Elemental analysis of biological matrices by laser ablation high resolution inductively coupled plasma mass spectrometry (LA-HR-ICP-MS) and high resolution inductively coupled plasma mass spectrometry (HR -ICP -MS)

The need for elemental analysis of biological matrices such as bone, teeth, and plant matter for sourcing purposes has emerged within the forensic and geochemical laboratories. Trace elemental analyses for the comparison of materials such as glass by inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS has been shown to offer a high degree of discrimination between different manufacturing sources. Unit resolution ICP-MS instruments may suffer from some polyatomic interferences including 40Ar16O+, 40Ar 16O1H+, and 40Ca 16O+ that affect iron measurement at trace levels. Iron is an important element in the analysis of glass and also of interest for the analysis of several biological matrices. A comparison of the analytical performance of two different ICP-MS systems for iron analysis in glass for determining the method detection limits (MDLs), accuracy, and precision of the measurement is presented. Acid digestion and laser ablation methods are also compared. Iron polyatomic interferences were reduced or resolved by using dynamic reaction cell and high resolution ICP-MS. MDLs as low as 0.03 μg g-1 and 0.14 μg g-1 for laser ablation and solution based analyses respectively were achieved. The use of helium as a carrier gas demonstrated improvement in the detection limits of both iron isotopes (56Fe and 57Fe) in medium resolution for the HR-ICP-MS and with a dynamic reaction cell (DRC) coupled to a quadrupole ICP-MS system. ^ The development and application of robust analytical methods for the quantification of trace elements in biological matrices has lead to a better understanding of the potential utility of these measurements in forensic chemical analyses. Standard reference materials (SRMs) were used in the development of an analytical method using HR-ICP-MS and LA-HR-ICP-MS that was subsequently applied on the analysis of real samples. Bone, teeth and ashed marijuana samples were analyzed with the developed method. ^ Elemental analysis of bone samples from 12 different individuals provided discrimination between individuals, when femur and humerus bones were considered separately. Discrimination of 14 teeth samples based on elemental composition was achieved with the exception of one case where samples from the same individual were not associated with each other. The discrimination of 49 different ashed plant (cannabis) samples was achieved using the developed method. ^

The association of depression and perceived stress with beta cell function between African and Haitian Americans with and without type 2 diabetes

Background: Diabetes and diabetes-related complications are major causes of morbidity and mortality in the United States. Depressive symptoms and perceived stress have been identified as possible risk factors for beta cell dysfunction and diabetes. The purpose of this study was to assess associations between depression symptoms and perceived stress with beta cell function between African and Haitian Americans with and without type 2 diabetes. Participants and Methods: Informed consent and data were available for 462 participants (231 African Americans and 231 Haitian Americans) for this cross-sectional study. A demographic questionnaire developed by the Primary Investigator was used to collect information regarding age, gender, smoking, and ethnicity. Diabetes status was determined by self-report and confirmed by fasting blood glucose. Anthropometrics (weight, and height and waist circumference) and vital signs (blood pressure) were taken. Blood samples were drawn after 8 10 hours over-night fasting to measure lipid panel, fasting plasma glucose and serum insulin concentrations. The homeostatic model assessment, version 2 (HOMA2) computer model was used to calculate beta cell function. Depression was assessed using the Beck Depression Inventory-II (BDI-II) and stress levels were assessed using the Perceived Stress Scale (PSS). Results: Moderate to severe depressive symptoms were more likely for persons with diabetes (p = 0.030). There were no differences in perceived stress between ethnicity and diabetes status (p = 0.283). General linear models for participants with and without type 2 diabetes using beta cell function as the dependent variable showed no association with depressive symptoms and perceived stress; however, Haitian Americans had significantly lower beta cell function than African Americans both with and without diabetes and adjusting for age, gender, waist circumference and smoking. Further research is needed to compare these risk factors in other race/ethnic groups.

