The role of the soluble fms-like tyrosine kinase-1/placental growth factor (sFlt-1/PIGF) - ratio in clinical practice in obstetrics: diagnostic and prognostic value.

OBJECTIVES: Analyze the diagnostic and prognostic value of the sFlt-1/PlGF ratio in pregnant women with at least one sign/symptom of suspected/diagnosed pre-eclampsia. METHODS: This retrospective observational study included 170 pregnant women with at least one sign/symptom of pre-eclampsia, who had sFlt-1/PlGF ratio values. The following information was evaluated: pregnant women's demographic data and clinical history; laboratory data (urine protein/creatinine ratio; sFlt-1/PlGF ratio); signs and symptoms presented; clinical outcome; fetal complications; data related to childbirth. Statistical analysis was performed by R Software Version 3.5.2. RESULTS: Among the 170 patients, 78 presented pre-eclampsia. The median sFlt-1/PlGF ratio was significantly higher [143.1 (2.2-2,927.1)] for women who presented pre-eclampsia than for women without pre-eclampsia [33.5 (0.8-400.2)]. The negative predictive value of sFlt-1/PlGF ratio <38 was 83.9 % (95 % CI, 71.7-92.4 %) and the positive predictive value of sFlt-1/PlGF ratio >85 or 110 (for late onset pre-eclampsia) was 76.4 % (95 % CI, 66.2-84.8 %). sFlt-1/PlGF >85 or 110 was associated with pre-eclampsia clinical development, fetal complications, shorter gestational age at birth, higher number of caesarean deliveries and lower birth weight. CONCLUSIONS: The sFlt-1/PlGF ratio, together with the standard diagnostic criteria, can be used to rule out pre-eclampsia, identify high-risk patients and predict the occurrence of adverse outcomes.

Kidney graft function before pregnancy as a predictor of graft, maternal and fetal outcomes in pregnant renal transplant recipients.

OBJECTIVES: Maternal and fetal complications can occur in pregnant kidney transplant recipients. Since these are high-risk pregnancies, they require a multidisciplinary follow-up to prematurely detect adverse events. Identifying factors that would affect fetal, maternal and graft outcomes is essential to further stratify the risk of pregnant kidney transplant recipients. METHODS: All pregnancies in kidney transplant recipients followed in a single center for 30 years were included. Data included previous transplant information and blood and urine tests performed before pregnancy. Impact of graft function on fetal, maternal and graft outcomes was evaluated. RESULTS: There were 41 pregnancies among 34 patients. Mean gestational age of 35 ± 3 weeks. Caesarean section was performed in 69.4% of patients. Five pregnancies were unsuccessful (12.2%). Four patients suffered an acute graft dysfunction (9.8%) and 12 (29.3%) had a serious maternal hypertensive disorder (preeclampsia, eclampsia or HELLP syndrome). Graft function before pregnancy showed significant correlation with adverse outcomes. CONCLUSIONS: A proteinuria >669 mg/g, serum creatinine >1.75 mg/dL and glomerular filtration rate <36.2 mL/min/1.73 m2 before pregnancy were correlated to graft dysfunction during pregnancy. Similar values of proteinuria were also associated with a risk of maternal hypertensive disorders and pregnancy failure. Therefore, in patients with proteinuria and graft dysfunction, follow-up should be stricter to quickly detect complications.

Transcript and metabolite analysis in Trincadeira cultivar reveals novel information regarding the dynamics of grape ripening.

