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A unique feature of Hodgkin lymphoma (HL) is the presence of CD4+ T cells that surround, protect, and promote survival of tumor cells. The adhesion molecules involved in this so-called T-cell rosetting are important components of the immunological synapse (IS). However, it is unknown whether this synapse is fully assembled and leads to T-cell activation by enabling interaction between the T-cell receptor (TCR) and human leukocyte antigen class II (HLA-II). We established a novel rosetting model by coculturing HLA-II–matched peripheral blood mononuclear cells with HL cell lines and showed IS formation with activation of rosetting T cells. HLA-II downregulation by class II transactivator knockout did not affect the extent of rosetting, but almost completely abrogated T-cell activation. Intriguingly, the level of CD58 expression correlated with the extent of rosette formation, and CD58 knockout or CD2 blockade reduced both rosette formation and T-cell activation. The extension of our findings to primary HL tissue by immunohistochemistry and proximity ligation assays showed interaction of CD2 with CD58 and of TCR-associated CD4 with HLA-II. In conclusion, T-cell rosetting in HL is established by formation of the IS, and activation of rosetting T cells critically depends on the interaction of both TCR-HLA-II and CD2-CD58.

Development of heterotic hybrids is the most efficient approach to solve the problem of increasing the yield of rapeseed ( Brassica napus L.), a leading oilseed crop. The cytoplasmic male sterility (CMS), widely used in rapeseed hybrid seed production, makes it possible to control hybridization between female and male lines. A review of publications on the nature of CMS systems in rapeseed and their utilization in breeding is presented. In rapeseed there are more than 10 known CMS systems of alloplasmic and homoplasmic origin. The male sterility character proved to be determined by chimeric mitochondrial genes, characterized by the presence of novel transcribed open reading frames ( orf ). Mitochondrial CMS genes associated with nap , pol , ogu and Nsa sterility types as well as nuclear Rf genes for pollen fertility restoration were identified. Molecular marker systems for identification of CMS-inducing and male fertility restoring genes were developed. The ogu , pol , MSL and inap CMS systems are commonly used for producing industrial rapeseed hybrids. The State Register of the Russian Federation for 2019 contains rapeseed hybrids of only foreign origin. Main achievements in domestic rapeseed hybrid production are highlighted. Research and breeding institutions developed new source material for rapeseed hete rotic hybrid breeding in various regions of the country. The sterility and fertility restoration sources were received from Canadian and French institutions as well as from domestic working collections. The yield structure traits did not deteriorate after transferring hybrid maternal lines to the sterile cytoplasm, while the glucosinolate content increased when pollen fertility restoring genes were transferred into paternal lines. Dihaploid (androclinium) lines and in vitro culture of unfertilized ovules were used to accelerate the breeding process. Experimental hybrids were developed using pol and ogu CMS.

Relevance. High lycopene fruit content has been regarded as a very important genetic trait in tomato breeding. Use lycopene molecular markers in combination with conventional breeding techniques allowed us to create hybrids with high lycopene accumulation, excellent organoleptic qualities, high yield production and resistance to pathogens, and to effectively optimize our breeding programmes for commercial greehouses production.Material and Methods. In this study tomato samples including selected lines and hybrids with various allelic combinations of genes determining carotene accumulation, and other genetic traits, such as disease resistance and yield production were tested. Introgression of spontaneous and induced mutations was used to increase carotenoid levels (og and hp) and improve fruit technological qualities (nor, alc, rin). The research material was tomato collection, mutants, breeding lines and hybrids listed in the State Register Russian Federation tomato hybrids of breeding SS Agrofirm "Ilyinichna" VNIIO branch of the All-Russian Scientific Research Institute of Vegetable Growing – Branch of the FSBSI Federal Scientific Vegetable Center. DNA typing of fruit quality genes was performed at the Institute of Genetics and Cytology of the National Academy of Sciences of Belarus.Results. New domestic hybrids for industrial greenhouses, which characterised by improved organoleptic qualities and technological traits were developed with the help of phasedcross-breeding that allowed to combine the genes nor, rin, alc, leading to an extension of the shelf life with the genes B, og, hp1, etc., contributing to an increase in carotenoid content in fruits. It was established that for targeted selection and hybridization, despite the negative influence of the nor, rin, alc genes it is possible to raise the level of carotenoids to average values. Correlation between lycopene concentration in fruits and high temperature and level of insolation was confirmed. It was shown that pink-fruited forms contain significantly more lycopenethanred-fruitedones. Different all eliccombinations of structural genes involved in carotenoids biosynthesis and regulatory genes that provided maximal accumulation of lycopene in hybrid swithred and pink fruits were revealed. Hybrids with the combination of high concentrations of sugar (° Brix), dry matter and maximal lycopene values, combined defining excellent taste were selected: Prekrasnaiya lady, Olya, Quadrille, Victoria. New F1 hybrids one for industrial greenhouses: G950, G956, G960, Magistral and pink fruited G12897, surpassed the Dutch standard in productivity up to 21%, and in tastes/organoleptic qualities for 1-1.8 points.

Growth arrest at the secondary growth plate, also known as the acrophysis, is a rare phenomenon with only very few known published case reports. We report on a case of formation of ghost secondary ossification centers at the acrophyses of the knee joint in a 14-year-old female, who survived early childhood acute lymphoblastic leukemia. The patient suffered from severe side effects from both disease and subsequent treatment strategies with a 10-month immobilization period as a consequence at the age of 3 years. The ghost secondary ossification centers were encountered on radiographs and MRI 10 years later, when she presented for evaluation of chronic pain in her left knee related to sports activities, due to a meniscal cyst. Awareness of this phenomenon is nevertheless important, because it seems that endochondral bone growth recovery at the acrophyses might be different from recovery in physes, because we found no concomitant sequelae of growth arrest in the metaphyses. ispartof: SKELETAL RADIOLOGY vol:49 issue:12 pages:2095-2099 ispartof: location:Germany status: published

Dinoflagellates in the family Symbiodiniaceae are obligate endosymbionts of diverse marine invertebrates, including corals, and impact the capacity of their hosts to respond to climate change-driven ocean warming. Understanding the conditions under which increased genetic variation in Symbiodiniaceae arises via sexual recombination can support efforts to evolve thermal tolerance in these symbionts and ultimately mitigate coral bleaching, the breakdown of the coral-Symbiodiniaceae partnership under stress. However, direct observations of meiosis in Symbiodiniaceae have not been reported, despite various lines of indirect evidence that it occurs. We present the first cytological evidence of sex in Symbiodiniaceae based on nuclear DNA content and morphology using Image Flow Cytometry, Cell Sorting and Confocal Microscopy. We show the Symbiodiniaceae species, Cladocopium latusorum, undergoes gamete conjugation, zygote formation, and meiosis within a dominant reef-building coral in situ. On average, sex was detected in 1.5% of the cells analyzed (N = 10,000–40,000 cells observed per sample in a total of 20 samples obtained from 3 Pocillopora colonies). We hypothesize that meiosis follows a two-step process described in other dinoflagellates, in which diploid zygotes form dyads during meiosis I, and triads and tetrads as final products of meiosis II. This study sets the stage for investigating environmental triggers of Symbiodiniaceae sexuality and can accelerate the assisted evolution of a key coral symbiont in order to combat reef degradation., DIANAS, DETECCIÓN INNOVADORA DE PROLIFERACIONES ALGALES TÓXICAS: UNA NECESIDAD FRENTE AL CALENTAMIENTO GLOBAL

Mesenchymal cells of diverse origins differ in gene and protein expression besides producing varying effects on their organ-matched epithelial cells’ maintenance and differentiation capacity. Co-culture with rodent’s tissue-specific pancreatic mesenchyme accelerates proliferation, self-renewal, and differentiation of pancreatic epithelial progenitors. Therefore, in our study, the impact of three-dimensional (3D) co-culture of human fetal pancreatic-derived mesenchymal cells (hFP-MCs) with human embryonic stem cell-derived pancreatic progenitors (hESC-PPs) development towards endocrine and beta cells was assessed. Besides, the ability to maintain scalable cultures combining hFP-MCs and hESC-PPs was investigated. hFP-MCs expressed many markers in common with bone marrow-derived mesenchymal stem cells (BM-MSCs). However, they showed higher expression of DESMIN compared to BM-MSCs. After co-culture of hESC-PPs with hFP-MCs, the pancreatic progenitor (PP) spheroids generated in Matrigel had higher expression of NGN3 and INSULIN than BM-MSCs co-culture group, which shows an inductive impact of pancreatic mesenchyme on hESC-PPs beta-cells maturation. Pancreatic aggregates generated by forced aggregation through scalable AggreWell system showed similar features compared to the spheroids. These aggregates, a combination of hFP-MCs and hESC-PPs can be applied as an appropriate tool for assessing endocrine-niche interactions and developmental processes by mimicking the pancreatic tissue.

