Validating Radiosensitivity with Pre-Exposure Differential Gene Expression in Peripheral Blood Predicting Survival and Non-Survival in a Second Irradiated Rhesus Macaque Cohort.

Radiosensitivity differs in humans and possibly in closely related nonhuman primates. The reasons for variation in radiosensitivity are not well known. In an earlier study, we examined gene expression (GE) pre-radiation in peripheral blood among male (n = 62) and female (n = 60) rhesus macaques (n = 122), which did or did not survive (up to 60 days) after whole-body exposure of 7.0 Gy (LD66/60). Eight genes (CHD5, CHI3L1, DYSF, EPX, IGF2BP1, LCN2, MBOAT4, SLC22A4) revealed significant associations with survival. Access to a second rhesus macaque cohort (males = 40, females = 23, total n = 63) irradiated with 5.8-7.2 Gy (LD29-50/60) and some treated with gamma-tocotrienol (GT3, a radiation countermeasure) allowed us to validate these gene expression changes independently. Total RNA was isolated from whole blood samples and examined by quantitative RT-PCR on a 96-well format. cycle threshold (Ct)-values normalized to 18S rRNA were analyzed for their association with survival. Regardless of the species-specific TaqMan assay, similar results were obtained. Two genes (CHD5 and CHI3L1) out of eight revealed a significant association with survival in the second cohort, while only CHD5 (involved in DNA damage response and proliferation control) showed mean gene expression changes in the same direction for both cohorts. No expected association of CHD5 GE with dose, treatment, or sex could be established. Instead, we observed significant associations for those comparisons comprising pre-exposure samples with CHD5 Ct values ≤ 11 (total n = 17). CHD5 Ct values ≤ 11 in these comparisons were mainly associated with increased frequencies (61-100%) of non-survivors, a trend which depending on the sample numbers, reached significance (P = 0.03) in males and, accordingly, in females. This was also reflected by a logistic regression model including all available samples from both cohorts comprising CHD5 measurements (n = 104, odds ratio 1.38, 95% CI 1.07-1.79, P = 0.01). However, this association was driven by males (odds ratio 1.62, 95% CI 1.10-2.38, P = 0.01) and CHD5 Ct values ≤ 11 since removing low CHD5 Ct values from this model, converted to insignificance (P = 0.19). A second male subcohort comprising high CHD5 Ct values ≥ 14.4 in both cohorts (n = 5) appeared associated with survival. Removing these high CHD5 Ct values converted the model borderline significant (P = 0.051). Based on the probability function of the receiver operating characteristics (ROC) curves, 8 (12.3%) and 5 (7.7%) from 65 pre-exposure RNA measurements in males, death and survival could be predicted with a negative and positive predictive value ranging between 85-100%. An associated odds ratio reflected a 62% elevated risk for dying or surviving per unit change (Ct-value) in gene expression, considering the before-mentioned CHD5 thresholds in RNA copy numbers. In conclusion, we identified two subsets of male animals characterized by increased (Ct values ≤ 11) and decreased (Ct values ≥ 14.4) CHD5 GE copy numbers before radiation exposure, which independently of the cohort, radiation exposure or treatment appeared to predict the death or survival in males.

Validating a Four-gene Set for H-ARS Severity Prediction in Peripheral Blood Samples of Irradiated Rhesus Macaques.

