Measurement of inter and intra fraction organ motion in radiotherapy using cone beam CT projection images.

A method is presented for extraction of intra and inter fraction motion of seeds/markers within the patient from cone beam CT (CBCT) projection images. The position of the marker is determined on each projection image and fitted to a function describing the projection of a fixed point onto the imaging panel at different gantry angles. The fitted parameters provide the mean marker position with respect to the isocentre. Differences between the theoretical function and the actual projected marker positions are used to estimate the range of intra fraction motion and the principal motion axis in the transverse plane. The method was validated using CBCT projection images of a static marker at known locations and of a marker moving with known amplitude. The mean difference between actual and measured motion range was less than 1 mm in all directions, although errors of up to 5 mm were observed when large amplitude motion was present in an orthogonal direction. In these cases it was possible to calculate the range of motion magnitudes consistent with the observed marker trajectory. The method was shown to be feasible using clinical CBCT projections of a pancreas cancer patient.

Shading correction algorithm for improvement of cone-beam CT images in radiotherapy.

Cone-beam CT (CBCT) images have recently become an established modality for treatment verification in radiotherapy. However, identification of soft-tissue structures and the calculation of dose distributions based on CBCT images is often obstructed by image artefacts and poor consistency of density calibration. A robust method for voxel-by-voxel enhancement of CBCT images using a priori knowledge from the planning CT scan has been developed and implemented. CBCT scans were enhanced using a low spatial frequency grey scale shading function generated with the aid of a planning CT scan from the same patient. This circumvents the need for exact correspondence between CBCT and CT and the process is robust to the appearance of unshared features such as gas pockets. Enhancement was validated using patient CBCT images. CT numbers in regions of fat and muscle tissue in the processed CBCT were both within 1% of the values in the planning CT, as opposed to 10-20% different for the original CBCT. Visual assessment of processed CBCT images showed improvement in soft-tissue visibility, although some cases of artefact introduction were observed.

Accuracy of radiotherapy dose calculations based on cone-beam CT: comparison of deformable registration and image correction based methods.

Radiotherapy dose calculations based on cone-beam CT (CBCT) images can be inaccurate due to unreliable Hounsfield units (HU) in the CBCT. Deformable image registration of planning CT images to CBCT, and direct correction of CBCT image values are two methods proposed to allow heterogeneity corrected dose calculations based on CBCT. In this paper we compare the accuracy and robustness of these two approaches. CBCT images for 44 patients were used including pelvis, lung and head & neck sites. CBCT HU were corrected using a 'shading correction' algorithm and via deformable registration of planning CT to CBCT using either Elastix or Niftyreg. Radiotherapy dose distributions were re-calculated with heterogeneity correction based on the corrected CBCT and several relevant dose metrics for target and OAR volumes were calculated. Accuracy of CBCT based dose metrics was determined using an 'override ratio' method where the ratio of the dose metric to that calculated on a bulk-density assigned version of the same image is assumed to be constant for each patient, allowing comparison to the patient's planning CT as a gold standard. Similar performance is achieved by shading corrected CBCT and both deformable registration algorithms, with mean and standard deviation of dose metric error less than 1% for all sites studied. For lung images, use of deformed CT leads to slightly larger standard deviation of dose metric error than shading corrected CBCT with more dose metric errors greater than 2% observed (7% versus 1%).

A method to calculate coverage probability from uncertainties in radiotherapy via a statistical shape model.

In this paper we describe a technique that may be used to model the geometric uncertainties that accrue during the radiotherapy process. Using data from in-treatment cone beam CT scans, we simultaneously analyse non-uniform observer delineation variability and organ motion together with patient set-up errors via the creation of a point distribution model (PDM). We introduce a novel method of generating a coverage probability matrix, that may be used to determine treatment margins and calculate uncertainties in dose, from this statistical shape model. The technique does not assume rigid body motion and can extrapolate shape variability in a statistically meaningful manner. In order to construct the PDM, we generate corresponding surface points over a set of delineations. Correspondences are established at a set of points in parameter space on spherically parameterized and canonical aligned outlines. The method is demonstrated using rectal delineations from serially acquired in-treatment cone beam CT image volumes of a prostate patient (44 image volumes total), each delineated by a minimum of two observers (maximum six). Two PDMs are constructed, one with set-up errors included and one without. We test the normality assumptions of the PDMs and find the distributions to be Gaussian in nature. The rectal PDM variability is in general agreement with data in the literature. The two resultant coverage probability matrices show differences as expected.

