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Research Paper Volume 8, Issue 5 pp 917—932
The effects of graded levels of calorie restriction: VII. Topological rearrangement of hypothalamic aging networks
- 1 Institute of Biological and Environmental Sciences, University of Aberdeen, Aberdeen, Scotland, AB24 2TZ, UK
- 2 Centre for Genome Enabled Biology and Medicine, University of Aberdeen, Aberdeen, Scotland, AB24 3RL, UK
- 3 State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Chaoyang, Beijing, 100101, China
- 4 Chinese Academy of Sciences Key Laboratory of Computational Biology, Chinese Academy of Sciences-Max Planck Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China
- 5 Key Laboratory of Systems Biology, Innovation Center for Cell Signaling Network, Institute of Biochemistry and Cell Biology, Shanghai Institute of Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China
- 6 Department of Pathology and Department of Biology, University of Washington, Seattle, WA 98195, USA
received: March 2, 2016 ; accepted: March 31, 2016 ; published: April 23, 2016 ;https://doi.org/10.18632/aging.100944
How to Cite
Connectivity in a gene-gene network declines with age, typically within gene clusters. We explored the effect of short-term (3 months) graded calorie restriction (CR) (up to 40 %) on network structure of aging-associated genes in the murine hypothalamus by using conditional mutual information. The networks showed a topological rearrangement when exposed to graded CR with a higher relative within cluster connectivity at 40CR. We observed changes in gene centrality concordant with changes in CR level, with Ppargc1a, and Ppt1 having increased centrality and Etfdh, Traf3 and Abcc1 decreased centrality as CR increased. This change in gene centrality in a graded manner with CR, occurred in the absence of parallel changes in gene expression levels. This study emphasizes the importance of augmenting traditional differential gene expression analyses to better understand structural changes in the transcriptome. Overall our results suggested that CR induced changes in centrality of biological relevant genes that play an important role in preventing the age-associated loss of network integrity irrespective of their gene expression levels.
Brain aging has an impact on cognitive function and forms a major risk for the development of neuro-degenerative disorders such as Parkinson's, Huntington's and Alzheimer's disease . In murine brain tissue, aging is associated with transcriptional changes in genes that induce an elevated inflammatory response and increased oxidative stress [2,3]. These transcriptomic patterns associated with aging may be established in the brain early in adolescent . Aging and aging-related diseases are thought to be centrally programmed by the hypothalamus [4,5]. Much evidence suggests that calorie restriction (CR), a non-invasive method to increase lifespan, has anti-inflammatory properties and reduces age-related oxidative stress [6–10]. A recent study suggests that CR leads to a neuroprotective transcriptomic profile in rodent brain and protects against aging related decline in memory function . A large number of interacting genes, forming an interaction network, alter their transcription in response to aging and CR. However, understanding the complex interactions between genes is a challenge that cannot be addressed by looking at gene expression alone . Network analyses of interactions between genes could further elaborate the complex mechanisms underlying both aging and CR. We can define interaction networks in several ways. Most often, we have represented gene interaction networks using correlations in gene expression. We can also derive from the same observations a measure of information shared between gene expressions  (reviewed in ). This flow of information between genes in a network therefore gives rise to new measurable characteristics (i.e. network behavior) which are absent in isolated components such as the expression levels of single genes (Alm 2003). Soltow et al. (2010) proposed that aging of a system can be generalized to four characteristics: (1) decline in capacity and utilization of energy, (2) decline in structure or metabolic organization, (3) decline in barrier functions and (4) decline in transfer of information . The decline in structure and information flow during aging can be assessed by evaluating quantitative changes in network topology.
The structure of a gene network is subject to a few simple principles and allows for the comparison and characterization of different complex networks. Empirical studies show that many cellular networks are scale-free which means that the degree distribution (or connectivity of nodes) follows a negative power law . In yeast exposed to CR, gene-regulatory networks were also found to be scale-free . Many scale-free networks also have a hierarchical structure with high clustering coefficients of the nodes following a negative relationship with the degree distribution. These networks are found to divide naturally into highly connected clusters, which generally correspond to specific biological functions . Biologically important genes play key topological roles in network function and structure, and influence network dynamic processes  enabling us to identify these central genes (network nodes) using network metrics such as node centrality. Central genes have different structural features such as having more connections, influencing more genes in the network through intermediary genes and playing a disproportionate role in information flow . Eigenvector centrality captures the relative importance of each gene for the overall network structure, by reflecting the contribution of the variance in interaction of each gene to the overall interaction heterogeneity over the entire network .
Age-associated changes in network structure have been identified which can help us pinpoint the potential genome wide mechanisms involved in aging [16,22,23]. Network system failure is often associated with information flow disruptions and therefore central genes, in a gene interaction network, are expected to be particular foci for negative effects during the aging process . Functionally-related genes should in principle co-express with each other and therefore gene co-expression networks, estimated using correlation in gene expression across individuals, should change with age. A key change in gene co-expression network structure associated with aging in mice was a loss in connectivity (26% decrease) over the entire network and a decrease in strength of correlations between genes within functional gene clusters . Biologically-related genes clustered together forming highly connected functional gene clusters. Within these functional gene clusters there were gene groups involved in aging-related pathways . Associations between interacting metabolites are also significantly altered under CR compared to ad libitum (AL) feeding in Drosophila melanogaster suggesting that network structure is modified under CR . Laye et al. (2015) postulated that these changes in network structure under CR are protective against the age-associated decline in metabolic homeostasis. Aging protein-protein interaction networks of D. melanogaster showed that the relationship between identified protein clusters could be modified by CR . These results suggest that aging-associated proteins might be key to maintaining stability in a network . Southworth et al. (2009) identified the transcription factor nuclear factor-kappa B (NF-ĸB) as a central gene involved in the loss of network connectivity with aging in mice. Under CR, it would be anticipated that the network should be protected against the aging-associated decline in interdependencies, and should result in a less fragmented network .
Correlations between gene expression levels as a measure of gene interdependencies has been widely used to construct gene co-expression networks . However, this method is limited in what can be inferred from these networks, as well as difficulties in identifying spurious correlations. Further, gene co-expression networks cannot separate direct interactions from transcriptional interactions that correlate with expression levels of many non-interacting genes. Therefore correlations cannot be used to reconstruct gene-gene interaction networks without additional and restrictive assumptions  and are heavily dependent on the associations being linear. In contrast, Mutual Information (MI) provides a measure of general dependence which is not necessarily assumed to be linear in form. MI provides an estimate of the information emanating from gene expression which is shared between any two given genes. This provides a mean to make inferences about interaction between genes in large networks [27,28]. The dependency found between two genes by MI-based methods represents a possible functional dependency, although causality cannot be inferred. Furthermore, MI is limited to only detect dependency between two genes. Conditional MI (CMI) estimates were developed, analogous to partial correlation coefficients for correlation measures, to discount the effects of the expression of other genes when estimating the MI for any gene pairs, hence accounting for false positives . CMI is able to detect joint regulations of more than two genes by exploiting the conditional dependency between genes of interest. Therefore, CMI better captures functional relationships between genes and allows us to asses network-wide functional changes under, in our case, different CR levels [27,30,31]. Changes in a CMI network therefore represent changes in functions in response to aging. We can therefore capture overall functional changes concurrently, allowing us to detect central genes involved with these changes.
Based on the prior knowledge that aged brains have increased oxidative stress and inflammation [2,3], we tested whether the network structure of genes associated with aging, oxidative stress response and inflammation changed when individuals were exposed to different levels of CR. We postulated that the gene-gene CMI of these aging-associated genes would alter under CR, representing changes in functionality and regulation. We predicted that the network structure, specifically its connectivity would be increased by CR, which should protect against the loss of network connectivity. Furthermore, we expected that the changes in connectivity would be found in gene clusters, as demonstrated by Southworth et al. (2009), where aging-associated decline was typically found in connections between genes within functional gene clusters. We expected to see a change in central genes in the networks according to CR level, reflecting a potential protection against aging. Lastly, we also used classic Spearman's rank correlation coefficients to measure associations, and compared these results to those found using CMI. To test these predictions, we used the hypothalamic transcriptome of mice (20 weeks of age at the beginning of the experiment) exposed to three months graded CR (at levels up to 40% in 10% increments) [10,32–34] and constructed gene CMI interaction networks and gene co-expression networks of aging-associated genes for each CR level. This study complements previous work estimating the change in gene expression with CR for the hypothalamic transcriptome .
