Turn-on, photostable, nontoxic and specific, iron(II) sensor.

The pressing need to develop a specific analytical sensor that can identify and quantify Fe(II) without a cytotoxic response was the major motivation drive in this work. The turn-on fluorescent sensor here described can successfully detect Fe(II) and discriminate this ion from other analytes that commonly act as interferents in biological media. Moreover, this reduced fluoresceinamine-based sensor has a high photostability and high dissociation constant, which is an indication that the complex obtained between reduced fluoresceinamine (RFL) and Fe(II) is highly stable. This fluorescence-based sensor has a binding mechanism of 1:1 and a positive cooperativity was found between analyte and sensor. The detection, quantification and sensitivity parameters of the sensor were determined: 21.6 ± 0.1 µM; 65.6 ± 0.1 µM and 48 ± 3 (×107) µM, respectively. To evaluate a possible cytotoxicity effect an erythrocyte assay was performed and the obtained data were evaluated considering CdTe Quantum Dots (QDs) passivated with mercaptoacetic acid has experimental control. According to the resulting data RFL is not cytotoxic even when used in high concentrations, 660 mM. On the other hand QDs are quite different. Indeed it was proven that these heavy metal-based nanoparticles are responsible for 40% erytrocytes hemolysis in concentrations of 600 mM.

Cytocompatible and Antibacterial Properties of Chitosan-Siloxane Hybrid Spheres.

Microporous spheres in a hybrid system consisting of chitosan and γ-glycidoxypropyltrimethoxysilane (GPTMS) have advantages in a range of applications, e.g., as vehicles for cell transplantation and soft tissue defect filling materials, because of their excellent cytocompatibility with various cells. In this study, microporous chitosan-GPTMS spheres were prepared by dropping chitosan-GPTMS precursor sols, with or without a cerium chloride, into liquid nitrogen using a syringe pump. The droplets were then freeze dried to give the pores of size 10 to 50 µm. The cell culture tests showed that L929 fibroblast-like cells migrated into the micropores larger than 50 µm in diameter, whereas MG63 osteoblast-like cells proliferated well and covered the granule surfaces. The spheres with cerium chloride showed antibacterial properties against both gram-negative and gram-positive bacteria.

Digestibility and nutritional value of fresh and stored pollen for honey bees (Apis mellifera scutellata).

Pollen, the main protein source for honey bees, is mixed with regurgitated nectar or honey during collection and then stored as 'bee bread' before its consumption, mainly by young nurse workers. It has been suggested that storage of pollen improves its nutritional value and digestibility, but there is little evidence for such changes. We fed two fresh pollen types of different protein content (aloe and sunflower), and two stored pollen types (sunflower and a mixed pollen), to young caged worker bees. We measured daily consumption of pollen and sucrose solution, and survival after 14 days. At day 14 we recorded ovarian activation and extraction efficiency, by counting empty pollen grains in the rectal contents. Extraction efficiency is a measure of pollen digestibility. Contrary to our predictions, bees did not consume more fresh sunflower pollen than fresh aloe pollen to compensate for the lower protein content of sunflower pollen. In addition, they did not consume less sucrose solution when fed stored pollen diets that are already enriched in sugar. Consumption of stored sunflower pollen resulted in a low protein to carbohydrate (P:C) intake. Survival and ovarian activation were higher on diets giving higher P:C intakes. Extraction efficiency was high (up to 99%) for all pollen diets, and comparison of fresh and stored sunflower pollen showed that storage did not make it easier to digest. Changes to pollen during storage do not confer obvious benefits to honey bees.

Characterization and degradation study of chitosan-siloxane hybrid microspheres synthesized using a microfluidic approach.

Chitosan microspheres can address challenges associated with poor bioavailability or unsustained drug release when used as drug delivery systems thanks to their mucoadhesiveness, which allows the drug dosage to be retained in the gastrointestinal track for extended periods. Chitosan-3-glycidoxypropyltrimethoxysilane-ß-glycerophosphate (chitosan-GPTMS-ß-GP) hybrid microspheres were synthetized through sol-gel processing using a microfluidic approach. Microspheres with uniform spherical shapes and sizes of approximately 650µm were obtained. The microstructures of the microspheres consisted of four different siloxane structures. The degradation behaviors of the hybrid microspheres were examined under acidic pH conditions mimicking those found in the gastrointestinal track. Microspheres with different GPTMS molar ratios were incubated under several pH conditions for 2weeks. The microspheres incubated at pH7.4 extended the lowest weight loss (27%-32%), whereas those incubated at pH1.7 and pH5.4 showed greater weight losses of 43-59% and 69-77%, respectively. The inhibition of the degradation at low pH was dependent on the siloxane network in the chitosan matrix. Phosphate was mostly released in early stages, and the released amount of silicon was dependent on the composition. GPTMS was released with a chitosan chain via the hydrolysis of a chitosan molecule. The pelargonidin was incorporated in the microspheres and the slow releasing was observed at acidic condition. The resistance of these hybrid microspheres to low-pH conditions for longer than a full digestion cycle is promising for gastrointestinal drug delivery applications.

