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Plant height and lodging resistance can affect rice yield significantly, but these traits have always conflicted in crop cultivation and breeding. The current study aimed to establish a rapid and accurate plant type evaluation mechanism to provide a basis for breeding tall but lodging-resistant super rice varieties. A comprehensive approach integrating plant anatomy and histochemistry was used to investigate variations in flexural strength (a material property, defined as the stress in a material just before it yields in a flexure test) of the rice stem and the lodging index of 15 rice accessions at different growth stages to understand trends in these parameters and the potential factors influencing them. Rice stem anatomical structure was observed and the lignin content the cell wall was determined at different developmental stages. Three rice lodging evaluation models were established using correlation analysis, multivariate regression and artificial radial basis function (RBF) neural network analysis, and the results were compared to identify the most suitable model for predicting optimal rice plant types. Among the three evaluation methods, the mean residual and relative prediction errors were lowest using the RBF network, indicating that it was highly accurate and robust and could be used to establish a mathematical model of the morphological characteristics and lodging resistance of rice to identify optimal varieties.

AIM To investigate the role of suppressor of cytokine signaling 1 (SOCS1) in regulating MET-mediated invasive potential of hepatocellular carcinoma (HCC) cells. METHODS Stable derivatives of mouse (Hepa1-6) and human (hep3B, HepG2) HCC cell lines expressing SOCS1 or control vector were evaluated for their ability to migrate towards hepatocyte growth factor (HGF) in the transwell migration assay, invade extracellular matrix in response to HGF stimulation in a 3-D invasion assay by confocal microscopy, and to undergo anchorage-independent proliferation in semisolid agar. Following intravenous and intrasplenic inoculation into NOD.scid.gamma mice, the ability of Hepa cells to form othotopic tumors was evaluated. Following HGF stimulation of Hepa and Hep3B cells, expression of proteins implicated in epithelial-to-mesenchymal transition was evaluated by western blot and qRT-PCR. RESULTS SOCS1 expression in mouse and human HCC cells inhibited HGF-induced migration through matrigel. In the 3-D invasion assay, HGF stimulation induced invasion of HCC cells across type-I collagen matrix, and SOCS1 expression significantly reduced the depth of invasion. SOCS1 expression also reduced the number and size of colonies formed by anchorage-independent growth in semisolid agar. Following intravenous inoculation, control Hepa cell formed large tumor nodules that obliterated the liver whereas the SOCS1-expressing Hepa cells formed significantly smaller nodules. Tumors formed by SOCS1-expressing cells showed reduced phosphorylation of STAT3 and ERK that was accompanied by reduced levels of MET protein expression. HGF stimulated Hepa cells expressing SOCS1 showed increased expression of E-cadherin and decreased expression of EGR1, SNAI1 and ZEB1. Comparable results were obtained with Hep3B cells. SOCS1 expressing HCC cells also showed reduced levels of EGR1 and SNAI1 transcripts. CONCLUSION Our findings indicate that loss of SOCS1-dependent control over epithelial-to-mesenchymal transition may contribute to MET-mediated migration, invasion and metastatic growth of HCC.

Cancer cell metabolism is heavily influenced by microenvironmental factors, including nutrient availability. Therefore, knowledge of microenvironmental nutrient levels is essential to understand tumor metabolism. To measure the extracellular nutrient levels available to tumors, we utilized quantitative metabolomics methods to measure the absolute concentrations of >118 metabolites in plasma and tumor interstitial fluid, the extracellular fluid that perfuses tumors. Comparison of nutrient levels in tumor interstitial fluid and plasma revealed that the nutrients available to tumors differ from those present in circulation. Further, by comparing interstitial fluid nutrient levels between autochthonous and transplant models of murine pancreatic and lung adenocarcinoma, we found that tumor type, anatomical location and animal diet affect local nutrient availability. These data provide a comprehensive characterization of the nutrients present in the tumor microenvironment of widely used models of lung and pancreatic cancer and identify factors that influence metabolite levels in tumors., eLife digest In the body, cancer cells can rely on different nutrients than normal cells, and they can use these nutrients in a different way. What cancer cells consume also depends on what is available in their immediate environment. In a tumor, cells grab nutrients from the ‘interstitial’ fluid that surrounds them, but what is present in this liquid may vary within tumors arising in different locations. Understanding what nutrients are ‘on the menu’ in specific tumors would help to target diseased cells while sparing healthy ones, but this knowledge has been difficult to obtain. To investigate this, Sullivan et al. used a technique called mass spectrometry to measure the amounts of 120 nutrients present in the interstitial fluid of mouse pancreas and lung tumors. Different levels of nutrients were found in the two types of tumors, and analyses showed that what was present in the interstitial fluid depended on the type of cancer cells, where the tumor was located, and what the animals ate. This suggests that cancer cells may have different needs because they are limited in what they have access to. It remains to be seen whether the nutrients levels found in mouse tumors are the same as those in humans. Armed with this knowledge, it may then be possible to feed cancer cells grown in the laboratory with the nutrient menu that they would have access to in the body. This could help identify new cancer treatments.