Essential tremor (ET) is a common movement disorder affecting the performance of various daily tasks, including drawing. While spiral-drawing task characteristics have been described among patients with ET, research about the significance of the drawing direction of both spiral and lines tasks on the performance process is scarce. This study mapped inter-group differences between people with ET and controls related to drawing directions and the intra-effect of the drawing directions on the tremor level among people with ET. Twenty participants with ET and eighteen without ET drew spirals and vertical and horizontal lines on a digitizer with an inking pen. Time-based outcome measures were gathered to address the effect of the drawing directions on tremor by analyzing various spiral sections and comparing vertical and horizontal lines. Significant group differences were found in deviation of the spiral radius from a filtered radius curve and in deviation of the distance curve from a filtered curve for both line types. Significant differences were found between defined horizontal and vertical spiral sections within each group and between both line types within the ET group. A significant correlation was found between spiral and vertical line deviations from filtered curve outcome measures. Achieving objective measures about the significance of drawing directions on actual performance may support the clinical evaluation of people with ET toward developing future intervention methods for improving their functional abilities.

Simple Summary Head and neck cancers can be divided in two major groups according to their risk factors, being high-risk human papillomavirus related (HPV-positive) and alcohol and tobacco related (HPV-negative) head and neck cancers. The majority of the locally advanced patients are treated with radiotherapy. However, up to 50% of these patients show local recurrences. The majority of these recurrences are linked to resistance to radiotherapy treatment. It is known that the response to DNA damage, also a process called the DNA damage response, is an important factor that determines the effectivity of radiotherapy. Here, we assessed the role of the DNA damage response in the resistance process to radiotherapy of head and neck cancers, by generating head and neck cancer cells resistant to radiotherapy. We show that the DNA damage response is differentially involved in the resistance process of HPV-positive and HPV-negative head and neck cancer cells. More specifically, HPV-positive radiotherapy-resistant cells showed increased ability to repair the DNA damage induced by radiotherapy. HPV-negative radiotherapy-resistant cells showed increased capacity to replicate after radiotherapy treatment. Despite this difference, inhibition of the DNA damage response enhanced the effect of radiotherapy in both groups. Abstract Radioresistance is a major cause of recurrences and radiotherapy (RT) failure in head and neck squamous cell carcinoma (HNSCC). DNA damage response (DDR) is known to be important for RT response, but its role in radioresistance is not fully understood. Here, we assessed the role of DDR in the radioresistance process of HNSCC by generating radioresistant clones from both HPV-positive SCC154 and HPV-negative SCC61 cells. We show that fractionated RT decreased RT response of HPV-positive and HPV-negative radioresistant clones in vitro and in vivo. Moreover, HPV-positive and HPV-negative radioresistant clones were characterized by differential DDR response. HPV-positive radioresistant clones showed less residual double-strand break damage and increased G2/M arrest recovery after RT, indicating an acquisition of increased DDR kinetics. In contrast, HPV-negative radioresistant clones showed less micronucleated cells after RT and increased survival upon checkpoint inhibition, indicating an increased replicative capacity. Inhibiting key factors of DDR in combination with RT rescued the radioresistant phenotype of both HPV-positive and HPV-negative radioresistant clones. Altogether, our results not only highlight the importance of DDR response in the radioresistance process of HPV-positive and HPV-negative HNSCC, but also provide possibilities for new therapies for HNSCC patients in recurrent settings.

Responses of an individual to food deprivation, such as a 16-h fast, are complex, and are influenced by environmental and genetic factors. Domestication is an ongoing process during which adaptations to changing environments occur over generations. Food deprivation by their caretakers is less for domestic chickens than for their junglefowl ancestors. Unlike domestic chicken, the junglefowl adapted over generations to periods of food deprivation, which may be reflected in differences in metabolic responses to brief periods without food. Here, we compared the blood glucose and plasma levels of non-esterified fatty acids (NEFA) among four populations when deprived of feed for 16 h. The four populations included a domestic White Rock experimental line (LWS) maintained for generations under ad libitum feeding, adult red junglefowl (RJF), and a reciprocal cross of the lines. Although there were significant differences in adult (31-week) body weight between the RJF (683 g) and LWS (1282 g), with the weight of F1 crosses being intermediate, the amount of abdominal fat relative to body weight was similar for all populations. Patterns for blood glucose responses to a glucose bolus after a 16-h fast were similar for the initial and final points in the parental and cross populations. However, RJF reached their peak faster than LWS, with the reciprocal cross intermediate to the parental populations. Plasma NEFA concentrations were higher after the 16-h fast than in fed states, with no population differences for the fasting state. However, in the fed state, NEFA levels were lesser for LWS than for others, which was reflected further in percentage change from fed to fasted. This larger change in LWS suggests differences in mobilization of energy substrates and implies that during domestication or development of the LWS line, thresholds for responses to acute stressors may have increased. Published version

Clubroot disease (causative organism Plasmodiophora brassicae Wor.) is among the most economically important and harmful diseases of the cole crops, and the damage due to this disease may reach up to 50-75% of the yield and even 100% in epiphytotics years. Even resistant varieties become susceptible over the years, because of appearance of the new pathogen races and change of climatic conditions in the main growing areas of the crop. In this context the Laboratory of Plant Immunity and Protection, of Federal State Budgetary Scientific Institution “Federal Scientific Vegetable Center” implements continuous phytoimmunological evaluation of collection and selection specimens and also directional material rather than just annual monitoring of causative organism dissemination in order to find new resistance sources. For this purpose an artificial infection background is used: compost obtained from decomposed nodules on the cabbage roots affected by clubroot disease (infection load 105-106 spores/cm3). The resistance of white cabbage varieties was evaluated during the harvesting period using five-point score of the root system damage, which formed the basis for categorization into resistance groups. For the analysis of artificial background intensity and specimen ranking the individual plants of the white cabbage variety Slava 1305, which is a susceptibility standard, were randomly planted in the entire area of the infection background. The impact of atmospheric conditions in the study year on the results of phytopathological evaluation of cabbage selection specimens against the infection background is demonstrated. Under unfavorable conditions for pathogen development (2014) the most specimens (74%) were categorized as relatively resistant, while in favourable for pathogen year 2015 relatively resistant specimens comprised only 5% of the total number of studied specimens. Since the same specimen may show different level of resistance depending on the year conditions, the stability of character manifestation is the important criterion for identification of the resistance resources. Phytopathological evaluation aimed on selection of clubroot-resistant forms in the Moscow region should last for at least three years even with the use of infection background. Long-lasting evaluation showed that the strains No 234/15,140/14,216/17 exhibiting high resistance to clubroot against artificial infection background regardless of the year conditions are the most valuable for selection. The resistance of white cabbage selection varieties to clubroot disease was studied against the infection background.

International audience; Congestion, related to pressures and/or fluid overload, plays a central role in the pathophysiology, presentation and prognosis of heart failure and is an important therapeutic target. While symptoms and physical signs of fluid overload are required to make a clinical diagnosis of heart failure, they lack both sensitivity and specificity, which might lead to diagnostic delay and uncertainty. Over the last decades, new ultrasound methods for the detection of elevated intracardiac pressures and/or fluid overload have been developed that are more sensitive and specific, thereby enabling earlier and more accurate diagnosis and facilitating treatment strategies. Accordingly, we considered that a state-of-the-art review of ultrasound methods for the detection and quantification of congestion was timely, including imaging of the heart, lungs (B-lines), kidneys (intrarenal venous flow), and venous system (inferior vena cava and internal jugular vein diameter). This article is protected by copyright. All rights reserved.