Maternal executive function, infant feeding responsiveness and infant growth during the first 3 months.

BACKGROUND: There is limited research in young infants, particularly <3 months of age, on maternal feeding practices in spite of increasing evidence that early weight gain velocity is a determinant of later obesity risk. OBJECTIVE: To examine associations between maternal executive function (cognitive control over one's own behaviour), maternal feeding decisions and infant weight and adiposity gains. METHODS: We used a checklist to assess cues mothers use to decide when to initiate and terminate infant feedings at 2 weeks and 3 months of age (N = 69). Maternal executive function was assessed using the NIH Toolbox Cognition Battery subtests for executive function and infant body composition using air displacement plethysmography. RESULTS: Mothers with higher executive function reported relying on fewer non-satiety cues at 2 weeks of age (ß = -0.29, p = 0.037) and on more infant hunger cues at 3 months of age (ß = 0.31, p = 0.018) in their decisions on initiating and terminating feedings. Responsive feeding decisions, specifically the use of infant-based hunger cues at 3 months, in turn were associated with lower gains in weight-for-length (ß = -0.30, p = 0.028) and percent body fat (ß = -0.2, p = 0.091; non-covariate adjusted ß = -0.27, p = 0.029). CONCLUSIONS: These findings show both an association between maternal executive function and responsive feeding decisions and an association between responsive feeding decisions and infant weight and adiposity gains. The causal nature and direction of these associations require further investigation.

Cone beam CT with zonal filters for simultaneous dose reduction, improved target contrast and automated set-up in radiotherapy.

Cone beam CT (CBCT) using a zonal filter is introduced. The aims are reduced concomitant imaging dose to the patient, simultaneous control of body scatter for improved image quality in the tumour target zone and preserved set-up detail for radiotherapy. Aluminium transmission diaphragms added to the CBCT x-ray tube of the Elekta Synergytrade mark linear accelerator produced an unattenuated beam for a central "target zone" and a partially attenuated beam for an outer "set-up zone". Imaging doses and contrast noise ratios (CNR) were measured in a test phantom for transmission diaphragms 12 and 24 mm thick, for 5 and 10 cm long target zones. The effect on automatic registration of zonal CBCT to conventional CT was assessed relative to full-field and lead-collimated images of an anthropomorphic phantom. Doses along the axis of rotation were reduced by up to 50% in both target and set-up zones, and weighted dose (two thirds surface dose plus one third central dose) was reduced by 10-20% for a 10 cm long target zone. CNR increased by up to 15% in zonally filtered CBCT images compared to full-field images. Automatic image registration remained as robust as that with full-field images and was superior to CBCT coned down using lead-collimation. Zonal CBCT significantly reduces imaging dose and is expected to benefit radiotherapy through improved target contrast, required to assess target coverage, and wide-field edge detail, needed for robust automatic measurement of patient set-up error.

Relationship of mammary tumor susceptibility, mammary cell-mediated mutagenesis, and metabolism of polycyclic aromatic hydrocarbons in four types of rats.

