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Rodent behavioral studies have largely focused on male animals, which has limited the generalizability and conclusions of neuroscience research. Working with humans and rodents, we studied sex effects during interval timing that requires participants to estimate an interval of several seconds by making motor responses. Interval timing requires attention to the passage of time and working memory for temporal rules. We found no differences between human females and males in interval timing response times (timing accuracy) or the coefficient of variance of response times (timing precision). Consistent with prior work, we also found no differences between female and male rodents in timing accuracy or precision. In female rodents, there was no difference in interval timing between estrus and diestrus cycle stages. Because dopamine powerfully affects interval timing, we also examined sex differences with drugs targeting dopaminergic receptors. In both female and male rodents, interval timing was delayed after administration of sulpiride (D2-receptor antagonist), quinpirole (D2-receptor agonist), and SCH-23390 (D1-receptor antagonist). By contrast, after administration of SKF-81297 (D1-receptor agonist), interval timing shifted earlier only in male rodents. These data illuminate sex similarities and differences in interval timing. Our results have relevance for rodent models of both cognitive function and brain disease by increasing represenation in behavioral neuroscience.

Lithium-ion batteries have become the primary battery technology used for electric and hybrid vehicles powertrains. Battery temperature, however, is a critical factor for these devices, as it influences battery performance life-time and safety and must be preferably kept in the 15-35 °C range. A dynamic electro-thermal model of a lithium iron phosphate battery was developed. The model predicts battery voltage and temperature evolution in different operating conditions. A battery equivalent circuit model (ECM) with an open circuit voltage source, an ohmic resistance and a capacitor-resistor pair in series is adopted. The state-of-charge is determined by the Coulomb counting approach and the battery temperature is computed by carrying out an energy balance for the cell. The balance takes into account the difference between the heat generated within the cell and the heat loss to the environment. Finally a controller, which cools or heats the battery in order to keep its temperature in the desired range, was developed. The case of the battery pack of a hybrid plug-in powertrain during a WLTP cycle is simulated and the result of different environmental conditions are presented.

Aim. To determine the prognostic significance PCA3 gene expression in urine sediment and exosomes in patients with localized prostate cancer (PC) and associated histologic changes in the peritumoral zone as a predictor of biochemical recurrence after radical prostatectomy (RPE). Materials and methods. Of 148 patients with localized PC, 96 (65%) had high-grade prostatic intraepithelial neoplasia (PIN-2) in the peritumoral zone. The other 52 (35%) had no pathologic tissue of the peritumoral zone. PDA3 expression in urine sediment and exosomes was determined with real-time PCR with respect to the reference gene KLK3. Results. The PCA3 gene expression level in urine exosomes in patients with PIN-2 in the prostatic peritumoral zone and synchronous pancreatic adenocarcinoma was higher among patients with subsequent disease recurrence. Increased PCA3 gene expression in the urine sediment was also predictive of the risk of recurrence of a prostatic tumor with PIN-2 in the peritumoral zone, although to a lesser degree than the results with urine exosomes. When the ∆Ct РСА3-KLK3 was ≥1,86 in the urine sediment, biochemical recurrence of PC and PIN-2 developed more frequently in the peritumoral zone (84% versus 51%, p = 0,013). Conclusions. Increased PCA3 gene expression in urine sediment and exosomes is a predictor of increased risk of biochemical recurrence after RPE in patients with localized PC and PIN-2 in the peritumoral zone.

Accurate estimation of the levels of non-compliance to fishing regulations is crucial in ensuring that long term sustainability goals of fisheries are met. When requesting information regarding sensitive behaviour, such as an angler’s non-compliance to regulations through direct questioning methods (DQM), their responses can be influenced by social desirability bias (SDB). Literature in human dimensions research on methods for controlling this bias is limited. There has been an emerging prevalence in the use of the random response technique (RRT), which is a method aimed at reducing SDB in questions regarding sensitive behaviour, although it has never been validated against observed data in an environmental resource use context. An alternative to the RRT, the use of a ballot box method (BBM) has been successfully implemented to reduce SDB in contingent valuation studies and is introduced in this paper as a method for reducing SDB in face-to-face survey responses regarding sensitive behaviour. The RRT, BBM and a baseline DQM were compared for accuracy in obtaining honest responses from non-compliant anglers in South Africa’s recreational marine based shore fishery (MBSF) who were covertly observed violating various regulations. A total of 79.6% of anglers who were interviewed using the BBM admitted to breaking the observed regulation. However, only 46.5% and 38.5% of the anglers who were interviewed using the DQM and RRT admitted to breaking the regulations. The length of the angler interview using the RRT was also significantly longer than the BBM and DQM. These results suggest that the BBM is the most accurate and practical method for reducing SDB in compliance surveys.

