Development and application of an analytical strategy for the determination of ²³²Th and ²²⁸Th in ivory based on the combined use of inductively coupled plasma mass spectrometry and α-spectrometry

In this work, the significance of the determination of the ratio of the activity concentrations of 228Th and 232Th in the context of age determination of ivory was discussed and the corresponding dating uncertainty was reduced. Calibration curves for the relevant isotopes for ivory dating, namely 14C, 90Sr and 228Th/232Th, were created by analyzing young ivory samples with known age. It was shown that the age determination of ivory via the thorium isotopes will become an indispensable and powerful technique in the future. The applicability of the bomb curve dating via the isotopes 14C and 90Sr will fade, due to ambitious findings for ivory, which will be poached within the future. Since the ratio 228Th/232Th is not affected by the atomic bomb tests in the sixties, unlike the isotopes 14C an 90Sr, the age determination via 228Th/232Th will also work in the future and the method will be able to identify illegal ivory, which is not older than 40 years. The validation of the created time-depending calibration curve of 228Th/232Th in ivory was done by repeated analysis of the same ivory samples with known age after half a year and by repeated analysis of an ivory sample within two years. The expected trend of the ratio change could be confirmed by these experiments. The activity concentration ratio 228Th/232Th rises after death of the elephant, reaches a maximum after roughly four years, decreases and assimilates to a value of one after 40 years. The uncertainty of the overall 228Th/232Th determination could be reduced by developing improved strategies for the determination of the 228Th and 232Th activities. 232Th showed low counting rates and high uncertainties for the determination via α-spectrometry, but 232Th was well accessible via inductively coupled plasma mass spectrometry (ICP-MS). An ICP-MS measurement was integrated in the common analytical procedure. The approach was validated by the analysis of 232Th standard solutions as well as ivory samples. It was shown that the ICP-MS and the α-spectrometry provided similar results. The benefit of the improved technique was the significant lower uncertainty of the 232Th determination by ICP-MS compared to α-spectrometry. The evaluation of a representative ivory sample showed that the uncertainty could be reduced to a value down to 17% of the uncertainty of the 232Th determination by α-spectrometry. This was reflected in an improved dating uncertainty of one year, instead of five years in this case. The determination of the 228Th activity could be improved by evaluating the signals of the decay products of 228Th via α-spectrometry. After extraction of thorium from the ivory matrix, the decay series 228Th, 224Ra, 220Rn, 216Po and 212Bi established a secular radioactive equilibrium. The formation of the secular radioactive equilibrium could be monitored by repeated measurements of the same extracted thorium after time periods. The signal ratios of these isotopes versus 228Th after an achieved equilibrium could be predicted and experimentally validated. A time period of 32 days for the establishment of the secular radioactive equilibrium was found. By using the signals of the decay products for the determination of the 228Th activity, the uncertainty of the former 228Th determination method could be reduced to a value down to 33% of the uncertainty of the evaluation without the decay products of 228Th. The ICP-MS was applied for yield determination for the thorium extraction from ivory. The strategy was developed for a simple matrix and applied to an ivory sample. Two ICP-MS measurements were made during the analytical procedure. One measurement was made after the matrix removal via a trioctlyphosphine oxide solid phase extraction procedure and one measurement was made prior to the electrodeposition of the thorium on a steel chip. The yield result matched well with the result provided by the common isotope dilution yield determination method. The substitution of the isotope dilution method, and the associated prevented addition of external activity in form of a yield tracer, with the yield determination via ICP-MS enables the benefit of an inactive analysis

CE-UV/VIS and CE-MS for monitoring organic impurities during the downstream processing of fermentative-produced lactic acid from second-generation renewable feedstocks

During the downstream process of bio-based bulk chemicals, organic impurities, mostly residues from the fermentation process, must be separated to obtain a pure and ready-to-market chemical. In this study, capillary electrophoresis was investigated for the non-targeting downstream process monitoring of organic impurities and simultaneous quantitative detection of lactic acid during the purification process of fermentatively produced lactic acid. The downstream process incorporated 11 separation units, ranging from filtration, adsorption and ion exchange to electrodialysis and distillation, and 15 different second-generation renewable feedstocks were processed into lactic acid. The identification of organic impurities was established through spiking and the utilization of an advanced capillary electrophoresis mass spectrometry syste

Response to primary chemoradiotherapy of locally advanced oropharyngeal carcinoma is determined by the degree of cytotoxic T cell infiltration within tumor cell aggregates

