Ninetyeast Ridge basalts do not represent a simple mixing of the Kerguelen plume and mid-ocean Ninetyeast Ridge basalt mantle.

Chemical composition of phillipsites from Core AKO26-35

Behavior of rare earth elements (REE) was examined in oceanic phillipsites collected from four horizons of eupelagic clay in the Southwest Basin of the Pacific Ocean. REE concentrations were determined in >50 ?m size fraction phillipsite samples by the ICP-MS method. Composition of separate phillipsite aggregates was studied by electron microprobe and secondary ion mass-spectrometry. Rare earth elements in phillipsite samples are related to admixture of ferrocalcium hydroxophosphates. Analysis of separate phillipsite aggregates reveals low (<0.1-18.1 ppm) REE(III) concentrations. Ce concentration varies between 2.7 and 140 ppm. The correlation analysis shows that REE(III) present in admixture of iron oxyhydroxides in separate phillipsite aggregates. Based on the REE(III) concentration in iron oxyhydroxides we can identify two generations of phillipsite aggregates. Massive rounded aggregates (phillipsite I) are depleted in REE, while pseudorhombic (phillipsite II) aggregates are enriched in REE and marked by a positive Ce anomaly. Oceanic phillipsites do not accumulate REE or inherit the REE signature of volcaniclastic material and oceanic deep water. Hence, REE distribution in phillipsites does not depend on sedimentation rate and composition of host sediments.