Fibrocyte localisation to the ASM bundle in asthma: bidirectional effects on cell phenotype and behaviour.

OBJECTIVES: Airway hyper-responsiveness and persistent airflow obstruction contribute to asthma pathogenesis and symptoms, due in part to airway smooth muscle (ASM) hypercontractility and increased ASM mass. Fibrocytes have been shown to localise to the ASM in asthma however it is not known whether fibrocytes localise to the ASM in nonasthmatic eosinophilic bronchitis (NAEB) and chronic obstructive pulmonary disease (COPD). In addition, the potential consequences of fibrocyte localisation to ASM as regards asthma pathophysiology has not been widely studied. METHODS: Fibrocytes and proliferating cells were enumerated in ASM in bronchial tissue using immunohistochemistry. The effects of primary ASM and fibrocytes upon each other in terms of phenotype and behaviour following co-culture were investigated by assessing cell number, size, apoptotic status, phenotype and contractility in in vitro cell-based assays. RESULTS: Increased fibrocyte number in the ASM was observed in asthma versus NAEB, but not NAEB and COPD versus controls, and confirmed in asthma versus controls. ASM proliferation was not detectably different in asthmatics versus healthy controls in vivo. No difference in proliferation, apoptotic status or size of ASM was seen following culture with/without fibrocytes. Following co-culture with ASM from asthmatics versus nonasthmatics, fibrocyte smooth muscle marker expression and collagen gel contraction were greater. Following co-culture, fibrocyte CD14 expression was restored with the potential to contribute to asthma pathogenesis via monocyte-mediated processes dependent on the inflammatory milieu. CONCLUSION: Further understanding of mechanisms of fibrocyte recruitment to and/or differentiation within the ASM may identify novel therapeutic targets to modulate ASM dysfunction in asthma.

Targeting Induced Local Lesions IN Genomes (TILLING) in Medicago truncatula.

TILLING is a reverse genetics strategy that combines the high density of point mutations provided by traditional chemical mutagenesis with rapid screening of DNA pools from a mutagenized population for induced mutations (McCallum et al., Nat Biotechnol 18:455-457, 2000). This high-throughput technique allows the identification of point mutations in any gene of interest.

Functional Genomics and Seed Development in Medicago truncatula: An Overview.

The study of seed development in the model species Medicago truncatula has made a significant contribution to our understanding of this process in crop legumes. Thanks to the availability of comprehensive proteomics and transcriptomics databases, coupled with exhaustive mutant collections, the roles of several regulatory genes in development and maturation are beginning to be deciphered and functionally validated. Advances in next-generation sequencing and the availability of a genomic sequence have made feasible high-density SNP genotyping, allowing the identification of markers tightly linked to traits of agronomic interest. A further major advance is to be expected from the integration of omics resources in functional network construction, which has been used recently to identify "hub" genes central to important traits.

Genome-wide association studies with proteomics data reveal genes important for synthesis, transport and packaging of globulins in legume seeds.

Improving nutritional seed quality is an important challenge in grain legume breeding. However, the genes controlling the differential accumulation of globulins, which are major contributors to seed nutritional value in legumes, remain largely unknown. We combined a search for protein quantity loci with genome-wide association studies on the abundance of 7S and 11S globulins in seeds of the model legume species Medicago truncatula. Identified genomic regions and genes carrying polymorphisms linked to globulin variations were then cross-compared with pea (Pisum sativum), leading to the identification of candidate genes for the regulation of globulin abundance in this crop. Key candidates identified include genes involved in transcription, chromatin remodeling, post-translational modifications, transport and targeting of proteins to storage vacuoles. Inference of a gene coexpression network of 12 candidate transcription factors and globulin genes revealed the transcription factor ABA-insensitive 5 (ABI5) as a highly connected hub. Characterization of loss-of-function abi5 mutants in pea uncovered a role for ABI5 in controlling the relative abundance of vicilin, a sulfur-poor 7S globulin, in pea seeds. This demonstrates the feasibility of using genome-wide association studies in M. truncatula to reveal genes that can be modulated to improve seed nutritional value.

The role of the testa during development and in establishment of dormancy of the legume seed.

