Effect of long-term organic and mineral fertilization strategies on rhizosphere microbiota assemblage and performance of lettuce

Long-term agricultural fertilization strategies gradually change soil properties including the associated microbial communities. Cultivated crops recruit beneficial microbes from the surrounding soil environment via root exudates. In this study, we aimed to investigate the effects of long-term fertilization strategies across field sites on the rhizosphere prokaryotic (Bacteria and Archaea) community composition and plant performance. We conducted growth chamber experiments with lettuce (Lactuca sativa L.) cultivated in soils from two long-term field experiments, each of which compared organic versus mineral fertilization strategies. 16S rRNA gene amplicon sequencing revealed the assemblage of a rhizosphere core microbiota shared in all lettuce plants across soils, going beyond differences in community composition depending on field site and fertilization strategies. The enhanced expression of several plant genes with roles in oxidative and biotic stress signalling pathways in lettuce grown in soils with organic indicates an induced physiological status in plants. Lettuce plants grown in soils with different fertilization histories were visibly free of stress symptoms and achieved comparable biomass. This suggests a positive aboveground plant response to belowground plant–microbe interactions in the rhizosphere. Besides effects of fertilization strategy and field site, our results demonstrate the crucial role of the plant in driving rhizosphere microbiota assemblage.     

Lysosomal Signaling Licenses Embryonic Stem Cell Differentiation via Inactivation of Tfe3

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Successful bone marrow transplantation in a patient with DNA ligase IV deficiency and bone marrow failure

Cirrhotic cardiomyopathy is the term used to describe a constellation of features indicative of abnormal heart structure and function in patients with cirrhosis. These include systolic and diastolic dysfunction, electrophysiological changes, and macroscopic and microscopic structural changes. The prevalence of cirrhotic cardiomyopathy remains unknown at present, mostly because the disease is generally latent and shows itself when the patient is subjected to stress such as exercise, drugs, hemorrhage and surgery. The main clinical features of cirrhotic cardiomyopathy include baseline increased cardiac output, attenuated systolic contraction or diastolic relaxation in response to physiologic, pharmacologic and surgical stress, and electrical conductance abnormalities (prolonged QT interval). In the majority of cases, diastolic dysfunction precedes systolic dysfunction, which tends to manifest only under conditions of stress. Generally, cirrhotic cardiomyopathy with overt severe heart failure is rare. Major stresses on the cardiovascular system such as liver transplantation, infections and insertion of transjugular intrahepatic portosystemic stent-shunts (TIPS) can unmask the presence of cirrhotic cardiomyopathy and thereby convert latent to overt heart failure. Cirrhotic cardiomyopathy may also contribute to the pathogenesis of hepatorenal syndrome. Pathogenic mechanisms of cirrhotic cardiomyopathy are multiple and include abnormal membrane biophysical characteristics, impaired β-adrenergic receptor signal transduction and increased activity of negative-inotropic pathways mediated by cGMP. Diagnosis and differential diagnosis require a careful assessment of patient history probing for excessive alcohol, physical examination for signs of hypertension such as retinal vascular changes, and appropriate diagnostic tests such as exercise stress electrocardiography, nuclear heart scans and coronary angiography. Current management recommendations include empirical, nonspecific and mainly supportive measures. The exact prognosis remains unclear. The extent of cirrhotic cardiomyopathy generally correlates to the degree of liver insufficiency. Reversibility is possible (either pharmacological or after liver transplantation), but further studies are needed.     

Identification and validation of colorectal neoplasia-specific methylation markers for accurate classification of disease

Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis.     

Solution Structure of C. elegans UNC-6: A Nematode Paralogue of the Axon Guidance Protein Netrin-1

UNCoordinated-6 (UNC-6) was the first member of the netrin family to be discovered in Caenorhabditis elegans. With homology to human netrin-1, it is a key signaling molecule involved in directing axon migration in nematodes. Similar to netrin-1, UNC-6 interacts with multiple receptors (UNC-5 and UNC-40, specifically) to guide axon migration in development. As a result of the distinct evolutionary path of UNC-6 compared to vertebrate netrins, we decided to employ an integrated approach to study its solution behavior and compare it to the high-resolution structure we previously published on vertebrate netrins. Dynamic light scattering and analytical ultracentrifugation on UNC-6 (with and without its C-domain) solubilized in a low-ionic strength buffer suggested that UNC-6 forms high-order oligomers. An increase in the buffer ionic strength resulted in a more homogeneous preparation of UNC-6, that was used for subsequent solution x-ray scattering experiments. Our biophysical analysis of UNC-6 ΔC solubilized in a high-ionic strength buffer suggested that it maintains a similar head-to-stalk arrangement as netrins ?1 and ?4. This phenomenon is thought to play a role in the signaling behavior of UNC-6 and its ability to move throughout the extracellular matrix.     

