Radiocarbon integrity of dissolved inorganic carbon (DIC) samples stored in plastic and glass bottles: implications for reliable groundwater age dating.

Various approaches based on the natural variations of carbon isotopes (14C and 13C) in dissolved inorganic carbon (DIC) are routinely used to study groundwater dynamics and to estimate recharge rates by deriving groundwater ages. However, differences in 14C activities in groundwater samples collected repeatedly from the same wells and discordantly young 14C groundwater ages compared to noble gases led some authors to question the validity of radiocarbon dating. Poor sampling protocols and storage effects (14C contamination) for radiocarbon analysis are a critical factor in explaining age determination discrepancies. We evaluated the impact of storage protocols on carbon isotope exchange with atmospheric carbon dioxide by comparing glass versus standard plastic field sampling bottles for various storage times before radiocarbon and 13C analyses. The 14C bias after 12 months in pre-evacuated glass vials was minimal and within analytical precision. However, storage of DIC samples in plastic sampling bottles led to marked changes in 14C and 13C contents (up to ∼15 pmC and ∼ 5 ‰, respectively, after 12 months), meaning contamination led to younger groundwater age estimations than it should have been. Protocols for sampling and storing DIC samples for radiocarbon using pre-evacuated glass bottles help avoid atmospheric 14CO2 contamination and microbial activity.

Advancements in the Design and Development of Dry Powder Inhalers and Potential Implications for Generic Development.

Dry powder inhalers (DPIs) are drug-device combination products where the complexity of the formulation, its interaction with the device, and input from users play important roles in the drug delivery. As the landscape of DPI products advances with new powder formulations and novel device designs, understanding how these advancements impact performance can aid in developing generics that are therapeutically equivalent to the reference listed drug (RLD) products. This review details the current understanding of the formulation and device related principles driving DPI performance, past and present research efforts to characterize these performance factors, and the implications that advances in formulation and device design may present for evaluating bioequivalence (BE) for generic development.

Considerations for the Forced Expiratory Volume in 1 Second-Based Comparative Clinical Endpoint Bioequivalence Studies for Orally Inhaled Drug Products.

Herein, we present the US Food and Drug Administration (FDA) Office of Research and Standards' current thinking, challenges, and opportunities for comparative clinical endpoint bioequivalence (BE) studies of orally inhaled drug products (OIDPs). Given the product-associated complexities of OIDPs, the FDA currently uses an aggregate weight-of-evidence approach to demonstrate that a generic OIDP is bioequivalent to its reference listed drug. The approach utilizes comparative clinical endpoint BE or pharmacodynamic BE studies, pharmacokinetic BE studies, and in vitro BE studies to demonstrate equivalence, in addition to formulation sameness and device similarity. For the comparative clinical endpoint BE studies, metrics based on forced expiratory volume in the first second (FEV1 ) are often the recommended clinical endpoints. However, the use of FEV1 can pose a challenge due to its large variability and a relatively flat dose-response relationship for most OIDPs. The utility of applying dose-scale analysis was also investigated by the FDA but often not recommended, due to either flat dose-response relationships or insufficient clinical study data. As a potential way to reduce sample size, we found adapting covariate analysis only explained a limited portion of the variation based on further investigation. The FDA continues to develop alternative methods to make BE assessment of OIDPs more cost- and time-efficient. Prospective generic drug applicants and academia are encouraged to participate in this effort by proposing new approaches in pre-abbreviated new drug application meeting requests and collaborating in the form of grants and contracts under the Generic Drug User Fee Amendments (GDUFA) Regulatory Science and Research Program.

Principles and uncertainties of 14C age estimations for groundwater transport and resource evaluation.

Radiocarbon (14C) is useful for estimating groundwater ages for transport and water resource exploitation assessment. If the 14C content of dissolved inorganic carbon (14CDIC) is known, the age of groundwater can be estimated by applying a radiocarbon decay equation combined with an appropriate geochemical correction model. However, age determinations are subject to uncertainties caused by parameters which need to be estimated or assumed. Here, we discuss the principles of 14C-based groundwater age estimations and the corrections and errors that affect age determinations differently. Generally, the two factors that impact the results of 14C groundwater age are Type-1 and Type-2 errors. Type-1 errors are pulse-type changes on derived groundwater ages that are independent of the water age. Type-2 errors cause gradual changes on derived groundwater 14C ages that depend on the water age. The cumulative impact of these errors substantively reduces the accuracy and confidence of 14C age determinations and corrections. When using 14C for groundwater age, consideration of both error types along with the use of samples having a range of 14CDIC contents helps practitioners recognize and minimize 14C age uncertainty, especially for groundwater ages of <1000 and >30,000 years B.P.