BACKGROUND: Grapes (Vitis vinifera L.) are economically the most important fruit crop worldwide. However, the complexity of molecular and biochemical events that lead to the onset of ripening of nonclimacteric fruits is not fully understood which is further complicated in grapes due to seasonal and cultivar specific variation. The Portuguese wine variety Trincadeira gives rise to high quality wines but presents extremely irregular berry ripening among seasons probably due to high susceptibility to abiotic and biotic stresses. RESULTS: Ripening of Trincadeira grapes was studied taking into account the transcriptional and metabolic profilings complemented with biochemical data. The mRNA expression profiles of four time points spanning developmental stages from pea size green berries, through véraison and mature berries (EL 32, EL 34, EL 35 and EL 36) and in two seasons (2007 and 2008) were compared using the Affymetrix GrapeGen® genome array containing 23096 probesets corresponding to 18726 unique sequences. Over 50% of these probesets were significantly differentially expressed (1.5 fold) between at least two developmental stages. A common set of modulated transcripts corresponding to 5877 unigenes indicates the activation of common pathways between years despite the irregular development of Trincadeira grapes. These unigenes were assigned to the functional categories of "metabolism", "development", "cellular process", "diverse/miscellanenous functions", "regulation overview", "response to stimulus, stress", "signaling", "transport overview", "xenoprotein, transposable element" and "unknown". Quantitative RT-PCR validated microarrays results being carried out for eight selected genes and five developmental stages (EL 32, EL 34, EL 35, EL 36 and EL 38). Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, amino acid and sugar metabolism as well as secondary metabolism. These results were integrated with transcriptional profiling obtained using genome array to provide new information regarding the network of events leading to grape ripening. CONCLUSIONS: Altogether the data obtained provides the most extensive survey obtained so far for gene expression and metabolites accumulated during grape ripening. Moreover, it highlighted information obtained in a poorly known variety exhibiting particular characteristics that may be cultivar specific or dependent upon climatic conditions. Several genes were identified that had not been previously reported in the context of grape ripening namely genes involved in carbohydrate and amino acid metabolisms as well as in growth regulators; metabolism, epigenetic factors and signaling pathways. Some of these genes were annotated as receptors, transcription factors, and kinases and constitute good candidates for functional analysis in order to establish a model for ripening control of a non-climacteric fruit.

Arginine Decarboxylase expression, polyamines biosynthesis and reactive oxygen species during organogenic nodule formation in hop.

Hop (Humulus lupulus L.) is an economically important plant species used in beer production and as a health-promoting medicine. Hop internodes develop upon stress treatments organogenic nodules which can be used for genetic transformation and micropropagation. Polyamines are involved in plant development and stress responses. Arginine decarboxylase (ADC; EC 4·1.1·19) is a key enzyme involved in the biosynthesis of putrescine in plants. Here we show that ADC protein was increasingly expressed at early stages of hop internode culture (12h). Protein continued accumulating until organogenic nodule formation after 28 days, decreasing thereafter. The same profile was observed for ADC transcript suggesting transcriptional regulation of ADC gene expression during morphogenesis. The highest transcript and protein levels observed after 28 days of culture were accompanied by a peak in putrescine levels. Reactive oxygen species accumulate in nodular tissues probably due to stress inherent to in vitro conditions and enhanced polyamine catabolism. Conjugated polyamines increased during plantlet regeneration from nodules suggesting their involvement in plantlet formation and/or in the control of free polyamine levels. Immunogold labeling revealed that ADC is located in plastids, nucleus and cytoplasm of nodular cells. In vacuolated cells, ADC immunolabelling in plastids doubled the signal of proplastids in meristematic cells. Location of ADC in different subcellular compartments may indicate its role in metabolic pathways taking place in these compartments. Altogether these data suggest that polyamines play an important role in organogenic nodule formation and represent a progress towards understanding the role played by these growth regulators in plant morphogenesis.

Organogenic nodule formation in hop: a tool to study morphogenesis in plants with biotechnological and medicinal applications.

The usage of Humulus lupulus for brewing increased the demand for high-quality plant material. Simultaneously, hop has been used in traditional medicine and recently recognized with anticancer and anti-infective properties. Tissue culture techniques have been reported for a wide range of species, and open the prospect for propagation of disease-free, genetically uniform and massive amounts of plants in vitro. Moreover, the development of large-scale culture methods using bioreactors enables the industrial production of secondary metabolites. Reliable and efficient tissue culture protocol for shoot regeneration through organogenic nodule formation was established for hop. The present review describes the histological, and biochemical changes occurring during this morphogenic process, together with an analysis of transcriptional and metabolic profiles. We also discuss the existence of common molecular factors among three different morphogenic processes: organogenic nodules and somatic embryogenesis, which strictly speaking depend exclusively on intrinsic developmental reprogramming, and legume nitrogen-fixing root nodules, which arises in response to symbiosis. The review of the key factors that participate in hop nodule organogenesis and the comparison with other morphogenic processes may have merit as a study presenting recent advances in complex molecular networks occurring during morphogenesis and together, these provide a rich framework for biotechnology applications.

Organogenic nodule development in hop (Humulus lupulus L.): transcript and metabolic responses.