BackgroundEffective anti-tumor immune responses are mediated by T cells and require organized, spatially coordinated interactions within the tumor microenvironment (TME). Understanding coordinated T-cell-behavior and deciphering mechanisms of radiotherapy resistance mediated by tumor stem cells will advance risk stratification of oropharyngeal cancer (OPSCC) patients treated with primary chemoradiotherapy (RCTx).MethodsTo determine the role of CD8 T cells (CTL) and tumor stem cells for response to RCTx, we employed multiplex immunofluorescence stains on pre-treatment biopsy specimens from 86 advanced OPSCC patients and correlated these quantitative data with clinical parameters. Multiplex stains were analyzed at the single-cell level using QuPath and spatial coordination of immune cells within the TME was explored using the R-package Spatstat.ResultsOur observations demonstrate that a strong CTL-infiltration into the epithelial tumor compartment (HR for overall survival, OS: 0.35; p<0.001) and the expression of PD-L1 on CTL (HR: 0.36; p<0.001) were both associated with a significantly better response and survival upon RCTx. As expected, p16 expression was a strong predictor of improved OS (HR: 0.38; p=0.002) and correlated with overall CTL infiltration (r: 0.358, p<0.001). By contrast, tumor cell proliferative activity, expression of the tumor stem cell marker CD271 and overall CTL infiltration, regardless of the affected compartment, were not associated with response or survival.ConclusionIn this study, we could demonstrate the clinical relevance of the spatial organization and the phenotype of CD8 T cells within the TME. In particular, we found that the infiltration of CD8 T cells specifically into the tumor cell compartment was an independent predictive marker for response to chemoradiotherapy, which was strongly associated with p16 expression. Meanwhile, tumor cell proliferation and the expression of stem cell markers showed no independent prognostic effect for patients with primary RCTx and thus requires further study

In vitro studies on the modification of low-dose hyper-radiosensitivity in prostate cancer cells by incubation with genistein and estradiol

<p>Abstract</p> <p>Background</p> <p>As the majority of prostate cancers (PC) express estrogen receptors, we evaluated the combination of radiation and estrogenic stimulation (estrogen and genistein) on the radiosensitivity of PC cells in vitro.</p> <p>Methods</p> <p>PC cells LNCaP (androgen-sensitive) and PC-3 (androgen-independent) were evaluated. Estrogen receptor (ER) expression was analyzed by means of immunostaining. Cells were incubated in FCS-free media with genistein 10 μM and estradiol 10 μM 24 h before irradiation and up to 24 h after irradiation. Clonogenic survival, cell cycle changes, and expression of p21 were assessed.</p> <p>Results</p> <p>LNCaP expressed both ER-α and ER-β, PC-3 did not. Incubation of LNCaP and PC-3 with genistein resulted in a significant reduction of clonogenic survival. Incubation with estradiol exhibited in low concentrations (0.01 μM) stimulatory effects, while higher concentrations did not influence survival. Both genistein 10 μM and estradiol 10 μM increased low-dose hyper-radiosensitivity [HRS] in LNCaP, while hormonal incubation abolished HRS in PC-3. In LNCaP cells hormonal stimulation inhibited p21 induction after irradiation with 4 Gy. In PC-3 cells, the proportion of cells in G2/M was increased after irradiation with 4 Gy.</p> <p>Conclusion</p> <p>We found an increased HRS to low irradiation doses after incubation with estradiol or genistein in ER-α and ER-β positive LNCaP cells. This is of high clinical interest, as this tumor model reflects a locally advanced, androgen dependent PC. In contrast, in ER-α and ER-β negative PC-3 cells we observed an abolishing of the HRS to low irradiation doses by hormonal stimulation. The effects of both tested compounds on survival were ER and p53 independent. Since genistein and estradiol effects in both cell lines were comparable, neither ER- nor p53-expression seemed to play a role in the linked signalling. Nevertheless both compounds targeted the same molecular switch. To identify the underlying molecular mechanisms, further studies are needed.</p

Monitoring the equilibration of 228Th and decay products over time after extraction from ivory

The worldwide population of elephants shows a rapid decline due to trafficking of ivory. Hence, a method is needed for the age determination to distinguish legal from illegal ivory. The analysis of Th-228 and Th-232 in ivory can be used for this purpose. This study provides a technique for the analysis of Th-228 in ivory. The thorium was extracted from ivory and measured after various time periods applying -spectrometry until a secular radioactive equilibrium between Th-228 and its decay products was established. An equilibration period of 32days was found. The uncertainty of the determination of the activity of Th-228 could be reduced by integrating the counts of the equilibrated decay products into the determination process

Development of an analytical strategy for the determination of 228Th and 232Th in ivory based on the combined use of ICP-MS and α-spectrometry

Elephants are endangered due to the value of their ivory tusks. Therefore, a reliable method for age determination of ivory is of interest for the law enforcement against elephant poachers. The ratio of the specific activities Th-228/Th-232 can be used for the age assessment of ivory. In this report, the combination of two complementary detection techniques, namely inductively coupled plasma-mass spectrometry (ICP-MS) and -spectrometry, for the determination of Th-228 and Th-232 in ivory is presented. Using ICP-MS in addition to -spectrometry, the uncertainty of the Th-232 determination could be reduced significantly which enabled a more accurate and effective ivory dating