Breast cancer is one of the most prevalent types of cancers worldwide and yet, its pathophysiology is poorly understood. Single-cell electrophysiological studies have provided evidence that membrane depolarization is implicated in the proliferation and metastasis of breast cancer. However, metastatic breast cancer cells are highly dynamic microscopic systems with complexities beyond a single-cell level. There is an urgent need for electrophysiological studies and technologies capable of decoding the intercellular signaling pathways and networks that control proliferation and metastasis, particularly at a population level. Hence, we present for the first time non-invasive in vitro electrical recordings of strongly metastatic MDA-MB-231 and weakly/non-metastatic MCF-7 breast cancer cell lines. To accomplish this, we fabricated an ultra-low noise sensor that exploits large-area electrodes, of 2 mm2, which maximizes the double-layer capacitance and concomitant detection sensitivity. We show that the current recorded after adherence of the cells is dominated by the opening of voltage-gated sodium channels (VGSCs), confirmed by application of the highly specific inhibitor, tetrodotoxin (TTX). The electrical activity of MDA-MB-231 cells surpasses that of the MCF-7 cells, suggesting a link between the cells’ bioelectricity and invasiveness. We also recorded an activity pattern with characteristics similar to that of Random Telegraph Signal (RTS) noise. RTS patterns were less frequent than the asynchronous VGSC signals. The RTS noise power spectral density showed a Lorentzian shape, which revealed the presence of a low-frequency signal across MDA-MB-231 cell populations with propagation speeds of the same order as those reported for intercellular Ca2+ waves. Our recording platform paves the way for real-time investigations of the bioelectricity of cancer cells, their ionic/pharmacological properties and relationship to metastatic potential.

Accurate quantification of drug effects is crucial for identifying pharmaceutically actionable cancer vulnerabilities. Current cell viability-based measurements often lead to biased response estimates due to varying growth rates and experimental artifacts that explain part of the inconsistency in high-throughput screening results. We developed an improved drug scoring model, normalized drug response (NDR), which makes use of both positive and negative control conditions to account for differences in cell growth rates, and experimental noise to better characterize drug-induced effects. We demonstrate an improved consistency and accuracy of NDR compared to existing metrics in assessing drug responses of cancer cells in various culture models and experimental setups. Notably, NDR reliably captures both toxicity and viability responses, and differentiates a wider spectrum of drug behavior, including lethal, growth-inhibitory and growth-stimulatory modes, based on a single viability readout. The method will therefore substantially reduce the time and resources required in cell-based drug sensitivity screening., Abhishekh Gupta et al. present a normalized drug response (NDR) metric for accurate quantification of drug sensitivity in cell-based high-throughput assays. They show that NDR captures both toxicity and viability responses to improve drug effect classification over existing methods.

We discovered Bowen emission arising from a strongly lensed (i.e., with magnification factor $��$>20) source hosted in the Sunburst arc at z=2.37. We claim this source is plausibly a transient stellar object and study the unique ultraviolet lines emerging from it. In particular, narrow ($��$_v ~ 40 km/s) ionisation lines of Fe fluoresce after being exposed to Lya radiation that pumps selectively their atomic levels. Data from VLT/MUSE, X-Shooter and ESPRESSO observations (the latter placed at the focus of the four UTs) at increasing spectral resolution of R=2500, 11400 and R=70000, respectively, confirm such fluorescent lines are present since at least 3.3 years (~ 1 year rest-frame). Additional Fe forbidden lines have been detected, while C and Si doublets probe an electron density n_e >~ $10^6$ cm$^{-3}$. Similarities with the spectral features observed in the circum-stellar Weigelt blobs of Eta-Carinae probing the circum-stellar dense gas condensations in radiation-rich conditions are observed. We discuss the physical origin of the transient event, which remains unclear. We expect such transient events (including also supernova or impostors) will be easily recognised with ELTs thanks to high angular resolution provided by adaptive optics and large collecting area, especially in modest ($��< 3$) magnification regime., 5 pages, 4 figures. MNRAS accepted

Background: For understanding injuries in paediatric elbow and checking the degree of reduction after manipulation, various radiological criteria using anthropometric parameters are used. Since anthropometric parameters of Caucasians are different from European and Mongoloids, their parameters cannot be applied on our population. Hence, there is a need to characterise anthropometric parameters of elbow among children in the Indian population. Materials and Methods: The study population comprised all cases of injury to the elbow joint between 3 and 13 years of age reporting to the Department of Orthopaedics during the study. The X-rays films were preserved, and the angles and lines (as anthropometric parameters) were drawn on the radiographs. Results: Mean ± (standard deviation [SD]) for Baumann angle in children included in this study was 75° ± 4.70°. Mean ± (SD) of Baumann angle in males was 76° ± 4.44° and females was 74° ± 5.37°. Mean ± (SD) for lateral capitellohumeral angle in children from 3 to 13 years of age was 49 ± 5.75. Conclusions: As the values of normal side have been found to affect the functional and cosmetic outcome of the affected extremity, slight changes in values of our population in comparison to that of the Western population can significantly affect the outcome.

Cidofovir (CDV) is an antiviral agent with antiproliferative properties. The aim of our study was to investigate the efficacy of CDV in HPV-positive and -negative head and neck squamous cell carcinoma (HNSCC) cell lines and whether it is caused by a difference in response to DNA damage. Upon CDV treatment of HNSCC and normal oral keratinocyte cell lines, we carried out MTT analysis (cell viability), flow cytometry (cell cycle analysis), (immuno) fluorescence and western blotting (DNA double strand breaks, DNA damage response, apoptosis and mitotic catastrophe). The growth of the cell lines was inhibited by CDV treatment and resulted in &gamma, H2AX accumulation and upregulation of DNA repair proteins. CDV did not activate apoptosis but induced S- and G2/M phase arrest. Phospho-Aurora Kinase immunostaining showed a decrease in the amount of mitoses but an increase in aberrant mitoses suggesting mitotic catastrophe. In conclusion, CDV inhibits cell growth in HPV-positive and -negative HNSCC cell lines and was more profound in the HPV-positive cell lines. CDV treated cells show accumulation of DNA DSBs and DNA damage response activation, but apoptosis does not seem to occur. Rather our data indicate the occurrence of mitotic catastrophe.

Tumor-associated cell-free DNAs (cfDNAs) are found to play some important roles at different stages of tumor progression; they are involved in the transformation of normal cells and contribute to tumor migration and invasion. DNase I is considered a promising cancer cure, due to its ability to degrade cfDNAs. Previous studies using murine tumor models have proved the high anti-metastatic potential of DNase I. Later circulating cfDNAs, especially tandem repeats associated with short-interspersed nuclear elements (SINEs) and long-interspersed nuclear elements (LINEs), have been found to be the enzyme's main molecular targets. Here, using Lewis lung carcinoma, melanoma B16, and lymphosarcoma RLS40 murine tumor models, we reveal that tumor progression is accompanied by an increase in the level of SINE and LINEs in the pool of circulating cfDNAs. Treatment with DNase I decreased in the number and area of metastases by factor 3-10, and the size of the primary tumor node by factor 1.5-2, which correlated with 5- to 10-fold decreasing SINEs and LINEs. We demonstrated that SINEs and LINEs from cfDNA of tumor-bearing mice are able to penetrate human cells. The results show that SINEs and LINEs could be important players in metastasis, and this allows them to be considered as attractive new targets for anticancer therapy.