BACKGROUND: Hop (Humulus lupulus L.) is an economically important plant forming organogenic nodules which can be used for genetic transformation and micropropagation. We are interested in the mechanisms underlying reprogramming of cells through stress and hormone treatments. RESULTS: An integrated molecular and metabolomic approach was used to investigate global gene expression and metabolic responses during development of hop's organogenic nodules. Transcript profiling using a 3,324-cDNA clone array revealed differential regulation of 133 unigenes, classified into 11 functional categories. Several pathways seem to be determinant in organogenic nodule formation, namely defense and stress response, sugar and lipid metabolism, synthesis of secondary metabolites and hormone signaling. Metabolic profiling using 1H NMR spectroscopy associated to two-dimensional techniques showed the importance of metabolites related to oxidative stress response, lipid and sugar metabolism and secondary metabolism in organogenic nodule formation. CONCLUSION: The expression profile of genes pivotal for energy metabolism, together with metabolites profile, suggested that these morphogenic structures gain energy through a heterotrophic, transport-dependent and sugar-degrading anaerobic metabolism. Polyamines and auxins are likely to be involved in the regulation of expression of many genes related to organogenic nodule formation. These results represent substantial progress toward a better understanding of this complex developmental program and reveal novel information regarding morphogenesis in plants.

Fungal transcript pattern during the preinfection stage (12 h) of ectomycorrhiza formed between Pisolithus tinctorius and Castanea sativa roots, identified using cDNA microarrays.

Transcriptional changes in Pisolithus tinctorius leading to ectomycorrhizal formation in P. tinctorius- Castanea sativa were investigated using a 12-h fungal interaction in vitro system. Using a 3107-cDNA clone microarray, 34 unique expressed sequence tags (ESTs) were found to be differentially expressed. These ESTs represent 14 known genes, 5 upregulated and 9 downregulated, and 20 orphan sequences. Some transcripts of upregulated genes (with unknown function) were previously identified in other mycorrhizal Pisolithus spp. associations. ESTs for S-adenosyl-L-homocysteine hydrolase and several orphan sequences were identified in our system. The identified transcript of downregulated genes involved hydrophobins, 5S, 18S, and 28S ribosomal RNA genes, large subunits of ribosomal RNA (mitochondrial gene), and two types of heat shock proteins. This study demonstrates the high complexity of molecular events involved in the preinfection steps and suggests the utilization of different fungal gene repertories before ectomycorrhizal formation. These data constitute a first contribution for the molecular understanding of early signaling events between P. tinctorius and C. sativa roots during ectomycorrhizal formation.

Production and characterization of recombinant cyprosin B in Saccharomyces cerevisiae (W303-1A) strain.

The Saccharomyces cerevisiae W303-1A strain transformed with a centromeric plasmid containing CYPRO11, which codifies the aspartic protease cyprosin B, was grown in a 3 l bioreactor under aerobic conditions. Expression of cyprosin B is directly dependent on the concentration of galactose used as the inducer and carbon source in 1% yeast extract, 2% bactopeptone, and 4% galactose in culture medium. For 4% of galactose, 209 mg.l(-1) total protein, and 1036 U.ml(-1) recombinant cyprosin B activity were obtained from 6.1 g dcw.l(-1) biomass. The recombinant cyprosin B, purified by two consecutive anion-exchange chromatographies (diethyl amino-ethyl [DEAE]-Sepharose and Q-Sepharose XK-16 columns), shows a specific activity of 62 x 10(3) U.mg(-1), corresponding to a purification degree of 12.5-fold and a recovery yield of 25.6% relative to that in fermentation broth. The proteolytic activity of recombinant cyprosin B is optimal at 42 degrees C and pH 4.5. The recombinant cyprosin B activity is 95% inhibited by pepstatin A, which confirms its aspartic protease nature. The pure recombinant cyprosin B is composed of two subunits, one with 14 and the other with 32 kDa. It exhibits clotting activity, similar to that of the natural enzyme from Cynara cardunculus flowers. The results reported here show that recombinant cyprosin B, the first clotting protease of plant origin produced in a bioreactor, can now be produced in large scale and may constitute a new and efficient alternative to enzymes of animal or fungal origin that are widely used in cheese making.

The Arabidopsis protein kinase PTI1-2 is activated by convergent phosphatidic acid and oxidative stress signaling pathways downstream of PDK1 and OXI1.