Aerobic glycolysis, or preferential fermentation of glucose-derived pyruvate to lactate despite available oxygen, is a hallmark of proliferative metabolism that is observed across many organisms and conditions. To better understand why aerobic glycolysis is associated with cell proliferation, we examined the metabolic consequence of activating the pyruvate dehydrogenase complex (PDH) to increase mitochondrial pyruvate oxidation at the expense of fermentation. We find that increasing PDH activity impairs cell proliferation by reducing the nicotinamide adenine dinucleotide cofactor ratio (NAD+/NADH). This change in NAD+/NADH ratio is caused by an increase in mitochondrial membrane potential that impairs mitochondrial electron transport and NAD+ regeneration. Uncoupling mitochondrial respiration from ATP synthesis or increasing ATP hydrolysis restores NAD+/NADH homeostasis and proliferation even when glucose oxidation is increased. These data suggest that when the demand for NAD+ to support oxidation reactions exceeds the demand for ATP consumption in cells, NAD+ regeneration by mitochondrial respiration becomes constrained, promoting fermentation despite available oxygen. This argues that cells engage in aerobic glycolysis when the cellular demand for NAD+ is in excess of the cellular demand for ATP.

A lack of understanding regarding the feeding biology of post-settlement mussels has considerably constrained the hatchery culture of many commercially important mussel species, including the Greenshell™ mussel, Perna canaliculus . Therefore, we evaluated how particles of different size (MySpat artificial diet full particle size range 2–60 μm, sieved MySpat diet with a size range of 2–15 μm; polystyrene beads, 3–15 μm, and microalgae, 4–6.5 μm) affected the retention and capture efficiency in post-settlement juveniles (PSJ). The response of PSJ of various sizes (PSJ-I: 0.3 mm shell length, PSJ-II: 1.0 mm, and PSJ-III: 7.0 mm) was different. Overall, smaller mussels (PSJ-I and PSJ-II) were unable to selectively capture particles by their size on all diets, conversely larger juveniles actively captured smaller particles between 2 and 8 μm. Independently of the PSJ size, the full particle size range MySpat artificial diet had a negative effect on the feeding activity of the mussels, with particles blocking the pallial organs in the non-selective PSJ-I and PSJ-II, and completely inhibiting the feeding activity in the more selective PSJ-III. These results provide useful information to understand the feeding biology and ecology of PSJ of this species that can be used for the improvement of hatchery feeding methods. Artificial and live food particle size can be chosen in order to avoid larger harmful particles (> 15 μm), and the exclusive use of smaller food particles should be considered for PSJ Statement of relevance This article provides information to better understand particle capture process in post-settlement juvenile mussel in order to improve hatchery/nursery feeding practices. The results of this research will allow making informed decisions regarding selection of food particle size in relation to the size of the juvenile mussels and their ability to deal with particles of different sizes. Furthermore will also help to decide the adequate size of transfer for grow-out as well as being useful for the development of suitable artificial diets.

Cultured cells convert glucose to lactate, and glutamine is the major source of tricarboxylic acid (TCA)-cycle carbon, but whether the same metabolic phenotype is found in tumors is less studied. We infused mice with lung cancers with isotope-labeled glucose or glutamine and compared the fate of these nutrients in tumor and normal tissue. As expected, lung tumors exhibit increased lactate production from glucose. However, glutamine utilization by both lung tumors and normal lung was minimal, with lung tumors showing increased glucose contribution to the TCA cycle relative to normal lung tissue. Deletion of enzymes involved in glucose oxidation demonstrates that glucose carbon contribution to the TCA cycle is required for tumor formation. These data suggest that understanding nutrient utilization by tumors can predict metabolic dependencies of cancers in vivo. Furthermore, these data argue that the in vivo environment is an important determinant of the metabolic phenotype of cancer cells., National Science Foundation (U.S.) (Grant T32GM007287)