The effect of graded CR on the transcriptomic profile of aging-associated genes
We initially used an orthogonal signal correction partial least squares discriminant analysis (O-PLS-DA) to classify 12 and 24 hr AL fed groups (12AL, 24AL) and CR groups based on the expression levels of genes involved in aging, inflammation and oxidative stress (n = 408) obtained from a priori defined gene lists curated by Ingenuity Pathway Analysis (IPA) (‘inflammation of the nervous system’ and ‘oxidative stress response’) and GenAge database (‘aging-associated genes’ in Mus musculus). Individuals were clustered according to CR level, indicating that gene expression levels could predict CR level (Fig 1). Indeed model validation indicated that 41.8% of the variance in gene expression levels was explained by the different treatment groups (n = 6) (Xvar = 41.8 ± 18.5, p-value < 0.001). Of these 408 genes, expression analysis identified 16 genes differentially expressed at 24AL, 10 genes at 10CR, 14 genes at 20CR, 14 genes at 30CR and 59 genes at 40CR relative to 12AL (Table S1).
Figure 1. Orthogonal signal correction partial least squares discriminant analysis (O-PLS-DA) demonstrating the effect of graded CR on gene expression levels of aging-associated genes. 24AL, 12AL, 10CR, 20CR, 30CR and 40CR refer respectively to 24h ad libitum feeding per day, 12h ad libitum feeding per day, 10 %, 20 %, 30 % and 40 % restriction.
The effect of graded CR on network structure and connectivity
We estimated CMI based networks of these aging-associated genes and also standard gene co-expression networks based on correlations. CMI networks were characterized as having either a hierarchical or scale-free structure. Theory predicts that hierarchical networks should display a negative relationship between the degree to which genes cluster together (estimated as a cluster coefficient) and the number of neighbors of a given gene (node degree) (Fig 2A and 2B) . The cluster coefficient did not decrease as node degree increased (Fig 2C), indicating that the networks were not hierarchical. Furthermore, node strength, a cumulative measure of node degree and the CMI of each gene, is predicted to follow a power distribution in scale-free networks (Fig 3A and 3B) . However, in all networks node strength was not power law distributed (Fig 3C). In addition, visualizing the node strength on a log-log scale did not result in a linear relationship (Fig S1).
Figure 2. Node strength plotted against clustering coefficient to assess hierarchical topology. (A) Visualization of a hierarchical network obtained from . (B) Hierarchical network following a negative relationship between cluster coefficient and node strength. (C) Plots to assess hierarchical topology of aging-associated genes networks. 24AL, 12AL, 10CR, 20CR, 30CR and 40CR refer respectively to 24h ad libitum feeding per day, 12h ad libitum feeding per day, 10 %, 20 %, 30 % and 40 % restriction.
Figure 3. Node strength frequency distribution. (A) Visualization of a scale free network obtained from . (B) Node strength in scale free network follow a power law distribution . (C) Plots to asses scale free topology of the aging-associated genes networks. 24AL, 12AL, 10CR, 20CR, 30CR and 40CR refer respectively to 24h ad libitum feeding per day, 12h ad libitum feeding per day, 10 %, 20 %, 30 % and 40 % restriction.
Another characterization of a network is the natural occurrence of gene clusters and the degree of connectivity of those clusters. This was estimated by modularity-based clusters of gene interactions and by assessing the strength of those interactions within these clusters. Modularity-based clusters of gene interactions changed and genes clustered differently at each CR level (Fig 4A) (Table S2). The modularity of the network, which represented the number of clusters, was also different at each CR level. The connectivity between genes in a cluster relative to the connections between clusters was measured by determining the modularity coefficient (Fig 4B). The highest modularity coefficient was observed at 10CR and 40CR, indicating that 10CR and 40CR had a higher connectivity between genes within clusters than between clusters (Fig 4C) (Table S2).
Figure 4. Modularity coefficients per conditional mutual information network at each treatment level were calculated and genes were assigned to clusters according to their mutual information. (A) Changes of clusters across CR level and colored for clusters found at 12AL. (B) Modularity coefficient for each CMI network at each CR level. (C) CMI networks colored according to clusters identified at each CR level. Edges between genes with strong CMI are colored in black, those with weak CMI in light grey. 24AL, 12AL, 10CR, 20CR, 30CR and 40CR refer respectively to 24h ad libitum feeding per day, 12h ad libitum feeding per day, 10 %, 20 %, 30 % and 40 % restriction.
The effects of graded CR on gene centrality
We identified gene centrality, which are genes playing a central role in connectivity, by estimating gene eigenvector centrality in each CMI network. The variance in eigenvector centrality was reduced for 20CR and 30CR (Table S2) and we observed well-defined groups of genes with disproportionally large eigenvector centrality at 24AL, 12AL, 10CR and 40CR (Fig 5A) . Central genes were identified based on the absolute maximum eigenvector centrality value (Table S2). At 24AL four genes were identified as central genes in the CMI network: excision repair cross-complementing rodent repair deficiency, complemen-tation group 4 (Ercc4), electron transferring flavoprotein, dehydrogenase (Etfdh), integrin alpha 9 (Itga9) and mastermind like 1 (Drosophila) (Maml1). One gene, ATP-binding cassette, sub-family C (CFTR/MRP), member 1 (Abcc1) was identified at 12AL, 10CR, and 30CR as a central gene. At 20CR three genes were identified as central genes: tumor necrosis factor (ligand) superfamily, member 10 (Tnfsf10), topoisomerase I binding, arginine/serine-rich (Topors), tripeptidyl peptidase II (Tpp2) and TNF receptor-associated factor 3 (Traf3). At 40CR the genes peroxisome proliferative activated receptor, gamma, coactivator 1 alpha (Ppargc1a) and palmitoyl-protein thioesterase 1 (Ppt1) were identified as central genes in the network. Ppargc1a and Ppt1 showed a clear graded increase in centrality, and Etfdh, Abbc1 and Traf3 a de-crease in centrality relative to CR level, but all genes did not show any simultaneous significant changes in gene expression levels relative to 12AL (Fig 5B). Furthermore at all levels of CR, genes with the highest eigenvector values were found in the same clusters (Fig 5C).
Figure 5. Node centrality was identified by calculating the eigenvector value for each gene in each conditional mutual information (CMI) network. (A) Eigenvector distribution visualized in a violin plot per CR level. (B) Changes of eigenvector values and gene expression levels relative to 12h ad libitum feeding of key genes visualized in a heat map. (C) CMI networks at each CR level with node sizes and color proportional to eigenvector values. Edges between genes with strong CMI are colored in black, those with weak CMI in light grey. 24AL, 12AL, 10CR, 20CR, 30CR and 40CR refer respectively to 24h ad libitum feeding per day, 12h ad libitum feeding per day, 10 %, 20 %, 30 % and 40 % restriction.
Comparison of networks based on CMI and correlation coefficients
We estimated Spearman's rank correlation coefficient between genes and constructed gene co-expression networks based on these correlations. The networks showed the same average number of edges between genes but their modularity and eigenvector centrality differed compared to CMI networks. Overall the gene co-expression network contained substantially more clusters compared to the CMI network with a higher modularity coefficient across CR. In contrast to the CMI based network, the modularity coefficient was the highest at 20CR (Table 1). The genes with the highest eigenvector centrality at 24AL, 20CR, 30CR and 40CR in the gene co-expression networks differed to CMI networks but were identical at 12AL and 10CR (Abcc1). Similar to the CMI networks, these genes were not significantly differentially expressed relative to 12AL. At 24AL angiotensinogen (serpin peptidase inhibitor, clade A, member 8) (Agt) was identified as central gene. In addition to Abcc1, four other genes were identified as central genes at 10CR: Agt, calcium/calmodulin-dependent protein kinase II gamma (Camk2g), CD200 antigen (Cd200), nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2 (Nfatc2) and transformation related protein 53 (Trp53). At 20 and 30CR the central gene was c-abl oncogene 1, non-receptor tyrosine kinase (Abl1). At 40CR the central gene was amyloid beta (A4) precursor protein (App).
Table 1. Comparison between gene co-expression networks and CMI networks
|Treatment||Gene co-expression network||CMI network|
|Modularity coefficient||Clusters||Modularity coefficient||Clusters|
Previous studies of gene expression networks in aging have mostly used gene co-expression networks to assess changes in network structure of individuals in relation to age and CR [16,22,23,36]. Here we used CMI because this measure provides a way to make inference about the amount of information shared between two genes when they are expressed. This is important for age-related studies for which we have a biological understanding that information flow at different cellular levels is disrupted by age. To our knowledge, CMI is used here for the first time to assess the changes in network topology associated with aging. We predicted that CR would result in a less fragmented regulatory network of aging associated genes compared to AL. We show that CR explained a significant proportion of the variability observed in the expression levels of aging-associated genes. Our CMI networks based on these genes suggest that (1) our networks were modular but did not have a hierarchical or scale-free topology; (2) the CMI network changed most at 40CR compared to 12AL with a higher connectivity between genes within clusters than between clusters; and (3) central genes for the regulatory network architecture showed a graded response to changing CR levels, with changes most pronounced at 40CR. This is consistent with previous studies at other biological levels for this experiment which showed a physiological state shift in mice exposed to 40CR [10,32–34,37]. Although the expression level of individual genes Ppargc1a, Ppt1, Etfdh, Abbc1 and Traf3 did not change , these genes were identified as ‘central genes’ indicating a network-wide role of these genes. We also showed that the networks exhibited a topological arrangement when exposed to graded CR.. It might be argued that when the networks are exposed to CR and close to a state shift, more non-monotonic relationships are formed which cannot be inferred using correlation methods.