Development of definitive endoderm from embryonic stem cells in culture

The cellular and molecular events regulating the induction and tissue-specific differentiation of endoderm are central to our understanding of the development and function of many organ systems. To define and characterize key components in this process, we have investigated the potential of embryonic stem (ES) cells to generate endoderm following their differentiation to embryoid bodies (EBs) in culture. We found that endoderm can be induced in EBs, either by limited exposure to serum or by culturing in the presence of activin A (activin) under serum-free conditions. By using an ES cell line with the green fluorescent protein (GFP) cDNA targeted to the brachyury locus, we demonstrate that endoderm develops from a brachyury(+) population that also displays mesoderm potential. Transplantation of cells generated from activin-induced brachyury(+) cells to the kidney capsule of recipient mice resulted in the development of endoderm-derived structures. These findings demonstrate that ES cells can generate endoderm in culture and, as such, establish this differentiation system as a unique murine model for studying the development and specification of this germ layer.     

5-Hydroxyindoleacetic acid and homovanillic acid concentrations in cerebrospinal fluid in patients with Alzheimer's disease, depression and mild cognitive impairment

In this study we investigated the cerebrospinal fluid (CSF) concentrations of 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) in Alzheimer (AD) patients (n=75), patients with mild cognitive impairment (MCI, n=9) and patients with depression (n=7). CSF HVA was significantly elevated in AD with depression (Geriatric Depression Scale, 15 point version GDS>5) in comparison to AD without depression (p<0.05, ANOVA) and CSF HVA showed a significant positive correlation with the GDS score of AD-patients (p=0.03, Spearman Rho: 0.38, Spearman Rank Correlation). In the group of AD patients CSF 5-HIAA was positively correlated with cerebrospinal fluid beta-amyloid 1-42 (Abeta42), p<0.05, Spearman Rho: 0.3, Spearman Rank Correlation, but not with CSF tau. Additionally, there was a significant positive correlation between cerebrospinal fluid 5-HIAA and HVA in the group of AD patients (p<0.0001, Rho: 0.47, Spearman Rank correlation). Neither 5-HIAA nor HVA in CSF could differentiate between mild cognitive impairment, depression and AD. The results of this study support the hypothesis that the serotonergic system plays a role in the course of AD. They further suggest an important role of dopamine metabolism in depression within AD patients.     

Differential RNA interference: replacement of endogenous with recombinant low density lipoprotein receptor-related protein (LRP)

The interpretation of experiments involving the overexpression of a recombinant cDNA is often hampered by the interference of mRNA expression from the endogenous gene locus. Unless cell lines from naturally occurring mutations or knockout mice are available, difficult and time-consuming gene targeting techniques are required to inhibit endogenous gene expression. Using a method we refer to as "differential RNA interference" we demonstrate that RNA interference can be used to selectively suppress endogenous gene expression without affecting the expression of a co-transfected recombinant version of the same protein. Functional analyses of recombinant low density lipoprotein receptor-related protein (LRP) to study its involvement in lipid metabolism have been shown to be extremely difficult due to its large cDNA and the unavailability of suitable LRP-deficient cell lines. We constructed an expression vector containing the full-length coding sequence of human LRP fused to EGFP and a vector expressing small hairpin RNA directed against the 3'-untranslated region of the wild-type human LRP mRNA (LRP-shRNA). When overexpressed, EGFP-tagged LRP colocalizes with endogenous LRP and stimulates the uptake of LRP ligands. Overexpression of LRP-shRNA vectors significantly inhibits LRP expression, as judged by quantitative RT-PCR, Western blot and immunofluorescence analysis, and it dramatically decreases receptor-associated protein (RAP) uptake. Finally, co-transfection of EGFP-LRP and LRP-shRNA vectors demonstrates selective inhibition of endogenous LRP expression without affecting simultaneous expression of recombinant LRP protein. Thus, utilization of "differential RNA interference" provides a new experimental approach to selectively study the function of any recombinant protein in any given cell line without interference of endogenous protein expression.     

Sequential roles of Cdc42, Par-6, aPKC, and Lgl in the establishment of epithelial polarity during Drosophila embryogenesis

How epithelial cells subdivide their plasma membrane into an apical and a basolateral domain is largely unclear. In Drosophila embryos, epithelial cells are generated from a syncytium during cellularization. We show here that polarity is established shortly after cellularization when Par-6 and the atypical protein kinase C concentrate on the apical side of the newly formed cells. Apical localization of Par-6 requires its interaction with activated Cdc42 and dominant-active or dominant-negative Cdc42 disrupt epithelial polarity, suggesting that activation of this GTPase is crucial for the establishment of epithelial polarity. Maintenance of Par-6 localization requires the cytoskeletal protein Lgl. Genetic and biochemical experiments suggest that phosphorylation by aPKC inactivates Lgl on the apical side. On the basolateral side, Lgl is active and excludes Par-6 from the cell cortex, suggesting that complementary cortical domains are maintained by mutual inhibition of aPKC and Lgl on opposite sides of an epithelial cell.