Behavior of prostate cancer cells in a nanohydroxyapatite/collagen bone scaffold.

Prostate cancer (PCa) is the second leading cause of death among men in Europe and U.S. The metastatic dissemination pattern of PCa is unique, developing bone metastasis as the only site of progression, consequently with a prognosis very poor. The cancer cells interactions within the surrounding bone environment are critical for tumor growth and progression. Secreted protein, acidic and rich in cysteine (SPARC) is described to be involved in PCa cells migration and invasion into bone. Three-dimensional (3D) in vitro systems that are able to closely resemble the in vivo microenvironment are recently taking importance in cancer research. Original nanohydroxyapatite/collagen scaffolds were designed to resemble bone microenvironment in order to be applied as substitutes in bone defects and as potential biomaterials to mimic skeletal tumors. In fact, these 3D structures were cytocompatible and able to support osteoblast (MC3T3-E1) colonization and to promote bone ingrowth. Additionally, SPARC adsorption onto the scaffolds affected PC3 and LNCaP PCa cell lines behavior. PC3 cells were found to adapt and colonize the scaffolds, differing from LNCaP where cells underwent morphogenic changes and grew as clusters. Furthermore, for the tested SPARC concentration, SPARC plays a role in retaining LNCaP cells at the latter time points while with PC3 cells no significant differences were observed. This characterization study is required to establish a bone model to provide new insights into the poorly understood PCa mechanisms of metastasis to bone and the generation of improved therapies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2035-2046, 2017.

An HcpR paralog of Desulfovibrio gigas provides protection against nitrosative stress.

Desulfovibrio gigas belongs to the group of sulfate reducing bacteria (SRB). These ubiquitous and metabolically versatile microorganisms are often exposed to reactive nitrogen species (RNS). Nonetheless, the mechanisms and regulatory elements involved in nitrosative stress protection are still poorly understood. The transcription factor HcpR has emerged as a putative regulator of nitrosative stress response among anaerobic bacteria. HcpR is known to orchestrate the expression of the hybrid cluster protein gene, hcp, proposed to be involved in cellular defense against RNS. According to phylogenetic analyses, the occurrence of hcpR paralog genes is a common feature among several Desulfovibrio species. Within the D. gigas genome we have identified two HcpR-related sequences. One of these sequences, hcpR1, was found in the close vicinity of the hcp gene and this finding prompted us to proceed with its functional characterization. We observed that the growth of a D. gigas strain lacking hcpR1 is severely impaired under nitrosative stress. An in silico search revealed several putative targets of HcpR1 that were experimentally validated. The fact that HcpR1 regulates several genes encoding proteins involved in nitrite and nitrate metabolism, together with the sensitive growth phenotype to NO displayed by an hcpR1 mutant strain, strongly supports a relevant role of this factor under nitrosative stress. Moreover, the finding that several Desulfovibrio species possess HcpR paralogs, which have been transmitted vertically in the evolution and diversification of the genus, suggests that these sequences may confer adaptive or survival advantage to these organisms, possibly by increasing their tolerance to nitrosative stress.

Interconnected network motifs control podocyte morphology and kidney function.