Mammary tumorigenesis by 7,12-dimethylbenz(a)anthracene (DMBA) was tested in two rat stocks and two rat strains: outbred Sprague-Dawley and Long-Evans and inbred Wistar/Furth (WF) and Fischer F344 (F344) rats. Both Sprague-Dawley and WF rats showed comparably high susceptibility to tumorigenesis, whereas Long-Evans and F344 rats proved relatively resistant to tumorigenesis. Cultured mammary stromal and parenchymal cell populations from these four types of rats were used in a cell-mediated mutagenesis assay, and all were found to produce comparable levels of mutagenic metabolites from DMBA. Metabolisms of DMBA and benzo(a)pyrene (BP) were also examined in these cell populations, and similar patterns were found in mammary cells from both susceptible and resistant rat types. The stromal cell populations metabolized both carcinogens at a faster rate than the epithelial cells, but high-pressure liquid chromatographic analysis of ethyl acetate-soluble BP metabolites indicated that both cell types produced similar profiles of BP metabolites in all four rat types. Epithelial cells generally maintained higher intracellular concentrations of carcinogen compared to those of stromal cell populations. The four types were indistinguishable in the amounts and identities of glucuronic acid conjugates of BP that were formed by the mammary cells. The data demonstrate that the rat type-specific susceptibility to polycyclic aromatic hydrocarbon-induced tumorigenesis does not reside in the ability of the mammary cells to metabolically activate these carcinogens.

Interspecies comparison of polycyclic aromatic hydrocarbon metabolism in human and rat mammary epithelial cells.

Our laboratory has developed optimized and uniform methods for the isolation and culture of normal mammary epithelial cells from both rats and humans. We have reported that, in a cell-mediated mutagenesis assay, treatment of rat mammary epithelial cells with 7,12-dimethylbenz(a)anthracene, but not benzo(a)pyrene, resulted in significant rates of mutagenesis in cocultured V-79 cells. An opposite mutation pattern was found with human cells under identical conditions. To determine the mechanism of this species-specific difference in polycyclic aromatic hydrocarbon-induced mutagenesis patterns, we then studied the abilities of the human and rat mammary epithelial cells to metabolize benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene. Quantitative levels of carcinogen metabolism were found to be highly dependent on the cell culture densities, although this factor appeared to have little qualitative effect. The most significant qualitative difference in polycyclic aromatic hydrocarbon metabolism between the two species was the ability of the rat, but not the human, mammary epithelial cells to conjugate significant amounts of either polycyclic aromatic hydrocarbon to glucuronic acid. Other aspects of carcinogen metabolism, including production of the precursors to known active metabolites of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene, were similar though not identical. These results, which address only primary metabolism of the polycyclic aromatic hydrocarbons, do not indicate a simple metabolic explanation for the species-specific pattern found in the mammary cell-mediated mutagenesis assay. They do suggest that the effects of cell culture density must be carefully considered in order to properly analyze either interindividual or species differences in carcinogen metabolism.

Human breast cell-mediated mutagenesis of mammalian cells by polycyclic aromatic hydrocarbons.

A system has been developed in which human breast cells activate chemical procarcinogens to mutagenic compounds. The degree of activation is quantitated by the estimation of induction of 6-thioguanine-resistant specific locus mutants in a cocultured Chinese hamster V-79 cell population which does not activate carcinogens. Both mammary stromal and parenchymal cells could activate the procarcinogen 7,12-dimethylbenz(a)anthracene. In addition, it is shown that the two mammary cell populations converted both 7,12-dimethylbenz(a)anthracene and benzo(a)pyrene to water-soluble metabolites. The stromal cells produced substantial amounts of glucuronic acid conjugates, but the parenchymal cells did not. Both cell types metabolize benzo(a)pyrene to the organic-soluble metabolites 9,10- and 7,8-dihydrodiol and both 9- and 3-hydroxybenzo(a)pyrene. These results suggest that the human breast may be a target for polycyclic aromatic hydrocarbon carcinogenesis.

Metabolism and macromolecular binding of carcinogenic and noncarcinogenic metabolites of benzo(a)pyrene by hamster embryo cells.

