YODA MAPK kinase kinase regulates a novel immunity pathway conferring broad-spectrum resistance to pathogens

Plant mitogen-activated protein kinase (MAPK) casca des transduce environmental molecular signals and developmental cues into cellular responses. Among these signals are the pathogen-associated molecular patterns (PAMPs) that upon recognition by plant pattern recognition receptors (PRR), including Receptor-Like Kinases (RLKs), activate MAPK cascades that regulate PAMP-triggered immunity responses (PTI).

Requirement for membrane lymphotoxin in natural killer cell development

Development of natural killer (NK) cells is thought to depend on interactions between NK progenitors and the bone marrow (BM) microenvironment; however, little is known about the molecular signals involved. Here we show that lymphotoxin (LT) provides an important signal for the development of both NK cells and NK/T cells. LTα−/− mice show marked reduction in splenic and BM NK and NK/T cell numbers and dramatically impaired NK and NK/T cell function. Mice deficient in either tumor necrosis factor receptor (TNFR)-I or TNFR-II have normal numbers of NK and NK/T cells, implying that neither of the TNFRs nor soluble LTα3 is required for development of these cell types. Reciprocal BM transfers between LTα−/− and wild-type mice suggest that close interactions between membrane LT-expressing NK cell precursors and LT-responsive radioresistant stromal cells are necessary for NK cell development. When LT-deficient BM cells are incubated with IL-15, NK cells are formed. In addition, LT-deficient BM cells produce IL-15 after activation. Thus, membrane LT appears to deliver a signal for NK cell development that is either independent of IL-15 or upstream in the IL-15 pathway. These results reveal a novel function for membrane LT in NK and NK/T cell development. They also support a cellular and molecular mechanism by which NK cell precursors themselves deliver essential signals, through the membrane ligand, that induce the microenvironment to promote further NK cell and NK/T cell development.

Excess “read-through” acetylcholinesterase attenuates but the “synaptic” variant intensifies neurodeterioration correlates

Acute stress increases the risk for neurodegeneration, but the molecular signals regulating the shift from transient stress responses to progressive disease are not yet known. The “read-through” variant of acetylcholinesterase (AChE-R) accumulates in the mammalian brain under acute stress. Therefore, markers of neurodeterioration were examined in transgenic mice overexpressing either AChE-R or the “synaptic” AChE variant, AChE-S. Several observations demonstrate that excess AChE-R attenuates, whereas AChE-S intensifies, neurodeterioration. In the somatosensory cortex, AChE-S transgenics, but not AChE-R or control FVB/N mice, displayed a high density of curled neuronal processes indicative of hyperexcitation. In the hippocampus, AChE-S and control mice, but not AChE-R transgenics, presented progressive accumulation of clustered, heat shock protein 70–immunopositive neuronal fragments and displayed a high incidence of reactive astrocytes. Our findings suggest that AChE-R serves as a modulator that may play a role in preventing the shift from transient, acute stress to progressive neurological disease.

Rational design of a bacterial transcriptional cascade for amplifying gene expression capacity

Cascade regulatory circuits have been described that control numerous cell processes, and may provide models for the design of artificial circuits with novel properties. Here we describe the design of a transcriptional regulatory cascade to amplify the cell response to a given signal. We used the salicylate-responsive activators of Pseudomonas putida NahR of the naphthalene degradation plasmid NAH7 and XylS2, a mutant regulator of the TOL plasmid for catabolism of m-xylene and their respective cognate promoters Psal and Pm. Control of the expression of xylS2 with the nahR/Psal system permitted either their selective activation with specific effectors for each protein or the simultaneous activation of both of them with salicylate. When cells face the common effector of the two regulators, both the increase in XylS2 concentration and the stimulation of its activity act synergistically on the Pm promoter, amplifying the gene expression capacity by at least one order of magnitude with respect to the individual systems. By changing the hierarchy of regulators, we showed that the specific features of the downstream regulator were crucial for the amplification effect. Directed changes in the effector profile of the regulators allowed the extension of the amplifying system to other molecular signals.