Podocytes are kidney cells with specialized morphology that is required for glomerular filtration. Diseases, such as diabetes, or drug exposure that causes disruption of the podocyte foot process morphology results in kidney pathophysiology. Proteomic analysis of glomeruli isolated from rats with puromycin-induced kidney disease and control rats indicated that protein kinase A (PKA), which is activated by adenosine 3',5'-monophosphate (cAMP), is a key regulator of podocyte morphology and function. In podocytes, cAMP signaling activates cAMP response element-binding protein (CREB) to enhance expression of the gene encoding a differentiation marker, synaptopodin, a protein that associates with actin and promotes its bundling. We constructed and experimentally verified a ß-adrenergic receptor-driven network with multiple feedback and feedforward motifs that controls CREB activity. To determine how the motifs interacted to regulate gene expression, we mapped multicompartment dynamical models, including information about protein subcellular localization, onto the network topology using Petri net formalisms. These computational analyses indicated that the juxtaposition of multiple feedback and feedforward motifs enabled the prolonged CREB activation necessary for synaptopodin expression and actin bundling. Drug-induced modulation of these motifs in diseased rats led to recovery of normal morphology and physiological function in vivo. Thus, analysis of regulatory motifs using network dynamics can provide insights into pathophysiology that enable predictions for drug intervention strategies to treat kidney disease.

Decoding information in cell shape.

Shape is an indicator of cell health. But how is the information in shape decoded? We hypothesize that decoding occurs by modulation of signaling through changes in plasma membrane curvature. Using analytical approaches and numerical simulations, we studied how elongation of cell shape affects plasma membrane signaling. Mathematical analyses reveal transient accumulation of activated receptors at regions of higher curvature with increasing cell eccentricity. This distribution of activated receptors is periodic, following the Mathieu function, and it arises from local imbalance between reaction and diffusion of soluble ligands and receptors in the plane of the membrane. Numerical simulations show that transient microdomains of activated receptors amplify signals to downstream protein kinases. For growth factor receptor pathways, increasing cell eccentricity elevates the levels of activated cytoplasmic Src and nuclear MAPK1,2. These predictions were experimentally validated by changing cellular eccentricity, showing that shape is a locus of retrievable information storage in cells.

ERK regulation of phosphodiesterase 4 enhances dopamine-stimulated AMPA receptor membrane insertion.

AMPA-type glutamate receptor (AMPAR) trafficking is essential for modulating synaptic transmission strength. Prior studies that have characterized signaling pathways underlying AMPAR trafficking have identified the cAMP/PKA-mediated phosphorylation of GluA1, an AMPAR subunit, as a key step in the membrane insertion of AMPAR. Inhibition of ERK impairs AMPAR membrane insertion, but the mechanism by which ERK exerts its effect is unknown. Dopamine, an activator of both PKA and ERK, induces AMPAR insertion, but the relationship between the two protein kinases in the process is not understood. We used a combination of computational modeling and live cell imaging to determine the relationship between ERK and PKA in AMPAR insertion. We developed a dynamical model to study the effects of phosphodiesterase 4 (PDE4), a cAMP phosphodiesterase that is phosphorylated and inhibited by ERK, on the membrane insertion of AMPAR. The model predicted that PKA could be a downstream effector of ERK in regulating AMPAR insertion. We experimentally tested the model predictions and found that dopamine-induced ERK phosphorylates and inhibits PDE4. This regulation results in increased cAMP levels and PKA-mediated phosphorylation of DARPP-32 and GluA1, leading to increased GluA1 trafficking to the membrane. These findings provide unique insight into an unanticipated network topology in which ERK uses PDE4 to regulate PKA output during dopamine signaling. The combination of dynamical models and experiments has helped us unravel the complex interactions between two protein kinase pathways in regulating a fundamental molecular process underlying synaptic plasticity.

Phylogeny and fruit evolution in Menispermaceae.

PREMISE OF THE STUDY: This work surveys endocarp morphology of Menispermaceae in the context of a well-supported molecular phylogeny. The study is important since menispermaceous endocarps appear often in the fossil record and indicate the presence of a wet forest ecosystem. • METHODS: Three chloroplast regions were used to derive phylogenies for 53 genera and 60 species. Endocarps of 47 genera and 92 species were dissected and morphological characters scored. Photographs of key features are presented. We superimposed our morphological matrix onto the phylogeny to explore character evolution. A detailed key to fruits is presented, allowing identification of extant and fossil specimens to the level of clade or genus. • KEY RESULTS: Menispermaceae consists of two major subfamilies: Tinosporoideae and Menispermoideae. Within Tinosporoideae, tribe Coscineae is basal. Within Menispermoideae, tribe Menispermeae is basal. Tinosporoideae consists mainly of taxa with apical style scars, bilateral curvature, subhemispherical condyles, and foliaceous cotyledons with divaricate or imbricate orientation. Menispermoideae consists almost entirely of taxa with basal or subbasal style scars, dorsoventral curvature, bilaterally and/or dorsoventrally compressed condyles, and subterete or fleshy cotyledons oriented dorsoventrally or laterally. • CONCLUSIONS: Several fruit characters differentiate major clades, and further synapomorphies are diagnostic of various subclades. Fruit characters that can be inferred as ancestral in the family are basal or subbasal stylar scars, endocarps with dorsoventral curvature, endocarp walls woody or bony, presence of a condyle, locule without ribs, sublateral vascular traces, presence of endosperm, and foliaceous or subterete cotyledons.