The metabolism and macromolecular binding of four metabolites of benzo(a)pyrene in hamster embryo fibroblasts has been studied. Two noncarcinogenic phenolic derivatives, 3-hydroxybenz(a)pyrene and 9-hydroxybenzo(a)pyrene, are rapidly metabolized, primarily to their respective glucuronic acid conjugates and other H2O-soluble conjugates (78.4 to 80.8% of total radioactivity). Water-soluble conjugates were also formed from the carcinogenic phenol, 2-hydroxybenzo(a)pyrene, and from 7,8-dihydro-7,8-dihydroxybenzo(a)pyrene, but in lower amounts (36.8 to 43.8% of total radioactivity. With each of the compounds, from 10 to 20% of the radioactivity was converted to ethyl acetate-soluble metabolites. The amount of unmetabolized 2-hydroxybenzo(a)pyrene recovered intracellularly was 20-fold higher than that recovered in incubations with the other phenols. Covalent binding to nuclear macromolecules was monitored after isopyknic separation. Binding of the three phenols tested was similar and was lower than the binding of benzo(a)pyrene to nuclear DNA, RNA, and protein. In contrast to the results with the monohydroxybenzo(a)pyrenes, high levels of covalent binding were observed with 7,8-dihydroxy-7,8-dihydrobenzo(a)pyrene; binding to DNA was 8-fold higher (315 pmol bound per mg DNA) than binding of benzo(a)pyrene to DNA.

Quantitation of benzo(a)pyrene and 7,12-dimethylbenz(a)anthracene binding to nuclear macromolecules in human and rat mammary epithelial cells.

Our laboratory has developed virtually identical techniques for the isolation and culture of mammary epithelial cells (MEC) from rats and humans. In a cell-mediated mutagenesis assay, rat MEC activated 7,12-dimethylbenz(a)anthracene (DMBA) but not benzo(a)pyrene [B(a)P] to mutagenic forms, and the opposite pattern was found with human MEC. These species-specific patterns were not readily explained by either qualitative or quantitative differences in Phase I metabolism of these compounds. In contrast, relative levels of covalent binding of these compounds to DNA in the human and rat cells under identical assay conditions generally parallel the pattern of the mutagenesis results, while not reflecting the absolute levels of metabolism in each system. The ability of the rat MEC to bind relatively higher levels of DMBA than B(a)P to nuclear DNA, and the reversed pattern in human MEC, was found at all incubation times tested between 6 and 48 h. Culture density was found to exert a greater effect on the levels of PAH-DNA binding in rat than in human cells, but in neither case did it affect the ratio of DMBA to B(a)P binding within a species. C2SO4 gradient separation of nuclear macromolecules from PAH-treated MEC revealed that the relative DNA binding levels of DMBA and B(a)P did not correlate with relative levels of nuclear protein binding. For both species, nuclear (DNA + protein) binding levels of B(a)P were approximately 2-fold higher than DMBA. However, these binding levels were 4 to 5-fold higher for both carcinogens in the human than in the rat MEC. The species-specific patterns of PAH activation shown by these cells suggest that caution should be used in extrapolating rodent carcinogenesis data to humans, for either quantitative or qualitative purposes.

Interspecies comparison of human and rat mammary epithelial cell-mediated mutagenesis by polycyclic aromatic hydrocarbons.

In order to compare the interactions of procarcinogens with mammary cells from humans and rats, a uniform set of mediated mutagenesis assays has been established. In these assays, species-specific mammary epithelial cells activate procarcinogens, and specific locus mutations are quantitated in cocultured V-79 cells. Mutations were induced in the rat mammary cell coculture system exposed to 7,12-dimethylbenz(a)anthracene but not benzo(a)pyrene. In contrast, in the human mammary cell coculture system benzo(a)pyrene was much more effective than 7,12-dimethylbenz(a)anthracene in the induction of mutations. These results suggest caution in extrapolating carcinogenesis data between rodents and humans. They also suggest that the relationship between the ubiquitous environmental xenobiotic benzo(a)pyrene and the etiology of human breast cancer requires further exploration.

Interspecies differences in the major DNA adducts formed from benzo(a)pyrene but not 7,12-dimethylbenz(a)anthracene in rat and human mammary cell cultures.