Despite the commercial aquaculture importance of the Greenshell™ mussel, Perna canaliculus , the morphological development of gill structure (ctenidial filaments) in relation to potential particle feeding abilities have not been described. Scanning electron microscopy (SEM) and light microscopy were used to examine the ontogenetic changes across five size classes of mussels from post-settlement larvae to adults ranging from 300 μm to > 100 mm in shell height. Key morphological characteristics were measured including filament width, the eulatero-frontal cirri (ELF) length, interfilamentary space (IFS) and the mean distance between adjacent ELF, d ELF . Overall the development of the ctenidial filaments in P. canaliculus , shared some similarities to other homorhabdic filibranch bivalves with differences in the timing of development. Filament width and ELF length followed sigmoidal growth curves in relation to increasing shell height of mussels. The morphological changes of the ctenidia, reflected in changes in the ELF/IFS ratios, together with the development of cilia in the ELF may help explain the differences in the capture efficiency of small particles between small individuals ( Statement of relevance –This article provides information regarding the gill ontogenetic development of the commercially important Greenshell™ mussel, Perna canaliculus . –The information given will help improve hatchery practices, especially regarding the selection of adequate particle size for feeding post-settlement P. canaliculus . –Considering that mussel aquaculture is a global phenomenon, these results could be used worldwide in order to help the industry find economically feasible alternatives for mussel juvenile supply. The New Zealand Greenshell™ industry has recently invested $20 M to build a large scale hatchery/nursery to address an on-going shortage with seed supply which has been constraining the growth of the industry. Shortages of mussel seed, caused by poor feeding and starvation, have resulted in seed shortages in the industry for the last three years, shaving off almost 20% production of the entire industry, in the order of $30 M lost production a year. Hence there is intense interest in better understanding the feeding biology of juvenile mussels and using this knowledge to resolve the major production issues faced by this aquaculture industry. From our international contacts, we understand that other countries with significant mussel farming industries face similar issues, such as Chile. Therefore, we believe that there is strong reader interest and value in the publication of this fundamental research.

The role of pyruvate kinase M2 (PKM2) in cell proliferation is controversial. A unique function of PKM2 proposed to be important for the proliferation of some cancer cells involves the direct activity of this enzyme as a protein kinase; however, a detailed biochemical characterization of this activity is lacking. Using [32P]-phosphoenolpyruvate (PEP) we examine the direct substrates of PKM2 using recombinant enzyme and in vitro systems where PKM2 is genetically deleted. Labeling of some protein species from [32P]-PEP can be observed; however, most were dependent on the presence of ADP, and none were dependent on the presence of PKM2. In addition, we also failed to observe PKM2-dependent transfer of phosphate from ATP directly to protein. These findings argue against a role for PKM2 as a protein kinase., Howard Hughes Medical Institute (International Student Research Fellowship), Vertex Pharmaceuticals Incorporated. Vertex Scholars Program, Massachusetts Institute of Technology. Department of Biology (Grant T32GM007287), David H. Koch Institute for Integrative Cancer Research at MIT (Graduate Student Fellowship), National Cancer Institute (U.S.) (Grants R01CA168653 and P30CA14051), Burroughs Wellcome Fund

SummaryMitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis.

Suppressor of cytokine signaling 1 (SOCS1) is considered as a tumor suppressor protein in hepatocellular carcinoma (HCC), but the underlying mechanisms remain unclear. Previously, we have shown that SOCS1-deficient hepatocytes displayed increased responsiveness to hepatocyte growth factor (HGF) due to enhanced signaling via the MET receptor tyrosine kinase. As aberrant MET activation occurs in many tumors including HCC, here we elucidated the mechanisms of SOCS1-mediated regulation. SOCS1 attenuated HGF-induced proliferation of human and mouse HCC cell lines and their growth as tumors in NOD.scid.gamma mice. Tumors formed by SOCS1 expressing HCC cells showed significantly reduced MET expression, indicating that SOCS1 not only attenuates MET signaling but also regulates MET expression. Mechanistically, SOCS1 interacted with MET via the Src homology 2 domain and this interaction was promoted by MET tyrosine kinase activity. The SOCS1-mediated reduction in MET expression does not require the juxtamembrane Y1003 residue implicated in Cbl-mediated downmodulation. Moreover, the proteasome inhibitor MG-132, but not the inhibitors of lysosomal degradation bafilomycin and chloroquine, reversed the SOCS1-mediated reduction in MET expression, indicating that this process is distinct from Cbl-mediated downmodulation. Accordingly, SOCS1 promoted polyubiquitination of MET via K48-dependent but not K63-mediated ubiquitin chain elongation. Furthermore, siRNA-mediated downmodulation of Cbl did not abolish SOCS1-mediated reduction in MET expression in HCC cells. SOCS1-dependent ubiquitination of endogenous MET receptor occurred rapidly following HGF stimulation in HCC cells, leading to proteasomal degradation of phosphorylated MET receptor. These findings indicate that SOCS1 mediates its tumor suppressor functions, at least partly, by binding to MET and interfering with downstream signaling pathways as well as by promoting the turnover of the activated MET receptor. We propose that loss of this control mechanism due to epigenetic repression of SOCS1 could contribute to oncogenic MET signaling in HCC and other cancers, and that MET inhibitors might be useful in treating these patients.