Germ cells enter meiosis in a rostro-caudal wave during development of the mouse ovary

Germ cells in the mouse embryo remain undifferentiated until about 13.5 days post-coitum (dpc), when male germ cells enter mitotic arrest and female germ cells enter meiosis. The molecular signals and transcriptional control mechanisms governing the differential fate of germ cells in males and females remain largely unknown. In order to gain insights into the behavior of germ cells around this period and into likely mechanisms controlling entry into meiosis, we have studied by wholemount in situ hybridization the expression pattern of two germ cell-specific markers, Oct4 and Sycp3, during mouse fetal gonad development. We observed a dynamic wave of expression of both genes in developing ovaries, with Oct4 expression being extinguished in a rostro-caudal wave and Sycp3 being upregulated in a corresponding wave, during the period 13.5-15.5 dpc. These results indicate that entry into meiosis proceeds in a rostro-caudal progression, in turn suggesting that somatically derived signals may contribute to the control of germ cell entry into meiosis in developing ovaries. (C) 2004 Wiley-Liss, Inc.

Identification and analysis of novel genes expressed in the mouse embryonic facial primordia

Craniofacial anomalies are a common feature of human congenital dysmorphology syndromes, suggesting that genes expressed in the developing face are likely to play a wider role in embryonic development. To facilitate the identification of genes involved in embryogenesis, we previously constructed an enriched cDNA library by subtracting adult mouse liver cDNA from that of embryonic day (E)10.5 mouse pharyngeal arch cDNA. From this library, 273 unique clones were sequenced and known proteins binned into functional categories in order to assess enrichment of the library (1). We have now selected 31 novel and poorly characterised genes from this library and present bioinformatic analysis to predict proteins encoded by these genes, and to detect evolutionary conservation. Of these genes 61% (19/31) showed restricted expression in the developing embryo, and a subset of these was chosen for further in silico characterisation as well as experimental determination of subcellular localisation based on transient transfection of predicted full-length coding sequences into mammalian cell lines. Where a human orthologue of these genes was detected, chromosomal localisation was determined relative to known loci for human congenital disease.

Advances in gastropod immunity from the study of the interaction between the snail Biomphalaria glabrata and its parasites: A review of research progress over the last decade

This review summarizes the research progress made over the past decade in the field of gastropod immunity resulting from investigations of the interaction between the snail Biomphalaria glabrata and its trematode parasites. A combination of integrated approaches, including cellular, genetic and comparative molecular and proteomic approaches have revealed novel molecular components involved in mediating Biomphalaria immune responses that provide insights into the nature of host-parasite compatibility and the mechanisms involved in parasite recognition and killing. The current overview emphasizes that the interaction between B. glabrata and its trematode parasites involves a complex molecular crosstalk between numerous antigens, immune receptors, effectors and anti-effector systems that are highly diverse structurally and extremely variable in expression between and within host and parasite populations. Ultimately, integration of these molecular signals will determine the outcome of a specific interaction between a B. glabrata individual and its interacting trematodes. Understanding these complex molecular interactions and identifying key factors that may be targeted to impairment of schistosome development in the snail host is crucial to generating new alternative schistosomiasis control strategies.

Interações e efeitos fisiológicos de bactérias diazotróficas e fungos micorrízicos arbusculares na mandioca.

Visando conhecer as interações entre fungos micorrízicos arbusculares e bactérias diazotróficas na cultura da mandioca (Manihot esculenta Crantz), foram conduzidos vários experimentos para avaliar os efeitos de exsudatos de mandioca e de bactérias nos fungos micorrízicos, e a produção de ácido indolacético (AIA) in vitro pelas bactérias diazotróficas. Os experimentos evidenciaram que as bactérias diazotróficas estimularam a colonização micorrízica de Glomus clarum a partir do 30o dia. Os exsudatos de mandioca e das bactérias não apresentaram efeito na germinação de Gigaspora gigantea, mas influenciaram seu crescimento micelial. A adição de exsudatos de mandioca estimulou o crescimento das bactérias diazotróficas in vitro, evidenciando que podem existir, nos exsudatos, substâncias que atuariam como sinais moleculares ou estimulantes do crescimento, e não como fatores nutricionais. As bactérias diazotróficas apresentam ainda capacidade de produzir AIA in vitro. Azospirillum lipoferum isolado da mandioca produziu até 130 mM de AIA após 48 horas de incubação, ao passo que Klebsiella sp. produziu cerca 60 mM e a Bactéria E, aproximadamente 20 mM.