Timing of seed germination is one of the key steps in plant life cycles. It determines the beginning of plant growth in natural or agricultural ecosystems. In the wild, many seeds exhibit dormancy and will only germinate after exposure to certain environmental conditions. In contrast, crop seeds germinate as soon as they are imbibed usually at planting time. These domestication-triggered changes represent adaptations to cultivation and human harvesting. Germination is one of the common sets of traits recorded in different crops and termed the "domestication syndrome." Moreover, legume seed imbibition has a crucial role in cooking properties. Different seed dormancy classes exist among plant species. Physical dormancy (often called hardseededness), as found in legumes, involves the development of a water-impermeable seed coat, caused by the presence of phenolics- and suberin-impregnated layers of palisade cells. The dormancy release mechanism primarily involves seed responses to temperature changes in the habitat, resulting in testa permeability to water. The underlying genetic controls in legumes have not been identified yet. However, positive correlation was shown between phenolics content (e.g., pigmentation), the requirement for oxidation and the activity of catechol oxidase in relation to pea seed dormancy, while epicatechin levels showed a significant positive correlation with soybean hardseededness. myeloblastosis family of transcription factors, WD40 proteins and enzymes of the anthocyanin biosynthesis pathway were involved in seed testa color in soybean, pea and Medicago, but were not tested directly in relation to seed dormancy. These phenolic compounds play important roles in defense against pathogens, as well as affecting the nutritional quality of products, and because of their health benefits, they are of industrial and medicinal interest. In this review, we discuss the role of the testa in mediating legume seed germination, with a focus on structural and chemical aspects.

Interferon gamma release assays and the NICE 2011 guidelines on the diagnosis of latent tuberculosis.

In this clinical audit, we assessed retrospectively the current practice of respiratory physicians with respect to interferon gamma (IFNγ) release assay (IGRA) testing for tuberculosis (TB), as recommended by the 2011 National Institute of Health and Care Excellence (NICE) guidelines for the diagnosis and management of TB. All IGRAs requested by respiratory physicians over a 3-year period were identified retrospectively, and both results and clinical indications analysed. Of the total number of IGRAs carried out, 90% formed part of investigations of suspected active TB. However, 89% of the patients had not had a documented Mantoux test and human immunodeficiency virus (HIV) status was unclear in the 35.2% of patients treated for active TB. Of patients with chest X-rays suggestive of TB, 92.3% were treated for active TB. Of the patients under the age of 35 with reactive IGRAs, 84.6% were treated for active or latent TB and 15.4% had justifiable reasons for not receiving chemoprophylaxis. Based on the results of our audit, IGRAs are commonly being utilised for the investigation of active TB, which is contrary to current guidance.

The role of the DNA-binding One Zinc Finger (DOF) transcription factor family in plants.

The DOF (DNA-binding One Zinc Finger) family of transcription factors is involved in many fundamental processes in higher plants, including responses to light and phytohormones as well as roles in seed maturation and germination. DOF transcription factor genes are restricted in their distribution to plants, where they are in many copies in both gymnosperms and angiosperms and also present in lower plants such as the moss Physcomitrella patens and in the alga Chlamydomonas reinhardtii which possesses a single DOF gene. DOF transcription factors bind to their promoter targets at the consensus sequence AAAG. This binding depends upon the presence of the highly conserved DOF domain in the protein. Depending on the target gene, DOF factor binding may activate or repress transcription. DOF factors are expressed in most if not all tissues of higher plants, but frequently appear to be functionally redundant. Recent next-generation sequencing data provide a more comprehensive survey of the distribution of DOF sequence classes among plant species and within tissue types, and clues as to the evolution of functions assumed by this transcription factor family. DOFs do not appear to be implicated in the initial differentiation of the plant body plan into organs via the resolution of meristematic zones, in contrast to MADS-box and homeobox transcription factors, which are found in other non-plant eukaryotes, and this may reflect a more recent evolutionary origin.

The proinflammatory environment in potential heart and lung donors: prevalence and impact of donor management and hormonal therapy.

