Molecular and biochemical characterization of postharvest senescence in broccoli

Postharvest senescence in broccoli (Brassica oleracea L. var Italica) florets results in phenotypic changes similar to those seen in developmental leaf senescence. To compare these two processes in more detail, we investigated molecular and biochemical changes in broccoli florets stored at two different temperatures after harvest. We found that storage at cooler temperatures delayed the symptoms of senescence at both the biochemical and gene expression levels. Changes in key biochemical components (lipids, protein, and chlorophyll) and in gene expression patterns occurred in the harvested tissue well before any visible signs of senescence were detected. Using previously identified senescence-enhanced genes and also newly isolated, differentially expressed genes, we found that the majority of these showed a similar enhancement of expression in postharvest broccoli as in developmental leaf senescence. At the biochemical level, a rapid loss of membrane fatty acids was detected after harvest, when stored at room temperature. However, there was no corresponding increase in levels of lipid peroxidation products. This, together with an increased expression of protective antioxidant genes, indicated that, in the initial stages of postharvest senescence, an orderly dismantling of the cellular constituents occurs, using the available lipid as an energy source. Postharvest changes in broccoli florets, therefore, show many similarities to the processes of developmental leaf senescence.

Depression of glutamate and GABA release by presynaptic GABAB receptors in the entorhinal cortex in normal and chronically epileptic rats

Presynaptic GABAB receptors (GABABR) control glutamate and GABA release at many synapses in the nervous system. In the present study we used whole-cell patch-clamp recordings of spontaneous excitatory and inhibitory synaptic currents in the presence of TTX to monitor glutamate and GABA release from synapses in layer II and V of the rat entorhinal cortex (EC)in vitro. In both layers the release of both transmitters was reduced by application of GABABR agonists. Quantitatively, the depression of GABA release in layer II and layer V, and of glutamate release in layer V was similar, but glutamate release in layer II was depressed to a greater extent. The data suggest that the same GABABR may be present on both GABA and glutamate terminals in the EC, but that the heteroreceptor may show a greater level of expression in layer II. Studies with GABABR antagonists suggested that neither the auto- nor the heteroreceptor was consistently tonically activated by ambient GABA in the presence of TTX. Studies in EC slices from rats made chronically epileptic using a pilocarpine model of temporal lobe epilepsy revealed a reduced effectiveness of both auto- and heteroreceptor function in both layers. This could suggest that enhanced glutamate and GABA release in the EC may be associated with the development of the epileptic condition. Copyright © 2006 S. Karger AG.

Species differential regulation of COX2 can be described by an NFκB-dependent logic AND gate

Cyclooxygenase 2 (COX2), a key regulatory enzyme of the prostaglandin/eicosanoid pathway, is an important target for anti-inflammatory therapy. It is highly induced by pro-inflammatory cytokines in a Nuclear factor kappa B (NFκB)-dependent manner. However, the mechanisms determining the amplitude and dynamics of this important pro-inflammatory event are poorly understood. Furthermore, there is significant difference between human and mouse COX2 expression in response to the inflammatory stimulus tumor necrosis factor alpha (TNFα). Here, we report the presence of a molecular logic AND gate composed of two NFκB response elements (NREs) which controls the expression of human COX2 in a switch-like manner. Combining quantitative kinetic modeling and thermostatistical analysis followed by experimental validation in iterative cycles, we show that the human COX2 expression machinery regulated by NFκB displays features of a logic AND gate. We propose that this provides a digital, noise-filtering mechanism for a tighter control of expression in response to TNFα, such that a threshold level of NFκB activation is required before the promoter becomes active and initiates transcription. This NFκB-regulated AND gate is absent in the mouse COX2 promoter, most likely contributing to its differential graded response in promoter activity and protein expression to TNFα. Our data suggest that the NFκB-regulated AND gate acts as a novel mechanism for controlling the expression of human COX2 to TNFα, and its absence in the mouse COX2 provides the foundation for further studies on understanding species-specific differential gene regulation.

The value of corporate reputation and the Defamation Act 2013:a brave new world or road to ruin?

Questions whether the focus on freedom of expression under the Defamation Act 2013 could undermine the value of corporate reputation as a commercial asset.