Comparison of radiobiological effects induced by ultra-high and standard dose rate of x-rays on a radio-resistant cell line

Radiotherapy (RT) has recently evolved with the emergence of heavy ion radiations or new fractionation schemes of photon therapy, which modify the dose rate of treatment delivery. The aim of the present study was then to evaluate the in vitro influence of a ultra-high dose rate comparing them with standard dose rate. In this regard, a radioresistant SK-MEL-28 cell line were irradiated with x-ray in order to have a total dose of 2 and 4 Gy, at two different dose rate. The ultra-high dose rate is a specific property of the dense plasma focus (DPF) device, which has pulsed operation and thus gives short and highly energetic pulses of multiple types of rays and particles, in this case, we focused our study on the influence of X-rays. While a low dose rate is obtained with conventional X-ray tube. In this study it results that a ultra-high dose rate enhances radiosensitivity of melanoma cells while reducing the adhesion, proliferation and migration ability of cells.

JNK regulates Fas receptor mediated apoptosis in prostate cancer cell lines

Prostate Cancer is a disease that primarily affects elderly men. The incidence of prostate cancer has been progressively increasing in the western world over the last two decades. Life expectancy and diet are believed to be the main factors contributing to this increase in prevalence. Prostate cancer is a slowly progressing disorder and patients often live for over 10 years after initially being diagnosed with prostate cancer. However, patients with hormone refractory prostate cancer have a poor prognosis and generally do not survive for longer than 2 or 3 years. Hormone refractory prostate cancer is responsible for over 200,000 deaths each year and current chemotherapeutic regimens are only useful as palliative agents. The long-term survival rate is poor and chemotherapy does not significantly increase this. Cell lines derived from hormone refractory tumours usually display elevated resistance to many cytotoxic drugs. The Fas receptor is a membrane bound protein capable of binding to a ligand called Fas ligand. Engagement of Fas receptor with Fas ligand results in clustering of Fas receptor on the plasma membrane of cells. A number of proteins responsible for initiating apoptosis are recruited to the plasma membrane and are activated in response to elevated local concentrations. This series of events initiates a proteolysis cascade and that culminates in the degradation of structural and enzymatic processes and the repackaging of cellular constituents within membrane bound vesicles that can be endocytosed and recycled by surrounding phagocytic cells. The Fas receptor is believed to be a key mechanism by which immune cells can destroy damaged cells. Consequently, resistance to Fas receptor mediated apoptosis often correlates with tumour progression. It has been reported that prostate cancer cell lines display elevated resistance to Fas receptor mediated apoptosis and this correlates with the stage of tumour from which the cell lines were isolated. JNK, a stress-activated protein kinase, has been implicated both with increased survival and increased apoptosis in prostate cancer. Elevated endogenous JNK activity has been demonstrated to correlate with prostate cancer progression. It has been shown that endogenous JNK activity increases the expression of anti-apoptotic proteins and can increase the resistance of prostate cancer cell lines to chemotherapy. In addition, elevated endogenous JNK activity is required for improved proliferation and transformation of a number of epithelial tumours. However, prolonged JNK activation in response to cytotoxic stimuli can increase the sensitivity of cells to apoptosis. Prolonged JNK activity appears to induce the expression of a separate set of genes responsible for promoting apoptosis. Our group has recently shown that activation of JNK by chemotherapeutic drugs can sensitise DU 145 prostate carcinoma cells to Fas receptor mediated apoptosis. In order toidentify novel targets for treating hormone refractory prostate cancer we have investigated the role of JNK in Fas receptor mediated apoptosis. We have demonstrated that prolonged JNK activation is defective in DU 145 cells in response to Fas receptor activation alone. Co-administering anisomycin, a JNK agonist, greatly enhances the ability of DU 145 cells to undergo apoptosis by increasing the rate of Caspase 8 cleavage. We also investigated the role of endogenous JNK activity in Fas receptor mediated.