[EN] Although docetaxel is currently broadly used in prostate cancer treatment, poor water solubility and systemic toxicity limit the dose and duration of therapy. In this context, although different nanoplatforms have been proposed to overcome these issues, selective therapy needs developing methodologies to target malignant cells and minimizing the impact on healthy tissue. We here present a novel drug delivery system obtained by covalent conjugation of docetaxel and an anti-prostate specific membrane antigen (PSMA) molecule (anti-FOLH1 monoclonal antibody, clone C803N) over mesoporous silica nanoparticles. This conjugate remains stable in physiological medium and shows high selectivity for LNCaP, a specific cell line that overexpresses PSMA. As a consequence, cell internalization is increased by 25%. Furthermore, cytotoxic activity of the targeted system increases by 2-fold with regard to nontargeted nanoparticles and by 2 orders with regard to the naked drug. Conversely, no targeting effect is observed over PC3, a nonbearing PSMA cell line. We expect that this therapeutic system shows strong potential for treating nonmetastatic prostate cancer, mostly through intraprostatic administration., Financial support from the Spanish Ministry of Economy and Competitiveness (projects MAT2015-66666-C3-2-R, TEC2016-80976-R, and SEV-2016-0683) and the Generalitat Valenciana (project PROMETEO/2017/060) is gratefully acknowledged. We appreciate the assistance of the Electron Microscopy Service of the Universitat Politecnica de Valencia.

[EN] The aim of the experiment was to compare 3 genetic groups, slaughtered at similar weights, to examine their productive and carcass traits and economic value. Three lines of the Pannon Breeding Programme, selected for different criteria, were examined in the experiment. Pannon Ka (PKa, maternal line) does were inseminated with semen of PKa, Pannon White (PWhite) or Pannon Large (PLarge, terminal line) bucks. The kits (PKa×PKa, PWhite×PKa, PLarge×PKa; n=60 in each genetic group) were weaned at 35 d of age and reared until 88, 83 and 79, respectively, when they reached similar body weights for slaughtering (2.8 kg). The weight gain of PLarge×PKa was the largest (51.0 g/d) and that of PKa×PKa was the smallest (47.2 g/d), while PWhite×PKa (41.8 g/d) was intermediate (P, This paper was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences, and by the GOP-1.1.1-11-2012-0132 project.

Two distinct series of the 3-amino-1,5-benzoxathiepin scaffold, derived from L-cysteine, were synthesized and evaluated for their anti-proliferative activity in the breast cancer MDA-MB-231 and MCF-7 cells, and in the ovarian carcinoma SKOV-3 cell line. (3R)-Amino-3,4-dihydro-2H-1,5-benzoxathiepin [(R)-10] was diversified into two forms: (a) by incorporating different amino acids at its position 3, through an amide bond; and (b) by construction of the purine ring to give 6-chloro-9-[2-(3,4-dihydro-2H-1,5-benzoxathiepin-(3R)-yl)]-9H-purine [(R)-28]. Nevertheless, when the introduction of iodine was tried at position 2 of the purine ring of (R)-28, 2-{[2-(6-chloro-2-iodo-9H-purin-9-yl)prop-2-en-1-yl]thio}phenol (34) was obtained. Compound 34 shows activity against cancer cells. Interestingly, 34 inhibits mammosphere formation at the micromolar range, demonstrating activity against cancer stem cells. Although further studies of its targets and mechanism of action are needed, these findings support the therapeutic potential of this compound in cancer., We thank the Ministerio de Economía y Competitividad (grant CTQ2013-40855-R), Gobierno de Aragón–Fondo Social Europeo (research group E40), and the Intramural CSIC (201530E011) for providing X-ray structural facilities for this work.

Background: Lettuce is a globally important leafy vegetable and a model plant for biotechnology due to its adaptability to tissue culture and stable genetic transformation. Lettuce is also crucial for functional genomics research in the Asteraceae which includes species of great agronomical importance. The development of transgenic events implies the production of a large number of shoots that must be differentiated between transgenic and non-transgenic through the activity of the selective agent, being kanamycin the most popular. Results: In this work we adjusted the selection conditions of transgenic seedlings to avoid any escapes, finding that threshold concentration of kanamycin was 75 mg/L. To monitor the selection system, we studied the morphological response of transgenic and non-transgenic seedlings in presence of kanamycin to look for a visual morphological marker. Several traits like shoot length, primary root length, number of leaves, fresh weight, and appearance of the aerial part and development of lateral roots were affected in non-transgenic seedlings after 30 d of culture in selective media. However, only lateral root development showed an early, qualitative and reliable association with nptII presence, as corroborated by PCR detection. Applied in successive transgenic progenies, this method of selection combined with morphological follow-up allowed selecting the homozygous presence of nptII gene in 100% of the analyzed plants from T2 to T5. Conclusions: This protocol allows a simplified scaling-up of the production of multiple homozygous transgenic progeny lines in the early generations avoiding expensive and time-consuming molecular assays. Inst. de Biotecnología Fil: Darqui, Flavia Soledad. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Radonic, Laura Mabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Lopez, Nilda Ester. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina Fil: Hopp, Horacio Esteban. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina Fil: López Bilbao, Marisa Gisela. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentina

BACKGROUND We previously established parthenogenetic mouse embryonic stem cells (ESCs) and this study was subsequently conducted for elucidating the influence of oocyte parthenogenesis on gene expression profile of ESCs. METHODS Gene expression of parthenogenetic ESC (pESC)-1 or pESC-2 was separately compared with that of two normally fertilized ESC (nfESC) lines (B6D2F1 and R1 strains), and quantification of mRNA expression was conducted for validating microarray data. RESULTS In two sets of comparison, reaction of 11 347 and 15 454 gene probes were altered by parthenogenesis, while strain difference changed the expression of 15 750 and 14 944 probes. Level of correlation coefficient was higher in the comparisons between normal fertilization and parthenogenesis (0.974-0.985) than in the comparisons between strains of nfESCs (0.97-0.971). Overall, the expression of 3276-3329 genes was changed after parthenogenesis, and 88% (96/109) of major functional genes differentially (P < 0.01) expressed in one comparison set showed the same change in the other. When we monitored imprinted genes, expression of nine paternal and eight maternal genes were altered after parthenogenesis and 88% (14/16) of these was confirmed by mRNA quantification. CONCLUSIONS The change in gene expression after parthenogenesis was similar to, or less than, the change induced by a strain difference under a certain genetic background. These results may suggest the clinical feasibility of parthenogenesis-derived, pluripotent cells

Current first-line treatment for Adult T-cell leukemia (ATL) includes combination therapy with interferon alpha (IFN-α) and zidovudine (AZT). The use of IFN-α in this treatment is mostly empirical in origin, whereas the therapeutic potential of interferon beta (IFN-β), has not yet been thoroughly explored in this context. Here we compare the effects of IFN-α and IFN-β treatment in short term ex vivo peripheral blood mononuclear cell (PBMC) cultures from 22 ATL patients. Using proliferation, apoptosis and antiviral bioassays, complemented with microarray and gene set analysis, we demonstrate that in this setting IFN-β has superior antiproliferative and pro‑apoptotic effects than IFN-α. Increased p53 signaling is observed under the IFN-β treatment, while the antiviral effects are equivalent to those of IFN-α treatment. Notably, the genes in a published in vivo AZT/IFN-α response profile are affected more strongly by IFN-β than by IFN-α stimulus in these ex vivo PBMCs. In conclusion, this first comprehensive analysis comparing the effects of IFN-α and IFN-β on ex vivo PBMCs of ATL patients demonstrates that IFN-β has a greater impact than IFN-α on biological processes which have been shown to be crucial in the treatment of ATL, making IFN-β an intriguing candidate for further in vivo testing. ispartof: Blood Cancer Journal vol:7 issue:1 ispartof: location:United States status: published

Botulinum neurotoxin type-A (BoNT-A) blocks the release of acetylcholine from peripheral cholinergic nerve terminals and is an important option for the treatment of disorders characterised by excessive cholinergic neuronal activity. Several BoNT-A products are currently marketed, each with unique manufacturing processes, excipients, formulation, and non-interchangeable potency units. Nevertheless, the effects of all the products are mediated by the 150 kDa BoNT-A neurotoxin. We assessed the quantity and light chain (LC) activity of BoNT-A in three commercial BoNT-A products (Dysport&reg, Botox&reg, Xeomin&reg, ). We quantified 150 kDa BoNT-A by sandwich ELISA and assessed LC activity by EndoPep assay. In both assays, we assessed the results for the commercial products against recombinant 150 kDa BoNT-A. The mean 150 kDa BoNT-A content per vial measured by ELISA was 2.69 ng/500 U vial Dysport&reg, 0.90 ng/100 U vial Botox&reg, and 0.40 ng/100 U vial Xeomin&reg, To present clinically relevant results, we calculated the 150 kDa BoNT-A/US Food and Drug Administration (FDA)-approved dose in adult upper limb spasticity: 5.38 ng Dysport&reg, (1000 U, 2 &times, 500 U vials), 3.60 ng Botox&reg, (400 U, 4 &times, 100 U vials), and 1.61 ng Xeomin&reg, 100 U vials). EndoPep assay showed similar LC activity among BoNT-A products. Thus, greater amounts of active neurotoxin are injected with Dysport&reg, at FDA-approved doses, than with other products. This fact might explain the long duration of action reported across multiple indications, which benefits patients, caregivers, clinicians, and healthcare systems.