Redefining 'media' using a 'media-as-a-constitutional-component' concept:an evaluation of the need for the European Court of Human Rights to alter its understanding of 'media' within a new media landscape

This paper considers the impact of new media on freedom of expression and media freedom within the context of the European Convention on Human Rights and European Court of Human Rights jurisprudence. Through comparative analysis of US jurisprudence and scholarship, this paper deals with the following three issues. First, it explores the traditional purpose of the media, and how media freedom, as opposed to freedom of expression, has been subject to privileged protection, within an ECHR context at least. Secondly, it considers the emergence of new media, and how it can be differentiated from the traditional media. Finally, it analyses the philosophical justifications for freedom of expression, and how they enable a workable definition of the media based upon the concept of the media-as-a-constitutional-component.

The isolation and characterization of tescalcin, a novel gene differentially expressed in the mouse testis during the early stages of gonadal differentiation

Gonadal development is an ideal model to study organogenesis because a variety of developmental processes can be studied during the differentiation of the bipotential primordium into testis or ovary. To better understand this process, Representational Difference Analysis of cDNA was used to identify genes that are differentially expressed in mouse gonads at 13.5 days post-coitus. The analysis led to the identification of three testis specific genes and a sequence that was only expressed in the ovary. The male genes identified: renin, Col9a3, and a novel gene termed tescalcin had patterns of expression that suggested a role in testis determination. ^ Studies of the tescalcin gene revealed that it is organized into eight exons and seven introns. The gene was located at 64 cM in mouse chromosome 5, where it spans approximately 35 Kb. Three mRNA variants resulting from alternative splicing of intron 5 were identified in mouse tissues. Gel mobility shift assays demonstrated that Sp1 and Sp3 from Y-1, msc-1, and MIN-6 cells nuclear extracts bind the GC-boxes within the tescalcin proximal promoter. Bisulfite sequencing analysis of tescalcin CpG island revealed that it is differentially methylated in male and female mouse embryonic gonads, and that hypermethylation of this region represses expression of tescalcin in the β-TC3 cell line. ^ The major tescalcin mRNA encodes a protein with 214 amino acids that contains a consensus EF-hand Ca2+-binding domain and an N-myristoylation motif. The amino acid sequence of tescalcin is highly conserved among various species, and it showed the highest homology with calcineurin B homologous proteins 1 and 2, and calcineurin B. Western blot analysis using antibodies generated against the tescalcin protein confirmed its presence in specific mouse tissues and cell lines. Immunohistochemical analysis of mouse embryos confirmed the pattern of expression of tescalcin mRNA in fetal testis. Using pull-down assays, glyceraidehydes-3-phosphate dehydrogenase was identified as an interacting and potential functional partner of tescalcin. ^ The identification and characterization of tescalcin as a novel embryonic testicular marker will contribute to the elucidation of the genetic pathways involved in testis development and likely to the understanding of pathological conditions such as sex reversal and infertility. ^

The representation of motherhood in contemporary Catalan fiction

This dissertation analyzes four twenty-first-century Catalan novels which present the complex positions occupied by mothers in the last seven decades. Its conceptual framework posits motherhood as both a changing social construction and a political institution in a constant state of flux. In Inma Monsó´s Todo un carácter (2001), Eva Piquer´s Una victoria diferente (2002), Carme Riera´s La mitad del alma (2004), and Najat El Hachmi´s El último patriarca (2008) motherhood is explored as a metaphorical act, a gender-constructing experience, as well as the locus of expression with regard to gender and power relations. During the dictatorship of Francisco Franco (1939–1975), the majority of women were excluded from public spaces, and forced to stay home to care for their husbands and children. Furthermore, the state criminalized abortion, made contraception and divorce illegal, and promoted an ideal of femininity based on silence, sacrifice, and self-denial. The political changes of the late 1970s allowed women greater personal autonomy, and many women writers began to challenge stereotypical views of women’s social roles. Yet in the 70s and 80s, the narratives of Esther Tusquets, Ana María Moix, and Montserrat Roig represent the mother as a repressive figure whom the daughter must reject in order to liberate herself and regain her voice. It is not until the 90s when the novelists Mercedes Abad, Maruja Torres, Carme Riera, Imma Monsó, Eva Piquer, and María Barbal rehumanize the mother figure, recovering their matrilineal heritage. However, far from suggesting a unified trend in representations of motherhood in Catalan fiction, the diverse points of view of the novels under discussion here reveal that differences in attitudes among women authors about mother-daughter conflict are far from resolved. The theoretical background for this dissertation draws mainly on the work of Adrienne Rich, Nancy Chodorow, and Julia Kristeva. It includes psychoanalytic studies as well as sociologically based essays by Anna López Puig, Amparo Acereda, Jacqueline Cruz, Barbara Zecchi, Ángeles de la Concha, and Raquel Osborne, among others.