By comparing differences in network topology we can make inferences about changes in the functional interaction between genes that would not have been identified using a gene-by-gene traditional expression analysis.
Regulatory networks of aging-associated genes exposed to graded CR have a modular topology
Many biological networks have a scale-free topology displaying a high degree of clustering which is suggested to be a consequence of a hierarchical organization of that network . The CMI networks of our mice were not scale-free and did not display a hierarchical topology. Instead we had a modular network characterized by highly interlinked modules (or clusters) which were connected to each other with relatively few links . Both the number of clusters and the gene composition of clusters changed in response to CR. These changes in gene cluster membership would suggest changes in functional interactions between genes in response to CR . In agreement with our results, a study with Caenorhabditis elegans found a structural reorganization of the transcriptomic network when glucose metabolism was impaired, and specifically in aging-relevant signaling pathways including the mammalian target of rapamycin mTOR pathway . We also found that as CR levels increased the number of connections between genes was higher within gene clusters compared to those between clusters. The changes in connectivity observed here indicate that at 10CR and 40CR the networks have a higher connectivity in these functional gene groups compared to the two AL groups. The differences observed in network behavior at 20CR and 30CR could be a representation of a phase transition resulting in a topological state shift occurring at 40CR . This would be in agreement with phenomic state shifts observed for these mice [33,37,86]. Prolonged “network stress” was found to induce a topological phase transition in cellular networks with an entirely different switch in cellular function (reviewed in ). Exposing mice to CR imposes a “network stress” and structural reorganization is necessary as an adaptive response. The adaptive response to CR is well established to increase longevity in many taxa and induce other beneficial anti-aging effects (reviewed in ). A “network stress” induced shift includes an initial decrease in average link density which might help the network to prevent propagation of damage . During prolonged “network stress”, the network undergoes a segregation between less important genes and more important genes (central genes) which results in these central genes displaying more and stronger links to their neighbors. This would lead to more highly connected clusters of genes and less connections between genes outside these clusters which was observed at 40CR. As the level of CR increased the level of “network stress” increased and segregation occurred resulting in a reorganization and higher connectivity within clusters at 40CR. The network reorganization and increased connectivity at 40CR suggests the network would be more protected against the aging-observed decline in connectivity .
Changes in gene centrality irrespective of gene expression levels
Topologically central genes have a disproportionate influence on the overall integrity of gene regulatory networks . Here, we identified central genes that played a central role in network structure and found a change in the connectivity of these genes in relation to the CR level. The central gene in the CMI networks at 12AL, 10CR and 30CR was Abcc1 or also known as MRP1. The importance of this gene in the network structure deceased in a graded manner with increasing CR level. Gene expression analysis of this gene showed no significant difference of this gene under CR compared to 12AL. This is in agreement with Laye et al. (2015) who demonstrated that the changes in correlation coefficient with age between metabolites in a co-expression network were independent of their mean levels . Although first identified as a drug transporter, MRP1 is also involved in inflammation and oxidative stress . MRP1 can also flux small peptides such as N-acetyl-leu-leu-norleucina (ANLL) which inhibits the 6S-proteasome activity which activates NF-ĸB . Furthermore TNF-α has been associated with MRP1 . We previously found that circulating TNF-α levels were reduced under CR  and that the transcription factor NF-ĸB in the hypothalamus was inhibited at all CR levels compared to AL, suggesting a reduced state of inflammation . Furthermore micro-inflammation in the hypothalamus involving elevated NF-ĸB was recently suggested to mediate whole body-aging  and therefore a reduced state of inflammation under CR would have a protective role against whole-body aging.
At 20CR four genes were identified as central genes in network structure: Tnfsf10, Traf3, Tpp2 and Topors. The genes Tnfsf10 and Traf3 are associated with TNF-α and we established previously that Tnfsf10 correlated positively with circulating TNF-α levels [10,34]. Interestingly in human centenarians dementia is associated with high levels of circulating TNF-α levels and suggests a role of TNF-α in age-associated brain pathology . Our results may indicate that reduced TNF-α signaling via Tnfsf10 and Traf3 plays a key role in network structure at 20CR. The other key gene, Tpp2, was shown to have a pro-longevity effect based on a mouse knockout study . Tpp2 knockout mice over 1 year old exhibited elevated mortality associated with an aged appearance. Furthermore, alterations were observed in p53 expression and NF-ĸB activation but no direct molecular link was found with Tpp2 . Tnfsf10 has also been found to mediate p53 dependent cell death . Similar to Tpp2, Topors was also identified as having a pro-longevity effect  and also found to act as a tumor suppressor . Topors knockout mice exhibited a significant reduction in mean and maximum lifespan and several mice had signs of premature aging .
The connectivity of the 40CR network differed substantially from the 12AL network. If our assertions are right, the 40CR network represents a network which receives the benefits of CR. Ppargc1a and Ppt1 were key topological central genes at 40CR which were involved in this overall difference in connectivity. They are important to protect the network from aging-associated loss in structure and fragmentation. A key change associated with aging was a loss in connectivity over the entire network and a decrease between genes within functional gene clusters . Central genes are therefore expected to be particular foci for negative effects during the aging process . Ppargc1a encodes PGC1α which is a major transcription factor contributing to gluconeogenesis, energy metabolism, lipid metabolism and functioning of the mitochondria . PGC1α was shown to be upregulated in the brain when exposed to long term CR  but not in the hypothalamus when fasted for 60h . Here, we did not find a significant regulation of Ppargc1a under CR compared to 12AL after 3 months of study. Brain aging was associated with a downregulation of the PGC1α mediated transcriptional pathway  and neuro-degenerative disorders such as Parkinson's disease are associated with lower levels of the target genes of PGC1α . In Alzheimer's patients PGC1α expression is reduced . Interestingly PGC1α overexpression in transgenic mice models for Alzheimer diseases exacerbates the neuropathological and behavioral deficits . PGC1α null mice are lean and are resistant to diet-induced obesity. These mice exhibit profound hyperactivity associated with lesions in the brain region that controls movement . When PGC1α was blocked locally within the central nervous system, fasted rats exhibited lower 24h food intake compared to those treated with a vehicle. Similar results were found for non-fasted high-fat induced obese rats but not for non-fasted low fat chow fed rats suggesting a greater role for neural PGC1α during fasting and obesity . Fasted neural PGC1α null mice show diminished hypothalamic expression levels of the neuropeptides agouti-related neuropeptide (AgRP) and neuropeptide Y (NPY) which would suggest a role of PGC1α in regulating energy metabolism via regulation of hypothalamic neuropeptides . The neuropeptides AgRP and NPY both increase hunger signaling and were upregulated in our transcriptomic dataset . Although shown in diet induced obese mice, PGC1α is also associated with the control of reactive oxygen species (ROS) by hypothalamic pro-opiomelanocortin (POMC) neurons and hypothalamic ROS levels are positively correlated with circulating leptin levels . Diano et al. (2011) postulated that the leptin resistance observed in these mice might be controlled by ROS manifested by reduced POMC and increased NPY/AgRP neuronal firing . Interestingly during CR, leptin levels are reduced and correlated positively with reduced expression levels of POMC and negatively with increased expression levels of NPY/AgRP in our dataset [10,34] which suggests an improved leptin sensitivity and reduced levels of ROS. However, because of a lack of tissue we were unable to measure hypothalamic ROS levels in this study but we can conclude that our mice exhibited an increased ‘hunger profile’ . Upon re-feeding, after a period of short-term (100 days) CR, the hyperphagic response suggests that hunger remained even after energy balance was re-established . This elevated hunger profile might be a major factor contributing to the beneficial effect of CR . A recent study has shown an abrogated effect on longevity when NPY−/− mice were exposed to 30% CR compared to the AL fed mice, key role for NPY to link CR to increased lifespan .
We and others have previously postulated that hypothalamic nutrient sensing signaling might mediate the observed increase in lifespan under CR treatment [34,66–68]. Mice lacking PGC1α have abnormal diurnal rhythms of activity and metabolic rate and this was related to abnormalities in expression of clock genes and energy metabolism associate genes . PGC1α also induces circadian clock resetting in restricted feeding and stimulates the clock gene aryl hydrocarbon receptor nuclear translocator-like (ARNTL) expression . transcriptomic analysis and others indeed suggests that under CR mice are protected against aging-associated desynchronization of the circadian clock [34,36,70,71]. Although Ppargc1a gene expression levels were not significantly correlated with the extent of restriction, its downstream effects were. Here we were able to elucidate the importance of this gene in metabolic regulation via network analysis which would have remained unidentified with simple gene by gene expression based approaches. Interestingly mice with Ppt1 deficiency, the other key gene that changed in relation to the degree of CR, exhibited disruption of adaptive energy metabolism and downregulation of PGC1α . Due to their key roles in network structure as central genes, Ppargc1a and Ppt1 play a major role in protecting the network from age-observed decline in connectivity [22,23].
Comparison of CMI and correlation coefficient based networks