BACKGROUND: Brain stem death can elicit a potentially manipulable cardiotoxic proinflammatory cytokine response. We investigated the prevalence of this response, the impact of donor management with tri-iodothyronine (T3) and methylprednisolone (MP) administration, and the relationship of biomarkers to organ function and transplant suitability. METHODS: In a prospective randomized double-blinded factorially designed study of T3 and MP therapy, we measured serum levels of interleukin-1 and -6 (IL-1 and IL-6), tumor necrosis factor-alpha (TNF-alpha), C-reactive protein, and procalcitonin (PCT) levels in 79 potential heart or lung donors. Measurements were performed before and after 4 hr of algorithm-based donor management to optimize cardiorespiratory function and +/-hormone treatment. Donors were assigned to receive T3, MP, both drugs, or placebo. RESULTS: Initial IL-1 was elevated in 16% donors, IL-6 in 100%, TNF-alpha in 28%, CRP in 98%, and PCT in 87%. Overall biomarker concentrations did not change between initial and later measurements and neither T3 nor MP effected any change. Both PCT (P =0.02) and TNF-alpha (P =0.044) levels were higher in donor hearts with marginal hemodynamics at initial assessment. Higher PCT levels were related to worse cardiac index and right and left ventricular ejection fractions and a PCT level more than 2 ng x mL(-1) may attenuate any improvement in cardiac index gained by donor management. No differences were observed between initially marginal and nonmarginal donor lungs. A PCT level less than or equal to 2 ng x mL(-1) but not other biomarkers predicted transplant suitability following management. CONCLUSIONS: There is high prevalence of a proinflammatory environment in the organ donor that is not affected by tri-iodothyronine or MP therapy. High PCT and TNF-alpha levels are associated with donor heart dysfunction.

The haemodynamic effects of adjunctive hormone therapy in potential heart donors: a prospective randomized double-blind factorially designed controlled trial.

AIMS: The aim of this study was to assess the haemodynamic effects of tri-iodothyronine (T3) and methylprednisolone in potential heart donors. METHODS AND RESULTS: In a prospective randomized double-blind trial, 80 potential cardiac donors were allocated to receive T3 (0.8 microg kg(-1) bolus; 0.113 microg kg(-1) h(-1) infusion) (n = 20), methylprednisolone (1000 mg bolus) (n = 19), both drugs (n = 20), or placebo (n = 21) following initial haemodynamic assessment. After hormone or placebo administration, cardiac output-guided optimization was initiated, using vasopressin as a pressor and weaning norepinephrine and inotropes. Treatment was administered for 5.9 +/- 1.3 h until retrieval or end-assessment. Cardiac index increased significantly (P < 0.001) but administration of T3 and methylprednisolone alone or in combination did not affect this change or the heart retrieval rate. Thirty-five per cent (14/40) of initially marginal or dysfunctional hearts were suitable for transplant at end-assessment. At end-assessment, 50% of donor hearts fulfilled criteria for transplant suitability. CONCLUSION: Cardiac output-directed donor optimization improves donor circulatory status and has potential to increase the retrieval rate of donor hearts. Tri-iodothyronine and methylprednisolone therapy do not appear to acutely affect cardiovascular function or yield.

Transcriptional regulation of storage protein synthesis during dicotyledon seed filling.

Seeds represent a major source of nutrients for human and animal livestock diets. The nutritive value of seeds is largely due to storage products which accumulate during a key phase of seed development, seed filling. In recent years, our understanding of the mechanisms regulating seed filling has advanced significantly due to the diversity of experimental approaches used. This review summarizes recent findings related to transcription factors that regulate seed storage protein accumulation. A framework for the regulation of storage protein synthesis is established which incorporates the events before, during and after seed storage protein synthesis. The transcriptional control of storage protein synthesis is accompanied by physiological and environmental controls, notably through the action of plant hormones and other intermediary metabolites. Finally, recent post-genomics analyses on different model plants have established the existence of a conserved seed filling process involving the master regulators (LEC1, LEC2, ABI3 and FUS3) but also revealed certain differences in fine regulation between plant families.

Gene expression profiling of M. truncatula transcription factors identifies putative regulators of grain legume seed filling.