Arabidopsis PDK1 activity is regulated by binding to the lipid phosphatidic acid (PA) resulting in activation of the oxidative stress-response protein kinase OXI1/AGC2-1. Thus there is an inferred link between lipid signaling and oxidative stress signaling modules. Among a panel of hormones and stresses tested, we found that, in addition to PA, the fungal elicitor xylanase activated PDK1, suggesting that PDK1 has a role in plant pathogen defense mechanisms. The downstream OXI1 was activated by additional stress factors, including PA, H(2)O(2), and partially by xylanase. We have isolated an interacting partner of OXI1, a Ser/Thr kinase (PTI1-2), which is downstream of OXI1. Its sequence closely resembles the tomato Pti kinase, which has been implicated in the hypersensitive response, a localized programmed cell death that occurs at the site of pathogen infection. PTI1-2 is activated by the same stresses/elicitors as OXI1 and additionally flagellin. We have used RNA interference to knock out the expression of PDK1 and OXI1 and to study the effects on PTI1-2 activity. We show that specific lipid signaling pathways converge on PTI1-2 via the PDK1-OXI1 axis, whereas H(2)O(2) and flagellin signals to OXI1-PTI1-2 via a PDK1-independent pathway. PTI1-2 represents a new downstream component that integrates diverse lipid and reactive oxygen stress signals and functions closely with OXI1.

Expression of allene oxide cyclase and accumulation of jasmonates during organogenic nodule formation from hop (Humulus lupulus var. Nugget) internodes.

A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its stereoisomeric precursor, cis-(+)-12-oxophytodienoic acid (OPDA), which is catalyzed by allene oxide cyclase (AOC, EC 5.3.99.6). A cDNA of AOC was isolated from Humulus lupulus var. Nugget. The ORF of 765 bp encodes a 255 amino acid protein, which carries a putative chloroplast targeting sequence. The recombinant protein without its putative chloroplast target sequence showed significant AOC activity. Previously we demonstrated that wounding induces organogenic nodule formation in hop. Here we show that the AOC transcript level increases in response to wounding of internodes, peaking between 2 and 4 h after wounding. In addition, Western blot analysis showed elevated levels of AOC peaking 24 h after internode inoculation. The AOC increase was accompanied by increased JA levels 24 h after wounding, whereas OPDA had already reached its highest level after 12 h. AOC is mostly present in the vascular bundles of inoculated internodes. During prenodule and nodule formation, AOC levels were still high. JA and OPDA levels decreased down to 10 and 118 pmol (g FW)(-1), respectively, during nodule formation, but increased during plantlet regeneration. Double immunolocalization analysis of AOC and Rubisco in connection with lugol staining showed that AOC is present in amyloplasts of prenodular cells and in the chloroplasts of vacuolated nodular cells, whereas meristematic cells accumulated little AOC. These data suggest a role of AOC and jasmonates in organogenic nodule formation and plantlet regeneration from these nodules.

Expression of genes encoding cell wall modifying enzymes is induced by cold storage and reflects changes in pear fruit texture.

Preclimacteric 'Rocha' pears stored under chilling conditions, had a larger increase of ACO (1-aminocyclopropane-1-carboxylate oxidase) activity and softened faster than those treated with ethylene. Non-treated fruit did not ripen or soften, acquired a rubbery texture, and showed barely detectable levels of ACO activity. The transcript accumulation of seven genes encoding cell wall modifying enzymes was followed during fruit growth, ripening, and senescence, and in fruit that failed to ripen, by quantitative real-time PCR. Transcripts from 'Rocha' pear polygalacturonase1 and 2 (PcPG1, PcPG2), beta-galactosidase (PcbetaGAL) and beta-xylosidase (PcXYL) genes accumulated up to 1000-fold at the climacteric onset, while low transcript levels were detected in growing fruit. In fruit that did not ripen, this transcript accumulation was lower compared with fruits that ripened normally. Transcripts for expansin1 and 2 (PcEXPA1, PcEXPA2) accumulated in growing fruit, but about 10-fold more in fruit after rewarming. Xyloglucan endotransglucosylase/hydrolase (PcXTH) had the highest basal expression levels in all samples, showing only a small increase during fruit growth and ripening. PcEXPA2 and PcXTH transcripts accumulated in untreated fruit, 21 d after harvest, to levels similar to those of fruit that ripened normally. Since in untreated fruit ACO activity was barely detectable, it is likely that the activation of these genes might occur at very low ethylene levels. Results suggest that PcXTH and PcEXPA2 gene induction might be associated with cell wall maintenance during 'Rocha' pear development and ripening, while PcEXPA1, PcPG1, PcPG2, PcbetaGAL, and PcXYL expression is likely to be related to cell wall disassembly and loosening.