Many mammalian cancer cell lines depend on glutamine as a major tri-carboxylic acid (TCA) cycle anaplerotic substrate to support proliferation. However, some cell lines that depend on glutamine anaplerosis in culture rely less on glutamine catabolism to proliferate in vivo. We sought to understand the environmental differences that cause differential dependence on glutamine for anaplerosis. We find that cells cultured in adult bovine serum, which better reflects nutrients available to cells in vivo, exhibit decreased glutamine catabolism and reduced reliance on glutamine anaplerosis compared to cells cultured in standard tissue culture conditions. We find that levels of a single nutrient, cystine, accounts for the differential dependence on glutamine in these different environmental contexts. Further, we show that cystine levels dictate glutamine dependence via the cystine/glutamate antiporter xCT/SLC7A11. Thus, xCT/SLC7A11 expression, in conjunction with environmental cystine, is necessary and sufficient to increase glutamine catabolism, defining important determinants of glutamine anaplerosis and glutaminase dependence in cancer., eLife digest Cancer cells need to consume certain nutrients in order to grow, and some cancer drugs work by affecting the ability of the cells to use these nutrients. For decades researchers have grown cancer cells in petri dishes with standard nutrient formulations (also known as tissue culture), but the nutrients available to cancer cells in tissue culture are not the same as those found in the body. Cancer cells growing in tissue culture consume large amounts of a nutrient called glutamine. These cells die when exposed to a class of drugs called glutaminase inhibitors that prevent them from processing glutamine. However, when these same cancer cells grow as tumors in animals, they process less glutamine and are not affected by glutaminase inhibitors. So what differences are there between growing cancer cells in tumors and tissue culture that explain this different reliance on glutamine? Muir et al. reasoned that changing the levels of nutrients available to cancer cells might change what these cells consume, and so grew human cancer cells in cow serum (which has a similar nutrient content to blood in humans and other mammals). Indeed, these cells consumed less glutamine and responded to glutaminase inhibitors in a way that is similar to how tumors in the body respond to these drugs. Comparing the nutrient content of cow serum and typical tissue culture formulations revealed that high levels of the nutrient cystine cause the cells to rely more on glutamine. The results presented by Muir et al. suggest that cancer cells in tumors could be made to consume more glutamine and that this would make them sensitive to glutaminase inhibitors – a possibility that will be studied in future work. Exposing cultured cancer cells to nutrient levels closer to those found in the body may also better predict how tumor cells use nutrients and respond to some treatments.

Metabolic reprogramming contributes to tumor development and introduces metabolic liabilities that can be exploited to treat cancer. Chemotherapies targeting metabolism have been effective cancer treatments for decades, and the success of these therapies demonstrates that a therapeutic window exists to target malignant metabolism. New insights into the differential metabolic dependencies of tumors have provided novel therapeutic strategies to exploit altered metabolism, some of which are being evaluated in pre-clinical models or clinical trials. Here, we review our current understanding of cancer metabolism and discuss how this might guide treatments targeting the metabolic requirements of tumor cells.

Many cancer cell lines depend on extracellular glutamine as a major tri-carboxylic acid (TCA) cycle anaplerotic substrate to support proliferationin vitro. However, recent studies have suggested that some cells that depend on glutamine anaplerosis in culture rely much less on glutamine catabolism to proliferatein vivo, with environmental differences between tumors and cell culture influencing the extent of glutamine catabolism. Here we sought to better understand the environmental differences that cause differential dependence on glutamine for TCA cycle anaplerosis. We find that cells cultured in adult bovine serum, a condition that more closely reflects the nutrients available to cellsin vivo, leads to decreased glutamine catabolism and reliance on glutamine anaplerosis compared to standard tissue culture conditions. By analyzing the nutrient differences between bovine serum and media, we find that levels of a single nutrient, cystine, can account for the differential dependence on glutamine in these different environmental contexts. Further, we show that cystine levels dictate glutamine dependence via the cystine/glutamate antiporter xCT/SLC7A11, and that environmental cystine levels in conjunction withxCT/SLC7A11expression is necessary and sufficient to drive increased glutamine anaplerosis, defining important determinants of glutamine anaplerosis and glutaminase dependence in cancer cells.

Altered cell metabolism is a characteristic feature of many cancers. Aside from well-described changes in nutrient consumption and waste excretion, altered cancer cell metabolism also results in changes to intracellular metabolite concentrations. Increased levels of metabolites that result directly from genetic mutations and cancer-associated modifications in protein expression can promote cancer initiation and progression. Changes in the levels of specific metabolites, such as 2-hydroxyglutarate, fumarate, succinate, aspartate and reactive oxygen species, can result in altered cell signalling, enzyme activity and/or metabolic flux. In this Review, we discuss the mechanisms that lead to changes in metabolite concentrations in cancer cells, the consequences of these changes for the cells and how they might be exploited to improve cancer therapy.