Legume seeds represent a major source of proteins for human and livestock diets. The model legume Medicago truncatula is characterized by a process of seed development very similar to that of other legumes, involving the interplay of sets of transcription factors (TFs). Here, we report the first expression profiling of over 700 M. truncatula genes encoding putative TFs throughout seven stages of seed development, obtained using real-time quantitative RT-PCR. A total of 169 TFs were selected which were expressed at late embryogenesis, seed filling or desiccation. The site of expression within the seed was examined for 41 highly expressed transcription factors out of the 169. To identify possible target genes for these TFs, the data were combined with a microarray-derived transcriptome dataset. This study identified 17 TFs preferentially expressed in individual seed tissues and 135 corresponding co-expressed genes, including possible targets. Certain of the TFs co-expressed with storage protein mRNAs correspond to those already known to regulate seed storage protein synthesis in Arabidopsis, whereas the timing of expression of others may be more specifically related to the delayed expression of the legumin-class storage proteins observed in legumes.

Early donor management increases the retrieval rate of lungs for transplantation.

BACKGROUND: Lung transplantation activity is frustrated by donor lung availability. We sought to examine the effect of active donor management and hormone administration on pulmonary function and yield in cadaveric heart-beating potential lung donors. METHODS: We studied 182 potential lung donors (arterial oxygen tension [PaO2]/fractional inspired oxygen [FIO2] ratio > or = 230). From this group, 60 patients (120 lungs) were allocated, within a randomized trial, to receive methylprednisolone (1 g), triiodothyronine (0.8 microg/kg bolus and 0.113 microg/kg/h infusion), both methylprednisolone and triiodothyronine, or placebo as soon as feasible after consent and initial assessment. Trial donors underwent protocol-guided optimization of ventilation and hemodynamics, lung water assessment, and bronchoscopy. Function was assessed by PaO2/FIO2 ratio, extravascular lung water index (EVLWI), and pulmonary vascular resistance (PVR). A nontrial group of 122 donors (244 lungs) received similar management without bronchoscopy, pulmonary artery flotation catheter monitoring, or lung water assessment. RESULTS: Within the trial, management commenced within a median of 2 hours (interquartile range, 0.5 to 3.5 hours) of consent and continued for an average of 6.9 +/- 1.2 hours. The PaO2/FIO2 ratio deteriorated (p = 0.028) from 397 +/- 78 (95% CL, 376 to 417) to 359 +/- 126 (95% CL, 328 to 390) and EVLWI from 9.7 +/- 4.5 mL/kg (95% CL, 8.6 to 10.9 mL/kg) to 10.8 +/- 5.2 mL/kg (95% CL, 9.4 to 12.2 mL/kg; p = 0.009). PVR remained unchanged (p = 0.28). At end management, 48 of 120 trial lungs (40%) were transplanted versus 66 of 244 nontrial lungs (27%; p = 0.016). Neither methylprednisolone and triiodothyronine nor T3 increased lung yield or affected PaO2/FIO2 or EVLWI; however, methylprednisolone attenuated the increase in EVLWI (p = 0.009). CONCLUSIONS: Early active management of lung donors increases yield. Steroid administration reduces progressive lung water accumulation.

Interaction of maize Opaque-2 and the transcriptional co-activators GCN5 and ADA2, in the modulation of transcriptional activity.

Maize Opaque-2 (ZmO2), a bZip class transcription factor has been shown to activate the transcription of a series of genes expressed in the maturation phase of endosperm development. Activation requires the presence of one or more enhancer binding sites, which confer the propensity for activation by ZmO2 on heterologous promoters and in heterologous plant cell types, such as tobacco mesophyll protoplasts. The region of ZmO2 required for conferring transcriptional activation has been localised to a stretch of acidic residues in the N-terminal portion of the ZmO2 sequence, which is conserved between O2-related bZip factor sequences. Previously we identified the maize homologues of yeast transcriptional co-activators GCN5 and ADA2 that are implicated in nucleosome modification and transcription. In the present study we have shown that transcriptional modulation by ZmO2 involves the intranuclear interaction of ZmO2 with ZmADA2 and ZmGCN5. Förster resonance energy transfer (FRET) based techniques have enabled us to estimate the intracellular site of these intermolecular interactions. As a functional readout of these intranuclear interactions, we used the ZmO2 responsive maize b-32 promoter to drive the beta-glucuronidase (GUS) in the presence and absence of ZmGCN5 and ZmADA2. Our results suggest that the likely recruitment of ZmADA2 and ZmGCN5 modulates the transactivation of b-32 promoter by ZmO2 and that there may be a competition between ZmGCN5 and ZmO2 for binding to the amino-terminal of ZmADA2. The results may be taken as a paradigm for other processes of transcriptional modulation in planta involving acidic activation domains.