Silence by Stealth: Freedom of the Press and Polarization in Latin America

The weak democratic systems that followed decades of military dictatorships in Latin America coupled with the emergence of new authoritarian regimes of the left have had a significant impact on the relationships between the governments and the media. The new populist leaders have challenged the media that have generally reflected the perspectives of the traditional elites. This ideological clash has renewed direct and indirect censorship, curtailing freedom of expression and thus, freedom of the press. In this context, this paper discusses the mechanisms used by Latin American governments, particularly the new authoritarianism of the left, to silence dissident voices. Many of these mechanisms are legal, found in laws related to personal injury and defamation. Others have been of constitutional nature, invoking states of emergency or national security concerns. Some governments have used institutional means to close down newspapers and other sources of information. Current media conditions in Latin America show growing polarization. This has led to considerable levels of violence and intimidation against editors, journalists, and news crews in several countries. It is precisely this type of deterioration of fundamental rights that leads to questioning the strength and sustainability of Latin American democracies.

An investigation of twentieth century observant Jewish fine artists

People of the Jewish faith base their belief on the written word of the Torah. Presented in this paper are fine artists that produce work within these laws. The Torah sets guidelines for life and morality. The belief system within this domain is that visual images have an impact on the viewers, and artists are accountable for what they produce. This is in opposition with art education, where freedom of expression takes precedence over morality. The results of this study will form the basis for a curriculum for the community college. The researcher's area of inquiry is directed to painting and sculpture made by artists of the Jewish faith who follow the Torah, meaning those who are observant of their faith and practices. Their skills and perceptions will be presented to educate the viewer about their visions. The research questions were posed to rabbinical authorities and artists in order to establish a clear and defined statement of what the Jewish law is regarding the fine arts. The evidence presented was obtained by questionnaires, personal interviews, articles, and opinions from Jewish scholars. Four rabbis were selected based on their erudition on Torah law, and their strong leadership positions in Jewish educational institutions. The ten artists were selected based on recommendations from art historians, and art and gallery directors. The artists and the rabbis were mailed questionnaires, which was followed by an interview. The conclusion from this study is that fine artists are encouraged to use their talents, this is supported by the Torah text, and rabbinic explanation. The restriction for the Jewish artist is in making a replication of a realistic full-scale figure, making a visual rendition of G-d, a nude, or violent image. Art is made by the observant Jew with the intention of enhancing the world with visions inspired by their belief in the Torah. A crucial belief in Judaism is that there is but one G-d, and all man-made images should reflect the majesty of G-d's creations.

On the use of transgenic mice and optogenetics to characterize genetically defined subpopulations of neurons

Neuroscientists have a variety of perspectives with which to classify different parts of the brain. With the rise of genetic-based techniques such as optogenetics, it is increasingly important to identify whether a group of cells, defined by morphology, function or anatomical location possesses a distinct pattern of expression of one or more genetic promoters. This would allow for better ways to study of these genetically defined subpopulations of neurons. In this work, I present a theoretical discussion and threeexperimental studies in which this was the main question being addressed. Paper I discusses the issues involved in selecting a promoter to study structures and subpopulations in the Ventral Tegmental Area. Paper II characterizes a subpopulation of cells in the Ventral Tegmental Area that shares the expression of a promoter and is anatomically very restricted, and induces aversion when stimulated. Paper III utilizes a similar strategy to investigate a subpopulation in the subthalamic nucleus that expresses PITX2 and VGLUT2 which, when inactivated, causes hyperlocomotion. Paper IV exploits the fact that a previously identified group of cells in the ventral hippocampus expresses CHRNA2, and indicates that this population may be necessary and sufficient for the establishment of the theta rhythm (2-8 Hz) in the Local Field Potential of anesthetized mice. All of these studies were guided by the same strategy of characterizing and studying the role of a genetically defined subpopulation of cells, and they demonstrate the different ways in which this approach can generate new discoveries.