Purpose: Induction of viral lytic cycle, which induces death of host cells, may constitute a useful adjunct to current therapeutic regimens for Epstein–Barr virus (EBV)-driven malignancies. Human telomerase reverse transcriptase (hTERT), essential for the oncogenic process, may modulate the switch from latent to lytic infection. The possible therapeutic role of hTERT inhibition combined with antiviral drugs was investigated. Experimental Design: EBV-negative BL41 and convertant EBV-positive BL41/B95.8 Burkitt's lymphoma cell lines and lymphoblastoid cell lines (LCL) were infected with retroviral vector encoding short hairpin RNA (shRNA) anti-hTERT and cultured with or without the prodrug ganciclovir. The effects on EBV lytic replication, cell proliferation, and apoptosis were characterized. Results: hTERT silencing by shRNA induced the expression of BZLF1, EA-D, and gp350 EBV lytic proteins and triggered a complete lytic cycle. This effect was associated with downregulation of BATF, a negative regulator of BZLF1 transcription. hTERT silencing also resulted in antiproliferative and proapoptotic effects. In particular, hTERT inhibition induced an accumulation of cells in the S-phase, an effect likely due to the dephosphorylation of 4E-BP1, an AKT1-dependent substrate, which results in a decreased availability of proteins needed for cell-cycle progression. Besides inducing cell death through activation of complete EBV lytic replication, hTERT inhibition triggered AKT1/FOXO3/NOXA–dependent apoptosis in EBV-positive and -negative Burkitt's lymphoma cells. Finally, ganciclovir enhanced the apoptotic effect induced by hTERT inhibition in EBV-positive Burkitt's lymphomas and LCLs. Conclusions: These results suggest that combination of antiviral drugs with strategies able to inhibit hTERT expression may result in therapeutically relevant effects in patients with EBV-related malignancies. Clin Cancer Res; 19(8); 2036–47. ©2013 AACR.

Catheter-associated bloodstream infections (CABSIs) are common complications encountered with cancer treatment. The aims of this study were to analyze the factors associated with recurrent infection and catheter removal in pediatric hematology-oncology patients. All cases of CABSIs in patients attending the Department of Pediatric Hematology-Oncology between January 2008 and December 2010 were reviewed. A total of 44 episodes of CABSIs, including multiple episodes involving the same catheter, were identified in 31 children with cancer. The overall CABSIs rate was 7.4 infections per 1000 central venous catheter (CVC) days. The most frequent organism isolated was coagulase-negative Staphylococcus (CONS). The CVC was removed in nine (20.4%) episodes. We found that hypotension, persistent bacteremia, Candida infection, exit-side infection, neutropenia, and prolonged duration of neutropenia were the factors for catheter removal. There were 23 (52.2%) episodes of recurrence or reinfection. Mortality rate was found to be 9.6% in children with CABSIs. In this study, we found that CABSIs rate was 7.4 infections per 1000 catheter-days. CABSIs rates in our hematology-oncology patients are comparable to prior reports. Because CONS is the most common isolated microorganism in CABSIs, vancomycin can be considered part of the initial empirical regimen.

Drug-induced hepatotoxicity is a leading cause of attrition for candidate pharmaceuticals in development. New preclinical screening methods are crucial to predict drug toxicity prior to human studies. Of all in vitro hepatotoxicity models, primary human hepatocytes are considered as 'the gold standard.' However, their use is hindered by limited availability and inter-individual variation. These barriers may be overcome by using primary mouse hepatocytes. We used differential in gel electrophoresis (DIGE) to study large-scale protein expression of primary mouse hepatocytes. These hepatocytes were exposed to three well-defined hepatotoxicants: acetaminophen, amiodarone, and cyclosporin A. Each hepatotoxicant induces a different hepatotoxic phenotype. Based on the DIGE results, the mRNA expression levels of deregulated proteins from cyclosporin A-treated cells were also analyzed. We were able to distinguish cyclosporin A from controls, as well as acetaminophen and amiodarone-treated samples. Cyclosporin A induced endoplasmic reticulum (ER) stress and altered the ER-Golgi transport. Moreover, liver carboxylesterase and bile salt sulfotransferase were differentially expressed. These proteins were associated with a protective adaptive response against cyclosporin A-induced cholestasis. The results of this study are comparable with effects in HepG2 cells. Therefore, we suggest both models can be used to analyze the cholestatic properties of cyclosporin A. Furthermore, this study showed a conserved response between primary mouse hepatocytes and HepG2 cells. These findings collectively lend support for use of omics strategies in preclinical toxicology, and might inform future efforts to better link preclinical and clinical research in rational drug development.

Imaging techniques such as immunofluorescence (IF) and the expression of fluorescent protein (FP) fusions are widely used to investigate the subcellular distribution of proteins. Here we report a systematic analysis of >500 human proteins comparing the localizations obtained in live versus fixed cells using FPs and IF, respectively. We identify systematic discrepancies between IF and FPs as well as between FP tagging at the N and C termini. The analysis shows that for 80% of the proteins, IF and FPs yield the same subcellular distribution, and the locations of 250 previously unlocalized proteins were determined by the overlap between the two methods. Approximately 60% of proteins localize to multiple organelles for both methods, indicating a complex subcellular protein organization. These results show that both IF and FP tagging are reliable techniques and demonstrate the usefulness of an integrative approach for a complete investigation of the subcellular human proteome. QC 20130521

Background While nanotechnology is advancing rapidly, nanosafety tends to lag behind since general mechanistic insights into cell-nanoparticle (NP) interactions remain rare. To tackle this issue, standardization of nanosafety assessment is imperative. In this regard, we believe that the cell type selection should not be overlooked since the applicability of cell lines could be questioned given their altered phenotype. Hence, we evaluated the impact of the cell type on in vitro nanosafety evaluations in a human and murine neuroblastoma cell line, neural progenitor cell line and in neural stem cells. Acute toxicity was evaluated for gold, silver and iron oxide (IO)NPs, and the latter were additionally subjected to a multiparametric analysis to assess sublethal effects. Results The stem cells and murine neuroblastoma cell line respectively showed most and least acute cytotoxicity. Using high content imaging, we observed cell type- and species-specific responses to the IONPs on the level of reactive oxygen species production, calcium homeostasis, mitochondrial integrity and cell morphology, indicating that cellular homeostasis is impaired in distinct ways. Conclusions Our data reveal cell type-specific toxicity profiles and demonstrate that a single cell line or toxicity end point will not provide sufficient information on in vitro nanosafety. We propose to identify a set of standard cell lines for screening purposes and to select cell types for detailed nanosafety studies based on the intended application and/or expected exposure. Electronic supplementary material The online version of this article (doi:10.1186/s12951-016-0220-y) contains supplementary material, which is available to authorized users.

Evodiamine (EVO) and rutaecarpine (RUT) are promising anti-tumor drug candidates. The evaluation of the anti-proliferative activity and cellular uptake of EVO and RUT in 3D multicellular spheroids of cancer cells would better recapitulate the native situation and thus better reflect an in vivo response to the treatment. Herein, we employed the 3D culture of MCF-7 and SMMC-7721 cells based on hanging drop method and evaluated the anti-proliferative activity and cellular uptake of EVO and RUT in 3D multicellular spheroids, and compared the results with those obtained from 2D monolayers. The drugs’ IC50 values were significantly increased from the range of 6.4–44.1 μM in 2D monolayers to 21.8–138.0 μM in 3D multicellular spheroids, which may be due to enhanced mass barrier and reduced drug penetration in 3D models. The fluorescence of EVO and RUT was measured via fluorescence spectroscopy and the cellular uptake of both drugs was characterized in 2D tumor models. The results showed that the cellular uptake concentrations of RUT increased with increasing drug concentrations. However, the EVO concentrations uptaken by the cells showed only a small change with increasing drug concentrations, which may be due to the different solubility of EVO and Rut in solvents. Overall, this study provided a new vision of the anti-tumor activity of EVO and RUT via 3D multicellular spheroids and cellular uptake through the fluorescence of compounds.