Aims/hypothesis Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. Methods The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m2 and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p 1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10−14), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10−11) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10−10). Conclusions/interpretation We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits. Electronic supplementary material The online version of this article (doi:10.1007/s00125-012-2756-1) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Background & Aims Frequent repression of the Socs1 (suppressor of cytokine signaling 1) gene in hepatocellular carcinoma (HCC) and increased susceptibility of SOCS1-deficient mice to hepatocarcinogens suggest a tumor suppressor role for SOCS1 in the liver, but the underlying mechanisms remain unclear. Here we investigated the role of SOCS1 in regulating hepatocyte proliferation following partial hepatectomy and HGF stimulation. Methods Because Socs1 −/− mice die prematurely due to deregulated IFNγ signaling, we used Socs1 −/− Ifng −/− mice to study the role of SOCS1 in liver regeneration following partial hepatectomy. We examined the activation of signaling molecules downstream of IL-6 and hepatocyte growth factor (HGF) receptors in the regenerating liver, primary hepatocytes, and in human hepatoma cells. We examined the interaction between SOCS1 and the HGF receptor c-Met by reciprocal immunoprecipitation. Results Socs1 −/− Ifng −/− mice displayed accelerated liver regeneration with increased DNA synthesis compared to Ifng −/− and wild type mice. The regenerating liver of Socs1 −/− Ifng −/− mice did not show increased IL-6 signaling, but displayed earlier phosphorylation of Gab1, a signaling adaptor downstream of c-Met. Following HGF stimulation, hepatocytes from Socs1 −/− Ifng −/− mice displayed increased phosphorylation of c-Met and Gab1, cell migration and proliferation. Accordingly, SOCS1 overexpression attenuated HGF-induced phosphorylation of c-Met, Gab1, and ERK1/2 in hepatoma cells, and decreased their proliferation and migration. SOCS1 interacted with the Tpr-Met, an oncogenic form of the Met receptor. Conclusions SOCS1 attenuates c-Met signaling and thus negative regulation of HGF signaling could be an important mechanism underlying the anti-tumor role of SOCS1 in the liver.

Metformin use is associated with reduced cancer mortality, but how metformin impacts cancer outcomes is controversial. Although metformin can act on cells autonomously to inhibit tumor growth, the doses of metformin that inhibit proliferation in tissue culture are much higher than what has been described in vivo. Here, we show that the environment drastically alters sensitivity to metformin and other complex I inhibitors. We find that complex I supports proliferation by regenerating nicotinamide adenine dinucleotide (NAD)+, and metformin's anti-proliferative effect is due to loss of NAD+/NADH homeostasis and inhibition of aspartate biosynthesis. However, complex I is only one of many inputs that determines the cellular NAD+/NADH ratio, and dependency on complex I is dictated by the activity of other pathways that affect NAD+ regeneration and aspartate levels. This suggests that cancer drug sensitivity and resistance are not intrinsic properties of cancer cells, and demonstrates that the environment can dictate sensitivity to therapies that impact cell metabolism. Keywords: cancer metabolism; metformin; biguanide; NAD+/NADH ratio; drug sensitivity; complex I; mitochondria; aspartate, National Institutes of Health (U.S.) (Grant P30CA1405141), National Institutes of Health (U.S.) (Grant GG006413), National Institutes of Health (U.S.) (Grant R01 CA168653), National Institutes of Health (U.S.) (Grant R01 CA201276)

The SOCS1 gene coding for suppressor of cytokine signaling 1 is frequently repressed in hepatocellular carcinoma (HCC), and hence SOCS1 is considered a tumor suppressor in the liver. However, the tumor-suppressor mechanisms of SOCS1 are not yet well understood. SOCS1 is known to inhibit pro-inflammatory cytokine production and signaling and to promote activation of the p53 tumor suppressor. However, we observed that SOCS1-deficient mice developed numerous and large liver tumor nodules following treatment with the hepatocarcinogen diethylnitrosamine (DEN) without showing increased interleukin-6 production or activation of p53. On the other hand, the livers of DEN-treated Socs1-null mice showed elevated levels of p21(CIP1/WAF1) protein (p21). Even though p21 generally functions as a tumor suppressor, paradoxically many cancers, including HCC, are known to express elevated levels of p21 that correlate with poor prognosis. We observed elevated p21 expression also in the regenerating livers of SOCS1-deficient mice and in cisplatin-treated Socs1-null hepatocytes, wherein the p21 protein showed increased stability. We show that SOCS1 interacts with p21 and promotes its ubiquitination and proteasomal degradation. Besides, the DEN-treated livers of Socs1-null mice showed increased nuclear and cytosolic p21 staining, and the latter was associated with growth factor-induced, phosphatidylinositol 3-kinase-dependent phosphorylation of p21 in SOCS1-deficient hepatocytes. Cytosolic p21 is often associated with malignancy and chemo-resistance in many cancers. Accordingly, SOCS1-deficient hepatocytes showed increased resistance to apoptosis that was reversed by shRNA-mediated p21 knockdown. In the regenerating livers of Socs1-null mice, increased p21 expression coincided with elevated cyclinD levels. Correspondingly, SOCS1-deficient hepatocytes showed increased proliferation to growth factor stimulation that was reversed by p21 knockdown. Overall, our findings indicate that the tumor-suppressor functions of SOCS1 in the liver could be mediated, at least partly, via regulation of the expression, stability and subcellular distribution of p21 and its paradoxical oncogenic functions, namely, resistance to apoptosis and increased proliferation.