Gene co-expression networks based on correlation coefficients have been widely used, for example in aging/CR research [16,23,36,73], to assess topological changes to networks in response to diseases [74–79], as characterization of a cellular state  and to understand plant cellular processes [81,82]. In contrast, CMI based networks have rarely been used despite recent theoretical advances in their application and interpretation and additional insight they can provide. Correlations assess ‘co-expression’ as the sum of the product of two random variables, while CMI specifically calculates the sum of the joint probabilities of two random variables from which inferences of causality can be made. Crucially, correlation assumes linear associations, or associations that change at the same rate with rank/order for non-parametric measures. CMI however, will detect associations whether they are monotonic or not. These measures are not antagonistic, they are complementary . However, a major hurdle arises when we try to address the issue that in a many-body system the association between any given pair of objects can be influenced by their own associations with other objects. In our case this represents our attempt to detect whether the co-expression of two genes is real or caused by their mutual co-expression with a third gene. CMI estimates are more robust than partial correlation estimates because we have a better understanding of how to define joint probability distributions. Therefore, CMI can be more advantageous, particularly for large networks . Finally correlation and CMI capture the shared information/covariance between two different measures of gene expression. For correlation, we try to understand the shared variance in gene expression. With CMI we try to estimate the joint information emanating from genes and thereby assuming causality. These two methods have previously been compared and an almost one-to-one correspondence between the two methods was found, the authors concluding that non-linear relationships did not play an important role in their gene networks [83,85]. The fundamental difference however lies in what can be inferred from these two methods. We indeed found similarities between our CMI and gene co-expression networks with respect to eigenvector centrality at 12AL and 10CR but we also found fundamental differences in modularity. In the CMI network the intermediate groups had fewer clusters but the amount of clusters at 40CR was more similar to 12AL while modularity differed. This was clearly shown by the modularity coefficient of the CMI based analysis while this was not the case for the correlation coefficient based networks. We can conclude from these results that gene co-expression based modularity coefficient estimates were relatively insensitive to CR whereas the CMI estimates were able to discriminate between 20CR and 30CR. It might be argued that when the networks are exposed to CR and close to a state shift, more non-monotonic relationships are formed which cannot be inferred using correlation methods. Hence CMI provides a measure for which we can directly test biologically relevant co-expression and given the particular focus we can measure changes in information flow and regulation with aging and CR.
In this study we found a structural reorganization of the gene regulatory network under graded levels of CR. Using the CMI network analysis approach we identified Ppargc1a, Ppargc1a, Ppt1, Etfdh, Traf3 and Abcc1 as key regulatory genes in network structure with changes in their centrality as the level of CR increased. Since expression levels of these genes did not change with CR, this study emphasizes the importance to move beyond gene by gene level analyses to better understand regulatory changes of the transcriptome when it is challenged (in our case by CR). Our results suggest that the network structure of aging-associated genes under graded CR was altered and might play an important role in preventing the loss of network structure observed with age.
Materials and Methods
To create a systems level description of graded CR responses, we performed a three month graded CR study on male C57BL/6 mice commencing at age 5 months [10,32,33,37,86]. Full details of the experimental protocol and rationale for the experiment are given in  and are elaborated briefly below. In this study, transcriptome sequencing or RNA-seq of the hypothalamus (see also ) was used to assess changes in network structure of aging-associated genes.
Animals and experimental manipulations
All procedures were approved by the University of Aberdeen ethical approval committee and carried out under the Animals (Scientific Procedures) Act 1986 Home Office license (PPL 60/4366 held by JRS). Forty nine male C57BL/6 mice (Mus musculus), purchased from Charles River (Ormiston, UK) were individually housed and free access to water was provided. Mice were exposed to 12 hour dark/light cycle (lights on at 0630h) and body mass and food intake were recorded daily, immediately prior to nocturnal feeding. At 20 weeks of age (resembling early adulthood in humans), mice had their food intake monitored for 2 weeks of baseline and were then randomly allocated into 6 different treatment groups: 24h ad libitum intake (24AL) (n=8), 12AL intake which had access to ad libitum food but for only for the 12 hours of darkness per day, sometimes also called ‘time restricted feeding’ (n=8), 10 CR (n=8), 20CR (n=8), 30CR (n=8) and 40CR (n=9). Mice exposed to 40CR indicates 40% fewer calories were provided than the individual intake measured over a baseline period of 14 days prior to introducing CR. This is a CR protocol rather than a caloric dilution experiment .
Animals fed completely AL (i.e., having 24 hours access to food) may potentially over feed, become obese and CR associated changes compared to 24AL would therefore most likely reflect the anti-obesity effect of CR [42,88]. To address this issue, 12AL was set as a reference and graded levels of CR were introduced to investigate a potential graded response mirroring the graded lifespan response to CR manipulation [87,89]. Detailed information on overall study design, diet composition and detailed rationale are described elsewhere .
RNA isolation, RNA-sequencing, alignment and analytical procedure
After culling by a terminal CO2 overdose, brains were removed, weighed and frozen in isopentane over dry ice and stored at −80°C for RNA isolation. The hypothalamus was carefully dissected at a later stage and RNA was isolated by homogenizing in Trizol (Sigma Aldrich, UK) according to manufacturer's instructions. Prior to RNA quantification, using the Agilent RNA 6000 Nano Kit, samples were denatured at 65°C.
Due to the very small size of the hypothalamus, some samples did not contain sufficient quantity of high quality RNA. In total, the RNA of 37 individual mice (12 h AL n=6, 24 h AL n=6, 10 % CR n=7, 20 % CR n=5, 30 % CR n=5, 40 % CR n=8) was successfully isolated and sent to Beijing Genomic Institute (BGI, Hong Kong) for RNA sequencing. Library preparation was done using a standard protocol of BGI and the library products were sequenced using an Illumina Hi-seq 2000, resulting in 50 bp single end reads. Standard primers and barcodes developed by BGI were used. Detailed information on library preparation for these samples and the bio-informatic pipeline has been described previously .
Network construction and biological interpretation
Based on the prior knowledge that aged brains have increased oxidative stress and inflammation [2,3], we curated a data set based on a prior defined gene lists from Ingenuity pathway analysis (IPA) (Ingenuity® Systems, http://www.ingenuity.com/products/ipa,version 2000-2015): ‘inflammation of the nervous system’ (n=196) and ‘oxidative stress response’ (n=155); and genes associated with aging derived from the online database GenAge (build 18, version 11/10/2015) (n=99) . Normalized read counts (for method see ) of these a priori identified gene were initially analyzed by an O-PLS-DA. The Devium package remove the first package with PLS and OPLS R command functions were used (https://github.com/dgrapov/devium.git) to validate the model in the statistical environment R (version 3.1.2) .
The Bioconductor package minet  was used to infer large transcriptomic networks using CMI. The package returns a network where the nodes are genes and the edges are statistical dependencies between genes (CMI matrix). Four different entropy estimators and four different inference methods are available of which we used the Miller-Madow asymptotic bias corrected empirical estimator  and the ARACNE method . CMI between genes was inferred in two steps: (1) MI is estimated between all pairs of genes and (2) the ARACNE inference algorithm applies a filter to remove estimated spurious links. ARACNE initially assigns to each pair of genes (i.e. nodes) a weight equal to MI and then removes any indirect interaction . A network based on Spearman correlation coefficients between gene expression levels were also estimated with minet.
Gene centrality measures were estimated for each network based on eigenvector values. Eigenvector centrality captures the relative importance of each gene for the overall interaction network structure by estimating the contribution of the variance in interaction of each gene to the overall interaction heterogeneity in the entire network . Gene eigenvector centrality was estimated by Eigen-decomposing each CMI network and using the elements of the eigenvector corresponding to the dominant eigenvalue from these decompositions. Gene strength per gene was calculated by the sum of the CMI between that gene and other genes in the network (row sums of the CMI matrix).
Weighted clustering coefficient estimates were used as described in . Clusters of genes were identified based on defining a parsimonious division of a network [94,95]. This approach maximizes the number of connections (edges) within a cluster and minimizes the number of connections between clusters. The modularity coefficient quantifies the modularity technique and is a sum of associations for all genes belonging to the same clusters minus its expected value if genes were associated at random, based on the strength of each gene (method based on ).
JRS conceptualized and designed the original graded CR experiment, raised the funding to execute it and was the HO project license holder. SEM performed all experimental procedures related to the study. DL, AD and DD performed the bio-informatic, statistical and network analysis. DL, AD, and DD interpreted the results. DD wrote the manuscript and AD, DL, SEM, JRS and DP revised it. All authors contributed to the analysis during discussions at joint meetings funded by BBSRC grant BB/JO20028/1 (China partnering award).
We would like to acknowledge the BSU staff for their invaluable help with caring for the animals.
The work was supported by the UK Biotechnology and Biological Sciences Research Council BBSRC (BB/G009953/1 and BB/J020028/1) of JRS and SEM and a studentship of DD supported by the Centre for Genome Enabled Biology and Medicine, Aberdeen, UK. Joint meetings were funded by BBSRC grant (China partnering award BB/JO20028/1).
Conflicts of Interest
Conflict of interest statement
Authors declare no conflict of interests.
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domain: medical