Frequency-domain stimulated and spontaneous light emission signals at molecular junctions

Using a diagrammatic superoperator formalism we calculate optical signals at molecular junctions where a single molecule is coupled to two metal leads which are held at different chemical potentials. The molecule starts in a nonequilibrium steady state whereby it continuously exchanges electrons with the leads with a constant electron flux. Expressions for frequency domain optical signals measured in response to continuous laser fields are derived by expanding the molecular correlation functions in terms of its many-body states. The nonunitary evolution of molecular states is described by the quantum master equation. (C) 2014 AIP Publishing LLC.

Response to ``Comment on `Frequency-domain stimulated and spontaneous light emission signals at molecular junctions''' J. Chem. Phys. 142, 137101 (2015)]

In a recent work [U. Harbola, B. K. Agrawalla, and S. Mukamel, J. Chem. Phys. 141, 074107 (2014)], we have presented a superoperator (Liouville space) diagrammatic formulation of spontaneous and stimulated optical signals from current-carrying molecular junctions. We computed the diagrams that contribute to the spontaneous light emission SLE (fluorescence and Raman) signal using a diagrammatic method which clearly distinguishes between the Raman and the fluorescence contributions. We pointed out some discrepancies with the work of Galperin, Ratner and Nitzan (GRN) [M. Galperin, M. A. Ratner and, A. Nitzan, J. Chem. Phys. 130, 144109 (2009)]. In their response [M. Galperin, M. A. Ratner and A. Nitzan, “Comment on‘ Frequency-domain stimulated and spontaneous light emission signals at molecular junctions’” [J. Chem. Phys. 141, 074107 (2014)], J. Chem. Phys. 142, 137101 (2015)] to our work, GRN have argued that there are no differences in the choice of Raman diagrams in both works. Here we reply to their points and show where the differences exist.

Molecular markers of obesity and diabetes

Recently it has been shown that the consumption of a diet high in saturated fat is associated with impaired insulin sensitivity and increased incidence of type 2 diabetes. In contrast, diets that are high in monounsaturated fatty acids (MUFAs) or polyunsaturated fatty acids (PUFAs), especially very long chain n-3 fatty acids (FAs), are protective against disease. However, the molecular mechanisms by which saturated FAs induce the insulin resistance and hyperglycaemia associated with metabolic syndrome and type 2 diabetes are not clearly defined. It is possible that saturated FAs may act through alternative mechanisms compared to MUFA and PUFA to regulate of hepatic gene expression and metabolism. It is proposed that, like MUFA and PUFA, saturated FAs regulate the transcription of target genes. To test this hypothesis, hepatic gene expression analysis was undertaken in a human hepatoma cell line, Huh-7, after exposure to the saturated FA, palmitate. These experiments showed that palmitate is an effective regulator of gene expression for a wide variety of genes. A total of 162 genes were differentially expressed in response to palmitate. These changes not only affected the expression of genes related to nutrient transport and metabolism, they also extend to other cellular functions including, cytoskeletal architecture, cell growth, protein synthesis and oxidative stress response. In addition, this thesis has shown that palmitate exposure altered the expression patterns of several genes that have previously been identified in the literature as markers of risk of disease development, including CVD, hypertension, obesity and type 2 diabetes. The altered gene expression patterns associated with an increased risk of disease include apolipoprotein-B100 (apo-B100), apo-CIII, plasminogen activator inhibitor 1, insulin-like growth factor-I and insulin-like growth factor binding protein 3. This thesis reports the first observation that palmitate directly signals in cultured human hepatocytes to regulate expression of genes involved in energy metabolism as well as other important genes. Prolonged exposure to long-chain saturated FAs reduces glucose phosphorylation and glycogen synthesis in the liver. Decreased glucose metabolism leads to elevated rates of lipolysis, resulting in increased release of free FAs. Free FAs have a negative effect on insulin action on the liver, which in turn results in increased gluconeogenesis and systemic dyslipidaemia. It has been postulated that disruption of glucose transport and insulin secretion by prolonged excessive FA availability might be a non-genetic factor that has contributed to the staggering rise in prevalence of type 2 diabetes. As glucokinase (GK) is a key regulatory enzyme of hepatic glucose metabolism, changes in its activity may alter flux through the glycolytic and de novo lipogenic pathways and result in hyperglycaemia and ultimately insulin resistance. This thesis investigated the effects of saturated FA on the promoter activity of the glycolytic enzyme, GK, and various transcription factors that may influence the regulation of GK gene expression. These experiments have shown that the saturated FA, palmitate, is capable of decreasing GK promoter activity. In addition, quantitative real-time PCR has shown that palmitate incubation may also regulate GK gene expression through a known FA sensitive transcription factor, sterol regulatory element binding protein-1c (SREBP-1c), which upregulates GK transcription. To parallel the investigations into the mechanisms of FA molecular signalling, further studies of the effect of FAs on metabolic pathway flux were performed. Although certain FAs reduce SREBP-1c transcription in vitro, it is unclear whether this will result in decreased GK activity in vivo where positive effectors of SREBP-1c such as insulin are also present. Under these conditions, it is uncertain if the inhibitory effects of FAs would be overcome by insulin. The effects of a combination of FAs, insulin and glucose on glucose phosphorylation and metabolism in cultured primary rat hepatocytes at concentrations that mimic those in the portal circulation after a meal was examined. It was found that total GK activity was unaffected by an increased concentration of insulin, but palmitate and eicosapentaenoic acid significantly lowered total GK activity in the presence of insulin. Despite the fact that total GK enzyme activity was reduced in response to FA incubation, GK enzyme translocation from the inactive, nuclear bound, to active, cytoplasmic state was unaffected. Interestingly, none of the FAs tested inhibited glucose phosphorylation or the rate of glycolysis when insulin is present. These results suggest that in the presence of insulin the levels of the active, unbound cytoplasmic GK are sufficient to buffer a slight decrease in GK enzyme activity and decreased promoter activity caused by FA exposure. Although a high fat diet has been associated with impaired hepatic glucose metabolism, there is no evidence from this thesis that FAs themselves directly modulate flux through the glycolytic pathway in isolated primary hepatocytes when insulin is also present. Therefore, although FA affected expression of a wide range of genes, including GK, this did not affect glycolytic flux in the presence of insulin. However, it may be possible that a saturated FA-induced decrease in GK enzyme activity when combined with the onset of insulin resistance may promote the dys-regulation of glucose homeostasis and the subsequent development of hyperglycaemia, metabolic syndrome and type 2 diabetes.