G Protein-regulated inducer of neurite outgrowth (GRIN) modulates Sprouty protein repression of mitogen-activated protein kinase (MAPK) activation by growth factor stimulation.

Gα(o/i) interacts directly with GRIN (G protein-regulated inducer of neurite outgrowth). Using the yeast two-hybrid system, we identified Sprouty2 as an interacting partner of GRIN. Gα(o) and Sprouty2 bind to overlapping regions of GRIN, thus competing for GRIN binding. Imaging experiments demonstrated that Gα(o) expression promoted GRIN translocation to the plasma membrane, whereas Sprouty2 expression failed to do so. Given the role of Sprouty2 in the regulation of growth factor-mediated MAPK activation, we examined the contribution of the GRIN-Sprouty2 interaction to CB1 cannabinoid receptor regulation of FGF receptor signaling. In Neuro-2A cells, a system that expresses all of the components endogenously, modulation of GRIN levels led to regulation of MAPK activation. Overexpression of GRIN potentiated FGF activation of MAPK and decreased tyrosine phosphorylation of Sprouty2. Pretreatment with G(o/i)-coupled CB1 receptor agonist attenuated subsequent FGF activation of MAPK. Decreased expression of GRIN both diminished FGF activation of MAPK and blocked CB1R attenuation of MAPK activation. These observations indicate that Gα(o) interacts with GRIN and outcompetes GRIN from bound Sprouty. Free Sprouty then in turn inhibits growth factor signaling. Thus, here we present a novel mechanism of how G(o/i)-coupled receptors can inhibit growth factor signaling to MAPK.

Modeling of spatial intracellular signaling events in neurons.

A neuron is able to seamlessly respond to a number of signals, in a timely and specific manner. This process, of integrating multiple inputs, relays on the orchestration of intracellular events by signaling networks. The inherent complexity of signaling networks has made computational modeling a useful approach to understand their underlying regulatory principles. Recent advances in imaging techniques have highlighted the nonhomogeneous nature of intracellular signaling and its significant contribution to the maintenance of signal specificity. Computational modeling can provide mechanistic insight into the origins of these inhomogeneous distributions of signaling components and their role in the integrative capabilities of the neuron.

Receptor heteromerization expands the repertoire of cannabinoid signaling in rodent neurons.

A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB1R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB1R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB1R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB1R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB1R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB1R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB1R desensitization. Additionally, presence of DOR facilitates signaling via a new CB1R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB1R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling.

Modeling of spatially-restricted intracellular signaling.

Understanding the signaling capabilities of a cell presents a major challenge, not only due to the number of molecules involved, but also because of the complex network connectivity of intracellular signaling. Recently, the proliferation of quantitative imaging techniques has led to the discovery of the vast spatial organization of intracellular signaling. Computational modeling has emerged as a powerful tool for understanding how inhomogeneous signaling originates and is maintained. This article covers the current imaging techniques used to obtain quantitative spatial data and the mathematical approaches used to model spatial cell biology. Modeling-derived hypotheses have been experimentally tested and the integration of modeling and imaging approaches has led to non-intuitive mechanistic insights.

Developing models in virtual cell.

This Teaching Resource provides lecture notes, slides, and a student assignment for a two-part lecture on mathematical modeling using the Virtual Cell environment. The lectures discuss the steps involved in developing and running simulations using Virtual Cell, with particular focus on spatial partial differential equation models. We discuss how to construct both ordinary differential equation models, in which the cytoplasm is considered a well-mixed cellular compartment, and partial differential equation models, which calculate how chemical species change as a function of both time and location. The Virtual Cell environment is especially well suited for models that explore spatial specificity of cellular reactions. Partial differential equation models in Virtual Cell can give rise to simulations using predefined cellular geometries, which enable direct comparison with imaging data. These models address questions regarding the regulatory capability arising from spatial organization of the cell. Examples are provided of studies that have successfully exploited the Virtual Cell software to address the spatial contribution to signaling.