Objective. Examination of the expression of genes responsible for hypoxia-dependent control of transcription, neoangiogenesis, and apoptosis in tumor tissue of the prostate, in patients with localized prostate cancer (РС) with biochemical recurrence (BR) and without recurrences after radical prostatectomy (RPE).Materials and methods. The main group included 56 patients with localized PC who had been diagnosed with BR within two years after RP. 60 patients with localized PC who did not relapse had a comparative group. 55 patients in whom operative biopsy specimens of the prostate gland were taken within healthy tissues with the removal of benign prostatic hyperplasia were combined into a control group. Determination of the expression level of the BAX, BCL2, VEGFA and HIF1α genes in tumor tissue was performed by real-time polymerase chain reaction.Results. In patients with localized PC after RPE, development of BR is associated with an increase in the expression of BCL2, VEGFA and HIF1α genes and a decrease in the expression of the BAX gene. In patients with localized PC and early recurrence of tumor tissue through a hypoxia-dependent factor that enhances transcritical processes in tumor cells, neoangiogenesis is activated, which is associated with inhibition of apoptosis of tumor cells by enhancing the expression of the antiapoptotic gene BCL2.Conclusion. Determination of the expression of BAX, BCL2, VEGFA and HIF1α genes in tumor tissue with localized PC allows further assessment of the risk of disease progression after surgical treatment.

Aim. To assess the prognostic significance of histopathological processes in the peritumoral zone with respect to the risk of biochemical recurrence in patients with localized prostate cancer after radical prostatectomy. Methods. Histomorphological studies were conducted in the perifocal area of surgical tissue samples from 309 patients with localized prostate cancer (T1c-2cN0M0) after a radical surgery using light microscopy. Four groups of patients were identified depending on the risk of recurrence. Enzyme immunoassay was used to determine the concentration of prostate-specific antigen in the serum at baseline and every 3 months for two years after the surgery to detect biochemical recurrence. Results. Histomorphological examination of the peritumoral zone made it possible to identify histopathological processes associated with adenocarcinoma in 257 out of 309 (83.2%) patients with localized prostate cancer. The risk of biochemical recurrence of a combination of prostatic adenocarcinoma and prostatic intraepithelial neoplasia-2 increased by 3.3 (p=0.02), and of a combination of adenocarcinoma, neoplasia and chronic inflammation in the perifocal zone increased by 4.5 times (p=0.005) compared to patients without histopathological changes of the peritumoral zone. In combination of prostate adenocarcinoma with neoplasia and chronic inflammation in the peritumoral zone, the number of patients with an intermediate risk of cancer recurrence after surgical treatment increased due to decrease of the proportion of patients with very low and low risk of recurrence of the oncologic disease. Conclusion. The combination of prostate cancer with high-grade prostatic intraepithelial neoplasia and chronic inflammation in the peritumoral zone modifies the risk of biochemical recurrence of cancer after radical prostatectomy.

NEW DIAGNOSTIC OPPORTUNITIES FOR THE STUDY OF THE EXPRESSION OF PROSTATSPECIFIC GENES IN DEPOSIT AND URINE EXOSOMOX

The only documentation on the building downwash algorithm in AERMOD (American Meteorological Society/U.S. Environmental Protection Agency Regulatory Model), referred to as PRIME (Plume Rise Model Enhancements), is found in the 2000 AWMA journal article by Schulman, Strimaitis and Scire. Recent field and wind tunnel studies have shown that AERMOD can overpredict concentrations by factors of 2 to 8 for certain building configurations. While a wind tunnel equivalent building dimension study (EBD) can be conducted to approximately correct the overprediction bias, past field and wind tunnel studies indicate that there are notable flaws in the PRIME building downwash theory. A detailed review of the theory supported by CFD (Computational Fluid Dynamics) and wind tunnel simulations of flow over simple rectangular buildings revealed the following serious theoretical flaws: enhanced turbulence in the building wake starting at the wrong longitudinal location; constant enhanced turbulence extending up to the wake height; constant initial enhanced turbulence in the building wake (does not vary with roughness or stability); discontinuities in the streamline calculations; and no method to account for streamlined or porous structures.This paper documents theoretical and other problems in PRIME along with CFD simulations and wind tunnel observations that support these findings. Although AERMOD/PRIME may provide accurate and unbiased estimates (within a factor of 2) for some building configurations, a major review and update is needed so that accurate estimates can be obtained for other building configurations where significant overpredictions or underpredictions are common due to downwash effects. This will ensure that regulatory evaluations subject to dispersion modeling requirements can be based on an accurate model. Thus, it is imperative that the downwash theory in PRIME is corrected to improve model performance and ensure that the model better represents reality.

Background and purpose : Autoimmune encephalitides (AE) include a spectrum of neurological disorders whose diagnosis revolves around the detection of neuronal antibodies (Abs). Consensus-based diagnostic criteria (AE-DC) allow clinic-serological subgrouping of AE, with unclear prognostic implications. The impact of AE-DC on patients’ management was studied, focusing on the subgroupofAb-negative-AE. Methods: This was a retrospective multicenter study on patients fulfilling AE-DC. All patients underwent Ab testing with commercial cell-based assays (CBAs) and, when available, in-house assays (immunohistochemistry, live/fixed CBAs, neuronal cultures) that contributed to defining final categories. Patients were classified as Ab-positive-AE [N-methyl-d-aspartate-receptor encephalitis (NMDAR-E), Ab-positive limbic encephalitis (LE), definite-AE] or Ab-negative-AE (Ab-negative-LE, probable-AE, possible-AE). Results: Commercial CBAs detected neuronal Abs in 70/118 (59.3%) patients. Testing 37/48 Ab-negative cases, in-house assays identified Abs in 11 patients (29.7%). A hundred and eighteen patients fulfilled the AE-DC, 81 (68.6%) with Ab-positive-AE (Ab-positive-LE, 40; NMDAR-E, 32; definite-AE, nine) and 37 (31.4%) with Ab-negative-AE (Ab-negative-LE, 17; probable/possible-AE, 20). Clinical phenotypes were similar in Ab-positive-LE versus Ab-negative-LE. Twenty-four/118 (20.3%) patients had tumors, and 19/118 (16.1%) relapsed, regardless of being Ab-positive or Ab-negative. Ab-positive-AE patients were treated earlier than Ab-negative-AE patients (P=0.045), responded more frequently to treatments (92.3% vs. 65.6%, P&nbsp