Mammary epithelial cells from rats and humans show both quantitative and qualitative species- and carcinogen-specific differences in their abilities to activate benzo(a)pyrene (B(a)P) and 7,12-dimethylbenz(a)anthracene (DMBA). Previous studies of the DNA binding of these compounds in mammary epithelial cells demonstrated that rat cells bound relatively more DMBA than B(a)P to DNA under identical treatment conditions, while the opposite pattern was exhibited by human mammary epithelial cells. The specific DNA adducts formed in these cells after 24-h incubations with [3H]DMBA and [3H]B(a)P were analyzed to determine if there were qualitative as well as quantitative differences in the amounts of individual adducts. Similar proportions of specific DMBA-DNA adducts were found in both rat and human cells, although the total amount of adducts formed was significantly higher in the rat cells. In contrast, an essentially qualitative species-specific difference was observed in the major B(a)P-DNA adduct present in the rat and human cells. The major B(a)P adduct formed in the human mammary epithelial cells was identified as the (+)-anti-B(a)P-7,8-dihydrodiol-9, 10-epoxide(BPDE)-deoxyguanosine adduct. However, this adduct was formed at very low levels in the rat mammary epithelial cells. The rat cells contained a large proportion of syn-BPDE adducts, and other unidentified B(a)P-DNA adducts. The high level of the (+)-anti-BPDE-deoxyguanosine adduct in the human but not the rat mammary cells is consistent with the potential role of (+)-anti-BPDE in the high mutagenic activity of B(a)P in the cell-mediated mutagenesis assays using the human mammary cells as activators, and the low mutagenic activity of B(a)P when rat cells were used as activators. The quantitative differences in the activation of DMBA by cells from these two species are also consistent with the cell-mediated mutagenic activities of DMBA using these cells as activators. These results suggest that the higher carcinogenic activity of DMBA compared to B(a)P in the rat mammary gland may not be indicative of the relative carcinogenic potencies of these compounds for human mammary cells.

Limonene chemoprevention of mammary carcinoma induction following direct in situ transfer of v-Ha-ras.

Monoterpenes, including limonene and its in vivo rat plasma metabolites, have been shown to be inhibitors of protein isoprenylation of small G proteins, including p21 ras. In addition, dietary limonene has been shown to be capable of preventing the development and causing the regression of chemically induced mammary carcinomas, many of which contain activated ras oncogenes. On the basis of these observations, it was hypothesized that a possible mechanism by which limonene exerts its effects on the chemoprevention and regression of mammary tumors involves the inhibition of protein isoprenylation of the small G protein p21. In the first study, we asked whether dietary limonene was able to prevent the development of mammary carcinomas which were induced using direct retroviral gene transfer of v-Ha-ras into the mammary parenchyma in situ. Limonene modified neither the rate of gene transfer nor the stability of gene expression. However, limonene did greatly inhibit the formation of mammary carcinomas induced by the insertion of activated ras. In a follow-up study, we asked whether chemoprevention by limonene was preferentially effective against a subset of chemically induced mammary carcinomas with activated ras. Rats were fed limonene to prevent the development of N-nitroso-N-methylurea-induced mammary tumors, a majority of which contain the activated Ha-ras oncogene. As expected, limonene administration increased the latency period and lowered the frequency of mammary carcinoma development as compared to controls. However, tumor characterization revealed that limonene treatment did not alter the percentage of carcinomas with activated ras. These studies are consistent with the above studies in that limonene is effective in preventing mammary carcinomas with activated ras. Interestingly, carcinomas without activated ras were prevented to the same extent as those with the activated oncogene.

Fast methods for training Gaussian processes on large datasets.

Gaussian process regression (GPR) is a non-parametric Bayesian technique for interpolating or fitting data. The main barrier to further uptake of this powerful tool rests in the computational costs associated with the matrices which arise when dealing with large datasets. Here, we derive some simple results which we have found useful for speeding up the learning stage in the GPR algorithm, and especially for performing Bayesian model comparison between different covariance functions. We apply our techniques to both synthetic and real data and quantify the speed-up relative to using nested sampling to numerically evaluate model evidences.