HEYL Regulates Neoangiogenesis Through Overexpression in Both Breast Tumor Epithelium and Endothelium.

Blocking tumor angiogenesis is an appealing therapeutic strategy, but to date, success has been elusive. We previously identified HEYL, a downstream target of Notch signaling, as an overexpressed gene in both breast cancer cells and as a tumor endothelial marker, suggesting that HEYL overexpression in both compartments may contribute to neoangiogenesis. Carcinomas arising in double transgenic Her2-neu/HeyL mice showed higher tumor vessel density and significantly faster growth than tumors in parental Her2/neu mice. Providing mechanistic insight, microarray-based mRNA profiling of HS578T-tet-off-HEYL human breast cancer cells revealed upregulation of several angiogenic factors including CXCL1/2/3 upon HEYL expression, which was validated by RT-qPCR and protein array analysis. Upregulation of the cytokines CXCL1/2/3 occurred through direct binding of HEYL to their promoter sequences. We found that vessel growth and migration of human vascular endothelial cells (HUVECs) was promoted by conditioned medium from HS578T-tet-off-HEYL carcinoma cells, but was blocked by neutralizing antibodies against CXCL1/2/3. Supporting these findings, suppressing HEYL expression using shRNA in MDA-MB-231 cells significantly reduced tumor growth. In addition, suppressing the action of proangiogenic cytokines induced by HEYL using a small molecule inhibitor of the CXCl1/2/3 receptor, CXCR2, in combination with the anti-VEGF monoclonal antibody, bevacizumab, significantly reduced tumor growth of MDA-MB-231 xenografts. Thus, HEYL expression in tumor epithelium has a profound effect on the vascular microenvironment in promoting neoangiogenesis. Furthermore, we show that lack of HEYL expression in endothelial cells leads to defects in neoangiogenesis, both under normal physiological conditions and in cancer. Thus, HeyL-/- mice showed impaired vessel outgrowth in the neonatal retina, while the growth of mammary tumor cells E0771 was retarded in syngeneic HeyL-/- mice compared to wild type C57/Bl6 mice. Blocking HEYL's angiogenesis-promoting function in both tumor cells and tumor-associated endothelium may enhance efficacy of therapy targeting the tumor vasculature in breast cancer.

A simple polymer electrolyte membrane system for enrichment of low-level tritium (3H) in environmental water samples.

Tritium (3H) is an essential tracer of the Earth's water cycle; yet widespread adoption of tritium in hydrologic studies remains a challenge because of analytical barriers to quantification and detection of 3H by electrolytic pre-concentration. Here, we propose a simple tritium electrolytic enrichment system based on the use of solid polymer electrolyte membranes (PEMs) that can be used to enrich 3H in 250-3000 mL environmental water samples to a 10-mL final volume. The IAEA PEM-3H system reported here can produce high enrichment factors (>70-fold) and, importantly, removes some of the deterrents to conventional 3H enrichments methods, including the use of toxic electrolysis and neutralization chemicals, spike standards, a complex electrolysis apparatus that requires extensive cooling and temperature controls, and improves precision by eliminating the need for tracking recovery gravimetrics. Preliminary results with varying operating conditions show 3H enrichments to 70-fold and higher are feasible, spanning a wide range of tritium activities from 5 to 150 TU with a precision of ∼4.5 %. Further work is needed to quantify inter-sample memory and to establish lower 3H detection limits. The IAEA PEM-3H system is open source, with 3-D CAD and design files made freely available for adoption and improvement by others.

Isotope fractionation of sandy-soil water during evaporation - an experimental study.

Soil samples containing water with known stable isotopic compositions were prepared. The soil water was recovered by using vacuum/heat distillation. The experiments were held under different conditions to control rates of water evaporation and water recovery. Recoveries, δ18O and δ2H values of the soil water were determined. Analyses of the data using a Rayleigh distillation model indicate that under the experimental conditions only loosely bound water is extractable in cases where the recovery is smaller than 100 %. Due to isotopic exchange between vapour and remaining water in the micro channels or capillaries of the soil matrix, isotopic fractionation may take place under near-equilibrium conditions. This causes the observed relationship between δ2H and δ18O of the extracted water samples to have a slope close to 8. The results of this study may indicate that, in arid zones when soil that initially contains water dries out, the slope of the relationship between δ2H and δ18O values should be close to 8. Thus, a smaller slope, as observed by some groundwater and soil water samples in arid zones, may be caused by evaporation of water before the water has entered the unsaturated zone.

