MWCNT-ruthenium oxide composite paste electrode as non-enzymatic glucose sensor

A non-enzymatic glucose sensor of multi-walled carbon nanotube-ruthenium oxide/composite paste electrode (MWCNT-RuO(2)/CPE) was developed. The electrode was characterized by using XRD, SEM, TEM and EIS. Meanwhile, cyclic voltammetry and amperometry were used to check on the performances of the MWCNT-RuO(2)/CPE towards glucose. The proposed electrode has displayed a synergistic effect of RuO(2) and MWCNT on the electrocatalytic oxidation of glucose in 3M NaOH. This was possible via the formation of transitions of two redox pairs, viz. Ru(VI)/Ru(IV) and Ru(VII)/Ru(VI). A linear range of 0.5-50mM glucose and a limit of detection of 33μM glucose (S/N=3) were observed. There was no significant interference observable from the traditional interferences, viz. ascorbic acid and uric acid. Indeed, results so obtained have indicated that the developed MWCNT-RuO(2)/CPE would pave the way for a better future to glucose sensor development as its fabrication was without the use of any enzyme.     

Is Opium a Real Risk Factor for Esophageal Cancer or Just a Methodological Artifact? Hospital and Neighborhood Controls in Case-Control Studies

Background

Control selection is a major challenge in epidemiologic case-control studies. The aim of our study was to evaluate using hospital versus neighborhood control groups in studying risk factors of esophageal squamous cell carcinoma (ESCC).

Methodology/Principal Findings

We compared the results of two different case-control studies of ESCC conducted in the same region by a single research group. Case definition and enrollment were the same in the two studies, but control selection differed. In the first study, we selected two age- and sex-matched controls from inpatient subjects in hospitals, while for the second we selected two age- and sex-matched controls from each subject''s neighborhood of residence. We used the test of heterogeneity to compare the results of the two studies. We found no significant differences in exposure data for tobacco-related variables such as cigarette smoking, chewing Nass (a tobacco product) and hookah (water pipe) usage, but the frequency of opium usage was significantly different between hospital and neighborhood controls. Consequently, the inference drawn for the association between ESCC and tobacco use did not differ between the studies, but it did for opium use. In the study using neighborhood controls, opium use was associated with a significantly increased risk of ESCC (adjusted OR 1.77, 95% CI 1.17–2.68), while in the study using hospital controls, this was not the case (OR 1.09, 95% CI 0.63–1.87). Comparing the prevalence of opium consumption in the two control groups and a cohort enrolled from the same geographic area suggested that the neighborhood controls were more representative of the study base population for this exposure.

Conclusions/Significance

Hospital and neighborhood controls did not lead us to the same conclusion for a major hypothesized risk factor for ESCC in this population. Our results show that control group selection is critical in drawing appropriate conclusions in observational studies.     

Uptake of copper from plasma proteins in cells where expression of CTR1 has been modulated

Plasma proteins rather than amino acid chelates are the direct sources of copper for mammalian cells. In continuing studies on the mechanisms by which albumin and transcuprein deliver copper and the potential involvement of CTR1, rates of uptake from these proteins and Cu-histidine were compared in cells with/without CTR1 knockdown or knockout. siRNA knocked down expression of CTR1 mRNA 60-85% in human mammary epithelial and hepatic cell models, but this had little or no effect on uptake of 1?μM Cu(II) attached to pure human albumin or alpha-2-macroglobulin. Mouse embryonic fibroblasts that did/did not express Ctr1 took up Cu(II) bound to albumin about as readily as from the histidine complex at physiological concentrations and by a single saturable process. Uptake from mouse albumin achieved a 2-4-fold higher Vmax (with a lower Km) than from heterologous human albumin. Maximum uptake rates from Cu(I)-histidine were >12-fold higher (with higher Km) than for Cu(II), suggesting mediation by a reductase. The presence of cell surface Cu(II) and Fe(III) reductase activity responding only slightly to dehydroascorbate was verified. Excess Fe(III) inhibited uptake from albumin-Cu(II). Ag(I) also inhibited, but kinetics were not or un-competitive. In general there was little difference in rates/kinetics of uptake in the Ctr1+/+ and -/- cells. Endocytosis was not involved. We conclude that plasma proteins deliver Cu(II) to homologous cells with greater efficiency than ionic copper at physiological concentrations, probably through the mediation of a Steap Cu(II)-reductase, and confirm the existence of an additional copper uptake system in mammalian cells.     