Neutrophil elastase (NE)-deficient mice demonstrate a nonredundant role for NE in neutrophil migration, generation of proinflammatory mediators, and phagocytosis in response to zymosan particles in vivo.

Neutrophil elastase (NE) remains a controversial player in the process of leukocyte transmigration and much of this controversy stems from conflicting reports on the effects of NE inhibitors. The availability of NE-deficient mice (NE(-/-)) provides a clean and elegant tool for the study of leukocyte migration in vivo. In this study, NE(-/-) mice were used to investigate the role of NE in leukocyte migration through cremasteric venules, as observed by intravital microscopy, induced by locally administered cytokines IL-1beta and TNF-alpha and the particulate stimulus, zymosan. Although no defects in leukocyte responses induced by the cytokines were observed, zymosan-induced leukocyte firm adhesion and transmigration was suppressed in NE(-/-) mice. These responses were also inhibited in wild-type mice when zymosan was coinjected with a specific NE inhibitor. Quantification of inflammatory mediator levels in homogenates of zymosan-stimulated tissues indicated reductions in levels of IL-1beta, KC, and macrophage inflammatory protein-1alpha in NE(-/-) mice. Furthermore, phagocytosis of fluorescent zymosan particles, as observed by intravital microscopy, was diminished in NE-deficient animals. Collectively, the findings of this study indicate a nonredundant role for NE in zymosan-induced leukocyte firm adhesion and transmigration, and that this defect is associated with impaired generation of proinflammatory mediators as well as phagocytosis of zymosan particles in vivo.

Murine CD93 (C1qRp) contributes to the removal of apoptotic cells in vivo but is not required for C1q-mediated enhancement of phagocytosis.

Human CD93 (known as C1qRp) has been shown to be a phagocytic receptor involved in the in vitro C1q-dependent enhancement of phagocytosis. However, binding of CD93 to C1q and its function remain controversial. In this study, we have generated CD93-deficient mice (CD93(-/-)) to investigate its biological role(s). The CD93(-/-) mice were viable and showed no gross abnormalities in their development. Thioglycolate-elicited peritoneal macrophages deficient in CD93 showed a similar enhancement in complement- and FcgammaR-dependent uptake of RBC to the wild-type macrophages when plated on C1q-coated surfaces suggesting that the lack of this receptor had no effect on these C1q-mediated events. There was no impairment in either complement- or FcgammaR-dependent phagocytic assays in vivo. By contrast, the CD93(-/-) mice had a significant phagocytic defect in the clearance of apoptotic cells in vivo (human Jurkat T cells and murine thymocytes: p=0.0006 and p=0.0079, respectively) compared with strain-matched controls. However, in vitro, the CD93(-/-) macrophages showed similar engulfment of apoptotic cells to wild-type macrophages. Furthermore, no supporting evidence for a role of CD93 as an adhesion molecule was found using intravital microscopy or analyzing peritoneal cell recruitment in response to three different inflammatory stimuli (thioglycolate, zymosan A, and IL-1beta). Thus, our findings indicate that murine CD93 is expressed on the peritoneal macrophage, especially on thioglycolate-elicited cells, but does not appear to play a key role in C1q-mediated enhancement of phagocytosis or in the intercellular adhesion events tested. However, our results suggest that it may contribute to the in vivo clearance of dying cells.

Proteomics of Medicago truncatula seed development establishes the time frame of diverse metabolic processes related to reserve accumulation.