Elemental Analysis of Biological Matrices by Laser Ablation High Resolution Inductively Coupled Plasma Mass Spectrometry (LA-HR-ICP-MS) and High Resolution Inductively Coupled Plasma Mass Spectrometry (HR-ICP-MS)

The need for elemental analysis of biological matrices such as bone, teeth, and plant matter for sourcing purposes has emerged within the forensic and geochemical laboratories. Trace elemental analyses for the comparison of aterials such as glass by inductively coupled plasma mass spectrometry (ICP-MS) and laser ablation ICP-MS has been shown to offer a high degree of discrimination between different manufacturing sources. Unit resolution ICP-MS instruments may suffer from some polyatomic interferences including 40Ar16O+, 40Ar16O1H+, and 40Ca16O+ that affect iron measurement at trace levels. Iron is an important element in the analysis of glass and also of interest for the analysis of several biological matrices. A comparison of the nalytical performance of two different ICP-MS systems for iron analysis in glass for determining the method detection limits (MDLs), accuracy, and precision of the measurement is presented. Acid digestion and laser ablation methods are also compared. Iron polyatomic interferences were reduced or resolved by using dynamic reaction cell and high resolution ICP-MS. MDLs as low as 0.03 ìg g-1 and 0.14 ìg g-1 for laser ablation and solution based analyses respectively were achieved. The use of helium as a carrier gas demonstrated improvement in the detection limits of both iron isotopes (56Fe and 57Fe) in medium resolution for the HR-ICP-MS and with a dynamic reaction cell (DRC) coupled to a quadrupole ICP-MS system. The development and application of robust analytical methods for the quantification of trace elements in biological matrices has lead to a better understanding of the potential utility of these measurements in forensic chemical analyses. Standard reference materials (SRMs) were used in the development of an analytical method using HR-ICP-MS and LA-HR-ICP-MS that was subsequently applied on the analysis of real samples. Bone, teeth and ashed marijuana samples were analyzed with the developed method. Elemental analysis of bone samples from 12 different individuals provided discrimination between individuals, when femur and humerus bones were considered separately. Discrimination of 14 teeth samples based on elemental composition was achieved with the exception of one case where samples from the same individual were not associated with each other. The discrimination of 49 different ashed plant (cannabis)samples was achieved using the developed method.

Chlorophyll-a, primary production rates, carbon concentrations and cell counts of coccolithophores, diatoms and microzooplankton measured in water samples from the North Atlantic during the M87/1 cruise in April 2012

The Deep Convection cruise repeatedly sampled two locations in the North Atlantic, sited in the Iceland and Norwegian Basins, onboard the RV Meteor (19 March - 2 May 2012). Samples were collected from multiple casts of a conductivity-temperature-depth (CTD) - Niskin rosette at each station. Water samples for primary production rates, community structure, chlorophyll a [Chl a], calcite [PIC], particulate organic carbon [POC] and biogenic silicic acid [BSi] were collected from predawn casts from six light depths (55%, 20%, 14%, 7%, 5% and 1% of incident PAR). Additional samples for community structure and ancillary parameters were collected from a second cast. Carbon fixation rates were determined using the 13C stable isotope method. Water samples for diatom and micro zooplankton counts, collected from the predawn casts, were preserved with acidic Lugol's solution (2% final solution) and counted using an inverted light microscope. Water samples for coccolithophore counts were collected onto cellulose nitrate filters and counted using polarising light microscopy. Water samples for Chl a analysis were filtered onto MF300 and polycarbonate filters and extracted in 90% acetone. PIC and BSi samples were filtered onto polycarbonate filters and analysed using an inductively coupled plasma emission optical spectrometer and a SEAL QuAAtro autoanalyser respectively.

Damaging effects of advanced glycation end product in the murine macrophages cell line J774A.1.