Perfringolysin O (PFO) is a member of the cholesterol-dependent cytolysin (CDC) family of bacterial pore-forming proteins, which are highly efficient in delivering exogenous proteins to the cytoplasm. However, the indiscriminate and potent cytotoxicity of PFO limits its practical use as an intracellular delivery system. In this study, we describe the design and engineering of a bispecific, neutralizing antibody against PFO, which targets reversibly attenuated PFO to endocytic compartments via receptor-mediated internalization. This PFO-based system efficiently mediated the endosomal release of a co-targeted gelonin construct with high specificity and minimal toxicity in vitro. Consequently, the therapeutic window of PFO was improved by more than 5 orders of magnitude. Our results demonstrating that the activity of pore-forming proteins can be controlled by antibody-mediated neutralization present a novel strategy for utilizing these potent membrane-lytic agents as a safe and effective intracellular delivery vehicle.

Item does not contain fulltext Cancer cells adapt their metabolic processes to support rapid proliferation, but less is known about how cancer cells alter metabolism to promote cell survival in a poorly vascularized tumour microenvironment. Here we identify a key role for serine and glycine metabolism in the survival of brain cancer cells within the ischaemic zones of gliomas. In human glioblastoma multiforme, mitochondrial serine hydroxymethyltransferase (SHMT2) and glycine decarboxylase (GLDC) are highly expressed in the pseudopalisading cells that surround necrotic foci. We find that SHMT2 activity limits that of pyruvate kinase (PKM2) and reduces oxygen consumption, eliciting a metabolic state that confers a profound survival advantage to cells in poorly vascularized tumour regions. GLDC inhibition impairs cells with high SHMT2 levels as the excess glycine not metabolized by GLDC can be converted to the toxic molecules aminoacetone and methylglyoxal. Thus, SHMT2 is required for cancer cells to adapt to the tumour environment, but also renders these cells sensitive to glycine cleavage system inhibition.

Histone deacetylase (HDAC) inhibitors (HDACi) cause cancer cell growth arrest and/or apoptosis in vivo and in vitro . The HDACi suberoylanilide hydroxamic acid (SAHA) is in phase I/II clinical trials showing significant anticancer activity. Despite wide distribution of HDACs in chromatin, SAHA alters the expression of few genes in transformed cells. p21 WAF1 is one of the most commonly induced. SAHA does not alter the expression of p27 KIPI , an actively transcribed gene, or globin, a silent gene, in ARP-1 cells. Here we studied SAHA-induced changes in the p21 WAF1 promoter of ARP-1 cells to better understand the mechanism of HDACi gene activation. Within 1 h, SAHA caused modifications in acetylation and methylation of core histones and increased DNase I sensitivity and restriction enzyme accessibility in the p21 WAF1 promoter. These changes did not occur in the p27 KIPI or ε-globin gene-related histones. The HDACi caused a marked decrease in HDAC1 and Myc and an increase in RNA polymerase II in proteins bound to the p21 WAF1 promoter. Thus, this study identifies effects of SAHA on p21 WAF1 -associated proteins that explain, at least in part, the selective effect of HDACi in altering gene expression.

