Monitoring modulators of platelet aggregation in a microtiter plate assay

Platelets play a central role in maintaining biological hemostasis. Inappropriate platelet activation is responsible for thrombotic diseases such as myocardial infarction and stroke. Therefore, novel agents that can inhibit platelet activation are necessary. However, assays that monitor platelet aggregation are generally time-consuming and require high volumes of blood and specialized equipment. Therefore, a medium- to high-throughput assay that can monitor platelet aggregation would be considered useful. Such an assay should be sensitive, comparable to the "gold standard" assay of platelet aggregometry, and able to monitor multiple samples simultaneously but with low assay volumes. We have developed such a microtiter assay. It can assay an average of 60 independent treatments per 60 ml blood donation and demonstrates greater sensitivity than the current gold standard assay, namely platelet aggregation in stirring conditions in a platelet aggregometer. The microtiter plate (MTP) assay can detect known inhibitors of platelet function such as indomethacin, aspirin, and ReoPro. It is highly reproducible when using standard doses of agonists such as thrombin receptor-activating peptide (20 microM) and collagen (0.19 mg/ml). Finally, the MTP assay is rapid and sensitive and can detect unknown platelet-modulating agents from a library of compounds.     

The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear

In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27^kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27^kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27^kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear.     

Sox2 Cooperates with Inflammation-Mediated Stat3 Activation in the Malignant Transformation of Foregut Basal Progenitor Cells

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Highlights? Sox2+ve basal stem/progenitor cells self-renew and differentiate in vitro and in vivo ? Conditional Sox2 overexpression promotes the expansion of basal progenitor cells ? Sox2 cooperates with an inflammatory microenvironment to induce squamous cancer ? Sox2 and Stat3 together transform mouse and human esophageal progenitor cells     

Serum netrin and VCAM-1 as biomarker for Egyptian patients with type IΙ diabetes mellitus

ObjectiveThis study aimed to evaluate the serum level of netrin and soluble vascular cell adhesion molecule 1 (VCAM-I) in patients with type IΙ diabetes mellitus (T2DM) and evaluate the association of their levels with the development of a diabetic complication.Patients and methodsThis study was carried out on type II diabetic patients with and without complications and healthy individuals served as controls. All subjects were submitted to the estimation of serum lipid profile, serum creatinine, urinary albumin/creatinine ratio (ACR), fasting blood glucose (FBG), glycated hemoglobin (HbA1c), visceral adiposity index (VAI), atherogenic index of plasma (AIP), lipid accumulation product (LAP) and detection of serum level of netrin1 and VCAM1.ResultsDiabetic patients with complications had significantly higher serum levels of creatinine, ACR, cholesterol, Triglyceride, low-density lipoprotein, netrin1, and VCAM1 than diabetic patients without complications. Likewise, the level of VAI and LAP as markers of excessive body fat were significantly higher in diabetic patients with complications than diabetic patients without complications. The netrin1 and VCAM1 were a significant discriminator of T2DM renal complications with a sensitivity of 96%, 90%, and specificity of 82.7%, 91.3% respectively.ConclusionIt can be concluded that serum netrin1 and VCAM1 correlated significantly with markers of excessive body fat, a renal complication in the patient with type 2 diabetes mellitus.     

Bioinformatic discovery of novel bioactive peptides

Short synthetic oligopeptides based on regions of human proteins that encompass functional motifs are versatile reagents for understanding protein signaling and interactions. They can either mimic or inhibit the parent protein's activity and have been used in drug development. Peptide studies typically either derive peptides from a single identified protein or (at the other extreme) screen random combinatorial peptides, often without knowledge of the signaling pathways targeted. Our objective was to determine whether rational bioinformatic design of oligopeptides specifically targeted to potentially signaling-rich juxtamembrane regions could identify modulators of human platelet function. High-throughput in vitro platelet function assays of palmitylated cell-permeable oligopeptides corresponding to these regions identified many agonists and antagonists of platelet function. Many bioactive peptides were from adhesion molecules, including a specific CD226-derived inhibitor of inside-out platelet signaling. Systematic screens of this nature are highly efficient tools for discovering short signaling motifs in molecular signaling pathways.     

An integrative study of a meromictic lake ecosystem in Antarctica

In nature, the complexity and structure of microbial communities varies widely, ranging from a few species to thousands of species, and from highly structured to highly unstructured communities. Here, we describe the identity and functional capacity of microbial populations within distinct layers of a pristine, marine-derived, meromictic (stratified) lake (Ace Lake) in Antarctica. Nine million open reading frames were analyzed, representing microbial samples taken from six depths of the lake size fractionated on sequential 3.0, 0.8 and 0.1 μm filters, and including metaproteome data from matching 0.1 μm filters. We determine how the interactions of members of this highly structured and moderately complex community define the biogeochemical fluxes throughout the entire lake. Our view is that the health of this delicate ecosystem is dictated by the effects of the polar light cycle on the dominant role of green sulfur bacteria in primary production and nutrient cycling, and the influence of viruses/phage and phage resistance on the cooperation between members of the microbial community right throughout the lake. To test our assertions, and develop a framework applicable to other microbially driven ecosystems, we developed a mathematical model that describes how cooperation within a microbial system is impacted by periodic fluctuations in environmental parameters on key populations of microorganisms. Our study reveals a mutualistic structure within the microbial community throughout the lake that has arisen as the result of mechanistic interactions between the physico-chemical parameters and the selection of individual members of the community. By exhaustively describing and modelling interactions in Ace Lake, we have developed an approach that may be applicable to learning how environmental perturbations affect the microbial dynamics in more complex aquatic systems.     

Social support for healthy behaviors: scale psychometrics and prediction of weight loss among women in a behavioral program

Social support could be a powerful weight-loss treatment moderator or mediator but is rarely assessed. We assessed the psychometric properties, initial levels, and predictive validity of a measure of perceived social support and sabotage from friends and family for healthy eating and physical activity (eight subscales). Overweight/obese women randomized to one of two 6-month, group-based behavioral weight-loss programs (N = 267; mean BMI 32.1 ± 3.5; 66.3% White) completed subscales at baseline, and weight loss was assessed at 6 months. Internal consistency, discriminant validity, and content validity were excellent for support subscales and adequate for sabotage subscales; qualitative responses revealed novel deliberate instances not reflected in current sabotage items. Most women (>75%) "never" or "rarely" experienced support from friends or family. Using nonparametric classification methods, we identified two subscales-support from friends for healthy eating and support from family for physical activity-that predicted three clinically meaningful subgroups who ranged in likelihood of losing ≥5% of initial weight at 6 months. Women who "never" experienced family support were least likely to lose weight (45.7% lost weight) whereas women who experienced both frequent friend and family support were more likely to lose weight (71.6% lost weight). Paradoxically, women who "never" experienced friend support were most likely to lose weight (80.0% lost weight), perhaps because the group-based programs provided support lacking from friendships. Psychometrics for support subscales were excellent; initial support was rare; and the differential roles of friend vs. family support could inform future targeted weight-loss interventions to subgroups at risk.