This study aimed to assess the suitability of the Berkowitz' (2005) social norms approach (SNA) for improving compliance behaviour amongst recreational fishers. A total of 138 recreational shore anglers were interviewed in Eastern Cape, South Africa and asked about their compliance, attitudes towards compliance, perceptions of compliance and the attitudes of other anglers. Results indicate that angler compliance for individual regulations was relatively high (75%–90%). Attitudes of anglers towards compliance was positive, with >80% feeling that “breaking any regulation is wrong.” Yet, as predicted by the SNA, interviewees often overestimated the non-compliance and negative attitudes of other anglers, particularly as their social proximity decreased. Interviewees with the greatest misperceptions were also less compliant. The social norms present in the Eastern Cape rock and surf fishery fulfil the criteria required for the application of the SNA, suggesting that this approach may provide a suitable normative intervention for improving compliance to be used in conjunction with instrumental approaches in recreational fisheries.

The work aims at analysing the performance of an innovative multi-source integrated energy system for small-scale combined heat and power (CHP) applications. The system is based on an organic Rankine cycle (ORC) coupled with a biodiesel internal combustion engine (ICE), a wind turbine (WT) and a photovoltaic system (PV). The engine exhaust gas provides energy to the bottoming ORC system, whereas the thermal energy of the ICE cooling and lubricating system is used for cogeneration purposes at low temperature. Both ORC and ICE systems operate in order to satisfy the thermal demand of a commercial centre. Wind and photovoltaic units work in parallel to increase the electric self-consumption rate. When the solar and/or wind sources are high, the biodiesel ICE-ORC can be switched-off or operated at partial load.A numerical thermodynamic model has been developed and a preliminary investigation has been performed to define the proper size of the system according to a thermal-driven strategy. An hourly energy balance has been carried on an annual basis. The analysis has been focused on a multi-variable optimisation and the proper trade-off between electric surplus and self-consumption has been found. The results reveal that the multi-source CHP system represents an efficient solution for smart-grid communities.

A clearer definition of the molecular determinants that drive the development and progression of prostate cancer (PCa) is urgently needed. Efforts to map recurrent somatic deletions in the tumor genome, especially homozygous deletions (HODs), have provided important positional information in the search for cancer-causing genes. Analyzing HODs in the tumors of 244 patients from two independent cohorts and 22 PCa xenografts using high-resolution single-nucleotide polymorphism arrays, herein we report the identification of CHD1, a chromatin remodeler, as one of the most frequently homozygously deleted genes in PCa, second only to PTEN in this regard. The HODs observed in CHD1, including deletions affecting only internal exons of CHD1, were found to completely extinguish the expression of mRNA of this gene in PCa xenografts. Loss of this chromatin remodeler in clinical specimens is significantly associated with an increased number of additional chromosomal deletions, both hemi- and homozygous, especially on 2q, 5q and 6q. Together with the deletions observed in HEK293 cells stably transfected with CHD1 small hairpin RNA, these data suggest a causal relationship. Downregulation of Chd1 in mouse prostate epithelial cells caused dramatic morphological changes indicative of increased invasiveness, but did not result in transformation. Indicating a new role of CHD1, these findings collectively suggest that distinct CHD1-associated alterations of genomic structure evolve during and are required for the development of PCa.

The level of protection to fauna provided by tree cavities during wildland fires is not well understood. Here we present a model for estimating the transport of combustion gases into cylindrical, single-entrance cavities during exposures caused by different wildland fire scenarios. In these shelters, the entrance occurs near the top of the cavity. This empirical model was developed from a suite of numerical experiments using the National Institute of Standards and Technology's Fire Dynamics Simulator, which spanned a range of entrance diameters, wind speeds, gas temperatures, and vertical angles of in- cidence. To evaluate the model's predictions, it was used to replicate, with great accuracy, a time series of carbon monoxide (CO) concentrations in a controlled experiment where a fabricated cylindrical cavity was exposed to combustion products. The time constant for cavity filling is proportional to the ratio of cavity volume to entrance area. Hot gases lead to signifi- cant stratification within the cavity during exposures. To demonstrate the model's potential use in predicting faunal expo- sures in the context of land management, we show that the model can be used to estimate dosage within red-cockaded woodpecker (Picoides borealis Vieillot, 1809) cavities without requiring temporally detailed, local measurements of wind speed and combustion product concentrations. Resume : Le degre de protection que procurent les cavites d'arbre pour la faune durant les feux de foret n'est pas bien connu. Nous presentons ici un modele pour l'estimation du deplacement des gaz de combustion dans des cavites cylindri- ques a une seule entree lors d'expositions correspondant a differents scenarios de feux de foret. Dans ces abris, l'entree se trouve pres du haut de la cavite. Ce modele empirique a ete developpe a partir d'une serie d'experiences numeriques reali- sees a l'aide du simulateur de dynamique du feu du National Institute of Standards and Technology, qui permet de tester une gamme de diametres d'entree, de vitesses du vent, de temperatures des gaz et d'angles verticaux d'incidence. Pour eva- luer les predictions du modele, le simulateur a ete utilise pour repeter, avec une grande exactitude, une serie temporelle de concentrations de monoxyde de carbone (CO) dans une experience en conditions controlees qui consistait a exposer une ca- vite cylindrique usinee a des produits de combustion. La constante de temps pour le remplissage de la cavite est proportion- nelle au ratio du volume de la cavite sur l'aire de l'entree. Les gaz chauds provoquent une importante stratification dans la cavite durant l'exposition. Pour demontrer la possibilite d'utiliser le modele pour predire les expositions de la faune dans un contexte d'amenagement du territoire, nous montrons que le modele peut etre utilise pour estimer les doses dans des cavites de pic a face blanche (Picoides borealis Vieillot, 1809) et ce, sans faire appel a des mesures detaillees dans le temps, a des mesures de vitesse locale du vent ou a des concentrations de produits de combustion. (Traduit par la Redaction)