Combined Treatment with Epigenetic, Differentiating, and Chemotherapeutic Agents Cooperatively Targets Tumor-Initiating Cells in Triple-Negative Breast Cancer.

Efforts to induce the differentiation of cancer stem cells through treatment with all-trans retinoic acid (ATRA) have yielded limited success, partially due to the epigenetic silencing of the retinoic acid receptor (RAR)-ß The histone deacetylase inhibitor entinostat is emerging as a promising antitumor agent when added to the standard-of-care treatment for breast cancer. However, the combination of epigenetic, cellular differentiation, and chemotherapeutic approaches against triple-negative breast cancer (TNBC) has not been investigated. In this study, we found that combined treatment of TNBC xenografts with entinostat, ATRA, and doxorubicin (EAD) resulted in significant tumor regression and restoration of epigenetically silenced RAR-ß expression. Entinostat and doxorubicin treatment inhibited topoisomerase II-ß (TopoII-ß) and relieved TopoII-ß-mediated transcriptional silencing of RAR-ß Notably, EAD was the most effective combination in inducing differentiation of breast tumor-initiating cells in vivo Furthermore, gene expression analysis revealed that the epithelium-specific ETS transcription factor-1 (ESE-1 or ELF3), known to regulate proliferation and differentiation, enhanced cell differentiation in response to EAD triple therapy. Finally, we demonstrate that patient-derived metastatic cells also responded to treatment with EAD. Collectively, our findings strongly suggest that entinostat potentiates doxorubicin-mediated cytotoxicity and retinoid-driven differentiation to achieve significant tumor regression in TNBC. Cancer Res; 76(7); 2013-24. ©2016 AACR.

Targeting Glutamine Metabolism in Breast Cancer with Aminooxyacetate.

PURPOSE: Glutamine addiction in c-MYC-overexpressing breast cancer is targeted by the aminotransferase inhibitor, aminooxyacetate (AOA). However, the mechanism of ensuing cell death remains unresolved. EXPERIMENTAL DESIGN: A correlation between glutamine dependence for growth and c-MYC expression was studied in breast cancer cell lines. The cytotoxic effects of AOA, its correlation with high c-MYC expression, and effects on enzymes in the glutaminolytic pathway were investigated. AOA-induced cell death was assessed by measuring changes in metabolite levels by magnetic resonance spectroscopy (MRS), the effects of amino acid depletion on nucleotide synthesis by cell-cycle and bromodeoxyuridine (BrdUrd) uptake analysis, and activation of the endoplasmic reticulum (ER) stress-mediated pathway. Antitumor effects of AOA with or without common chemotherapies were determined in breast cancer xenografts in immunodeficient mice and in a transgenic MMTV-rTtA-TetO-myc mouse mammary tumor model. RESULTS: We established a direct correlation between c-MYC overexpression, suppression of glutaminolysis, and AOA sensitivity in most breast cancer cells. MRS, cell-cycle analysis, and BrdUrd uptake measurements indicated depletion of aspartic acid and alanine leading to cell-cycle arrest at S-phase by AOA. Activation of components of the ER stress-mediated pathway, initiated through GRP78, led to apoptotic cell death. AOA inhibited growth of SUM159, SUM149, and MCF-7 xenografts and c-myc-overexpressing transgenic mouse mammary tumors. In MDA-MB-231, AOA was effective only in combination with chemotherapy. CONCLUSIONS: AOA mediates its cytotoxic effects largely through the stress response pathway. The preclinical data of AOA's effectiveness provide a strong rationale for further clinical development, particularly for c-MYC-overexpressing breast cancers.

The Notch pathway inhibits TGFß signaling in breast cancer through HEYL-mediated crosstalk.

Acquired resistance to TGFß is a key step in the early stages of tumorigenesis. Mutations in TGFß signaling components are rare, and little is known about the development of resistance in breast cancer. On the other hand, an activated Notch pathway is known to play a substantial role in promoting breast cancer development. Here, we present evidence of crosstalk between these two pathways through HEYL. HEYL, a basic helix-loop-helix transcription factor and a direct target of Notch signaling, is specifically overexpressed in breast cancer. HEYL represses TGFß activity by binding to TGFß-activated Smads. HeyL(-/-) mice have defective mammary gland development with fewer terminal end buds. On the other hand, HeyL transgenic mice show accelerated mammary gland epithelial proliferation and 24% of multiparous mice develop mammary gland cancer. Therefore, repression of TGFß signaling by Notch acting through HEYL may promote initiation of breast cancer.

A portable membrane contactor sampler for analysis of noble gases in groundwater.