Single-Cell Transcriptome Analysis of Olfactory Sensory Neurons

Olfactory sensory neurons (OSNs), which detect a myriad of odorants, are known to express one allele of one olfactory receptor (OR) gene (Olfr) from the largest gene family in the mammalian genome. The OSNs expressing the same OR project their axons to the main olfactory bulb where they converge to form glomeruli. This “One neuron-one receptor rule” makes the olfactory epithelium (OE), which consists of a vast number of OSNs expressing unique ORs, one of the most heterogeneous cell populations. However, the mechanism of how the single OR allele is chosen remains unclear along with the question of whether one OSN only expresses a single OR gene, a hypothesis that has not been rigorously verified while we performed the experiments. Moreover, failure of axonal targeting to single glomerulus was observed in MeCP2 deficient OSNs where delayed development was proposed as an explanation for the phenotype. How Mecp2 mutation caused this aberrant targeting is not entirely understood.

In this dissertation, we explored the transcriptomes of single and mature OSNs by single-cell RNA-Seq to reveal their heterogeneity and further studied the OR gene expression from these isolated OSNs. The singularity of sequenced OSNs was ensured by the observation of monoallelic expression of X-linked genes from the hybrid samples from crosses between mice of different strains where strain-specific polymorphisms could be used to track the allelic origins of SNP-containing reads. The clustering of expression profiles from triplicates that originated from the same cell assured that the transcriptomic identities of OSNs were maintained through the experimental process. The average gene expression profiles of sequenced OSNs correlated well to the conventional transcriptome data of FACS-sorted Omp-positive cells, and the top-ranked expression of OR was conceded in the single-OSN transcriptomes. While exploring cellular diversity, in addition to OR genes, we revealed nearly 200 differentially expressed genes among the sequenced OSNs in this study. Among the 36 sequenced OSNs, eight cells (22.2%) showed multiple OR gene expression and the presences of additional ORs were not restricted to the neighbor loci that shared the transcriptional effect of the primary OR expression, suggesting that the “One neuron-one receptor rule” might not be strictly true at the transcription level. All of the inferable ORs, including additional co-expressed ORs, were shown to be monoallelic. Our sequencing of 21 Mecp2308 mutant OSNs, of which 62% expressed more than one OR genes, and the expression levels of the additional ORs were significantly higher than those in the wild-type, suggested that MeCP2 plays a role in the regulation of singular OR gene expression. Dual label in situ hybridization along with the sequence data revealed that dorsal and ventral ORs were co-expressed in the same Mecp2 mutant OSN, further implying that MeCP2 might be involved in regulation of OR territories in the OE. Our results suggested a new role of MeCP2 in OR gene choice and ratified that this multiple-OR expression caused by Mecp2 mutation did not accompany delayed OSN development that has been observed in the previous studies on the Mecp2 mutants.

Seawater carbonate chemistry and Strongylocentrotus purpuratus body length and gene expression pattern changes during experiments, 2011

Extensive use of fossil fuels is leading to increasing CO2 concentrations in the atmosphere and causes changes in the carbonate chemistry of the oceans which represents a major sink for anthropogenic CO2. As a result, the oceans' surface pH is expected to decrease by ca. 0.4 units by the year 2100, a major change with potentially negative consequences for some marine species. Because of their carbonate skeleton, sea urchins and their larval stages are regarded as likely to be one of the more sensitive taxa. In order to investigate sensitivity of pre-feeding (2 days post-fertilization) and feeding (4 and 7 days post-fertilization) pluteus larvae, we raised Strongylocentrotus purpuratus embryos in control (pH 8.1 and pCO2 41 Pa e.g. 399 µatm) and CO2 acidified seawater with pH of 7.7 (pCO2 134 Pa e.g. 1318 µatm) and investigated growth, calcification and survival. At three time points (day 2, day 4 and day 7 post-fertilization), we measured the expression of 26 representative genes important for metabolism, calcification and ion regulation using RT-qPCR. After one week of development, we observed a significant difference in growth. Maximum differences in size were detected at day 4 (ca. 10 % reduction in body length). A comparison of gene expression patterns using PCA and ANOSIM clearly distinguished between the different age groups (Two way ANOSIM: Global R = 1) while acidification effects were less pronounced (Global R = 0.518). Significant differences in gene expression patterns (ANOSIM R = 0.938, SIMPER: 4.3% difference) were also detected at day 4 leading to the hypothesis that differences between CO2 treatments could reflect patterns of expression seen in control experiments of a younger larva and thus a developmental artifact rather than a direct CO2 effect. We found an up regulation of metabolic genes (between 10 to 20% in ATP-synthase, citrate synthase, pyruvate kinase and thiolase at day 4) and down regulation of calcification related genes (between 23 and 36% in msp130, SM30B, SM50 at day 4). Ion regulation was mainly impacted by up regulation of Na+/K+-ATPase at day 4 (15%) and down regulation of NHE3 at day 4 (45%). We conclude that in studies in which a stressor induces an alteration in the speed of development, it is crucial to employ experimental designs with a high time resolution in order to correct for developmental artifacts. This helps prevent misinterpretation of stressor effects on organism physiology.