Faunal injury and mortality in wildland fires is a concern for wildlife and fire management although little work has been done on the mechanisms by which exposures cause their effects. In this paper, we use an integral plume model, field measurements, and models of carbon monoxide and heat effects to explore risk to tree-roosting bats during prescribed fires in mixed-oak forests of southeastern Ohio and eastern Kentucky. Tree-roosting bats are of interest primarily because of the need to mitigate risks for the endangered Indiana bat ( Myotis sodalis ), our focal species. Blood carboxyhemoglobin concentrations predicted from carbon monoxide data supplemented by model output only approached critical levels just above flames in the most intense fires. By contrast, an ear-heating model driven by plume model output suggested that injury to the bat’s thermally thin ears would occur up to heights similar to those of foliage necrosis, an effect for which predictive relationships exist. Risks of heat injury increase with fireline intensity and decrease with both roost height and ambient wind. Although more information is needed on bat arousal from torpor and behavior during fires, strategies for reducing the risk of heat injury emerge from consideration of the underlying causal processes.

Current operational methods for predicting tree mortality from fire injury are regression-based models that only indirectly consider underlying causes and, thus, have limited generality. A better understanding of the physiological consequences of tree heating and injury are needed to develop biophysical process models that can make predictions under changing or novel conditions. As an illustration of the benefits that may arise from including physiological processes in models of fire-caused tree mortality, we develop a testable, biophysical hypothesis for explaining pervasive patterns in conifer injury and functional impairment in response to fires. We use a plume model to estimate vapor pressure deficits (D) in tree canopies during surface fires and show that D are sufficiently high to cause embolism in canopy branches. The potential implications of plume conditions and tree response are discussed.

The maximum temperatures of thermocouples, temperature-sensitive paints, and calorimeters exposed to flames in wildland fires are often called “fire temperatures” but are determined as much by the properties and deployment of the measurement devices as by the fires themselves. Rather than report device temperatures that are not generally comparable among studies, we show that maximum and time-integrated temperatures of relatively thick (4.8 mm diameter) type-K thermocouple probes (TCPs) can be calibrated to estimate fuel consumption and fire line intensity in surface fires. Although reporting standard fire characteristics is an improvement over reporting device temperatures, TCPs are not ideal instruments for monitoring surface fires, because they provide only point estimates of fire behavior and must be calibrated for different fire environments, TCP characteristics, and deployments. To illustrate how TCPs respond to fires and to point the way towards a more general calibration method, we report results from a numerical model that accurately simulated TCP response to a spreading surface fire.

Data from 69 experimental, small-plot fires are used to describe relationships among fire intensity, bark-surface heat flux, and depth of necrosis in stem tissue for red maple (Acer rubrum L.) and chestnut oak (Quercus prinus L.). A tetrazolium staining technique was used to determine the depth of necrosis in tree boles subjected to fires with intensities of 20 to 2000 kW/m. Over a range of bark moistures (28%–83%) and bole diameters (3–20 cm), depth of necrosis appears to be primarily a function of fire intensity, flame residence time at the stem, and the corresponding time-integrated heat flux at the bark surface. Our results, along with known relations between bole diameter and bark thickness, and improved models of fire behavior and heat transfer, may be useful for estimating tree mortality resulting from prescribed fires.

Under normal conditions genomic CpG islands are not methylated. Hypermethylation of a CpG island in the 5' regulatory region of a gene has the capacity to silence gene transcription. Recently, hypermethylation of a CpG island at D17S5 on chromosome 17P13.3 has been shown to be a frequent tumor-specific event. When it has been observed, hypermethylation of D17S5 occurs solely in neoplastic tissues. Consequently, it has been hypothesized that hypermethylation of D17S5 may be an important carcinogenic event in the organs in which it occurs (colon, kidney, and brain). In this study we examine D17S5 hypermethylation in DNA from the prostate, a gland which is unique in that it undergoes hyperplastic or neoplastic growth or both in virtually all aging men.The methylation sensitive restriction enzyme Notl, a cDNA probe specific for the D17S5 locus, and Southern blotting were used to assay for hypermethylation of D17S5 in DNA derived from normal, benign hyperplastic and malignant prostate tissues.We find that methylation of Notl restriction sites at D17S5 is a very common occurrence in prostate cancers (25 of 26 cases examined). Surprisingly, we found that methylation of these sites at D17S5 also occurred in histologically normal prostate and benign hyperplastic (BPH) tissue from glands which both did and did not contain cancer. In contrast, seminal vesicle, an androgen-dependent male sex accessory tissue that rarely undergoes pathological overgrowth, was devoid of hypermethylation at this locus. CONCLUSIONS. These data demonstrate that hypermethylation of D17S5 is a tissue-specific event in prostate DNA, and we hypothesize that methylation of this and/or related loci may play a role in the extreme predilection of this gland to neoplastic growth.