Increased radiological and nuclear threats require preparedness. Our earlier work identified a set of four genes (DDB2, FDXR, POU2AF1 and WNT3), which predicts severity of the hematological acute radiation syndrome (H-ARS) within the first three days postirradiation In this study of 41 Rhesus macaques (Macaca mulatta, 27 males, 14 females) irradiated with 5.8-7.2 Gy (LD29-50/60), including some treated with gamma-tocotrienol (GT3, a radiation countermeasure) we independently validated these genes as predictors in both sexes and examined them after three days. At the Armed Forces Radiobiology Research Institute/Uniformed Services University of the Health Sciences, peripheral whole blood (1 ml) of Rhesus macaques was collected into PAXgene® Blood RNA tubes pre-irradiation after 1, 2, 3, 35 and 60 days postirradiation, stored at -80°C for internal experimental analyses. Leftover tubes from these already ongoing studies were kindly provided to Bundeswehr Institute of Radiobiology. RNA was isolated (QIAsymphony), converted into cDNA, and for further gene expression (GE) studies quantitative RT-PCR was performed. Differential gene expression (DGE) was measured relative to the pre-irradiation Rhesus macaques samples. Within the first three days postirradiation, we found similar results to human data: 1. FDXR and DDB2 were up-regulated, FDXR up to 3.5-fold, and DDB2 up to 13.5-fold in the median; 2. POU2AF1 appeared down regulated around tenfold in nearly all Rhesus macaques; 3. Contrary to human data, DDB2 was more up-regulated than FDXR, and the difference of the fold change (FC) ranged between 2.4 and 10, while the median fold changes of WNT3, except days 1 and 35, were close to 1. Nevertheless, 46% of the Rhesus macaques showed down-regulated WNT3 on day one postirradiation, which decreased to 12.2% on day 3 postirradiation. Considering the extended phase, there was a trend towards decreased fold changes at day 35, with median-fold changes ranging from 0.7 for DDB2 to 0.1 for POU2AF1, and on day 60 postirradiation, DGE in surviving animals was close to pre-exposure values for all four genes. In conclusion, the diagnostic significance for radiation-induced H-ARS severity prediction of FDXR, DDB2, and POU2AF1 was confirmed in this Rhesus macaques model. However, DDB2 showed higher GE values than FDXR. As shown in previous studies, the diagnostic significance of WNT3 could not be reproduced in Rhesus macaques; this could be due to the choice of animal model and methodological challenges.

PUM1 and PGK1 are Favorable Housekeeping Genes over Established Biodosimetry-related Housekeeping Genes such as HPRT1, ITFG1, DPM1, MRPS5, 18S rRNA and Others after Radiation Exposure.

In gene expression (GE) studies, housekeeping genes (HKGs) are required for normalization purposes. In large-scale inter-laboratory comparison studies, significant differences in dose estimates are reported and divergent HKGs are employed by the teams. Among them, the 18S rRNA HKG is known for its robustness. However, the high abundance of 18S rRNA copy numbers requires dilution, which is time-consuming and a possible source of errors. This study was conducted to identify the most promising HKGs showing the least radiation-induced GE variance after radiation exposure. In the screening stage of this study, 35 HKGs were analyzed. This included selected HKGs (ITFG1, MRPS5, and DPM1) used in large-scale biodosimetry studies which were not covered on an additionally employed pre-designed 96-well platform comprising another 32 HKGs used for different exposures. Altogether 41 samples were examined, including 27 ex vivo X-ray irradiated blood samples (0, 0.5, 4 Gy), six X-irradiated samples (0, 0.5, 5 Gy) from two cell lines (U118, A549), as well as eight non-irradiated tissue samples to encompass multiple biological entities. In the independent validation stage, the most suitable candidate genes were examined from another 257 blood samples, taking advantage of already stored material originating from three studies. These comprise 100 blood samples from ex vivo X-ray irradiated (0-4 Gy) healthy donors, 68 blood samples from 5.8 Gy irradiated (cobalt-60) Rhesus macaques (RM) (LD29/60) collected 0-60 days postirradiation, and 89 blood samples from chemotherapy-(CTx) treated breast tumor patients. CTx and radiation-induced GE changes in previous studies appeared comparable. RNA was isolated, converted into cDNA, and GE was quantified employing TaqMan assays and quantitative RT-PCR. We calculated the standard deviation (SD) and the interquartile range (IQR) as measures of GE variance using raw cycle threshold (Ct) values and ranked the HKGs accordingly. Dose, time, age, and sex-dependent GE changes were examined employing the parametrical t-test and non-parametrical Kruskal Wallis test, as well as linear regression analysis. Generally, similar ranking results evolved using either SD or IQR GE measures of variance, indicating a tight distribution of GE values. PUM1 and PGK1 showed the lowest variance among the first ten most suitable genes in the screening phase. MRPL19 revealed low variance among the first ten most suitable genes in the screening phase only for blood and cells, but certain comparisons indicated a weak association of MRPL19 with dose (P = 0.02-0.09). In the validation phase, these results could be confirmed. Here, IQR Ct values from, e.g., X-irradiated blood samples were 0.6 raw Ct values for PUM1 and PGK1, which is considered to represent GE differences as expected due to methodological variance. Overall, when compared, the GE variance of both genes was either comparable or lower compared to 18S rRNA. Compared with the IQR GE values of PUM1 and PGKI, twofold-fivefold increased values were calculated for the biodosimetry HKG HPRT1, and comparable values were calculated for biodosimetry HKGs ITFG1, MRPS5, and DPM1. Significant dose-dependent associations were found for ITFG1 and MRPS5 (P = 0.001-0.07) and widely absent or weak (P = 0.02-0.07) for HPRT1 and DPM1. In summary, PUM1 and PGK1 appeared most promising for radiation exposure studies among the 35 HKGs examined, considering GE variance and adverse associations of GE with dose.