GNRH significantly inhibits proliferation of a proportion of cancer cell lines by activating GNRH receptor (GNRHR)-G protein signaling. Therefore, manipulation of GNRHR signaling may have an under-utilized role in treating certain breast and ovarian cancers. However, the precise signaling pathways necessary for the effect and the features of cellular responses remain poorly defined. We used transcriptomic and proteomic profiling approaches to characterize the effects of GNRHR activation in sensitive cells (HEK293-GNRHR, SCL60)in vitroandin vivo, compared to unresponsive HEK293. Analyses of gene expression demonstrated a dynamic response to the GNRH superagonist Triptorelin. Early and mid-phase changes (0.5–1.0 h) comprised mainly transcription factors. Later changes (8–24 h) included a GNRH target gene,CGA, and up- or downregulation of transcripts encoding signaling and cell division machinery. Pathway analysis identified altered MAPK and cell cycle pathways, consistent with occurrence of G2/M arrest and apoptosis. Nuclear factor kappa B (NF-κB) pathway gene transcripts were differentially expressed between control and Triptorelin-treated SCL60 cultures. Reverse-phase protein and phospho-proteomic array analyses profiled responses in cultured cells and SCL60 xenograftsin vivoduring Triptorelin anti-proliferation. Increased phosphorylated NF-κB (p65) occurred in SCL60in vitro, and p-NF-κB and IκBε were higher in treated xenografts than controls after 4 days Triptorelin. NF-κB inhibition enhanced the anti-proliferative effect of Triptorelin in SCL60 cultures. This study reveals details of pathways interacting with intense GNRHR signaling, identifies potential anti-proliferative target genes, and implicates the NF-κB survival pathway as a node for enhancing GNRH agonist-induced anti-proliferation.

Objective The success of personalised therapy depends on identification and inhibition of the oncogene(s) on which that tumour is dependent. We aimed to determine whether a receptor tyrosine kinase (RTK) array could be used to select the most effective therapeutic strategies in molecularly heterogeneous oesophago-gastric adenocarcinomas. Design Gene expression profiling from oesophago-gastric tumours (n=75) and preinvasive stages (n=57) identified the active signalling pathways, which was confirmed using immunohistochemistry (n=434). RTK arrays on a cell line panel (n=14) determined therapeutic targets for in vitro cytotoxic testing. Feasibility of this personalised approach was tested in tumour samples (n=46). Results MAPK was the most frequently activated pathway (32/75 samples (42.7%)) with progressive enrichment in preinvasive disease stages (p

Platinum-based chemotherapies such as cisplatin are used as first-line treatment for many cancers. Although there is often a high initial responsiveness, the majority of patients eventually relapse with platinum-resistant disease. For example, a subset of testicular cancer patients still die even though testicular cancer is considered a paradigm of cisplatin-sensitive solid tumors, but the mechanisms of chemoresistance remain elusive. Here, we have shown that one key determinant of cisplatin-resistance in testicular embryonal carcinoma (EC) is high cytoplasmic expression of the cyclin-dependent kinase (CD K) inhibitor p21. The EC component of the majority of refractory testicular cancer patients exhibited high cytoplasmic p21 expression, which protected EC cell lines against cisplatin-induced apoptosis via CDK2 inhibition. Localization of p21 in the cytoplasm was critical for cisplatin resistance, since relocalization of p21 to the nucleus by Akt inhibition sensitized EC cell lines to cisplatin. We also demonstrated in EC cell lines and human tumor tissue that high cytoplasmic p21 expression and cisplatin resistance of EC were inversely associated with the expression of Oct4 and miR-106b seed family members. Thus, targeting cytoplasmic p21, including by modulation of the Oct4/miR-106b/p21 pathway, may offer new strategies for the treatment of chemoresistant testicular and other types of cancer.

Ascites syndrome is a metabolic disorder found in modern broilers that have insufficient pulmo- nary vascular capacity. Commercial breeding programs have heavily focused on high growth rate, which led to fast-growing chickens, but as a negative consequence, the incidence of ascites syndrome increased. However, not all birds with a high growth rate will suffer from ascites syndrome, which might indicate a genetic sus- ceptibility to ascites. Information on blood gas param- eters measured early in life and their relation to ascites susceptibility is expected to contribute to identification on the cause of ascites syndrome. In this study, several physiological parameters, such as blood gas parameters (pH, partial pressure of CO2 in venous blood (pvCO2), and partial pressure of O2 in venous blood), hematocrit, electrolytes (Na + , Ca 2+ , and K + ), metabolites (lactate and glucose), were measured at d 11 to 12 of age from 100 female and 100 male broilers. From d 14 onward, the birds were challenged to provoke the development of ascites syndrome. Our results showed that high pvCO 2 values together with low pH values (males) or high pH values (females) in the venous blood of juvenile broilers coincided with ascites. Therefore, blood pvCO 2 and pH in both juvenile male and female broilers seem to be critical factors in ascites pathophysiology and can be used as phenotypic traits to predict ascites susceptibil- ity in juvenile broilers at d 11 to 12. A prediction model was built on a subpopulation of the broilers without any loss in sensitivity (0.52) and specificity (0.78) when applied to the validation population. The parameter sex was included in the prediction model because levels of pvCO2 and pH that associated with ascites suscep- tibility are different between males and females. Com- mercial breeders can include these phenotypic traits in their genetic selection programs to reduce the incidence of ascites syndrome.

Bone marrow (BM)-derived mesenchymal stem cells (MSCs) have been implicated in tumor progression, making MSCs important targets for anti-cancer strategies. In this study, we show that MSCs promote tumor growth in vivo in a lymphoma xenograft model. We show that MSCs provide direct cell-cell contact interactions and, to a lesser extend, soluble factors that promote tumor cell proliferation and survival in vitro. PTK787/ZK 222584 reduces tumor growth-promoting effects of MSCs both in vitro and in vivo. Our results address the importance of targeting the MSCs for future anti-cancer strategies.

Background: Pigmentary demarcation lines (PDL) are physiological abrupt transition lines between hyperpigmented skin and lighter areas. Recent evidence suggests that they involve the face. Aims: To survey facial PDL in Saudi females referred to general dermatology clinics for various complaints and determine any associated risks. Methods: Screening for facial lines was done in general dermatology clinics over a year. Whenever a patient was found to have facial PDL, a detailed questionnaire and examination were undertaken. Results: Out of 1033 patients screened, 144 patients (14%) were found to have at least one of the facial PDLs. The median age of onset was 16 years. The most common line was F with 76 patients (53%). Family history was positive in 51 patients (35%). Conclusion: Facial PDL is a common and chronic pigmentary problem in Saudi women. It should be recognized and differentiated from other similar diseases like melasma. A significant proportion of patients have a milder presentation.

Predicting the individual vulnerability to immune mediated disease is one of the main challenges of modern biomedical research. However, the question of individual behavioral and physiological characteristics that might predict this vulnerability has been subject of research and debate for a long time. This paper will argue that animal models aimed at individual vulnerability should consider the biological function of variation in nature. An increasing number of studies show the ecological significance of variation within a species. Based on behavioral studies in several vertebrate species two coping style can be distinguished. Variation in coping style appears to play a role in the population dynamics and the evolutionary fitness of the species. Coping styles are reflected in a stable differentiation in the behavioral and physiological stress responsiveness over time and across situations. Based on the observations that the individual level of offensive aggressive behavior (i.e., the tendency to defend the home territory) is strongly related to the way animals react to various other environmental challenges, it is argued that the individual's level of offensiveness is an important indicator and component of a more trait-like behavioral and physiological response pattern (coping style) to environmental demands. The coping style of aggressive animals is principally aimed at a (pro)active prevention or manipulation of a stressor whereas the non-aggressive individuals tend to passively accept or react to it. Proactive coping is associated with high sympathetic reactivity to stressors; whereas the more passive or reactive coping style generally has a higher HPA axis reactivity. In view of the immune modulating nature of these major neuroendocrine stress systems, one might expect that coping styles will be reflected in a differential vulnerability to immune mediated disease as well. Indeed, several studies have demonstrated such a relationship, indicating that the functional variation in coping style and related neuroendocrine stress reactivity, as it occurs in nature, might be a good standard for studies aimed at understanding individual vulnerability. This is in agreement with more recent views that also in humans stress reactivity may be the best predictor for the individual vulnerability to immune mediated diseases. This asks for a more fundamental and translational approach of individual disease vulnerability based on a common biological basis of individual differentiation in behavior and physiology in humans and animals. (c) 2007 Elsevier Inc. All rights reserved.