Molecular biologic studies have now identified a number of important genetic and epigenetic mechanisms that cause alterations in growth and differentiation genes in prostate cancer. In addition to DNA deletion and point mutation, DNA methylation represents a new paradigm for the inactivation of tumor suppressor or growth suppressor genes. The identification of new genes, including a prostate cancer susceptibility locus, may furnish further insight into the molecular characteristics of prostate cancer and permit the early identification of affected individuals.

A new Taylor-Galerkin finite element method and adaptive, time-iterative scheme are developed for simulating single-carrier submicron-scale semiconductor device transport with the hydrodynamic model under the assumptions of parabolic energy bands. Boundary conditions are applied using characteristic projections. Numerical studies are conducted to investigate the sensitivity of the given model to some of the parameters contained in typical heat flux and relaxation time models for a one-dimensional, representative test problem.

A number of genetic changes have been documented in prostate cancer, ranging from allelic loss to point mutations and changes in DNA methylation patterns (summarized in Fig. 1). To date, the most consistent changes are those of allelic loss events, with the majority of tumors examined showing loss of alleles from at least one chromosomal arm. The short arm of chromosome 8, followed by the long arm of chromosome 16, appear to be the most frequent regions of loss, suggesting the presence of novel tumor suppressor genes. Deletions of one copy of the Rb and p53 genes are less frequent, as are mutations of the p53 gene, and accumulating evidence suggests the presence of an additional tumor suppressor gene on chromosome 17p, which is frequently inactivated in prostate cancer. Alterations in the E-cadherin/alpha-catenin-mediated cell-cell adhesion mechanism appear to be present in almost half of all prostate cancers and may be critical to the acquisition of metastatic potential of aggressive prostate cancers. Finally, altered DNA methylation patterns have been found in the majority of prostate cancers examined, suggesting widespread alterations in methylation-modulated gene expression. The presence of multiple changes in these tumors is consistent with the multistep nature of the transformation process. Finally, efforts to identify prostate cancer susceptibility loci are under way and may elucidate critical early events in prostatic carcinogenesis.

The effects of reducing external Na+ ([Na+]o) and increasing internal Na+ ([Na+]i) on evoked tension in arterial rings were correlated with intracellular Ca2+ transients evoked in cultured A7r5 cells. Contractions were elicited in rings of rat aorta and a small mesenteric artery branch by serotonin (5-HT), arginine vasopressin (AVP), or a high K+ concentration ([K+]o). Contractions were augmented by reduction of [Na+]o (replaced by Li+ or N-methylglucamine) or inhibition of the Na+ pump (to raise [Na+]i). These effects were dependent on external Ca2+. Ca2+ transients were measured by digital imaging of fura-2/AM-loaded A7r5 cells. The apparent free Ca2+ concentration [( Ca2+]app) in eight unstimulated cells was 110 +/- 10 nM in the peripheral cytoplasm. Activation of A7r5 cells by 5-HT, AVP, or high [K+]o evoked transient increases in [Ca2+]app. Lowering [Na+]o or raising [Na+]i elevated [Ca2+]app significantly in unstimulated cells and increased the amplitude and duration of the Ca2+ transients. These effects required external Ca2+. Caffeine (5-10 mM) reduced [Ca2+]app in resting cells and attenuated the vasoconstrictor-evoked Ca2+ transients in low [Na+]o. The data imply that Na+ gradient reduction promotes Ca2+ entry and slows Ca2+ extrusion via Na(+)-Ca2+ exchange. This increases cytosolic free Ca2+ and the intracellular Ca2+ stores so that more Ca2+ is delivered to the contractile apparatus during cell activation. Such augmented Ca2+ transients could induce the amplified vasoconstrictor-evoked contractions observed in arterial rings under reduced Na+ gradient conditions.