Transcript identification and profiling during salt stress and recovery of Populus euphratica.

Populus euphratica Oli. is a salt-tolerant species that can cope with up to 450 mM NaCl under hydroponic conditions and can tolerate high accumulations of Na+ and Cl- in roots and leaves when grown in 300 mM NaCl. Transcript responses to salt stress and recovery were monitored by microarray hybridization of 315 cDNAs preselected by suppression subtractive hybridization. Transcripts of a heat-shock protein and a hydroxyproline-rich glycoprotein accumulated 1.5 and 3 h, respectively, after adding 300 mM NaCl to the culture medium. Transcripts significantly up-regulated by salt stress included ionic and osmotic homeostasis elements such as magnesium transporter-like protein, syntaxin-like protein, seed imbibition protein and plasma membrane intrinsic protein; metabolism regulators like cytochrome P450, zinc finger protein, cleavage factor and aminotransferase; and the photosynthesis-activating enzyme Rubisco activase and photorespiration-related glycolate oxidase. Several photosynthesis-related transcripts were down-regulated in response to 72 h of salt stress but were up-regulated after long-term recovery (48 h). Sucrose synthase, ABC transporter, calmodulin, Pop3 peptide and aquaporin appeared to be actively involved in the process of plant recovery from salt stress. Several transcripts encoding proteins of unknown function were regulated by salt stress. Selected transcripts exhibiting altered transcript profiles in response to salt stress were also analyzed by real-time quantitative PCR. Transcript analysis during salt stress and recovery of this woody species revealed several genes and corresponding proteins deserving special attention in future studies of salt tolerance in woody species.

Knock-out of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects.

IRT1 and IRT2 are members of the Arabidopsis ZIP metal transporter family that are specifically induced by iron deprivation in roots and act as heterologous suppressors of yeast mutations inhibiting iron and zinc uptake. Although IRT1 and IRT2 are thought to perform redundant functions as root-specific metal transporters, insertional inactivation of the IRT1 gene alone results in typical symptoms of iron deficiency causing severe leaf chlorosis and lethality in soil. The irt1 mutation is characterized by specific developmental defects, including a drastic reduction of chloroplast thylakoid stacking into grana and lack of palisade parenchyma differentiation in leaves, reduced number of vascular bundles in stems, and irregular patterns of enlarged endodermal and cortex cells in roots. Pulse labeling with 59Fe through the root system shows that the irt1 mutation reduces iron accumulation in the shoots. Short-term labeling with 65Zn reveals no alteration in spatial distribution of zinc, but indicates a lower level of zinc accumulation. In comparison to wild-type, the irt1 mutant responds to iron and zinc deprivation by altered expression of certain zinc and iron transporter genes, which results in the activation of ZIP1 in shoots, reduction of ZIP2 transcript levels in roots, and enhanced expression of IRT2 in roots. These data support the conclusion that IRT1 is an essential metal transporter required for proper development and regulation of iron and zinc homeostasis in Arabidopsis.

Studies on callose and cutin during the expression of competence and determination for organogenic nodule formation from internodes of Humulus lupulus var. Nugget.

Callose and cutin deposition were followed by staining with Aniline Blue and Nile Red and by immunolocalization using antibodies raised against callose. Along with morphogenesis induction from internodes of Humulus lupulus var. Nugget, a temporal and spatial differential deposition of callose and cutin was observed. A cutin layer showing bright yellow autofluorescence appears, surrounding cells or groups of cells committed to express morphogenic competence. This cutin layer that evolves to a randomly organized network appeared underneath a callose layer and may create a specific cellular environment with altered permeability and altered receptors providing conditions for entering the cell cycle. The incipient callose accumulation in control explants cultured on basal medium suggests the involvement of callose in the initiation of the morphogenic programme leading to nodule formation. A scanning electron microscopic study during the organogenic process showed that before shoot bud regeneration, the cutin layer increases in thickness and acquires a smooth texture. This cutin layer is specific to nodular organogenic regions and disappeared with plantlet regeneration. This layer may control permeability to water and solute transfer throughout plantlet regeneration.