An assessment of clinically optimal gold marker length and diameter for pelvic radiotherapy verification using an amorphous silicon flat panel electronic portal imaging device.

Verification of target organ position is essential for the accurate delivery of conformal radiotherapy. Megavoltage electronic portal imaging with flat panel amorphous silicon detectors delivers high quality images that can be used for verification of bony landmark position. Gold markers implanted into the target organ can be visualized and used as a surrogate of actual organ position. On-line compensation for marker displacement, by adjusting patient position, can reduce geometric errors associated with radiation delivery. This study assesses the optimal marker length and diameter to be used with an amorphous silicon (a-Si) flat panel detector and electronic portal images (EPIs), prior to implementation of a clinical programme of gold marker insertion in prostate cancer patients. Seven marker sizes varying from 3 mm to 8 mm in length and 0.8 mm to 1.1 mm in diameter were investigated in a group of patients undergoing pelvic radiotherapy using an 8 MV Elekta SL20 linear accelerator. Markers were placed on the skin entry and exit sites of the treatment beam and EPIs in both lateral and anterior pelvic views were acquired. Three observers independently assessed visibility success and failure using a subjective scoring system. Markers less than 5 mm in length or 0.9 mm in diameter were poorly visualized (<70% visualization success in lateral EPIs). The marker measuring 0.9 mm x 5 mm appears to be clinically optimal in pelvic radiotherapy patients (80% visualization success in lateral EPIs) and will be used for actual organ implantation.

Antagonism of corticotrophin-releasing factor type 1 receptors attenuates caloric intake of free feeding subordinate female rhesus monkeys in a rich dietary environment.

Social subordination in macaque females is a known chronic stressor and previous studies have shown that socially subordinate female rhesus monkeys consume fewer kilocalories than dominant animals when a typical laboratory chow diet is available. However, in a rich dietary environment that provides access to chow in combination with a more palatable diet (i.e. high in fat and refined sugar), subordinate animals consume significantly more daily kilocalories than dominant conspecifics. Substantial literature is available supporting the role of stress hormone signals in shaping dietary preferences and promoting the consumption of palatable, energy-dense foods. The present study was conducted using stable groups of adult female rhesus monkeys to test the hypothesis that pharmacological treatment with a brain penetrable corticotrophin-releasing factor type 1 receptor (CRF1) antagonist would attenuate the stress-induced consumption of a palatable diet among subordinate animals in a rich dietary environment but would be without effect in dominant females. The results show that administration of the CRF1 receptor antagonist significantly reduced daily caloric intake of both available diets among subordinate females compared to dominant females. Importantly, multiple regression analyses showed that the attenuation in caloric intake in response to Antalarmin (Sigma-Aldrich, St Louis, MO, USA) was significantly predicted by the frequency of submissive and aggressive behaviour emitted by females, independent of social status. Taken together, the findings support the involvement of activation of CRF1 receptors in the stress-induced consumption of excess calories in a rich dietary environment and also support the growing literature concerning the importance of CRF for sustaining emotional feeding.

Controlling colloidal stability of silica nanoparticles during bioconjugation reactions with proteins and improving their longer-term stability, handling and storage.