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in vascular smooth muscle (VSM). We employed the fluorescent Ca2(+)-indicator, Fura-2, and digital imaging microscopy to study the spatial distribution of intracellular Ca2+ in cultured A7r5 cells and the changes evoked by activation with 5-HT. Several methodological considerations that affect the temporal and spatial resolution of Ca2+ images have been addressed. These include: cytoplasmic distribution of Fura-2, wavelength selection for ratio imaging, signal:noise ratio measurement and the effect of [Ca2+] on the limits of detectability under conditions in which [Ca2+] is changing. The distribution of apparent free Ca2+, [Ca2+]App, in A7r5 cells was heterogeneous. This reflects, in part, different pools of intracellular Ca2+. [Ca2+]App was lowest in the nucleus (113 +/- 14 nM; n = 20 cells) and highest in the organelle-rich perinuclear region (228 +/- 12; n = 20), while the surrounding cytoplasmic area (containing relatively few organelles) had intermediate [Ca2+]app levels (150 +/- 13; n = 20). 5-HT (1 microM) evoked transient increases in [Ca2+]App that began within 11 s as relatively modest elevations of [Ca2+]App in the periphery, near the sarcolemma, and subsequently spread to the entire cell, reaching a peak within 18-24 s. At the peak of the Ca2+ transients, [Ca2+]App was highest in the perinuclear region where it sometimes exceeded the maximal detectable levels of the system (1.9 microM). The average peak Ca2+ transient amplitude in the non-nuclear cytoplasm was 1083 +/- 208 nM (1 microM 5-HT; n = 20 cells). Despite the continued presence of 5-HT following the Ca2+ transients, [Ca2+]App then returned to pre-stimulation levels within 5 min. These observations indicate that digital imaging microscopy enables the study of subcellular regulation of intracellular Ca2+ in VSM. The results provide new insights into the role of localized changes in Ca2+ in the regulation of VSM contractility.

Cancers emerge from an on-going Darwinian evolutionary process, often leading to multiple competing subclones within a single primary tumour. This evolutionary process culminates in the formation of metastases, which is the cause of 90% of cancer-related deaths. However, despite its clinical importance, little is known about the principles governing the dissemination of cancer cells to distant organs. Although the hypothesis that each metastasis originates from a single tumour cell is generally supported, recent studies using mouse models of cancer demonstrated the existence of polyclonal seeding from and inter-clonal cooperation between multiple subclones. In this study, we sought definitive evidence for the existence of polyclonal seeding in human malignancy and to establish the clonal relationship among different metastases in the context of androgen-deprived metastatic prostate cancer. Using whole genome sequencing, we characterised multiple metastases arising from prostate tumours in ten patients. Integrated analyses of subclonal architecture revealed the patterns of metastatic spread in unprecedented detail. Metastasis-to-metastasis spread was found to be common, either through de novo monoclonal seeding of daughter metastases or, in five cases, through the transfer of multiple tumour clones between metastatic sites. Lesions affecting tumour suppressor genes usually occur as single events, whereas mutations in genes involved in androgen receptor signalling commonly involve multiple, convergent events in different metastases. Our results elucidate in detail the complex patterns of metastatic spread and further our understanding of the development of resistance to androgen deprivation therapy in prostate cancer. Citation Format: Gunes Gundem, Peter Van Loo, Barbara Kremeyer, Ludmil B. Alexandrov, Jose M.C. Tubio, Elli Papaemmanuil, Daniel S. Brewer, Heini Kallio, Gunilla Högnäs, Matti Annala, Kati Kivinummi, Victoria Goody, Calli Latimer, Sarah O'Meara, Kevin J. Dawson, William Isaacs, Michael R. Emmert-Buck, Matti Nykter, Christopher Foster, Zsofia Kote-Jarai, Douglas Easton, Hayley C. Whitaker, David E. Neal, Colin S. Cooper, Rosalind A. Eeles, Tapio Visakorpi, Peter J. Campbell, Ultan McDermott, David C. Wedge, G. S. Bova. The evolutionary history of lethal metastatic prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 956. doi:10.1158/1538-7445.AM2015-956

The bulk of valuable molecular research published in prostate cancer to date is based on radical prostatectomy specimens from men with prostate cancer, or on metastatic prostate cancer tissues collected at surgery or autopsy. The majority of normal control tissues for such studies come from the same radical prostatectomy specimens, and less commonly from TURP (transurethral resection of the prostate) samples from men with benign prostatic hyperplasia, cystoprostatectomy specimens in men with bladder cancer, or rarely from autopsy samples collected no sooner than 12-24 hours after death, although documentation is usually absent or scant2-5. Using noncancerous areas of radical prostatectomy specimens or samples from men with other GU diseases as normal control tissue has critical drawbacks. Prostate cancer is often multifocal when it arises6-11, and there is strong evidence that precancerous changes occur gradually over a period of years in men predisposed to the disease5,12-14. Based on these observations, the noncancerous areas of prostates of men with prostate cancer are probably in a state more prone to the development of cancer than men of the same age and race without prostate cancer. Therefore, having a population-based sample of prostates from men of various ages and races is essential for us to understand whether this critical difference exists.

Recent advances in prostate biology suggest that stromal cells surrounding prostate epithelia may play a key role in permitting or stimulating epithelial cells to lose control and form precancerous and cancerous lesions. The goal and purpose of this Hypothesis Development project is to obtain preliminary data sufficient to begin to explore the role of prostate stromal cells in prostate carcinogenesis under conditions as rigorously controlled as current technology allows. This hypothesis development project tests the feasibility of identification, laser capture microdissection, and expression analysis of prostate-stroma specific cells in normal and cancerous prostates, and aims to develop preliminary data sufficient to identify potential differences in stromal RNA expression in normal and cancerous prostate tissue. The studies found that it is difficult but not impossible to histologically identify prostate zones with an acceptable degree of confidence in frozen tissues, thus eliminating the need to attempt expression studies in fixed tissues with their attendant biases. Laser capture microdissection of stromal tissue was completed for 5 normal prostates from men across the age range and from two racial groups (African Americans and Caucasians), and for prostates from men of similar ages with adenocarcinoma identified distant from the area of dissection. High Quality RNA was isolated, and duplicate Affymetrix Plus 2.0 chip analysis was recently completed. RNA expression data analysis has just begun. Selective validation testing of these analyzed data is planned and will be the final step of this painstaking hypothesis development project. The key research accomplishments during this period were as follows: identification of appropriate normal tissues for study, determination of frozen tissues as the best foundation for study, completion of tedious laser capture microdissections, and completion of Affymetrix Gene Chip hybridizations.