The interaction of reducing sugars, such as aldose, with proteins and the subsequent molecular rearrangements, produces irreversible advanced glycation end-products (AGEs), a heterogeneous class of non-enzymatic glycated proteins or lipids. AGEs form cross-links, trap macromolecules and release reactive oxygen intermediates. AGEs are linked to aging, and increase in several related diseases. The aim of this study was to assess, in a murine macrophage cell line, J774A.1, the effects of 48 h of exposure to glycated serum containing a known amount of pentosidine, a well-known AGE found in the plasma and tissues of diabetic and uremic subjects. Fetal bovine serum was incubated with ribose (50 mm) for 7 days at 37 °C to obtain about 10 nmol/ml of pentosidine. The cytotoxic parameters studied were cell morphology and viability by neutral red uptake, lactate dehydrogenase release and tetrazolium salt test. In the medium and in the intracellular compartment, bound and free pentosidine were evaluated by HPLC, as sensitive and specific glycative markers, and thiobarbituric acid reactive substances (TBARs), as index of the extent of lipid peroxidation. Our results confirm that macrophages are able to take up pentosidine. It is conceivable that bound pentosidine is degraded and free pentosidine is released inside the cell and then into the medium. The AGE increase in the medium was combined with an increase in TBARs, meaning that an oxidative stress occurred; marked cytotoxic effects were observed, and were followed by the release of free pentosidine and TBARs into the culture medium.

Antimicrobial activity of prodigiosin is attributable to plasma-membrane damage

The bacterial pigment prodigiosin has various biological activities; it is, for instance, an effective antimicrobial. Here, we investigate the primary site targeted by prodigiosin, using the cells of microbial pathogens of humans as model systems: Candida albicans, Escherichia coli, Staphylococcus aureus. Inhibitory concentrations of prodigiosin; leakage of intracellular K+ ions, amino acids, proteins and sugars; impacts on activities of proteases, catalases and oxidases; and changes in surface appearance of pathogen cells were determined. Prodigiosin was highly inhibitory (30% growth rate reduction of C. albicans, E. coli, S. aureus at 0.3, 100 and 0.18 μg ml−1, respectively); caused leakage of intracellular substances (most severe in S. aureus); was highly inhibitory to each enzyme; and caused changes to S. aureus indicative of cell-surface damage. Collectively, these findings suggest that prodigiosin, log Poctanol–water 5.16, is not a toxin but is a hydrophobic stressor able to disrupt the plasma membrane via a chaotropicity-mediated mode-of-action.

Particle-in-cell simulation study of a lower-hybrid shock

The expansion of a magnetized high-pressure plasma into a low-pressure ambient medium is examined with particle-in-cell simulations. The magnetic field points perpendicular to the plasma's expansion direction and binary collisions between particles are absent. The expanding plasma steepens into a quasi-electrostatic shock that is sustained by the lower-hybrid (LH) wave. The ambipolar electric field points in the expansion direction and it induces together with the background magnetic field a fast E cross B drift of electrons. The drifting electrons modify the background magnetic field, resulting in its pile-up by the LH shock. The magnetic pressure gradient force accelerates the ambient ions ahead of the LH shock, reducing the relative velocity between the ambient plasma and the LH shock to about the phase speed of the shocked LH wave, transforming the LH shock into a nonlinear LH wave. The oscillations of the electrostatic potential have a larger amplitude and wavelength in the magnetized plasma than in an unmagnetized one with otherwise identical conditions. The energy loss to the drifting electrons leads to a noticeable slowdown of the LH shock compared to that in an unmagnetized plasma.

Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.

Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.

Establishing an EGFR mutation screening service for non-small cell lung cancer - sample quality criteria and candidate histological predictors.