Functioning of families with primary school-age children conceived using anonymous donor sperm.

STUDY QUESTION: How do families with children conceived using donor sperm operate as the children grow up? SUMMARY ANSWER: Families with children aged 5-13 years conceived through anonymous donor sperm function well, when compared with other family types with children of the same developmental stage. WHAT IS KNOWN ALREADY: Previous studies on family relationships after donor sperm conception have been reassuring. However, these studies have suffered from methodological limitations due to small sample sizes, respondent biases and absence of appropriate controls. STUDY DESIGN, SIZE, DURATION: This study was an observational study comparing 79 'donor insemination' (DI) families with 987 'couple' families, 364 'single mother' and 112 'step-father' families as part of the Australian Institute of Family Studies Children and Family Life (CFL) study. CFL involved the collection of data on family functioning and child wellbeing from all resident parents through a Family and Child Questionnaire for the 'primary' parent (FACQ-P1) and a Family Relationship Questionnaire (FRQ-P2) for the 'other' parent. PARTICIPANTS/MATERIALS, SETTING, METHODS: All questionnaires were coded with the identity known only to the researchers. The outcomes studied included parent psychological adjustment, family functioning, couple relationship, parenting and parent-child relationship. Family types were compared, separately for mothers' and fathers' reports. The results presented are the estimated means for each family type based on the final model for each outcome: post hoc comparisons between family types are reported with 95% confidence limits. MAIN RESULTS AND THE ROLE OF CHANCE: With all of the outcomes considered, there was not one result where the DI families showed poorer functioning on average than the comparison groups. LIMITATIONS, REASON FOR CAUTION: The final sample size of DI families is 79 with an excellent response rate of nearly 80%. However, there remains some scope for response bias. WIDER IMPLICATIONS OF THE FINDINGS: This study further reassures us that families conceived with anonymous donor sperm do not function any differently from other family types.

The development and implementation of quality assurance programs to support nutritional measurements.

The National Institute of Standards and Technology administers quality assurance programs devoted to improving measurements of nutrients and related metabolites in foods, dietary supplements, and serum and plasma samples. These programs have been developed in collaboration with the National Institutes of Health to assist measurement communities in their efforts to achieve accurate results that are comparable among different laboratories and over time. Targeted analytes include micronutrients, botanical markers, nutritional elements, contaminants, fatty acids, and vitamin D metabolites.

Development and certification of green tea-containing standard reference materials.

A suite of three green tea-containing Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST): SRM 3254 Camellia sinensis (Green Tea) Leaves, SRM 3255 Camellia sinensis (Green Tea) Extract, and SRM 3256 Green Tea-Containing Solid Oral Dosage Form. The materials are characterized for catechins, xanthine alkaloids, theanine, and toxic elements. As many as five methods were used in assigning certified and reference values to the constituents, with measurements carried out at NIST and at collaborating laboratories. The materials are intended for use in the development and validation of new analytical methods, and for use as control materials as a component in the support of claims of metrological traceability.