We utilized a proteomic approach to investigate seed development in Medicago truncatula, cv Jemalong, line J5 at specific stages of seed filling corresponding to the acquisition of germination capacity and protein deposition. One hundred twenty proteins differing in kinetics of appearance were subjected to matrix-assisted laser desorption ionization time of flight mass spectrometry. These analyses provided peptide mass fingerprint data that identified 84 of them. Some of these proteins had previously been shown to accumulate during seed development in legumes (e.g. legumins, vicilins, convicilins, and lipoxygenases), confirming the validity of M. truncatula as a model for analysis of legume seed filling. The study also revealed proteins presumably involved in cell division during embryogenesis (beta-tubulin and annexin). Their abundance decreased before the accumulation of the major storage protein families, which itself occurs in a specific temporal order: vicilins (14 d after pollination [DAP]), legumins (16 DAP), and convicilins (18 DAP). Furthermore, the study showed an accumulation of enzymes of carbon metabolism (e.g. sucrose synthase, starch synthase) and of proteins involved in embryonic photosynthesis (e.g. chlorophyll a/b binding), which may play a role in providing cofactors for protein/lipid synthesis or for CO2 refixation during seed filling. Correlated with the reserve deposition phase was the accumulation of proteins associated with cell expansion (actin 7 and reversibly glycosylated polypeptide) and of components of the precursor accumulating vesicles, which give rise to a trypsin inhibitor on maturation. Finally, we revealed a differential accumulation of enzymes involved in methionine metabolism (S-adenosyl-methionine synthetase and S-adenosylhomo-cysteine hydrolase) and propose a role for these enzymes in the transition from a highly active to a quiescent state during seed development.

Alteration of GCN5 levels in maize reveals dynamic responses to manipulating histone acetylation.

The role played by histone acetyltransferase (HAT), GCN5, in transcriptional co-activation has been analysed in detail in yeast and mammals. Here, we present the cloning and expression pattern of Zmgcn5, the maize homologue. The enzymatic activity of the recombinant ZmGCN5 was analysed with histone and nucleosome substrates. In situ hybridisation of developing maize kernels using Zmgcn5 as probe shows that the transcript is concentrated in rapidly dividing cells. To investigate the role of ZmGCN5 in the transcription of specific plant genes, direct protein-protein interactions were tested. A cDNA clone encoding a putative interacting partner in GCN5-adapter complexes, ZmADA2, was isolated and the interaction between ZmGCN5 and ZmADA2 was confirmed by a GST-spin down experiment. Co-immunoprecipitation of the plant transcriptional activator Opaque-2 and ZmADA2 in nuclear extracts suggests ADA2/GCN5-containing complexes to mediate transcriptional activation by binding of this bZIP factor. For a more general analysis of the effects of histone acetylation on plant gene expression, 2500 ESTs spotted on filters were hybridised with cDNA probes derived either from maize cell lines treated with Trichostatin A (TSA), or from a transgenic line expressing the ZmGCN5 antisense transcript. Several sequences showing marked changes in abundance were confirmed by RNA blot analysis. Inhibition of histone deacetylation with TSA is accompanied by a decrease in the abundance of ZmGCN5 acetylase protein, but by increases in mRNAs for histones H2A, H2B, H3 and H4. The elevated histone mRNA levels were not reflected in increasing histone protein concentrations, suggesting hyperacetylated histones arising from TSA treatment may be preferentially degraded and substituted by de novo synthesised histones. The ZmGCN5 antisense material showed suppression of the endogenous ZmGCN5 transcript and the profiling analysis revealed increased mRNA levels for H2A, H2B and H4. Furthermore, in the antisense line, a reduction in the amount of the RPD3-type HD1B-I histone deacetylase protein was observed. A model for linked regulation of histone acetylation and histone mRNA transcription is discussed.

Post-phloem protein trafficking in the maize caryopsis: zmTRXh1, a thioredoxin specifically expressed in the pedicel parenchyma of Zea mays L., is found predominantly in the placentochalaza.

The pedicel of the maize (Zea mays L.) caryopsis is a complex structure consisting of layers of specialized cell-types involved with solute transfer into the developing kernel. A molecular marker for one of these cell layers, the phloem parenchyma, has been obtained by differential screening of a cDNA library from 7 days after pollination (DAP) kernels. The clone encodes a novel processed type of thioredoxin, ZmTRXh1, with a variant active site sequence. The transcript is exclusively present in phloem parenchyma cells of the pedicel. The protein accumulates predominantly in the adjacent placentochalazal layer up to 21 DAP, declining thereafter until, at 31 DAP, only traces remain. ZmTRXh1, which is catalytically active, is present in the cytosol, and is restricted to a fraction of the placentochalazal cells at 12 DAP, where it accumulates to high levels. ZmTRXh1 represents the first example of post-phloem protein trafficking in the pedicel. The reasons for this transfer and possible functions for the protein in the placentochalaza are discussed.