INTRODUCTION: EGFR screening requires good quality tissue, sensitivity and turn-around time (TAT). We report our experience of routine screening, describing sample type, TAT, specimen quality (cellularity and DNA yield), histopathological description, mutation result and clinical outcome. METHODS: Non-small cell lung cancer (NSCLC) sections were screened for EGFR mutations (M+) in exons 18-21. Clinical, pathological and screening outcome data were collected for year 1 of testing. Screening outcome alone was collected for year 2. RESULTS: In year 1, 152 samples were tested, most (72%) were diagnostic. TAT was 4.9 days (95%confidence interval (CI)=4.5-5.5). EGFR-M+ prevalence was 11% and higher (20%) among never-smoking women with adenocarcinomas (ADCs), but 30% of mutations occurred in current/ex-smoking men. EGFR-M+ tumours were non-mucinous ADCs and 100% thyroid transcription factor (TTF1+). No mutations were detected in poorly differentiated NSCLC-not otherwise specified (NOS). There was a trend for improved overall survival (OS) among EGFR-M+ versus EGFR-M- patients (median OS=78 versus 17 months). In year 1, test failure rate was 19%, and associated with scant cellularity and low DNA concentrations. However 75% of samples with poor cellularity but representative of tumour were informative and mutation prevalence was 9%. In year 2, 755 samples were tested; mutation prevalence was 13% and test failure only 5.4%. Although samples with low DNA concentration (2.2 ng/μL), the mutation rate was 9.2%. CONCLUSION: Routine epidermal growth factor receptor (EGFR) screening using diagnostic samples is fast and feasible even on samples with poor cellularity and DNA content. Mutations tend to occur in better-differentiated non-mucinous TTF1+ ADCs. Whether these histological criteria may be useful to select patients for EGFR testing merits further investigation.

Multi-purpose utility of circulating plasma DNA testing in patients with advanced cancers.

Tumor genomic instability and selective treatment pressures result in clonal disease evolution; molecular stratification for molecularly targeted drug administration requires repeated access to tumor DNA. We hypothesized that circulating plasma DNA (cpDNA) in advanced cancer patients is largely derived from tumor, has prognostic utility, and can be utilized for multiplex tumor mutation sequencing when repeat biopsy is not feasible. We utilized the Sequenom MassArray System and OncoCarta panel for somatic mutation profiling. Matched samples, acquired from the same patient but at different time points were evaluated; these comprised formalin-fixed paraffin-embedded (FFPE) archival tumor tissue (primary and/or metastatic) and cpDNA. The feasibility, sensitivity, and specificity of this high-throughput, multiplex mutation detection approach was tested utilizing specimens acquired from 105 patients with solid tumors referred for participation in Phase I trials of molecularly targeted drugs. The median cpDNA concentration was 17 ng/ml (range: 0.5-1600); this was 3-fold higher than in healthy volunteers. Moreover, higher cpDNA concentrations associated with worse overall survival; there was an overall survival (OS) hazard ratio of 2.4 (95% CI 1.4, 4.2) for each 10-fold increase in cpDNA concentration and in multivariate analyses, cpDNA concentration, albumin, and performance status remained independent predictors of OS. These data suggest that plasma DNA in these cancer patients is largely derived from tumor. We also observed high detection concordance for critical 'hot-spot' mutations (KRAS, BRAF, PIK3CA) in matched cpDNA and archival tumor tissue, and important differences between archival tumor and cpDNA. This multiplex sequencing assay can be utilized to detect somatic mutations from plasma in advanced cancer patients, when safe repeat tumor biopsy is not feasible and genomic analysis of archival tumor is deemed insufficient. Overall, circulating nucleic acid biomarker studies have clinically important multi-purpose utility in advanced cancer patients and further studies to pursue their incorporation into the standard of care are warranted.

Refining the diagnosis and EGFR status of non-small cell lung carcinoma in biopsy and cytologic material, using a panel of mucin staining, TTF-1, cytokeratin 5/6, and P63, and EGFR mutation analysis.