Obtaining and estimating kinetic parameters from the literature.

This Teaching Resource provides lecture notes, slides, and a student assignment for a lecture on strategies for the development of mathematical models. Many biological processes can be represented mathematically as systems of ordinary differential equations (ODEs). Simulations with these mathematical models can provide mechanistic insight into the underlying biology of the system. A prerequisite for running simulations, however, is the identification of kinetic parameters that correspond closely with the biological reality. This lecture presents an overview of the steps required for the development of kinetic ODE models and describes experimental methods that can yield kinetic parameters and concentrations of reactants, which are essential for the development of kinetic models. Strategies are provided to extract necessary parameters from published data. The homework assignment requires students to find parameters appropriate for a well-studied biological regulatory system, convert these parameters into appropriate units, and interpret how different values of these parameters may lead to different biological behaviors.

Plant DNA sequencing for phylogenetic analyses: from plants to sequences.

DNA sequences are important sources of data for phylogenetic analysis. Nowadays, DNA sequencing is a routine technique in molecular biology laboratories. However, there are specific questions associated with project design and sequencing of plant samples for phylogenetic analysis, which may not be familiar to researchers starting in the field. This chapter gives an overview of methods and protocols involved in the sequencing of plant samples, including general recommendations on the selection of species/taxa and DNA regions to be sequenced, and field collection of plant samples. Protocols of plant sample preparation, DNA extraction, PCR and cloning, which are critical to the success of molecular phylogenetic projects, are described in detail. Common problems of sequencing (using the Sanger method) are also addressed. Possible applications of second-generation sequencing techniques in plant phylogenetics are briefly discussed. Finally, orientation on the preparation of sequence data for phylogenetic analyses and submission to public databases is also given.

Can antidepressants prevent interferon-alpha-induced depression? A review of the literature.

OBJECTIVE: To review the literature about the efficacy of antidepressant prophylaxis during interferon-alpha (IFN-alpha) therapy. METHOD: We have performed a database search in PUBMED and ISI Web of Knowledge (1980-August 2009) for the available literature. The keywords "prevention" or "prophylaxis", and "depression", and "interferon", and "antidepressant" or "antidepressive agents" were used. RESULTS: The six eligible studies comprise three randomized controlled trials, two in hepatitis C virus (HCV) patients and one in individuals with melanoma, and three open-label studies with HCV patients. The results of the randomized controlled trials suggest that antidepressant prophylaxis may blunt the magnitude of depressive symptoms in HCV patients and raise the rates of treatment completion. In melanoma patients, this preventive strategy may reduce the incidence of depression during IFN-alpha treatment. In addition, the open-label studies with HCV patients suggest that this strategy may reduce the onset of major depression in specific samples (current psychiatric diagnosis, major depression in remission, past history of IFN-alpha-induced depression) on IFN-alpha (re-)treatment. CONCLUSIONS: In the face of so few trials about the usefulness of prophylaxis with antidepressants before IFN-alpha treatment, there is not enough information to sufficiently and widely support this strategy to prevent depression. However, this approach may, nonetheless, bring some beneficial outcomes, if applied to specific patient groups.

Is the interferon-α-triggered depressive episode a self-limited kind of depression? Four cases of persistent affective symptoms after antiviral treatment in HCV-infected individuals.

OBJECTIVES: To discuss relevant aspects in a series of cases in which interferon-α-triggered depressive symptoms persisted up to 4 years after therapy cessation in HCV-infected patients. METHODS: Two experienced psychiatrists (AGA and LCQ) identified these four cases in a systematic evaluation program of HCV patients in the Hepatology Unit of the Teaching Hospital at the Federal University of Bahia, Brazil. Lifetime psychiatric diagnoses were confirmed by the Mini International Neuropsychiatric Interview (MINI Plus), and a questionnaire was submitted in order to gather clinical and sociodemographic characteristics. RESULTS: In three out of the four cases identified, major depression diagnosis was reached after more than 12 months of interferon-α therapy interruption and, in one case, depression recurred 6 months after antiviral treatment cessation in a patient on antidepressants. The only case that referred a past history of psychiatric diagnosis reported no offer of mental health care despite the presence of a major depressive episode with psychotic features and suicidal behaviour during the cytokine usage. CONCLUSIONS: Interferon-α-triggered depression may remain undiagnosed even in tertiary university hospitals, may persist years after the antiviral therapy cessation, and may recur even in patients on adequate antidepressant treatment.