Paroxysmal cold hemoglobinuria: a case report.

A 15-month-old white male child was admitted to the pediatric intensive care unit with symptoms of upper respiratory tract infection, increased somnolence, pallor, jaundice, fever, and decreased activity level. The purpose of this case study is to report the clinical findings associated with the patient's clinical symptoms and differential laboratory diagnosis.

Certification of vitamins and carotenoids in SRM 3280 multivitamin/multielement tablets.

A new multivitamin/multielement dietary supplement Standard Reference Material (SRM) has been issued by the National Institute of Standards and Technology (NIST), with certified and reference concentration values for 13 vitamins, 24 elements, and 2 carotenoids. The constituents have been measured by multiple analytical methods with data contributed by NIST and by collaborating laboratories. This effort included the first use of isotope dilution mass spectrometry for value assignment of both fat-soluble vitamins (FSVs) and water-soluble vitamins (WSVs). Excellent agreement was obtained among the methods, with relative expanded uncertainties for the certified concentration values typically ranging from <2% to 15% for vitamins.

An Adaptive Multigrid Algorithm for Simulating Solid Tumor Growth Using Mixture Models.

In this paper we give the details of the numerical solution of a three-dimensional multispecies diffuse interface model of tumor growth, which was derived in (Wise et al., J. Theor. Biol. 253 (2008)) and used to study the development of glioma in (Frieboes et al., NeuroImage 37 (2007) and tumor invasion in (Bearer et al., Cancer Research, 69 (2009)) and (Frieboes et al., J. Theor. Biol. 264 (2010)). The model has a thermodynamic basis, is related to recently developed mixture models, and is capable of providing a detailed description of tumor progression. It utilizes a diffuse interface approach, whereby sharp tumor boundaries are replaced by narrow transition layers that arise due to differential adhesive forces among the cell-species. The model consists of fourth-order nonlinear advection-reaction-diffusion equations (of Cahn-Hilliard-type) for the cell-species coupled with reaction-diffusion equations for the substrate components. Numerical solution of the model is challenging because the equations are coupled, highly nonlinear, and numerically stiff. In this paper we describe a fully adaptive, nonlinear multigrid/finite difference method for efficiently solving the equations. We demonstrate the convergence of the algorithm and we present simulations of tumor growth in 2D and 3D that demonstrate the capabilities of the algorithm in accurately and efficiently simulating the progression of tumors with complex morphologies.

Neural substrates of intermanual transfer of a newly acquired motor skill.

In healthy humans, the two cerebral hemispheres show functional specialization to a degree unmatched in other animals, and such strong hemispheric specialization contributes to unimanual skill acquisition [1, 2]. When most humans learn a new motor skill with one hand, this process results in performance improvements in the opposite hand as well [3-6]. Despite the obvious adaptive advantage of such intermanual transfer, there is no direct evidence identifying the neural substrates of this form of skill acquisition [7-9]. Here, we used functional magnetic resonance imaging (fMRI) to study brain regions activated during intermanual transfer of a learned sequence of finger movements. First, we found that the supplementary motor area (SMA) has more activity when a skill has transferred well than when it has transferred poorly. Second, we found that fMRI activity in the ventrolateral posterior thalamic nucleus correlated with successful future intermanual transfer, whereas activity in the ventrolateral anterior thalamic nucleus correlated with past intermanual transfer. Third, we found that repetitive transcranial magnetic stimulation applied over the SMA blocked intermanual transfer without affecting skill acquisition. These findings provide direct evidence for an SMA-based mechanism that supports intermanual transfer of motor-skill learning.

Nonlinear modelling of cancer: bridging the gap between cells and tumours.