Influence of culture conditions on the molecular signature of mesenchymal stem cells

Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.

A Bias in ML Estimates of Branch Lengths in the Presence of Multiple Signals

Sequence data often have competing signals that are detected by network programs or Lento plots. Such data can be formed by generating sequences on more than one tree, and combining the results, a mixture model. We report that with such mixture models, the estimates of edge (branch) lengths from maximum likelihood (ML) methods that assume a single tree are biased. Based on the observed number of competing signals in real data, such a bias of ML is expected to occur frequently. Because network methods can recover competing signals more accurately, there is a need for ML methods allowing a network. A fundamental problem is that mixture models can have more parameters than can be recovered from the data, so that some mixtures are not, in principle, identifiable. We recommend that network programs be incorporated into best practice analysis, along with ML and Bayesian trees.

Multiple novel prostate cancer susceptibility signals identified by fine-mapping of known risk loci among Europeans

Genome-wide association studies (GWAS) have identified numerous common prostate cancer (PrCa) susceptibility loci. We have fine-mapped 64 GWAS regions known at the conclusion of the iCOGS study using large-scale genotyping and imputation in 25 723 PrCa cases and 26 274 controls of European ancestry. We detected evidence for multiple independent signals at 16 regions, 12 of which contained additional newly identified significant associations. A single signal comprising a spectrum of correlated variation was observed at 39 regions; 35 of which are now described by a novel more significantly associated lead SNP, while the originally reported variant remained as the lead SNP only in 4 regions. We also confirmed two association signals in Europeans that had been previously reported only in East-Asian GWAS. Based on statistical evidence and linkage disequilibrium (LD) structure, we have curated and narrowed down the list of the most likely candidate causal variants for each region. Functional annotation using data from ENCODE filtered for PrCa cell lines and eQTL analysis demonstrated significant enrichment for overlap with bio-features within this set. By incorporating the novel risk variants identified here alongside the refined data for existing association signals, we estimate that these loci now explain ∼38.9% of the familial relative risk of PrCa, an 8.9% improvement over the previously reported GWAS tag SNPs. This suggests that a significant fraction of the heritability of PrCa may have been hidden during the discovery phase of GWAS, in particular due to the presence of multiple independent signals within the same region.