A journalist in Zimbabwe known for exposing corruption during the novel coronavirus pandemic was denied bail on Friday amid fears he would incite street protests on July 31.
Hopewell Chin’ono was arrested Monday but only charged Wednesday for inciting public violence, with his bail plea hindered by curfew orders as court staff adjourned early on Wednesday and Thursday.
Chin’ono’s lawyer, Beatrice Mtetwa, expressed dismay over this development.
“There is nothing that Hopewell has done including twitting, that is wrong, even if any of you here journalists wears a mask written #ZANUPFMUSTGO, that’s not a crime,” said Mtetwa.
“I am convinced they are persecuting him for exposing corruption,” she added.
Tendai Biti, another lawyer and deputy leader of the main opposition Movement for Democratic Change (MDC), slammed the bail judgement.
“For someone who was in court following the proceedings, it is surprising the court has denied Hopewell bail,
“Everything is pointing to fact that government is persecuting him for the exposure of massive looting,” Biti said.
Following the exposure involving $60 million of personal protective equipment that implicated President Emmerson Mnangagwa’s family, the government arrested and fired Health Minister Obadiah Moyo.
Meanwhile, leader of opposition group Transform Zimbabwe Jacob Ngarivhume was also denied bail on Thursday. Ngarivhume, along with Chin’ono, called for protests on July 31 against corruption and economic decay.
He is facing similar charges as Chin of inciting public violence.
Separately, Harare Mayor Herbert Gomba, who is facing corruption and abuse of office charges in a land scandal in the capital, has also been denied bail.
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Create a homey vibe by putting your favorite photos on display. Actress Illeana Douglas has done just that in her Los Angeles home. "If I?m working on a movie, no matter where or when, I have to take pictures," says Douglas. She then displays them around her home so she can relive those memories at a glance.
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Food and Drug Administration (FDA) has taken a bold step by approving the first heroine overdose treatment device. Until now, treatment for overdoses was in the hands of authorities and health professionals.
Drug problem in America has grown exponentially over the past few years. Every year, tens of thousands of Americans lose their lives at the hands of drug overdoses. Keeping this in view, FDA’s decision can help save a huge number of lives.
However, the device approved by FDA is called Evzio. It is a tiny, credit-card sized dispenser that has a needle and can be used to directly administer naloxene to the sufferer. Naloxene is an antidote that is quite effective in countering a heroine overdose.
Evzio is surprisingly easy to use, given that it starts giving verbal instructions when you turn it on. The instructions guide a user on how to inject a person with naloxene. In fact, to allow the masses to get a hang of it first, the device comes with a trainer device. The trainer device is a bogus set for practice, carrying no needles or medicine. Once people get familiar with the trainer, they can use the actual device to administer shots.
FDA has stated that Evzio is, by no means, an alternative to proper medical care that is required in the event of a heroine overdose. While many have opposed FDA’s decision, the regulatory agency says that such solutions can help save countless lives lost to drug habit each year. According to FDA Commissioner, “While the larger goal is to reduce the need for products like these by preventing opioid addiction and abuse, they are extremely important innovations that will help to save lives.”