Despite major scientific, medical and technological advances over the last few decades, a cure for cancer remains elusive. The disease initiation is complex, and including initiation and avascular growth, onset of hypoxia and acidosis due to accumulation of cells beyond normal physiological conditions, inducement of angiogenesis from the surrounding vasculature, tumour vascularization and further growth, and invasion of surrounding tissue and metastasis. Although the focus historically has been to study these events through experimental and clinical observations, mathematical modelling and simulation that enable analysis at multiple time and spatial scales have also complemented these efforts. Here, we provide an overview of this multiscale modelling focusing on the growth phase of tumours and bypassing the initial stage of tumourigenesis. While we briefly review discrete modelling, our focus is on the continuum approach. We limit the scope further by considering models of tumour progression that do not distinguish tumour cells by their age. We also do not consider immune system interactions nor do we describe models of therapy. We do discuss hybrid-modelling frameworks, where the tumour tissue is modelled using both discrete (cell-scale) and continuum (tumour-scale) elements, thus connecting the micrometre to the centimetre tumour scale. We review recent examples that incorporate experimental data into model parameters. We show that recent mathematical modelling predicts that transport limitations of cell nutrients, oxygen and growth factors may result in cell death that leads to morphological instability, providing a mechanism for invasion via tumour fingering and fragmentation. These conditions induce selection pressure for cell survivability, and may lead to additional genetic mutations. Mathematical modelling further shows that parameters that control the tumour mass shape also control its ability to invade. Thus, tumour morphology may serve as a predictor of invasiveness and treatment prognosis.

Microarchitecture and deposition of gastropod nacre.

The microarchitecture of gastropod nacre reveals deposition of aragonite in the form of crystal stacks and differs markedly from the characteristic microstructure of pelecypod nacre. The structure permits direct observation of both organic and mineral phases of deposition on a growth surface topography that is ideal for the study of processes of mollusk shell calcification. The stack mode of deposition is functionally useful to the animal because it increases the number of crystals which can form and develop on the growth surface at one time.

Scleractinian coral exoskeletons: surface microarchitecture and attachment scar patterns.

Scanning electron microscopic studies have revealed the configurations of the growth surfaces of scleractinian coral exoskeletons. Skeletal surfaces exhibit profuse growths of minute elongate aragonite crystals which, on basal and mural surfaces, are punctuated by scars. It is suggested that these scars are sites of attachment for the specialized processes that connect the living tissues of polyps to the nonliving skeleton. Patterns formed by the attachment scars are taxonomically significant.

Behavioral neurophysiology: insights into seeing and grasping.

One marvels at a batter's ability to hit a baseball traveling at 150 kilometers per hour or a monkey's skill in snatching a flying insect. Indeed, the ability of many animals to reach out, grasp, and manipulate objects is a feat of biological engineering unmatched by even state-of-the-art robots. But how are the objects of our attention chosen and how are the eyes and hands directed to it? Recent progress in behavioral neurophysiology has clarified some of the brain mechanisms at work.

Opiate receptor distribution in the cerebral cortex of the Rhesus monkey.

The distribution of opiate receptors in the cerebral cortex of the rhesus monkey (Macaca mulatta) was determined by autoradiographic visualization of [3H]naloxone binding to tissue sections. Naloxone was bound in relatively large amounts to the cortical laminae containing the cell bodies of output neurons, to a varying set of additional laminae in different cortical fields, to fields closer to more primitive types of cortex, and to polysensory cortical fields. From these laminar and areal variations in distribution, it appears that opiate receptors play a role in specific aspects of cortical function.

Opaline sediments of the southeastern coastal plain and horizon a: biogenic origin.

Scanning electron microscope techniques show that Eocene opaline claystones (fuller's earth and buhrstone) of the Atlantic and Gulf Coastal Plain, deposits long considered volcanic in origin, are actually highly altered diatomites formed as transgressive facies in normal marine continental shelf environmnents. These findings are in agreement with a biogenic origin for time-equivalent horizon A and A deep-sea cherts of the North Atlantic and Caribbean.

Photoluminescence spectroscopy of anthrathiophenes and benzonaphthothiophenes in Shpol'skii matrixes.