Language, Logos, and Social Ontology: Naturalist and Post-Naturalist Narratives of Human Rationality and Social Reality

I distinguish two ways that philosophers have approached and explained the reality and status of human social institutions. I call these approaches “naturalist” and “post-naturalist”. Common to both approaches is an understanding that the status of mind and its relation to the world or “nature” has implications on a conception of the status of institutional reality. Naturalists hold that mind is explicable within a scientific frame that conceives of mind as a fundamentally material process. By proxy, social reality is also materially explicable. Post-naturalists critique this view, holding instead that naturalism is parasitic on contemporary science—it therefore is non-compulsory and distorts how we ought to understand mind and social reality. A comparison of naturalism and post-naturalism will comprise the content of the first chapter. The second chapter turns to tracing out the dimensions of a post-naturalist narrative of mind and social reality. Post-naturalists conceive of mind and its activity of thought as sui generis, and it transpires from this that social institutions are better understood as a rational mind’s mode of the expression in the world. Post-naturalism conceives of social reality as a necessary dimension of thought. Thought requires a second person and thereby a tradition or context of norms that come to both structure its expression and become the products of expression. This is in contrast to the idea that social reality is a production of minds, and thereby derivative. Social reality, self-conscious thought, and thought of the second person are therefore three dimensions of a greater unity.

Media diversity as a guarantee to cultural diversity: the promotion of identities from an international law approach

This article deals with several international instruments which provide legal guarantees for media diversity, which is essential for the promotion of cultural diversity. Based on several articles of the Convention of cultural diversity, the General Comment of the Committee on Economic, Social and Cultural Rights No. 21 on the right to take part in cultural life, as well as the work of the UN Independent Expert on Cultural Rights, this article aims to identify legal tools for the establishing of measures promoting cultural diversity in the media. This article looks at the case study of Honduran Garifuna community radios. It emphasizes the importance of taking into account the economic aspects of cultural and communicational rights.

HER2 in high-risk rectal cancer patients treated in EXPERT-C, a randomized phase II trial of neoadjuvant capecitabine and oxaliplatin (CAPOX) and chemoradiotherapy (CRT) with or without cetuximab.

BACKGROUND: HER2 is an established therapeutic target in breast and gastric cancers. The role of HER2 in rectal cancer is unclear, as conflicting data on the prevalence of HER2 expression in this disease have been reported. We evaluated the prevalence of HER2 and its impact on the outcome of high-risk rectal cancer patients treated with neoadjuvant CAPOX and CRT±cetuximab in the EXPERT-C trial. PATIENTS AND METHODS: Eligible patients with available tumour tissue for HER2 analysis were included. HER2 expression was determined by immunohistochemistry (IHC) in pre-treatment biopsies and/or surgical specimens (score 0-3+). Immunostaining was scored according to the consensus panel recommendations on HER2 scoring for gastric cancer. Tumours with equivocal IHC result (2+) were tested for HER2 amplification by D-ISH. Tumours with IHC 3+ or D-ISH ratio ≥2.0 were classified as HER2+. The impact of HER2 on primary and secondary end points of the study was analysed. RESULTS: Of 164 eligible study patients, 104 (63%) biopsy and 114 (69%) surgical specimens were available for analysis. Only 3 of 104 (2.9%) and 3 of 114 (2.6%) were HER2+, respectively. In 77 patients with paired specimens, concordance for HER2 status was found in 74 (96%). Overall, 141 patients were assessable for HER2 and 6 out of 141 (4.3%) had HER2 overexpression and/or amplification. The median follow-up was 58.6 months. HER2 was not associated with a difference in the outcome for any of the study end points, including in the subset of 90 KRAS/BRAF wild-type patients treated±cetuximab. CONCLUSIONS: Based on the low prevalence of expression as recorded in the EXPERT-C trial, HER2 does not appear to represent a useful therapeutic target in high-risk rectal cancer. However, the role of HER2 as a potential predictive biomarker of resistance to anti-EGFR-based treatments and a therapeutic target in anti-EGFR refractory metastatic colorectal cancer (CRC) warrants further investigation. TRIAL REGISTRATION: ISRCTN Register: 99828560.