Identification and characterization of the rhizosphere phosphate-solubilizing bacterium <Emphasis Type="Italic">Pseudomonas frederiksbergensis</Emphasis> JW-SD2 and its plant growth-promoting effects on poplar seedlings

The rhizospheric bacterium JW-SD2 was identified as Pseudomonas frederiksbergensis based on phenotypic features, the Biolog Identification System and 16S rRNA sequence analysis. The phosphate-solubilizing activity, acidification in culture media, growth rate and organic acid secretion of JW-SD2 were investigated during 192 h of cultivation. The phosphate solubilized by JW-SD2 reached 7.75 mM. The decrease of pH and increase of titratable acidity were closely correlated (Pearson’s r?=??0.953 and 0.969, respectively) with the phosphate-solubilizing activity. High concentrations of gluconic, 2-ketogluconic, pyruvic, maleic and malic acids were detected before 96 h of culture, when the strain displayed a high level of phosphate-solubilizing activity, indicating that these organic acids were efficient components in phosphate solubilization. However, acetic acid did not affect phosphate solubilization as shown by a remarkable increase at 144 h of culture when the phosphate-solubilizing activity decreased. The phosphate-solubilizing ability of JW-SD2 was significantly (P?<?0.01) affected by environmental factors. Over a broad ranges of temperature (20?35 °C), pH (4?9), salinity (0?3.0 %), and volume of medium (1/5?3/5 of flask volume), the phosphate solubilized by JW-SD2 remained above 4.00 mM, demonstrating good potential in adapting to a changing environment. The inoculation experiments indicated that JW-SD2 could significantly (P?<?0.05) promote growth of poplar (Populus euramericana cv. NL-895) in both sterilized and non-sterilized soils. The effects of plant growth promotion were greater in non-sterilized than in sterilized soil. During the 150 days of the trial, the effects of plant growth promotion by JW-SD2 first increased then decreased over time, suggesting that, in field applications, the periodic supplementation of the strain into the rhizosphere should be considered.     

Vascular endothelial growth factor receptor-2 expression is down-regulated by 17beta-estradiol in MCF-7 breast cancer cells by estrogen receptor alpha/Sp proteins

17beta-Estradiol (E2) induces and represses gene expression in breast cancer cells; however, the mechanisms of gene repression are not well understood. In this study, we show that E2 decreases vascular endothelial growth factor receptor 2 (VEGFR2) mRNA levels in MCF-7 cells, and this gene was used as a model for investigating pathways associated with E2-dependent gene repression. Deletion analysis of the VEGFR2 promoter indicates that the proximal GC-rich motifs at -58 and -44 are critical for the E2-dependent decreased response in MCF-7 cells. Mutation or deletion of these GC-rich elements results in loss of hormone responsiveness and shows that the -60 to -37 region of the VEGFR2 promoter is critical for both basal and hormone-dependent decreased VEGFR2 expression in MCF-7 cells. Western blot, immunofluorescent staining, RNA interference, and EMSAs support a role for Sp proteins in hormone-dependent down-regulation of VEGFR2 in MCF-7 cells, primarily through estrogen receptor (ER)alpha/Sp1 and ERalpha/Sp3 interactions with the VEGFR2 promoter. Using chromatin immuno-precipitation and transient transfection/RNA interference assays we show that the ERalpha/Sp protein-promoter interactions are accompanied by recruitment of the co-repressors SMRT (silencing mediator of retinoid and thyroid hormone receptor) and NCoR (nuclear receptor corepressor) to the promoter and that SMRT and NCoR knockdown reverse E2-mediated down-regulation of VEGFR2 expression in MCF-7 cells. This study illustrates that both SMRT and NCoR are involved in E2-dependent repression of VEGFR2 in MCF-7 cells.     

Peptide inhibitors of dengue virus NS3 protease. Part 2: SAR study of tetrapeptide aldehyde inhibitors

With the aim of discovering potent and selective dengue NS3 protease inhibitors, we systematically synthesized and evaluated a series of tetrapeptide aldehydes based on lead aldehyde 1 (Bz-Nle-Lys-Arg-Arg-H, K(i)=5.8 microM). In general, we observe that interactions of P(2) side chain are more important than P(1) followed by P(3) and P(4). Tripeptide and dipeptide aldehyde inhibitors also show low micromolar activity. Additionally, an effective non-basic, uncharged replacement of P(1) Arg is identified.     

Progesterone Receptor Membrane Component 1 Is a Functional Part of the Glucagon-like Peptide-1 (GLP-1) Receptor Complex in Pancreatic β Cells