domain: news


text:
Our faithful Elf on the Shelf, Riddle, returned for another year of fun and antics. Last year, his hiding spots were for beginners, BUT THIS YEAR, Riddle showed his mischievous side and got into a bit of trouble. Emerson loved waking up every morning, searching for Riddle and finding the fun and funny things he had done the night before. Since we didn't want to forget any of his clever tomfoolery, I decided to take a picture each morning. We were sad to see him go the night of December 23rd, but knew he needed to get back to Santa to help him and all the other elves prepare for Christmas. Hope you all will enjoy the many Adventures of Riddle. We are anxiously awaiting his return next December 1st!!!
March on to Great Flash Fiction….
6 hours ago

domain: entertainment


text:
The #1 source for fashion inspiration from real people around the world.
More pics on my blog Fashionably Social and a new/updated design! xx
Green skinny jeans, Zara. Purple tank top, Forever21. Shoes, Steve Madden. Gold leaf necklace and clutch, gift from Mom. Nail polish, Essie Tart Deco.

domain: design


Sony appears to be fighting a losing battle in preventing users from jailbreaking their PlayStation 3 consoles. In a recent Australian court ruling, a judge made permanent a previous ban on the distribution of the PSJailbreak dongle only to watch the software code behind a similar hack released for free into the wild. Naturally, Sony responded with a minor firmware update blocking these bits of code from working their mojo, but it's an ongoing cat and mouse game at best.
What we find most interesting, however, is how insanely easy it is to jailbreak a PS3. This is Sony's flagship console, after all, yet users have been able to jailbreak the device with everything from a Palm Pre to a calculator.
That's right, we said a calculator. Gizmodo has put together a collection of clips showing various devices cracking the PS3, and one of them includes the above mentioned hack (known as PSGroove) run from a TI-84. Pretty rad, no? Check it out below.
Homebrew prodigy Comex is best known for developing the only browser-based jailbreak for Apple's iOS devices. A Flash port for iOS called Frash also figures prominently on his list of achievements. Now, the latest version of Frash supports jailbroken iPhone 4s as well. In fact, the Flash port is only compatible with iOS devices with ARMv7 architecture-based processors: iPhone 3GS, iPod Touch 3G, iPad, iPhone 4. iOS4 / iPad 3.2.x.
Having a jailbroken iOS device is the first step on the road to installing Frash. Tech blog Redmond Pie has a helpful guide detailing the rest of the journey. But be warned that Frash is still not the most stable piece of code and only supports limited Flash content.
The newest jailbreak for Apple's iOS platform has exposed a serious exploit that could allow a remote attacker to compromise the device. The exploit is present in all iPhones, iPads, and iPod Touches running version 3.1.2 and higher. The exploit doesn't even require any particular user intervention, just opening a malicious PDF document.
The user is just required to visit a web address in mobile Safari that will load a PDF document. The PDF contains malicious code hidden in a font. The font will cause a stack overflow, allowing the code to be run on the device. A hacker could conceivably do anything at that point. Anything from deleting files, to installing spyware in the background.
This is similar to an exploit early in the iPhone's existence that used TIFF images. But this time around there are many more iPhones in the world, so we expect Apple to take this pretty seriously. Users are cautioned to avoid any PDFs for the time being.
We're still celebrating the decision by U.S. regulators to add some much needed exemptions to the Digital Millennium Copyright Act which, among other things, makes it perfectly legal for users to jailbreak their iPhones and other mobile phones, and boy is that pissing off Apple.
"Apple's goal has always been to ensure that our customers have a great experience with their iPhone and we know that jailbreaking can severely degrade the experience," an Apple spokeswoman said in a statement. "As we've said before, the vast majority of customers do not jailbreak their iPhone as this can violate the warranty and can cause the iPhone to become unstable and not work reliably."
We bolded the warranty tidbit ourselves, because really, that's the part that some will construe as a veiled threat from Apple, and others will take as affirmation that, yes, your warranty goes out the window the minute you alter Jobs' magical handset in ways his Cupertino company doesn't approve.
Nobody really knows exactly how many jailbroken iPhones are out in the wild, though some estimates peg the number at around 10 million. And that was before this landmark ruling. With the Library of Congress handing the keys over to users, you can bet an increasing number of iPhone owners will drive off into what's no longer the Forbidden Zone.
Sure, Apple can kill your warranty for doing so, but that's all the company can do, right? Maybe not. If you haven't already, check out our latest edition of Murphy's Law, in which David Murphy explains why he thinks this is just the beginning of a frightening war between Apple and those would dare jailbreak their iPhones.
Huzzah! Throw up the flags! Send off the fireworks! Summon the townspeople! Apple has lost! The people have won! Huzzah!
I’m referring, of course, to Monday’s ruling by The Library of Congress, which explicitly carves out a legal exception for those looking to jailbreak their iPhones. No longer will industrious little hackers (or those who downloaded a one-button jailbreak app off the Interwebs) be subject to Digital Millennium Copyright Act smack-downs over their choice of Cydia instead of the App Store.
In short, so long as you’re jailbreaking your iPhone to make it work with a third-party application that, itself, isn’t kosher on a vanilla iPhone, you’re in the clear. I’m not quite sure what you would do with a jailbroken phone otherwise—perhaps smash it with a hammer to test its durability or something--but there you have it.
Now, we’ve won, right? The choice of how and why you use your iPhone has finally been wrested out of the turtleneck-laden hands of Apple CEO Steve Jobs. The people are in control now, and we all have carte blanche to do with our handheld devices as we please! Yay!
Score a victory for Joe Consumer, who according to the U.S. government, is fully within his legal right to unlock his iPhone, or any other mobile phone, without having to look over his shoulder for Johnny Law. Not that Apple or anyone else would ever go crying to the cops for trivial matters (wait a tick), but it doesn't matter now anyway.
Federal regulators approved a bunch of new exemptions to a federal law that prohibits circumventing technical measures companies put into place to prevent unauthorized use of copyrighted material. Apple has always taken the position that jailbreaking is an unauthorized modification of its software and violates copyright law, but under the new regulation, iPhone owners are within their legal right to unlock their mobile device and install third-party apps.
Regulators also approved the practice of unlocking cell phones to use on an unapproved carrier, another practice that already exists (scores of iPhone owners roll with unlocked phones on T-Mobile's network, even though the iPhone is available exclusively through AT&T) and is now out of the legal gray area.
There's not a whole lot to like about the Digital Millennium Copyright Act (DMCA), but one of the more onerous provisions of the law is a ban on circumvention of DRM and similar "technical protection measures". The decision handed down today from the Copyright Office and Librarian of Congress has resulted in three exemptions in this law. That is, three situations where it is now acceptable to break digital protection schemes.
The first exemption allows users to "Jailbreak" an iPhone or other handset in order to run legally obtained, but unapproved software. Apple had previously hinted that this activity could be illegal under the DMCA. This change was done to enable consumers to increase the interoperability of their devices. The EFF also secured new protections for artists that make fair use of copyrighted content in video remixes, or mashups. Noncomercial artists are now permitted to break digital protection for this purpose. Get ready for some YouTube celebration mashups.
The last ruling is not a new provision, but rather a renewal of an existing exemption. The Librarian of Congress reaffirmed a 2006 rule that protects cell phone unlocking from prosecution under the DMCA. The locking of a phone to one carrier makes it harder to use or resell later. It's important to note that none of these new exemptions mean that companies have to stop using DRM, just that we are actually allowed to break it in more situations.
In his tirade against Adobe's Flash platform, one of the reasons Steve Jobs says Apple doesn't allow the popular plug-in to run on Apple's iPhones, iPods, and iPads is because "Flash has not performed well on mobile devices." In fact, Steve Jobs claims his Cupertino company has "routinely asked Adobe to show us [Apple] Flash performing well on a mobile device, any mobile device, for a few years now." Anyone think a demo of Flash running smooth on the iPad would change his mind?
Probably not, but that didn't stop iPhone hacker "comex" from demonstrating it anyway. This is the same guy who developed the Spirit untethered jailbreak tool for the iPhone, iPad, and iPod touch, and now he's gone and ported a version of Adobe Flash runtime for Android to run on the iPad using a compatibility layer, which he's calling "Frash."
"Frash can currently run most Flash programs natively in the MobileSafari browser," reads a description of the YouTube video showing Frash in action. "Frash currently only runs on the iPad, but support for other devices (3GS+ only due to technical restrictions" is planned, as well as support for iOS 4."
Comex says he'll release Frash when it's fully stable, and in the meantime, "developers are welcome to join the effort at http://github.com/comex/frash -- fork it an send a pull request with your patches."
On Saturday morning, Apple's iPad left the stable and went on sale. A little over 24 hours later, MuscleNerd of the iPhone Dev-Team said he had cracked the iPad by exploiting unpatched security flaws that migrated from iPhone OS 3.1.3 (the iPad uses an updated iPhone OS, version 3.2).
According to MuscleNerd'sTwitter update, it appears he used a variation of the same "Spirit" jailbreak recently applied to iPhone OS 3.1.3, taking advantage of the same browser-based exploit in order to gain root access and let unsigned apps run on the tablet.
It doesn't come as any surprise that someone managed to jailbreak the iPad, especially considering Apple neglected to plug a handful of security holes in between firmware releases. What is surprising, however, is how quickly this was done. Whether or not this hack will be made into an automated program remains to be seen.
Apple told us jailbreaking wasn’t a good idea. Sure, we mocked them at the time, but it is looking a little less safe these days. The first iPhone worm has been discovered affecting iPhones in Australia. The virus takes advantage of a massive security hole in the SSH client for jailbroken phones. The “ikee” worm is fairly benign, simply changing the user’s wallpaper to a picture of Rick Astley of “Rickroll” fame.
As it turns out, the default password for the SSH client is ‘alpine’. The worm accesses the phone via this route, and then attempts to infect other phones on the network. The worm’s creator, a 21 year-old student, said in an interview, “The virus itself is not malicious and is not out to hurt people. It's just poking fun and hoping waking people up a little.”
Un-jailbroken phones, and jailbroken phones that don’t have SSH installed are not vulnerable. Jailbreakers should head to the Cydia store, and use the Mobile Terminal app to change their default password. With a zillion iPhones out there, it was only a matter of time.