Eukaryotic cells compartmentalize biochemical processes in different organelles, often relying on metabolic cycles to shuttle reducing equivalents across intracellular membranes. NADPH serves as the electron carrier for the maintenance of redox homeostasis and reductive biosynthesis, with separate cytosolic and mitochondrial pools providing reducing power in each respective location. This cellular organization is critical for numerous functions but complicates analysis of metabolic pathways using available methods. Here we develop an approach to resolve NADP(H)-dependent pathways present within both the cytosol and the mitochondria. By tracing hydrogen in compartmentalized reactions that use NADPH as a cofactor, including the production of 2-hydroxyglutarate by mutant isocitrate dehydrogenase enzymes, we can observe metabolic pathway activity in these distinct cellular compartments. Using this system we determine the direction of serine/glycine interconversion within the mitochondria and cytosol, highlighting the ability of this approach to resolve compartmentalized reactions in intact cells., National Institutes of Health (U.S.) (NIH grant P30CA147882), National Institutes of Health (U.S.) (NIH grant U54- CA121852-09), National Institutes of Health (U.S.) (NIH grant R01CA168653), David H. Koch Institute for Integrative Cancer Research at MIT (Koch Institute/DFHCC Bridge Project), David H. Koch Institute for Integrative Cancer Research at MIT (Koch Institute Frontier Research), Burroughs Wellcome Fund, Damon Runyon Cancer Research Foundation, Kathy and Curt Marble Cancer Research Fund, American Cancer Society (grant IRG #70-002), United States. Department of Defense (DOD grant W81XWH-13-1-0105), University of California, San Diego (University of California Cancer Research Coordinating Committee grant), Searle Scholars Program (Award)

The SOCS1 gene is a frequent target of epigenetic repression in hepatocellular carcinoma. Many other types of cancer also harbor methylated SOCS1 gene. Besides, recent studies implicate microRNAs targeting SOCS1 in cancer progression. These findings suggest a broad tumor suppressor role of SOCS1 and have stimulated the quest to elucidate the underlying molecular mechanisms. The essential physiological function of SOCS1 is to attenuate interferon gamma signaling in immune cells. SOCS1 binds activated JAK kinases and the receptor chains of several cytokines, some of which are implicated in cancer progression. SOCS1 also facilitates ubiquitination and proteasomal degradation of many signaling molecules downstream of cytokine and growth factor receptors. We have shown that SOCS1 inhibits signaling via the hepatocyte growth factor receptor c-MET in hepatocytes. Aberrant MET signaling, implicated in the progression of many types of cancers, also contributes to the development of chemoresistance to tyrosine kinase inhibitors and drugs targeting other oncogenic signaling pathways. Here, we discuss the SOCS1-dependent regulation of MET signaling as an important mechanism underlying the tumor suppressor role of SOCS1 that is relevant not only to hepatocellular carcinoma but also to other types of cancers.

Four hundred and thirty-eight endosperm-derived plants of kiwifruit (Actinidia chinensis) were observed. The plants were field planted in 1982 and all had flowered annually since 1986. A sample of these plants was assessed for leaf morphology, growth characteristics, flowering, sex expression, chromosome number, and fruit quality characteristics. The chromosome number of the endosperm plants varied from 58 to 146; most were aneuploid. A few triploid plants (2n=3x=87) were obtained; none of these were parthenocarpic. Segregation of sex expression was observed in progeny from one endosperm-derived callus.

Background While the association between ACPA and RA has been established, the impact of citrullination at the level of the protein is not well-defined. PAD enzymes remove an imine moiety from arginine to form citrulline. This enzymatic process results in the loss of a positive charge which can lead to a change in protein structure, consequential function or susceptibility to proteolytic processing.1 In an analogous fashion to the phosphorylome (phosphoproteins), to fully elucidate the role of citrullination for applied basic and clinical research, the field requires a laboratory developed pan reactive citrullination specific antibody (Ab) that identifies members of the “citrullinome” and site-specific citrullination antibodies that are generated to each target epitope since individual citrullination sites within a protein might elicit a gain or loss in function for the protein. These types of tools have clear advantages over other identification approaches like mass spectrometry, in that they are highly reproducible, can easily be integrated into multiple assay formats and are cost effective. Objectives The objective of this study was to 1) develop a pan reactive citrullination specific antibody and 2) an assay that can detect citrullinated 14-3-3η in patient serum. Methods 5 cit peptides and their corresponding arg peptides were synthesized and conjugated to BSA. 4 of the 5 peptides corresponded to putative 14-3-3η cit sites. One of the peptides, Peptide 4, was modelled on a PAD consensus motif. Polyclonal Abs to Peptide 4 were reproducibly raised in two independent species; twice in rabbits and once in goats with 3 animals per study. Reactivity was assessed by examining titres to both the citrullinated and arginylated forms of Peptide 4. Sera selected for purification underwent a two-step purification process utilizing the arg and cit peptide. Purified Cit-Ab was tested by examining reactivity to the other 4 cit-peptides and the corresponding arg forms. The Cit-Ab was tested by immunoblot analysis using cit 14-3-3η and cit vimetin that was generated using recombinant PAD4 as well as by 14-3-3η ELISA in seven 14-3-3η positive rheumatoid arthritis patients with levels >20 ng/ml. Results Five of the 6 rabbits and 2 of the 3 goats elicited immune responses that preferentially reacted with the cit form of Peptide 4. In a serial dilution study (1:50,000 to 1:250,000) the goat sera selected for purification exhibited 20X greater reactivity for the cit versus arg form of peptide 4. Purified Ab preferentially reacted with 3 of the 5 peptides based on a 2X ratio, and detected cit 14-3-3η and vimentin over the corresponding non-modified forms of these 2 proteins. As demonstrated in the [table][1] below, the burden of citrullination varied amongst patients despite having serum 14-3-3η levels >20ng/ml. View this table: Conclusions The pan reactive cit-specific Ab can be reliably reproduced and binds to multiple cit sites within a protein and across different proteins. The ELISA data reveals that the burden of citrullination varies amongst RA patients despite having similar levels of 14-3-3η protein. Citrullination of the “14-3-3η protein” is being clinically investigated in the context of disease outcomes. References 1. International Journal of Biochemistry and Cell Biology Disclosure of Interest Y. Gui Employee of: Augurex Life Sciences Corp, M. Murphy Employee of: Augurex Life Sciences Corp, W. Maksymowych Consultant for: Augurex Life Sciences Corp, (Co-inventor of 14-3-3η), A. Marotta Employee of: Augurex Life Sciences Corp, (Co-inventor of 14-3-3η) [1]: #T1