To enable a wider use of dissolved noble gas concentrations and isotope ratios in groundwater studies, we have developed an efficient and portable sampling device using a commercially available membrane contactor. The device separates dissolved gases from a stream of water and collects them in a small copper tube (6 mm in diameter and 100 mm in length with two pinch-off clamps) for noble gas analysis by mass spectrometry. We have examined the performance of the sampler using a tank of homogeneous water prepared in the laboratory and by field testing. We find that our sampling device can extract heavier noble gases (Ar, Kr, and Xe) more efficiently than the lighter ones (He and Ne). An extraction time of about 60 min at a flow rate of 3 L/min is sufficient for all noble gases extracted in the sampler to attain equilibrium with the dissolved phase. The extracted gas sample did not indicate fractionation of helium ((3) He/(4) He) isotopes or other noble gas isotopes. Field performance of the sampling device was tested using a groundwater well in Vienna and results were in excellent agreement with those obtained from the conventional copper tube sampling method.

ADP ribosylation by PARP-1 suppresses HOXB7 transcriptional activity.

Interactions with cofactors regulate transcriptional activity and also help HOX proteins to achieve the specificity required for transcriptional regulation of target genes. In this study, we describe a novel protein/protein interaction of HOXB7 with poly (ADP-ribose) polymerase-1 (PARP-1) that involves the homeodomain of HOXB7 and the first zinc finger domain of PARP-1. Upon binding to PARP-1, HOXB7 undergoes poly(ADP-ribosyl)altion resulting in a reduction of its transcriptional activity. Since aspartic acid and glutamic acid residues are acceptors of the ADP ribose moiety transferred by PARP-1, deletion of the evolutionarily conserved C-terminal Glu-rich tail of HOXB7 dramatically attenuates ADP-ribosylation of HOXB7 by PARP-1. Further, a mutant of HOXB7 without the Glu-rich tail loses the ability to be negatively regulated by PARP-1 and becomes transcriptionally more active in luciferase reporter assays. Since the homeodomain is highly conserved among HOX proteins, five other HOX proteins were tested. All six showed interaction with, and were poly(ADP-ribosyl)ated by PARP-1. However, among them, this modification altered the DNA binding activity of only HOXA7 and HOXB7. In summary, this study identifies a new interacting partner of HOX proteins. More importantly, this study reveals a novel mechanism whereby polyADP-ribosylation regulates transcriptional activities of HOX proteins such as HOXB7 and HOXA7.

Epigenetic inactivation of the potential tumor suppressor gene FOXF1 in breast cancer.

The expression of several members of the FOX gene family is known to be altered in a variety of cancers. We show that in breast cancer, FOXF1 gene is a target of epigenetic inactivation and that its gene product exhibits tumor-suppressive properties. Loss or downregulation of FOXF1 expression is associated with FOXF1 promoter hypermethylation in breast cancer cell lines and in invasive ductal carcinomas. Methylation of FOXF1 in invasive ductal carcinoma (37.6% of 117 cases) correlated with high tumor grade. Pharmacologic unmasking of epigenetic silencing in breast cancer cells restored FOXF1 expression. Re-expression of FOXF1 in breast cancer cells with epigenetically silenced FOXF1 genes led to G(1) arrest concurrent with or without apoptosis to suppress both in vitro cell growth and in vivo tumor formation. FOXF1-induced G(1) arrest resulted from a blockage at G(1)-S transition of the cell cycle through inhibition of the CDK2-RB-E2F cascade. Small interfering RNA-mediated depletion of FOXF1 in breast cancer cells led to increased DNA re-replication, suggesting that FOXF1 is required for maintaining the stringency of DNA replication and genomic stability. Furthermore, expression profiling of cell cycle regulatory genes showed that abrogation of FOXF1 function resulted in increased expression of E2F-induced genes involved in promoting the progression of S and G(2) phases. Therefore, our studies have identified FOXF1 as a potential tumor suppressor gene that is epigenetically silenced in breast cancer, which plays an essential role in regulating cell cycle progression to maintain genomic stability.

DNA methylation regulates MicroRNA expression.

MicroRNAs (miRNAs), an important class of small regulatory molecules for gene expression, are transcribed by RNA polymerase II. But little is known about the mechanisms that control miRNA expression. Comparing miRNA expression profiles between colon cancer cell line HCT 116 and its derivative, DNA methyltransferase 1 and 3b (DNMT1 and DNMT3b) double knockout cell line, we found that the expression of about 10% miRNAs was regulated by DNA methylation. In addition, neither 5-aza-2'-deoxycytidine treatment nor deletion of DNMT1 alone recapitulated miRNA expression profile seen in the double knockout cell line, suggesting that miRNA expression was tightly controlled by DNA methylation and partial methylation reduction was not sufficient for miRNA reexpression. We also found that HOXA3 and HOXD10 were putative targets of mir-10a, one of the differentially expressed miRNAs that is located in HOX gene cluster.