Design: Leonardo Cerliani, Titta Kotilainen, Matthew Robson
Data preparation and modelling: Leonardo Cerliani, Titta Kotilainen, Pedro Aphalo, Matthew Robson
R/Shiny Implementation: Leonardo Cerliani
AOD and Precipitable Water Data: NASA Aeronet
Total Ozone column: NASA Aura
Clara 2.0 satellite Surface Incoming Shortwave Radiation data, available through CM SAF
Measurements of spectral radiation transmitted by screens and filter materials carried out by Titta Kotilainen and Matthew Robson
The sum of solar irradiance, expressed as the daily light integral (DLI), varies across geographical locations and with time of the year.
Greenhouse and tunnel covering materials developed for a range of purposes modify the spectral ratios received by plants.
Knowledge of light conditions under these materials will allow growers to choose those that improve plant growth and yield through manipulation of the spectral quality of sunlight.
To make information on DLI and light quality readily accessible to growers we have designed an easy-to-use app. The app provides information on: (1) Solar DLI as a global map for a selected month and a monthly time series of DLI; (2) The effect on photon ratios of sunlight transmitted by +80 commercially available screens
DLI is calculated from the Clara 2.0 satellite surface incoming shortwave radiation data (200-4000 nm), available through EUMETSAT CM SAF Climate Monitoring. The user obtains monthly DLIs at a desired location averaged over the period of 2005-2015.
The effect of the covering material is calculated at <1 nm resolution and displayed as biologically meaningful spectral photon ratios of Red to Far-Red (R:FR), Blue to Green (B:G) and Blue to ed (B:R). For this, monthly data is estimated using a linear model based on the historical data (last 10-20 years) of total ozone column depth (NASA Aura), precipitable water vapour and aerosol optical density (NASA Aeronet AOD 2.0).
Using dropdown-menus for “Manufacturer” and “Filter Type”, the user can select the best material for their desired purpose (e.g. climate screen or shade net). The app provides calculated spectral ratios of sunlight transmitted under the selected materials, and plots this information for easy visual comparison, together with an image of each material.
Spectal photon ratios are calculated as follows: blue:green (B:G) 420–490 nm/500-570 nm, blue:red (B:R) 420–490 nm/620-680 nm, red:far-red (R:FR) 655–665 nm/725–735 nm. B:G and B:R are according to Sellaro et al. (2010) and R:FR according to Smith (1982).
Sellaro R., Crepy M., Trupkin S.A., Karayekov E., Buchovsky A.S., Rossi,C., Casal J.J. (2010) Cryptochrome as a sensor of the blue/green ratio of natural radiation in Arabidopsis. Plant Physiol. 154, 401–9. https://doi.org/10.1104/pp.110.160820
Smith H. (1982) Light quality, photoperception and plant strategy. Annu. Rev. Plant Physiol. 33, 481–518.
DLI is calculated from the surface incoming shortwave radiation data according to Faust & Logan (2018).
Faust J.E. & Logan J. (2018) Daily light integral: A research review and high-resolution maps of the Unites States. HortScience 53, 1250-1257.
For detailed description about the materials and the complete data set of those measurements: https://zenodo.org/record/1472634#.XfE3MZMzapq
1: Leonardo Cerliani, Netherlands Institute for Neuroscience, Amsterdam, The Netherlands
Copyright © 2021, Leonardo Cerliani, Titta Kotilainen, Matthew Robson
THE SOFTWARE IS PROVIDED “AS IS”, WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.
Made with R/Shiny


text:
From: Wong kwok keong [mailto:[email protected]] Sent: Sunday, March 20, 2016 7:05 PM To: ..
April 2015 Two weeks from Hanoï to the mountain valleys of Vietnam’s far north, home to ..
Licence of tour operator
TOUR OPERATOR LICENSE
Khoaviet Travel is an international tour operator company under the Licence number 01-266/LHQT, recognized by Vietnam National Administration of Tourism belong to Ministry of Sport, Culture and Tourism (for more informations, see at the bottom or you can check our company at www.vietnamtourism.com).
With the original fund of 2 billions VND, according to Vietnam legal Policies, the company has an another account of 250.000.000 VND at ACB Bank (Asia Comercial Bank) in order to guarantee our citizen’s duties toward the tourists.
Khoaviet Travel company's licence (page 1)
Khoaviet Travel company's licence (page 2)

domain: travel


text:
|Reviews for From Ruins|
| B1ackAshes chapter 21 . 3/21
This was an outstanding chapter. I'm glad the misunderstandings have been worked out and that Robin and Lucina are closer than before. You also did great job with the various characters.
| Aspectofthesecond chapter 20 . 3/11
Wow. Another great chapter. I read it, waited, red it again, and here's my review. I especially like how it's nicely laid out that Robin is entirely devoted to lucina, at this point, with the fire grabbing. And how equally devoted she is to him, as indicated both with the now shared snarky mannerisms, as well as straight up telling Gerome that 'no, I don't intend to tell you if Robin presents a threat'. And in spite of all that, Lucina still continues to go through the 'can never be together for his happiness' emo bit. Can't wait for her never have to go emo on us again.
Oh, and the bit with tharja practicing was wonderful. I thought maybe the evil dopplerobin had paid a visit.
Keep up the good work. Thanks for the read.
| Pious Mage chapter 21 . 2/26
I've been meaning to leave this review for a while but it took me a bit to process and refine what I wanted to say until I finally just said fuck it and went with what I am going to say.
This is a very very interesting twist on the typical Awakening novelization. I love the introduction of characters both first gen and second gen and you're take on the Robin/Lucina romance which I am a sucker for.
I also like how the whole slow burn and infiltration of Valm. Morgan being Valms tactician and how she will interact with Robin. Again the very slow yet satisfying burn of Robin and Lucina and the buddy cop bromance of Robin and Chrom. While I'm not personally a fan of Chrom/Olivia and a bigger fan of the canonical pairing. I gotta say you make it work and make it hilarious doing so.
I look forward heavily to the rest of the fic and wish you good luck! Cheers.
| thepkrmgc chapter 21 . 2/25
it should be cool to see where this story goes from here!
| thepkrmgc chapter 20 . 2/24
nice job on the banter between tharja and lucina, it really highlights their characters well
| thepkrmgc chapter 19 . 2/24
gotta love that dramatic irony, nice job subverting it with an honest conversation though!
| thepkrmgc chapter 18 . 2/24
if the "robin wakes up in the field again" epilogue is true then this becomes a lot more tragic in retrospect
| thepkrmgc chapter 17 . 2/24
well, thats one way to have a family reunion
| thepkrmgc chapter 16 . 2/23
props to lucina and lon'qu for seeing beneath the humor to the damaged man beneath
| thepkrmgc chapter 15 . 2/23
it should be cool to see the father daughter tactics showdown!
| thepkrmgc chapter 14 . 2/23
props to robin for staying above the influence!
| thepkrmgc chapter 12 . 2/23
so you legitly surprised me here, i thought the rescue party was going to stumble in on the wedding and complicate things
| Guest chapter 2 . 2/21
Upon rereading your work, i realised that you altered and added some details.
I am glad that you are spending time to change the fic to be more to your liking. Please continue your great work.
| thepkrmgc chapter 10 . 2/21
never underestimate the power of a humble bard!
| thepkrmgc chapter 9 . 2/21
well, thats certainly one way to slay a kraken