Osteoblastic metastases are common in lethal prostate cancer. Effective therapy for bone metastases is lacking. Thus, developing an appropriate in vitro screening system is critical to prioritize which of the newly developed agents should undergo additional expensive and time-consuming in vivo evaluation in bone metastases animal models. In the past, such in vitro screening evaluated the response of prostate cancer cells to chemotherapeutic agents in monoculture without the presence of osteoblasts. In such monoculture, prostate cancer cells have a high (i.e.,90%) proliferative growth fraction. In contrast, the growth fraction (i.e., mean: 7.1 +/- 0.8%; median: 3.1%) in 117 metastatic sites of prostate cancer obtained from 11 androgen ablation failing patients at "warm" autopsy was found to be10-fold lower. To better mimic the lower growth fraction observed clinically, LNCaP human prostate cancer cells were cocultured with membrane-separated hFOB human osteoblasts. Such coculturing significantly lowered the growth fraction of the LNCaP cells (i.e., from90 to30%) without enhancing their low rate (i.e.,5%) of apoptosis. This lowering of the growth fraction was documented using flow cytometry, Ki-67 immunohistochemistry, and 5-bromo-2-deoxyuridine incorporation. Using RNase protection assays, it was documented that coculture with osteoblasts causes enhanced p53, p27, and p21 expression leading to a decrease in the number of LNCaP cells entering the cell cycle (i.e., enhanced number of LNCaP cells in G(0)-G(1) and a decrease in S and G(2)-M and thus the growth fraction). This osteoblast-induced enhanced G(0)-G(1) checkpoint control affected the chemosensitivity of LNCaP cells. This was documented by coculturing LNCaP cells with hFOB cells to condition the medium for 3 days to lower the growth fraction to30% before exposing the LNCaP cells for 48 h to various concentrations of Taxol, doxorubicin, or thapsigargin (TG). In standard high (i.e.,90%) growth fraction cultures (i.e., cultures in the absence of osteoblast-conditioned medium), there was a dose-dependent and significant (P0.05) increase in apoptosis of LNCaP cells exposed to Taxol or doxorubicin. In contrast, even the highest dose of Taxol (1 microM) did not enhance apoptosis of lower growth fraction LNCaP cells cultured in osteoblast-conditioned medium. Similarly, only the highest concentration of doxorubicin (1 microM) enhanced apoptosis in lower growth fraction cells. In contrast, 100 nM TG induced high levels of apoptosis in both lower and high-growth fraction LNCaP cultures. These results demonstrate that the osteoblast/LNCaP coculture system is a better in vitro screen than monoculture to identify proliferation-independent agents for the treatment of prostate cancer bone metastases, and TG is such an agent.

We used RDA to identify homozygously deleted DNA clones within metastatic prostate cancer xenograft DNA, and found that all of the homozygously deleted clones mapped to a single region of chromosome 12p. We confirmed that the metastatic lesions from this patient also contained this homozygous deletion, and then analyzed this region in a series of 41 metastatic tumors from 19 patients, where we found that 50% of patients' tumors show loss. This 12p deletion occurs with similar frequency in caucasians and african-americans based on our small sample. We mapped this genomic region, and found that two previously identified potential candidate genes ETV(tel) and CDKN1B(p27) are located there. Analysis of these genes has revealed no sequence changes consistent with an important tumor suppressor role. Extensive analysis of p27 for possible mutational or methylation changes was negative. Major observations from our work include the finding of a homozygous deletion on chromosome 12p in metastatic prostate cancer, considered to be a major indicator that an important gene is nearby on chromosome 12p. Further analysis of this region revealed unexpected high degree of allelic loss in this region. Specific genes in the region were analyzed, and p27 appears to be the likely target, inactivated by loss of one copy. Further analysis was curtailed due to lack of funding.

E-cadherin and alpha-catenin are components of adherens junctions which mediate calcium-dependent, cell-cell adhesion in a homotypic manner. Both these molecules have been defined as useful tumor markers as their altered expression correlates with increased tumor aggressiveness and dedifferentiation. More recently, alterations of a third component of adherens junctions, beta-catenin, have been observed to play a role in several human cancers. Dysregulation of beta-catenin, either by direct mutation or by defects in interacting pathways/regulators, can result in its cytoplasmic accumulation and nuclear translocation. In the nucleus, beta-catenin forms a transcriptional complex capable of upregulating target genes, many of which encode proliferative factors. Given its oncogenic activity and connection to human cancer, we examined the beta-catenin gene and its expression in prostate cancer.By single-stranded conformational polymorphism (SSCP) and DNA sequencing analyses, we screened exon 3 of beta-catenin from a panel of 81 primary tumors obtained at radical prostatectomy, 22 lymph node metastases from untreated patients, and a unique set of 61 metastatic tissues from 19 patients who died of hormone-refractory disease.We found putative activating mutations (missense and deletion) at a rate of 5% (7/138). One patient had the same 72 base pair deletion in each of nine separate metastases examined, indicating that this change was associated with a clonal population of metastatic cells.Immunohistological staining of mutation-positive tumors demonstrated beta-catenin accumulation and nuclear localization in a heterogeneous fashion. Consistent with this in vivo finding, our in vitro analyses demonstrate that certain mutations can result in increased beta-catenin nuclear activity in prostate cancer cell lines. These data implicate the beta-catenin signaling pathway in the development of a subset of prostate cancers.