Despite the potential of antibody-coated nanoparticles (Ab-NPs) in many biological applications, there are very few successful, commercially available examples in which the carefully engineered nanomaterial has made it beyond the laboratory bench. Herein we explore the robustness and cost of protein-nanoparticle conjugation. Using multivalent polyamidoamine (PAMAM) dendrimers and dextran as crosslinkers, it was possible to retain colloidal stability during (i) NP-linker binding and (ii) the subsequent conjugation reaction between linker-coated NPs and proteins to generate monodisperse Ab-NPs. This was attributed to the physicochemical properties of the linkers, which were inherited by the NPs and thus benefited colloidal stability. Attaching negatively charged, EDC/sulfo-NHS-activated PAMAM to the NPs contributed to overall negative charge of particles, and in turn led to high electrostatic attraction between the protein and PAMAM-coated NPs during the reaction conditions. In contrast, using an uncharged, EDC/NHS-activated PAMAM dendrimer led to NP aggregation and lower protein binding efficiency. Dextran as a cost-effective, uncharged macromolecule allowed for steric repulsions between neighbouring particles during protein binding, thus inducing NP stability in solution, and also produced monodisperse Ab-NPs. By freeze-drying Ab-NPs from a 1% BSA solution it is possible to reconstitute the solid-form colloid back to a stable state by adding solvent and simply shaking the sample vial by hand. The consequences of the different surface chemistries and freeze-drying stabilizers on the colloidal stability of the NPs were probed by dynamic light scattering. The performance of Ab-NPs was compared in a simple fluorescence linked immunoassay in whole serum. Interestingly, the signal-to-noise ratios were similar for Ab-NPs using PAMAM and dextran, despite dextran binding fewer Abs per NP. We believe this work provides researchers with the tools and strategies for reliably generating Ab-NPs that can be used for a variety of biological applications.

Perinatal Administration of a Selective Serotonin Reuptake Inhibitor Induces Impairments in Reproductive Function and Follicular Dynamics in Female Rat Offspring.

INTRODUCTION: Up to 10% of pregnant women take antidepressants, of which selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed. Using a rodent model, we investigated the reproductive impacts of perinatal SSRI treatment on reproductive cyclicity and function in female offspring. METHODS: Virgin Wistar rats were given oral vehicle (n = 10) or fluoxetine hydrochloride (FLX, 10 mg/kg/d; n = 11) from 2 weeks prior to mating until weaning. Pubertal onset and reproductive cyclicity in offspring were assessed. Blood and ovarian tissues were collected for measures of reproductive function. RESULTS: Perinatal FLX tends to induce irregular reproductive cycles in adult offspring, which most commonly manifest as a prolonged estrus phase (FLX 34% vs control [CON] 10%) relative to CON offspring. The FLX offspring tended to have longer cycles (P = .052), had more secondary follicles (P = .0067), more total follicles (P = .0310), and increased apoptotic ovarian cells (P < .001). Prenatally exposed FLX offspring demonstrated elevated ovarian messenger RNA (mRNA) levels of ERß (P = .008), Cry1 (P = .043), and tryptophan hydroxylase 2 (P = .024), independent of stage of cycle. Ovarian mRNA levels of brain and muscle Arnt-like protein 1 (P = .046) and Pet-1 (P = .021) were increased in FLX offspring a manner that was reproductive cycle stage dependent. CONCLUSIONS: This is the first study to investigate the postnatal effects of maternal perinatal exposure to FLX on adult offspring reproduction. We show that genes that regulate serotonin signaling and action in the ovary are altered in prenatally FLX-exposed offspring, which when coupled with increased expression of components of the core Circadian Locomotor Output Cycles Kaput (CLOCK) gene regulatory loop may suggest an interaction between serotonergic signaling and clock gene signaling pathways leading to the altered reproductive phenotype.

Dark green islands in plant virus infection are the result of posttranscriptional gene silencing.

Dark green islands (DGIs) are a common symptom of plants systemically infected with a mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow, virus-infected tissue. We report here on two lines of evidence showing that DGIs are caused by posttranscriptional gene silencing (PTGS). First, transcripts of a transgene derived from the coat protein of Tamarillo mosaic potyvirus (TaMV) were reduced in DGIs relative to adjacent yellow tissues when the plants were infected with TaMV. Second, nontransgenic plants coinfected with TaMV and a heterologous virus vector carrying TaMV sequences showed reduced titers of the vector in DGIs compared with surrounding tissues. DGIs also were compared with recovered tissue at the top of transgenic plants because recovery has been shown previously to involve PTGS. Cytological analysis of the cells at the junction between recovered and infected tissue was undertaken. The interface between recovered and infected cells had very similar features to that surrounding DGIs. We conclude that DGIs and recovery are related phenomena, differing in their ability to amplify or transport the silencing signal.