Development and application of a method for ivory dating by analyzing radioisotopes to distinguish legal from illegal ivory

The age determination of elephant ivory provides necessary and crucial information for all criminal prosecution authorities enforcing the Convention on International Trade in Endangered Species of Wild Fauna and Flora. The knowledge of the age of ivory allows to distinguish between pre-convention, hence legal material and ivory deriving from recent, illegal poaching incidents. The commonly applied method to determine the age of ivory is radiocarbon dating in the form of bomb pulse dating, which however will fade out soon. This work provides an enhancement of the radiocarbon dating method by supplementary determination of the isotope profile of 90-Sr and the two thorium isotopes 228-Th and 232-Th. This combined analysis allows for a precise and unambiguous age determination of ivory. We provided calibration curves for all involved radionuclides by analyzing ivory samples with known age and investigated a new method for the extraction of strontium from ivory. (c) 2018 Elsevier B.V. All rights reserved

CE-UV/VIS and CE-MS for monitoring organic impurities during the downstream processing of fermentative-produced lactic acid from second-generation renewable feedstocks

Background: During the downstream process of bio-based bulk chemicals, organic impurities, mostly residues from the fermentation process, must be separated to obtain a pure and ready-to-market chemical. In this study, capillary electrophoresis was investigated for the non-targeting downstream process monitoring of organic impurities and simultaneous quantitative detection of lactic acid during the purification process of fermentatively produced lactic acid. The downstream process incorporated 11 separation units, ranging from filtration, adsorption and ion exchange to electrodialysis and distillation, and 15 different second-generation renewable feedstocks were processed into lactic acid. The identification of organic impurities was established through spiking and the utilization of an advanced capillary electrophoresis mass spectrometry system. Results: A total of 53 % of the organic impurities were efficiently removed via bipolar electrodialysis; however, one impurity, pyroglutamic acid, was recalcitrant to separation. It was demonstrated that the presence of pyroglutamic acid disrupts the polymerization of lactic acid into poly lactic acid. Pyroglutamic acid was present in all lactic acid solutions, independent of the type of renewable resource or the bacterium applied. Pyroglutamic acid, also known as 5-oxoproline, is a metabolite in the glutathione cycle, which is present in all living microorganisms. pyroglutamic acid is found in many proteins, and during intracellular protein metabolism, N-terminal glutamic acid and glutamine residues can spontaneously cyclize to become pyroglutamic acid. Hence, the concentration of pyroglutamic acid in the lactic acid solution can only be limited to a certain amount. Conclusions: The present study proved the capillary electrophoresis system to be an important tool for downstream process monitoring. The high product concentration encountered in biological production processes did not hinder the capillary electrophoresis from separating and detecting organic impurities, even at minor concentrations. The coupling of the capillary electrophoresis with a mass spectrometry system allowed for the straightforward identification of the remaining critical impurity, pyroglutamic acid. Although 11 separation units were applied during the downstream process, the pyroglutamic acid concentration remained at 12,900 ppm, which was comparatively high. All organic impurities found were tracked by the capillary electrophoresis, allowing for further separation optimization

Analysis of vascularization in thyroid gland nodes with superb microvascular imaging (SMI) and CD34 expression histology: a pilot study

Background!#!The Doppler sonography technique known as 'superb microvascular imaging' (SMI) is advancing sonographic micro vascularization imaging in various disciplines. In this study, we aimed to determine whether SMI could reliably reproduce the blood flow in thyroid nodes and whether malignancy could be diagnosed, based on vascularization properties. Immunhistochemical staining by CD34 and SMI where used to determine the vascularization of nodes in terms of quantified vascularization parameters gained by computational evaluation.!##!Methods!#!We used image analysis programs to investigate whether the quantitative value for vascularization strength in the thyroid node, measured with SMI, was correlated with the actual degree of vascularization, determined microscopically. We included 16 patients that underwent thyroid resections. We prepared thyroid gland tissue slices for immunohistochemistry and labelled endothelial cells with CD34 to visualize blood vessels microscopically. We used image analysis programs, ImageJ, to quantify SMI Doppler sonographic measurements and CellProfiler to quantify CD34 expression in histological sections. We evaluated the numeric values for diagnostic value in node differentiation. Furthermore, we compared these values to check for correlations.!##!Results!#!Among the 16 nodes studied, three harboured malignant tumours (18.75%): two papillary and one follicular carcinoma. Among the 13 benign lesions (81.25%), four harboured follicular adenomas. Malignant and benign nodes were not significantly different in sonographic (0.88 ± 0.89 vs. 1.13 ± 0.19; p = 0.2790) or immunohistochemical measurements of vascularization strength (0.05 ± 0.05 vs. 0.08 ± 0.06; p = 0.2260).!##!Conclusion!#!We found a positive, significant correlation (r = 0.55588; p = 0.0254) between SMI (quantitative values for vascularization strength) and immunohistochemistry (CD34 staining) evaluations of thyroid nodes