Advanced hormone-independent prostate cancer is characterized by a significant loss of androgen receptor (AR) expression in 20-30% of the tumors. The transcriptional block underlying this phenomenon is not known, but we have proposed that methylation of CpG sites in the AR promoter may reversibly inactivate transcription of the AR (D. F. Jarrard et al, Cancer Res., 58: 5310-5314, 1998). In this study, detailed methylation analysis using bisulfite sequencing was performed on a series of AR expression-positive and -negative prostate cancer cells. We found that methylation of several consensus sequences in the AR promoter (from -131 to -121 and +44 to +54) are tightly linked to the loss of AR expression in metastatic hormone-independent prostate cancer cell lines. These consensus sites of methylation correlate with the minimal promoter region critical for AR transcription. In human tissues, no methylation was demonstrated in normal or primary prostate cancers that express the AR. Four of 15 tumors obtained from men who had died from hormone-independent prostate cancer demonstrated a significant loss of AR expression immunohistochemically and two (50%) of these AR-negative tumors contained AR methylation. We conclude that the AR promoter contains specific CpG methylation hot spots that are markers for gene silencing. Furthermore, AR methylation may represent a phenotype important in the development of hormone independence in a subset of advanced prostate cancer in which AR expression is lost. The finding of AR methylation also represents the first report of aberrant methylation on an X-linked gene associated with a somatic male cancer.

Our laboratory has recently identified a 1 to 2 Mb homozygous deletion at 12p12-13 in a prostate cancer specimen and determined that the p27/kip1 gene lies within the deletion. While immunohistochemical analysis has implicated p27/kip1 in prostate carcinoma, no previous studies had identified genetic abnormalities at this locus. Here, we examined primary and metastatic prostate tumors to determine if allelic loss occurs at this locus in localized disease and if it increases the risk of metastatic, high stage or high-grade disease.DNA was extracted from prostate tumors and normal tissue of 99 patients. 60 tumors were primary, 20 were metastatic pelvic lymph nodes, and 19 were distant metastases. Multiple metastases were analyzed from 11 of 19 patients with metastatic disease. Polymorphic markers spanning our region of interest were PCR amplified from tumor and normal DNA. PCR products were then scored for allelic loss.Loss of heterozygosity (LOH) was identified in 14/60 (23%) primary tumors, 6/20 (30%) lymph node metastasis, and 9/19 (47%) distant metastases. The difference between primary and distant metastatic disease was statistically significant (p = 0.045, Fisher's exact test). The pattern of LOH was identical in all metastatic sites obtained from individual patients, indicating that genetic loss occurred prior to metastasis. Subset analysis of the 60 primary tumors demonstrated no association between LOH and adverse pathological feature [nodal involvement, seminal vesicle invasion, margin positivity, high Gleason score (7-10)].Demonstrating that 12p12-13 LOH is a prominent feature of primary prostate tumors and that multiple metastatic foci have an identical LOH pattern, provides evidence that gene inactivation in this region occurs prior to metastasis. In addition, the strong association between LOH and distant metastasis raises the possibility that mutational inactivation of a gene at 12p12-13, possibly p27/kip1, plays a pivotal role in the development of metastatic disease.

The identification of homozygous deletions in malignant tissue is a powerful tool for the localization of tumor suppressor genes. Representational difference analysis (RDA) uses selective hybridization and the polymerase chain reaction (PCR) to isolate regions of chromosomal loss and has facilitated the identification of tumor suppressor genes, such as BRCA2 and PTEN. We have recently identified a 1-5-cM homozygous deletion on 12p12-13 in a prostate cancer xenograft and found that 47% of patients who died of prostate carcinoma demonstrate focal loss of heterozygosity (LOH) in this region in metastatic deposits. We have now characterized the region of interest by assembling a yeast artificial chromosome (YAC) contig spanning the homozygous deletion and identifying which known genes and expressed sequence tags (EST) lie within the homozygous deletion. A rib metastasis was harvested at autopsy and placed subcutaneously in a male SCID mouse. Genomic DNA from this xenograft and from the patient's normal renal tissue was extracted. Multiplex PCR, with the xenograft and normal DNA used as template, was performed using primers for loci on the Whitehead contig 12.1 believed to be near our region of interest. We found that our deletion lay in a 1-2-Mb interval between WI-664 and D12S358. We then used the same primers to construct a YAC contig across the homozygous deletion. PCR amplification of YAC DNA, using primers for the genomic sequences of known genes and ESTs reported to lie on 12p12-13, was used to identify candidate genes that lay within the deletion. Duplex PCR, with control primers known not to be deleted in the xenograft, was used to confirm that both the CDKN1B and ETV6 genes were homozygously deleted in the xenograft. Mutations in either or both of these genes may play an important role in metastatic prostate carcinoma.