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Because of their direct stimulation of insulin secretion from pancreatic β cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells or MIN6 β cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with the GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, and Rab5c) were further validated through co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer, and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1-induced insulin secretion (GIIS) with the most robust effect. Knockdown of PGRMC1 in β cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS, whereas its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibition of adenylyl cyclase/EPAC signaling or the EGF receptor–PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGF receptor signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of β cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1.Glucagon-like peptide-1 (GLP-1)¹ is a gastrointestinal hormone secreted by intestinal L cells upon food intake that is best known for its role in controlling glucose homeostasis. Acting through its cognate glucagon-like peptide-1 receptor (GLP-1R), GLP-1 has several important physiological and pharmacological functions. GLP-1 is best known for enhancing glucose-stimulated insulin secretion (GSIS) from the pancreatic β cells. Importantly, the insulinotropic properties of GLP-1 are maintained in patients with type 2 diabetes (1), which is characterized by insufficient insulin secretion from pancreatic β cells and an inability to maintain glucose homeostasis. Therefore, therapeutic strategies targeting GLP-1R have been developed to treat type 2 diabetes (2, 3). In addition to augmenting insulin secretion, GLP-1 has been known to improve glucose sensing, proinsulin biosynthesis, survival, and proliferation of β cells (3, 4) in a variety of experimental models. GLP-1 also has several extrapancreatic effects, including actions on the central nervous system to inhibit food intake (5), the stomach to decrease gastric emptying and gastric acid secretion (6), and the lungs to stimulate secretion of macromolecules from airways (7). Additionally, GLP-1 has an effect on the heart and possibly the kidney to modulate blood pressure and heart rate (8, 9).The GLP-1R is a member of the B1 family of G protein–coupled receptors (secretin receptor family). In mammals, GLP-1R is expressed in multiple tissues, including pancreatic β cells and δ cells (10), hypothalamus, lung, stomach, heart, kidney (11), and thyroid (12), which in part explains its diverse actions. Upon ligand binding, the GLP-1R is capable of coupling to diverse cell signal transduction pathways, but it is best known for its actions on G protein Gs α and adenylate cyclase activity to increase intracellular cAMP. It is known that other proteins can affect GLP-1R activity in addition to G proteins, including β-arrestin and caveolin, which affect receptor internalization and trafficking. β-Arrestin 1 is also required for proper GLP-1-stimulated cAMP production (13–15). More recently, it was shown that another B1 family member, gastric inhibitory polypeptide receptor heterodimerizes with GLP-1R, decreasing GLP-1-induced β-arrestin recruitment and mobilization (16). Very recently, our group identified several novel potential GLP-1R interactors using a membrane-based split-ubiquitin yeast two-hybrid (MYTH) assay (17). Three β cell–expressing membrane-bound interactors, solute carrier family 15 member 4 (SLC15A4), amyloid β A4 precursor-like protein 1 (APLP1), and adaptor-related protein complex 2 subunit mu (AP2M1), were further selected for individual knockdown in mouse insulinoma (MIN6) β cells using small interfering RNAs (siRNAs). GLP-1-induced insulin secretion was significantly enhanced when these genes were silenced, suggesting that these interactor proteins attenuate GLP-1R activity. These findings demonstrated that GLP-1R protein interactions are complex and the interactors can have measurable effects on receptor trafficking and downstream signaling. Such interactions may in part explain the diverse tissue-specific effects of GLP-1 and offer avenues for controlling GLP-1 actions in a tissue-selective manner.Although the MYTH system is well established (18) and has been applied to study G protein–coupled receptor interactomes (17), it is limited on two fronts. Firstly, it must be performed in yeast which is not an ideal representation of the mammalian system. Secondly, it is technically difficult to activate the receptor in MYTH, thus, effects of ligand stimulation on the receptor interactome cannot be assessed. Recently, affinity purification–mass spectrometry (AP-MS) has become a powerful tool for discovering and examining novel protein–protein interactions, including those between membrane-bound proteins in mammalian cells (19–21). In the current study, we applied AP-MS to discover novel GLP-1R interactors and employed a human GLP-1R harboring a FLAG^® epitope. GLP-1R-Flag was expressed in either Chinese hamster ovary (CHO) cells or MIN6 β cells, and interactors were studied in the presence or absence of GLP-1.     

Validation of a Consensus Method for Identifying Delirium from Hospital Records

Background

Delirium is increasingly considered to be an important determinant of trajectories of cognitive decline. Therefore, analyses of existing cohort studies measuring cognitive outcomes could benefit from methods to ascertain a retrospective delirium diagnosis. This study aimed to develop and validate such a method for delirium detection using routine medical records in UK and Ireland.

Methods

A point prevalence study of delirium provided the reference-standard ratings for delirium diagnosis. Blinded to study results, clinical vignettes were compiled from participants'' medical records in a standardised manner, describing any relevant delirium symptoms recorded in the whole case record for the period leading up to case-ascertainment. An expert panel rated each vignette as unlikely, possible, or probable delirium and disagreements were resolved by consensus.

Results

From 95 case records, 424 vignettes were abstracted by 5 trained clinicians. There were 29 delirium cases according to the reference standard. Median age of subjects was 76.6 years (interquartile range 54.6 to 82.5). Against the original study DSM-IV diagnosis, the chart abstraction method gave a positive likelihood ratio (LR) of 7.8 (95% CI 5.7–12.0) and the negative LR of 0.45 (95% CI 0.40–0.47) for probable delirium (sensitivity 0.58 (95% CI 0.53–0.62); specificity 0.93 (95% CI 0.90–0.95); AUC 0.86 (95% CI 0.82–0.89)). The method diagnosed possible delirium with positive LR 3.5 (95% CI 2.9–4.3) and negative LR 0.15 (95% CI 0.11–0.21) (sensitivity 0.89 (95% CI 0.85–0.91); specificity 0.75 (95% CI 0.71–0.79); AUC 0.86 (95% CI 0.80–0.89)).

Conclusions

This chart abstraction method can retrospectively diagnose delirium in hospitalised patients with good accuracy. This has potential for retrospectively identifying delirium in cohort studies where routine medical records are available. This example of record linkage between hospitalisations and epidemiological data may lead to further insights into the inter-relationship between acute illness, as an exposure, for a range of chronic health outcomes.