Introduction Information obtained on the IL-2 receptor status of tumour infiltrating lymphocytes in patients suffering from squamous cell carcinoma of the head and neck (SSCHN) before and after IL-2 treatment may lead to a better understanding of the immunological changes and related kinetics induced at the tumour level and ultimately to a strategy that allows selection of those patients that will benefit from IL-2 therapy. This study set out to assess the relationship between (123)I-IL2 single-photon emission computed tomography (SPECT) findings and the presence of IL-2 receptors (CD25 staining) on tumour-infiltrating lymphocytes as well as on SCCHN tumour cells in patients suffering from SCCHN.Materials and methods Seventeen consecutive patients (12 men; mean age, 57 years) highly suspected to suffer from SSCHN were prospectively included in the study. All patients underwent planar and whole body (123)I-IL2 scintigraphy and underwent surgery or had a biopsy taken within 1 week from imaging. Surgical resected primary lesions as well as biopsy material from primary tumours were histologically analysed with respect to the presence and intensity of CD25 expression on tumour infiltrating lymphocytes and tumour cells (HSCORE). Tumor-to-background (T/N) ratios of the primary tumour derived from planar and tomographic (123)I-IL2 scintigraphy were related to the results derived from histology.Results All patients suffered from SSCHN. T/N ratios derived from SPECT images were significantly correlated with CD25 lymphocyte HSCOREs (r=0.66; p=0.03), but not with CD25 tumour cell HSCOREs.Conclusiond (123)I-IL-2 SPECT imaging allows for non-invasive imaging of the relative amount of IL-2 receptors present on tumour infiltrating lymphocytes in SCCHN.

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells. Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5(+)) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5(+) fraction are subsequently stained with alpha GalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 10(8) iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice.

Hadrontherapy is an advanced form of radiotherapy that uses beams of charged particles (such as protons and carbon ions). Compared with conventional radiotherapy, the main advantages of carbon ion therapy are the precise absorbed dose localization, along with an increased relative biological effectiveness (RBE). This high ballistic accuracy of particle beams deposits the maximal dose to the tumor, while damage to the surrounding healthy tissue is limited. Currently, hadrontherapy is being used for the treatment of specific types of cancer. Previous in vitro studies have shown that, under certain circumstances, exposure to charged particles may inhibit cell motility and migration. In the present study, we investigated the expression of four motility-related genes in prostate (PC3) and colon (Caco-2) cancer cell lines after exposure to different radiation types. Cells were irradiated with various absorbed doses (0, 0.5 and 2 Gy) of accelerated (13)C-ions at the GANIL facility (Caen, France) or with X-rays. Clonogenic assays were performed to determine the RBE. RT-qPCR analysis showed dose- and time-dependent changes in the expression of CCDC88A, FN1, MYH9 and ROCK1 in both cell lines. However, whereas in PC3 cells the response to carbon ion irradiation was enhanced compared with X-irradiation, the effect was the opposite in Caco-2 cells, indicating cell-type-specific responses to the different radiation types.

Background: Systematic approaches to functionally identify key players in microRNA (miRNA)-target networks regulating prostate cancer (PCa) proliferation are still missing. Objective: To comprehensively map miRNA regulation of genes relevant for PCa proliferation through phenotypic screening and tumor expression data. Design, setting, and participants: Gain-of-function screening with 1129 miRNA molecules was performed in five PCa cell lines, measuring proliferation, viability, and apoptosis. These results were integrated with changes in miRNA expression from two cohorts of human PCa (188 tumors in total). For resulting miRNAs, the predicted targets were collected and analyzed for patterns with gene set enrichment analysis, and for their association with biochemical recurrence free survival. Outcome measurements and statistical analysis: Rank product statistical analysis was used to evaluate miRNA effects in phenotypic screening and for expression differences in the prostate tumor cohorts. Expression data were analyzed using the significance analysis of microarrays (SAM) method and the patient material was subjected to Kaplan-Meier statistics. Results and limitations: Functional screening identified 25 miRNAs increasing and 48 miRNAs decreasing cell viability. Data integration resulted in 14 miRNAs, with aberrant expression and effect on proliferation. These miRNAs are predicted to regulate >3700 genes, of which 28 were found up-regulated and 127 down-regulated in PCa compared with benign tissue. Seven genes,. FLNC,. MSRB3,. PARVA,. PCDH7,. PRNP,. RAB34, and. SORBS1, showed an inverse association to their predicted miRNA, and were identified to significantly correlate with biochemical recurrence free survival in PCa patients. Conclusions: A systematic in vitro screening approach combined with in vivo expression and gene set enrichment analysis provide unbiased means for revealing novel miRNA-target links, possibly driving the oncogenic processes in PCa. Patient summary: This study identified novel regulatory molecules, which impact on PCa proliferation and are aberrantly expressed in clinical tumors. Thus, our study reveals regulatory nodes with potential for therapy. A comprehensive approach of functional microRNA screening and expression analyses, identifies miR-19a, miR-32, miR-124a, miR-130b, miR-148a, and miR-583 as potential regulators of. FLNC,. MSRB3,. PARVA,. PCDH7,. PRNP,. RAB34, and. SORBS1, which correlate with prostate specific antigen-relapse in prostate cancer patients.

Purpose The aim of this study was to evaluate the antiproliferative activity in breast cancer cells and the inhibition of tumorigenesis in pre-neoplastic cells of a new apple cultivar with reddish pulp, called the Pelingo apple. Methods The antiproliferative activity was evaluated in MCF-7 and MDA-MB-231 human breast cancer cells. The inhibition of tumorigenesis was performed in JB6 promotion-sensitive (P+) cells. Results Results showed that Pelingo apple juice is characterized by a very high polyphenol content and strongly inhibited breast cancer cell proliferation. Its antiproliferative activity was found to be higher than the other five apple juices tested. Pelingo juice induced cell accumulation in the G2/M phase of the cell cycle and autophagy through overexpression of p21, inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) activity and an increase in lipidated microtubule-associated protein-1 light chain-3 beta (LC3B). Remarkably, Pelingo juice inhibited the 12-o-tetra-decanoyl-phorbol-13-acetate (TPA)-induced tumorigenesis of JB6 P+ cells, suppressing colony formation in semi-solid medium and TPA-induced ERK1/2 phosphorylation. Conclusions Our data indicate that the Pelingo apple is rich in food components that can markedly inhibit in vitro tumorigenesis and growth of human breast cancer cells and could provide natural bioactive non-nutrient compounds, with potential chemopreventive activity.

Glucocorticoid (GC) resistance is an adverse prognostic factor in childhood acute lymphoblastic leukemia (ALL), but little is known about causes of GC resistance. Up-regulation of the glucocorticoid receptor (GR) has been suggested as an essential step to the induction of apoptosis in leukemic cells. In this study we investigated whether baseline mRNA expression levels of the 5 different GR promoter transcripts (1A1, 1A2, 1A3, 1B, and 1C) or differences in the degree of regulation of the GR or GR promoter transcripts upon GC exposure are related to GC resistance. Therefore, mRNA levels of the 5 GR promoter transcripts and of the GR were measured by quantitative real-time reverse transcriptase–polymerase chain reaction (RT-PCR; Taqman) technology in primary ALL cells prior to and after 3, 8, and 24 hours of prednisolone exposure. GR expression is induced upon GC exposure in primary ALL patient samples, which is opposite to what is found in tissues in which GCs do not induce apoptosis. GC resistance in childhood ALL cannot be attributed to an inability of resistant cells to up-regulate the expression of the GR upon GC exposure, nor to differences in GR promoter usage (at baseline and upon GC exposure).