Residence times and age distributions of spring waters at the Semmering catchment area, Eastern Austria, as inferred from tritium, CFCs and stable isotopes.

The groundwater system in the mountainous area of Semmering, Austria, was studied by environmental tracers in several karst springs. The tracers used included stable isotopes ((18)O, (2)H), tritium ((3)H) and chlorofluorocarbons (CFCs). The tracers provided valuable information in regard to (1) the mean altitude of the spring catchment areas; (2) the residence time and age distribution of the spring waters; and (3) the interconnection of the springs to a sinkhole. The combination of the stable isotopic data and the topography/geology provided the estimates of the mean altitudes of the catchment areas. Based on the stable isotopic data the recharge temperature of the spring waters was estimated. The smoothing of precipitation's isotopic signal in spring discharge provided information on the minimum transit time of the spring waters. Due to short observation time, (3)H data alone cannot be used for describing the mean residence time of the karst waters. CFCs, though useful in recognizing the co-existence of young (post-1993) water with old (CFC-free) water, could not be used to resolve age distribution models. It is shown in this article, however, that the combined use of tritium and CFCs can provide a better assessment of models to account for different groundwater age distributions. In Appendix A, a simplified method for collecting groundwater samples for the analysis of CFCs is described. The method provides a real facilitation for fieldwork. Test data are given for this sampling method in regard to potential contamination by atmospheric CFCs.

Reliable determination of oxygen and hydrogen isotope ratios in atmospheric water vapour adsorbed on 3A molecular sieve.

The isotope ratio of atmospheric water vapour is determined by wide-ranging feedback effects from the isotope ratio of water in biological water pools, soil surface horizons, open water bodies and precipitation. Accurate determination of atmospheric water vapour isotope ratios is important for a broad range of research areas from leaf-scale to global-scale isotope studies. In spite of the importance of stable isotopic measurements of atmospheric water vapour, there is a paucity of published data available, largely because of the requirement for liquid nitrogen or dry ice for quantitative trapping of water vapour. We report results from a non-cryogenic method for quantitatively trapping atmospheric water vapour using 3A molecular sieve, although water is removed from the column using standard cryogenic methods. The molecular sieve column was conditioned with water of a known isotope ratio to 'set' the background signature of the molecular sieve. Two separate prototypes were developed, one for large collection volumes (3 mL) and one for small collection volumes (90 microL). Atmospheric water vapour was adsorbed to the column by pulling air through the column for several days to reach the desired final volume. Water was recovered from the column by baking at 250 degrees C in a dry helium or nitrogen air stream and cryogenically trapped. For the large-volume apparatus, the recovered water differed from water that was simultaneously trapped by liquid nitrogen (the experimental control) by 2.6 per thousand with a standard deviation (SD) of 1.5 per thousand for delta(2)H and by 0.3 per thousand with a SD of 0.2 per thousand for delta(18)O. Water-vapour recovery was not satisfactory for the small volume apparatus.

[Blood lead level of children in the urban areas in China].

OBJECTIVE: The purpose of this study was to investigate the blood lead level of 3 - 5 year old children living in the cities in China and to provide scientific data for making policy on environmental lead pollution for children health protection. METHODS: Six thousand five hundred and two vein blood samples from 3 - 5 year old children in nineteen cities of nine provinces were sampled. Inductively coupled plasma-mass spectrometry (ICP-MS) were employed to determine lead level in whole blood after microwave digestion for sample preparation and questionnaire survey was also performed. Data were analyzed with multiple regression on factors which affecting blood lead levels. RESULTS: Results showed that mean blood lead level was 88.3 micro g/L for 3 - 5 year old children living in the cities in China and mean blood lead level of boys (91.1 micro g/L) was higher than that of girls (87.3 micro g/L). Twenty-nine point nine one per cent of the children's blood lead level exceeded 100 micro g/L. The research finding showed: (1) higher blood lead levels had negative effects on children's physical growth, language ability etc. (2) behavior of parents had certain effects on children's blood lead levels. (3) blood lead levels of children were affected by unhealthy habits. CONCLUSIONS: Problem of childhood lead poisoning in China has become more serious. During the past ten years, blood lead levels of children has been increased in China while decreasing in developed countries. Blood lead levels of children in China are higher than that of developed countries, which called for special concern by government and society.