Coptis chinensis F. (family Ranunculaceae) is a perennial herbaceous plant, 10 to 13 cm in height. Its stem is rhizomatic and often branched. It is an endemic species distributed in Anhui, Guizhou, Jiangsu, Jiangxi, Hubei, Hunan, Shaanxi, Sichuan, Zhejiang Provinces of China. It is a very important medicinal plant in China. The rhizomes of the plant contain berberine, coptine, and coptisine. These compounds are reputed to result in cooling of the blood, reducing stress and fatigue, removing of toxic substances, and purging sthenic fever and emotions as well as promoting salivation (Wang 1964). Considerable work has been done on the micropropagation and production of medicinal compounds in Coptis species (Ikuta et al. 1975; Yamamoto et al. 1981; Ikuta and Itokawa 1989).

Corn is widely cultivated, and has a great potential for increased yield. The Hybrids obtained by crossing self lines have long been an effective approach to improve the yield, but it usually takes 4–6 years to breed a pure line. However, through anther and pollen culture, this time period can be considerably reduced, which enables the early release of varieties. By using this method, haploids have been produced in a large number of crops (see Bajaj 1983, 1990). The production of haploid plants from cultured anthers is considered an effective method for obtaining inbred lines of maize, and it has therefore attracted the attention of maize breeders. In 1972 Murakami et al. for the first time obtained callus from anthers, but did not achieve the regeneration of plants. In China, pollen plants of maize have been obtained since 1975 (Research Group 401 1975; Institute of Maize Research 1977; Kuo et al. 1978, 1986, 1990). These plants have both male and female sex organs, and produce ears with good seeds through self-pollination (Institute of Maize Research 1977). during the past few years, significant progress has been made in the cytological studies of androgenesis, chromosome doubling, and the selection and culture of the progeny (Wu et al. 1980). More than 100 pure lines have been obtained from 30 different combinations (Wu et al. 1983). Moreover, hundreds of cross-combinations have also been made from these pure lines, and most of them express obvious heterosis with good prospects, (also see Chaps. III.3, 4, this Vol.).

Mature embryos of Acanthopanax senticosus explanted on Murashige and Skoog (MS) medium with 0.5 mg/1 2,4-D developed somatic embryos directly from swollen cotyledon and embryo axes within one to two months. When the somatic embryos were transferred to medium supplemented with 2,4-D (0.5 mg/1) or IAA (1–3 mg/1) or Zeatin (0.5 mg/1) and NAA (0.2 mg/1), additional somatic embryos developed. Most (93%) embryos germinated on the above medium without 2,4-D. Sixty-two percent of the plantlets survived in soil. Histological observations revealed that the somatic embryos originated from cell masses of epidermal and sub-epidermal origin. There was no cytological separation zone between the somatic embryos and cultured expiants. Consequently, embryos were difficult to separate from their expiant tissue.

Surgical stress animal models were established by performing laparotomies on mice. It was found that this type of stress could suppress the natural killer (NK) cytotoxicity and the proliferation of spleen cells induced by conA or lipopolysaccharide (LPS). Dynamic changes showed that the stress-mediated immunosuppression was reversible, as the responses to conA and LPS would be restored with time. The sensitivity to the stress-mediated suppression was different according to variations in immunological parameters. Furthermore, the macrophages in spleen were tested by isolation by the plastic-adherent procedure. The results showed clearly that these adherent cells (Plastic Adherent Cells, PAC) possessed an immunosuppressive effect on mitogen-induced lymphocyte proliferation in post-operative mice, but not in normal mice. Treatment of mice with indomethacin blocked the PAC-mediated immunosuppression. Surgical stress appeared to increase the level of prostaglandins, which in turn induced the production of suppressor PAC.