This experiment was conducted to investigate whether feed efficiency, as measured by residual feed intake as a phenotypic trait, affects energy partitioning in pullets that have received Salmonella inoculation as an immune challenge. In each of 8 trials, energy partitioning was measured during 5 wk in 15-wk-old efficient (R-) and nonefficient (R+) pullets, which were housed per efficiency group in 2 identical climate respiration chambers. After 1 wk of adaptation, the pullets in 4 trials were orally inoculated with 10(8) cfu of Salmonella enteritidis; pullets in the remaining trials were not inoculated and served as controls. Heat production was calculated from continuous recordings of O(2) consumption and CO(2) production. Energy and N partitioning were recorded on a weekly basis. Blood samples for analyses on thyroid hormones were taken at 16, 17, and 19 wk of age. There were no interactions between efficiency type and Salmonella treatment or Salmonella treatment effects in energy partitioning, except for a short-term increase in heat production in inoculated pullets. Nonefficient pullets had higher gross energy and ME intake, higher estimated ME for maintenance, lower ME:gross energy ratio, and higher total heat production and nonactivity-related heat production compared with R- pullets. Triiodothyronine levels in R+ pullets were higher at 16 and 17 wk but were lower at 19 wk of age compared with R- pullets. Thyroxine levels were higher in R- at 16 wk and showed interactions between efficiency type and Salmonella treatment at 17 and 19 wk of age. Body weights and spleen weights did not differ between efficiency groups. Nonefficient pullets had higher heart, liver, and ovary weights and more large yellow follicles than R- pullets. There were no Salmonella effects on body and organ weights. We conclude that R+ pullets have a faster running energy metabolism and that they put more resources into organ development than R- pullets. Inoculation with Salmonella has a short-term effect on nonactivity-related heat production but does not affect energy partitioning, regardless of efficiency type.

The intracellular level of the proto-oncoprotein beta-catenin is a parameter for the activity of the Wnt pathway, which has been linked to carcinogenesis. The paper introduces a novel sandwich-based ELISA for the determination of the beta-catenin concentration in lysates from cells or tissues. The advantages of the method were proven by determining beta-catenin levels in cell lines and in cells after activation of the Wnt pathway. Analysis revealed high beta-catenin concentrations in the cell lines HeLa, KB, HT1080, MCF-7, U-87 and U-373, which had not been described before. beta-Catenin concentrations were compared in HEK293 and C57MG cells after activation of the Wnt pathway. The beta-catenin concentrations increased by different factors depending on whether the Wnt pathway was activated by incubation with LiCl or with Wnt-3a-conditioned medium. This finding indicated that the beta-catenin level depends on the way and level of Wnt pathway activation. The quantitative analysis of beta-catenin in colorectal tumours revealed high beta-catenin levels in tumours with truncating mutations in the APC gene.

Epidermal growth factor receptor-1 (EGFR) and EGFR-2 (HER2) have become major targets for cancer treatment. Blocking antibodies and small-molecule inhibitors are being used to silence the activity of these receptors in different tumors with varying efficacy. Thus, a better knowledge on the signaling pathways activated by EGFR and HER2 may help unravel novel therapeutic targets and molecular markers of response. Here, we show that treatment of breast cancer cell lines with blocking antibodies against EGFR (cetuximab) or HER2 (trastuzumab) promotes the specific induction of proapoptotic Bnip3L and chemosensitization. Moreover, we found that the Bnip3L gene is transcriptionally activated by FoxO3a. Trastuzumab-mediated induction of Bnip3L and nuclear translocation of FoxO3a was also shown in pleural effusion cells from a breast cancer patient. Transfection of breast cancer cells with constitutively active FoxO3a or with Bnip3L promotes sensitization to chemotherapy-induced apoptosis. On the contrary, blockade of Bnip3L expression by a small interfering RNA strategy significantly diminished the chemosensitizing effect of cetuximab. We found also an inverse correlation between EGFR and Bnip3L expression in surgical specimens from patients with breast cancer. Therefore, blockading EGFR or HER2 specifically up-regulates Bnip3L, which is required for chemosensitization of breast cancer cells. This novel pathway provides also the rationale for therapeutic strategies aimed to induce the expression of Bnip3L.

1. Several studies have shown that genetic variation exists in response to various Salmonella strains in mammals and poultry. In the current study immunocompetence traits related to natural resistance to Salmonella were measured in 7 genetic groups of meat-type chickens (in total 296 chickens involved). 2. Variables were measured of both innate (phagocytic activity) and adaptive immune responses that are important after a natural or experimental Salmonella enteritidis infection. Two traditional Old Dutch Breeds (groups 1 and 2), four commercial broiler groups (groups 3 to 6), and one experimental broiler group (group 7) were used. In two periods, birds of each group were killed for examination at ages between 14 and 35 d post hatch. 3. Significant differences between groups were found for most immune variables measured, with significant correlations between several of them. All groups produced an adequate immune response, of either the innate or the adaptive type. 4. In the current study, group 2 showed the highest overall natural resistance, though none of the groups was uniformly superior with respect to all traits measured. 5. In conclusion, for reliable measurements of general immunocompetence or resistance to Salmonella, for example, it is important to measure several aspects of the immune system.

There is a distinct difference in prognosis between childhood versus adult acute lymphoblastic leukaemia (ALL). To define whether multidrug resistance (MDR) genes might contribute to this distinction, the expression and functional activity of P-glycoprotein (P-gp) and MDR associated proteins (MRP) were determined with RT-PCR (MDR-1, MRP1, MRP2, MRP3) and flow cytometry (P-gp and MRP). Patient samples were obtained from 36 children and 35 adults with de novo ALL. Of these patients, 38 showed a T-lineage and 33 showed a B-lineage immunophenotype. In the samples, large variability in P-gp activity (0.8-4.9) and MRP activity (1.1-13.9) was observed. Most T-ALL patients with high P-gp activity were adults (89%). The mRNA expression of MDR-1 correlated weakly with P-gp activity. In contrast, MRP activity did not correlate with the mRNA expression of MRP1, MRP2 and MRP3. In T-ALL, a worse overall survival and event-free survival was observed with increasing P-gp activity. P-gp activity had no prognostic impact in B-lineage ALL. In addition, high MRP activity did not influence treatment outcome in either T- or B-lineage ALL. Multivariate Cox regression analysis, showed P-gp activity to be the only unfavourable prognostic factor for overall survival in T-ALL. In conclusion, this study demonstrates the prognostic relevance of P-gp activity in T-ALL. Since the majority of the patients with high P-gp activity were adults, P-gp might contribute to the poor prognosis of adult T-ALL.

Self-renewal and the maintenance of pluripotency of mouse embryonal stem (ES) cells in vitro requires exogenous leukemia inhibitory factor (LIF). Mouse ES cells can be cultured and kept undifferentiated in the absence of embryonal feeder-cell layers when exogenous LIF concentrations are maintained above a threshold concentration. An important downstream target of LIF signal transduction in mouse ES cells is the transcription factor signal transducer and activator of transcription 3 (STAT3). In contrast to mouse ES cells, human ES cells are unresponsive to LIF and depend on feeder cells for undifferentiated growth. Here, we investigated the activation patterns of LIF-downstream effectors in mouse and human embryonal carcinoma (EC) cells. We report that LIF induces both ERK-1 as well as STAT3 activation in mouse P19 EC cells. LIF enhances the proliferation rate of P19 EC cells, which depends on ERK activity but does not require activation of STAT3. In contrast, LIF does not activate STAT3, ERK, or the gp130 receptor in human N tera-2/D1 EC cells, although all receptor components are expressed. The negative feedback protein suppressor of cytokine signaling 1 (SOCS-1) is constitutively expressed in N tera-2/D1 EC cells, suggesting that LIF signal transduction is inhibited by elevated levels of SOCS-1 expression. (C) 2002 Elsevier Science (USA).