INTRODUCTION: The dichotomization of non-small cell carcinoma (NSCLC) subtype into squamous (SQCC) and adenocarcinoma (ADC) has become important in recent years and is increasingly required with regard to management. The aim of this study was to determine the utility of a panel of commercially available antibodies in refining the diagnosis on small biopsies and also to determine whether cytologic material is suitable for somatic EGFR genotyping in a prospectively analyzed series of patients undergoing investigation for suspected lung cancer. METHODS: Thirty-two consecutive cases of NSCLC were first tested using a panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, 34betaE12, and a D-PAS stain for mucin, to determine their value in refining diagnosis of NSCLC. After this test phase, two further pathologists independently reviewed the cases using a refined panel that excluded 34betaE12 because of its low specificity for SQCC, and refinement of diagnosis and concordance were assessed. Ten cases of ADC, including eight derived from cytologic samples, were sent for EGFR mutation analysis. RESULTS: There was refinement of diagnosis in 65% of cases of NSCLC to either SQCC or ADC in the test phase. This included 10 of 13 cases where cell pellets had been prepared from transbronchial needle aspirates. Validation by two further pathologists with varying expertise in lung pathology confirmed increased refinement and concordance of diagnosis. All samples were adequate for analysis, and they all showed a wild-type EGFR genotype. CONCLUSION: A panel comprising cytokeratin 5/6, P63, thyroid transcription factor-1, and a D-PAS stain for mucin increases diagnostic accuracy and agreement between pathologists when faced with refining a diagnosis of NSCLC to SQCC or ADC. These small samples, even cell pellets derived from transbronchial needle aspirates, seem to be adequate for EGFR mutation analysis.

Gene scanning of VDJH-amplified segments is a clinically relevant technique to detect contaminating tumor cells in the apheresis products of multiple myeloma patients undergoing autologous peripheral blood stem cell transplantation.

Contaminating tumour cells in apheresis products have proved to influence the outcome of patients with multiple myeloma (MM) undergoing autologous stem cell transplantation (APBSCT). The gene scanning of clonally rearranged VDJ segments of the heavy chain immunoglobulin gene (VDJH) is a reproducible and easy to perform technique that can be optimised for clinical laboratories. We used it to analyse the aphereses of 27 MM patients undergoing APBSCT with clonally detectable VDJH segments, and 14 of them yielded monoclonal peaks in at least one apheresis product. The presence of positive results was not related to any pre-transplant characteristics, except the age at diagnosis (lower in patients with negative products, P = 0.04). Moreover, a better pre-transplant response trended to associate with a negative result (P = 0.069). Patients with clonally free products were more likely to obtain a better response to transplant (complete remission, 54% vs 28%; >90% reduction in the M-component, 93% vs 43% P = 0.028). In addition, patients transplanted with polyclonal products had longer progression-free survival, (39 vs 19 months, P = 0.037) and overall survival (81% vs 28% at 5 years, P = 0.045) than those transplanted with monoclonal apheresis. In summary, the gene scanning of apheresis products is a useful and clinically relevant technique in MM transplanted patients.

The Calreticulin gene and myeloproliferative neoplasms

The Philadelphia negative myeloproliferative neoplasms include polycythaemia vera (PV), essential thrombocytopenia (ET) and primary myelofibrosis (PMF). Patients with these conditions were mainly thought to harbour JAK2V617F mutations or an Myeloproliferative leukaemia (MPL) substitution. In 2013, two revolutionary studies identified recurrent mutations in a gene that encodes the protein calreticulin (CALR). This mutation was detected in patients with PMF and ET with non-mutated JAK2 or MPL but was absent in patients with PV. The CALR gene encodes the calreticulin protein, which is a multifactorial protein, mainly located in the endoplasmic reticulum in chromosome 19 and regulates calcium homeostasis, chaperones and has also been implicated in multiple cellular processes including cell signalling, regulation of gene expression, cell adhesion, autoimmunity and apoptosis. Somatic 52 bp deletions and recurrent 52 bp insertion mutations in CALR were detected and all resulted in frameshift and clusters in exon 9 of the gene. This review will summarise the current knowledge on the CALR gene and mutation of the gene in pathological conditions and patient phenotypes.