domain: literature


text:
The majority of patients with early stage unfavorable Hodgkin lymphoma respond well enough to a current standard regimen of 4 cycles of chemotherapy, and can skip the additional radiotherapy that is normally included in the combined modality treatment, say experts reporting the final results from an international phase 3 randomized trial dubbed HD17.
"Most patients with this disease will not need radiotherapy any longer," concluded first author Peter Borchmann, MD, assistant medical director in the Department of Hematology/Oncology at the University Hospital of Cologne, Germany.
Borchmann was speaking online as part of the virtual edition of the European Hematology Association (EHA) 25th Annual Congress 2020.
"Importantly, the mortality of patients with early stage unfavorable Hodgkin lymphoma in the HD17 study did not differ from the normal healthy German population, and this is the first time we have had this finding in one of our studies," he emphasized.
Borchmann added that positron emission tomography (PET) imaging is key in deciding which patients can skip radiation.
"We conclude from the HD17 trial that the combined modality concept can and should be replaced by a PET-guided omission of radiotherapy for patients with newly diagnosed early stage unfavorable Hodgkin lymphoma," he said.
"The vast majority of early stage unfavorable Hodgkin lymphoma patients can be treated with the brief and highly effective 2+2 chemotherapy alone," he added.
Therefore, he continued, "PET-guided 2+2 chemotherapy is the new standard of care for the German Hodgkin study group," which conducted the trial.
The use of both chemotherapy and radiation has long been a standard approach to treatment, and this combined modality treatment is highly effective, Borchmann explained. But it can cause long-term damage, and the known longer-term negative effects of radiotherapy, such as cardiovascular disease and second malignancies, are a particular concern because patients with early stage Hodgkin lymphoma are relatively young, with a median age of around 30 years at disease onset.
An expert approached for comment said that the momentum to skip radiotherapy when possible is an ongoing issue, and importantly, this study adds to those efforts.
"The treatment of Hodgkin lymphoma has moved for many years now to less radiation therapy, and this trend will continue with the results of this study," commented John G. Gribben, MD, director of the Stem Cell Transplantation Program and medical director of the North East London Cancer Research Network Centre at Barts Cancer Center of Excellence and The London School of Medicine, UK.
"We have moved to lower doses and involved fields with the intent of decreasing toxicity, and particularly long-term toxicity from radiotherapy," he told Medscape Medical News.
HD17 Study Details
For the multicenter, phase 3 HD17 trial, Borchmann and colleagues turned to PET to identify patients who had and had not responded well to chemotherapy (PET-negative and PET-positive) and to determine if those who had responded well could safely avoid radiotherapy without compromising efficacy.
“We wanted to determine if we could reduce the treatment intensity by omission of radiotherapy in patients who respond very well to the systemic treatment, so who have a complete metabolic remission after the chemotherapy,” Borchmann said.
The 2+2 treatment approach includes 2 cycles of eBEACOPP (bleomycin, etoposide, doxorubicin, cyclophosphamide, vincristine, procarbazine, and prednisone) and 2 subsequent cycles of ABVD (doxorubicin, bleomycin, vinblastine, and dacarbazine).
The trial enrolled 1100 patients with newly diagnosed Hodgkin lymphoma between January 2012 and March 2017. Of these, 979 patients had confirmed PET results, with 651 (66.5%) found to be PET-negative, defined as having a Deauville score (DS) of less than 3 (DS3); 238 (24.3%) were DS3 and 90 (9.2%) were DS4.
The study met its primary endpoint of noninferiority in progression-free survival (PFS) at 5 years, with a PFS of 95.1% in the PET-guided group (n = 447) compared with 97.3% in the standard combined-modality treatment group (n = 428), over a median observation time of 45 months, for a difference of 2.2% (P = .12).
"We found that the survival levels were very high and we can safely conclude the noninferiority of the PET-guided approach in PET-negative patients," Borchmann said.
A further analysis showed that the 597 PET-negative patients who did not receive radiotherapy due to their PET status had 5-year PFS that was noninferior to the combined modality group (95.9% vs 97.7%, respectively; P = .20).
And among 646 patients who received the 2+2 regimen plus radiotherapy, of those confirmed as PET-positive (n = 328), the estimated 5-year PFS was significantly lower (94.2%) compared with those determined to be PET-negative (n = 318; 97.6%; hazard ratio, 3.03).
A cut-off of DS4 for positivity was associated with a stronger effect, with a lower estimated 5-year PFS of 81.6% vs 98.8% for DS3 patients and 97.6% for DS < 3 (P < .0001).
"Only DS4 has a prognostic impact, but not DS3," Borchmann said. "DS4 positivity indicates a relevant risk for treatment failure, however, there are few patients in this risk group (9.2% in this trial)."
The 5-year overall survival rates in an intent-to-treat analysis were 98.8% in the standard combined modality group and 98.4% in the PET-guided group.
With a median observation time of 47 months, there have been 10 fatal events in the trial out of 1100 patients, including two Hodgkin lymphoma-related events and one treatment-related death.
"Overall, Hodgkin lymphoma or treatment-related mortality rates were extremely low," Borchmann said.
The study was funded by Deutsche Krebshilfe. Borchmann and Gribben have reported no relevant financial relationships.
EHA 2020 Congress. Presented June 12, 2020. Abstract S101.
Medscape Medical News © 2020 WebMD, LLC
Send comments and news tips to email@example.com.
Cite this: Safe to Skip Radiotherapy With Negative PET in Hodgkin Lymphoma - Medscape - Jun 15, 2020.


text:
Luise Stauss is a Creative Director and Photography Consultant based in New York. After having worked at the launch of T Magazine, and at the New York Times Magazine as a Senior Photo Editor for a decade, Luise started the studio with Ayanna Quint in 2014, taking her deep knowledge of photography and design to work with clients in both the publishing and advertising worlds. The studio creates imagery for design firms like Pentagram and brands like Apple, and collaborates with a wide range of international magazines and publishing houses. Luise has authored ‘Domains’, a design column for The New York Times Magazine and was a faculty member at the School of Visual Arts (SVA) where she taught Design and Photography. A graduate of Central Saint Martin’s College of Art in London, Luise holds a degree in Fashion and Communication Design. She has co-edited the book ‘Drawing Fashion, a century of Fashion Illustration’ and is currently working on a book about the illustrator Renee Gruau.
Ayanna Quint is a partner at Stauss & Quint. Before founding Stauss & Quint in 2014, Ayanna was consulting Photo Editor for a variety of print publications, including The Atlantic, The Ritz Carlton Magazine, GQ and Radar. She also worked as an Associate Producer on films such as The Toe Tactic, Ashes and The Wannabe and as a Photo Editor on documentaries such as About Face and The Out List for HBO. Soon after graduating from The Rhode Island School of Design with a degree in Photography, Ayanna was named Photo Editor at Out, which led to her roles as a staff Photo Editor at W and Teen People.

domain: artistic


text:
The budget Comfort Inn Farmington offers non-smoking rooms in proximity to Aztec Museum and Pioneer Village and Farmington Museum.
Located in a recreational area with casinos and restaurants, guests will see why Comfort Inn Farmington is admired by many tourists. The distance between Farmington city center and the property is 3 km.
The hotel is situated near a museum and a college.
Comfort Inn Farmington is within a 5-minute drive to Municipal airport.
This property counts 59 guestrooms featuring First-Floor Double Room, One-Room Suite and Double Room. They come with coffee/tea makers, a microwave and a fridge as well as bathrooms with a shower, a bathtub and a hairdryer.
Eat & Drink
A hot breakfast is served daily. The venue is set next to a bus stop and just 100 meters from Denny's and Tequilas with a broad selection of dishes.
A safety deposit box, free parking and a vending machine for general use and a photocopier and a business center for work can be found on-site.
Wireless internet is available in the entire hotel for free.
Free public parking is possible on site.