The dispersion of harmful oil components into the ocean waters could pose long-term risks to flora and fauna. Due to the complexity of oil-contaminated sites, the unambiguous identification and quantitation of environmental pollutants often requires the sequence of high-performance liquid chromatography and gas chromatography-mass spectrometry. A classic example is the analysis of polycyclic aromatic hydrocarbons. This article tackles a different aspect of environmental analysis as it focuses on the Shpol'skii spectroscopy of polycyclic aromatic sulfur heterocycles, specifically those belonging to the subgroups often known as anthrathiophenes and benzonaphthothiophenes. Photoluminescence measurements were made with a commercial spectrofluorimeter equipped with a continuous wave excitation source for steady state measurements and a pulsed excitation source for time-resolved measurements in the phosphorescence time domain. To the extent of our literature search, this is the first report on the 4.2 K fluorescence and phosphorescence spectra of anthrathiophenes and benzonaphthothiophenes, and the 77 K and 4.2 K phosphorescence lifetimes of benzonaphthothiophenes. 77 K and 4.2 K analytical figures of merit revealed the possibility to detect the studied compounds at the parts-per-billion (ng mL-1) concentration levels. The spectral and lifetime data gathered in this article provides the required information to choose an appropriate photoluminescence technique for the analysis of four-ring polycyclic aromatic sulfur heterocycles in complex environmental extracts.

External Quality Assessment of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) assays.

The discovery that mutations of the CYP24A1 gene are a cause of idiopathic infantile hypercalcemia (IIH) has revived interest in measuring serum 24,25(OH)2D3. Several studies have also suggested that a high 25-hydroxyvitamin D3(25-OHD3):24,25(OH)2D3 ratio might provide additional diagnostic information in the investigation of vitamin D deficiency. Measurement of 24,25(OH)2D3 is necessarily restricted to laboratories with mass spectrometry methods although cross reactivity of the metabolite in immunoassays for 25-OHD is a potential cause of misleading results. The international External Quality Assessment (EQA) scheme for vitamin D metabolites (DEQAS) was set up in 1989. In 2013 DEQAS became an accuracy based EQA for 25-OHD with 'target values' assigned by the National Institute of Standards and Technology (NIST) Reference Measurement Procedure (RMP). A pilot scheme for serum 24,25(OH)2D3 was started in 2015 and participants were asked to measure the metabolite on each of the 5 samples sent out for 25-OHD. Inter-laboratory agreement was poor but this may reflect methodological differences, in particular different approaches to assay standardization. An important potential contribution to reducing variability among assays was the development by NIST of a 24,25(OH)2D3 RMP and its use in assigning values to SRMs 972a, 2973 and 2971, supported by the NIH Office of Dietary Supplements (ODS) as part of the Vitamin D Standardization Program (VDSP) effort.

Three-dimensional multispecies nonlinear tumor growth--I Model and numerical method.

This is the first paper in a two-part series in which we develop, analyze, and simulate a diffuse interface continuum model of multispecies tumor growth and tumor-induced angiogenesis in two and three dimensions. Three-dimensional simulations of nonlinear tumor growth and neovascularization using this diffuse interface model were recently presented in Frieboes et al. [2007. Computer simulation of glioma growth and morphology. NeuroImage S59-S70], but that paper did not describe the details of the model or the numerical algorithm. This is done here. In this diffuse interface approach, sharp interfaces are replaced by narrow transition layers that arise due to differential adhesive forces among the cell species. Accordingly, a continuum model of adhesion is introduced. The model is thermodynamically consistent, is related to recently developed mixture models, and thus is capable of providing a detailed description of tumor progression. The model is well-posed and consists of fourth-order nonlinear advection-reaction-diffusion equations (of Cahn-Hilliard-type) for the cell species coupled with reaction-diffusion equations for the substrate components. We demonstrate analytically and numerically that when the diffuse interface thickness tends to zero, the system reduces to a classical sharp interface model. Using a new fully adaptive and nonlinear multigrid/finite difference method, the system is simulated efficiently. In this first paper, we present simulations of unstable avascular tumor growth in two and three dimensions and demonstrate that our techniques now make large-scale three-dimensional simulations of tumors with complex morphologies computationally feasible. In part II of this study, we will investigate multispecies tumor invasion, tumor-induced angiogenesis, and focus on the morphological instabilities that may underlie invasive phenotypes.