Antimicrobial Activity of Artemisia absinthium Against Surgical Wounds Infected by Staphylococcus aureus in a Rat Model

The wound infection is one of the frequent complications in patients undergoing surgical operations. Staphylococcus aureus is the most common cause of surgical wounds. Artemisia absinthium has been shown to bear strong antimicrobial activity, especially against Gram-positive pathogens. This study was designed to investigate the antimicrobial effects of A. absinthium against surgical wounds infected by S. aureus in a rat model. Twenty male Sprague–Dawley rats were divided randomly into two equal groups of treated and control rats. A circular incision was created on the dorsal inter-scapular region of each rat. After skin wounding, rats were inoculated locally with 1 × 10⁴ CFU of S. aureus at sites of skin wounds. The extract was applied topically twice a day throughout the experiment. Animals of the control group were left untreated. Results have revealed that topical application of A. absinthium extract on the infected wound sites produced significant antibacterial activity against S. aureus.     

Testing the biocompatibility of a glutathione-containing intra-ocular irrigation solution by using an isolated perfused bovine retina organ culture model - an alternative to animal testing

The effects of a glutathione-containing intra-ocular irrigation solution, BSS Plus?, on retinal function and on the survival of ganglion cells in whole-mount retinal explants were studied. Evidence is provided that the perfused ex vivo bovine retina can serve as an alternative to in vivo animal testing. Isolated bovine retinas were prepared and perfused with an oxygen-saturated standard irrigation solution, and an electroretinogram was recorded to assess retinal function. After stable b-waves were detected, the isolated retinas were perfused with BSS Plus for 45 minutes. To investigate the effects of BSS Plus on photoreceptor function, 1mM aspartate was added to the irrigation solution in order to obtain a-waves, and the ERG trace was monitored for 75 minutes. For histological analysis, isolated whole retinal mounts were stored for 24 hours at 4°C, in the dark. The percentages of cell death in the retinal ganglion cell layer and in the outer and inner nuclear layers were estimated by using an ethidium homodimer-1 stain and the TUNEL assay. General swelling of the retina was examined with high-resolution optical coherence tomography. During perfusion with BSS Plus, no significant changes in a-wave and b-wave amplitudes were recorded. Retinas stored for 24 hours in BSS Plus showed a statistically significant smaller percentage (52.6%, standard deviation [SD] = 16.1%) of cell death in the retinal ganglion cell layer compared to the control group (69.6%, SD = 3.9, p = 0.0031). BSS Plus did not seem to affect short-term retinal function, and had a beneficial effect on the survival of retinal ganglion cells. This method for analysing the isolated perfused retina represents a valuable alternative for testing substances for their retinal biocompatibility and toxicity.     

Dab2ip Regulates Neuronal Migration and Neurite Outgrowth in the Developing Neocortex

Dab2ip (DOC-2/DAB2 interacting protein) is a member of the Ras GTPase-activating protein (GAP) family that has been previously shown to function as a tumor suppressor in several systems. Dab2ip is also highly expressed in the brain where it interacts with Dab1, a key mediator of the Reelin pathway that controls several aspects of brain development and function. We found that Dab2ip is highly expressed in the developing cerebral cortex, but that mutations in the Reelin signaling pathway do not affect its expression. To determine whether Dab2ip plays a role in brain development, we knocked down or over expressed it in neuronal progenitor cells of the embryonic mouse neocortex using in utero electroporation. Dab2ip down-regulation severely disrupts neuronal migration, affecting preferentially late-born principal cortical neurons. Dab2ip overexpression also leads to migration defects. Structure-function experiments in vivo further show that both PH and GRD domains of Dab2ip are important for neuronal migration. A detailed analysis of transfected neurons reveals that Dab2ip down- or up-regulation disrupts the transition from a multipolar to a bipolar neuronal morphology in the intermediate zone. Knock down of Dab2ip in neurons ex-vivo indicates that this protein is necessary for proper neurite development and for the expression of several major neuronal microtubule associated proteins (MAPs), which are important for neurite growth and stabilization. Thus, our study identifies, for the first time, a critical role for Dab2ip in mammalian cortical development and begins to reveal molecular mechanisms that underlie this function.     

Structural basis for ubiquitin recognition by the Otu1 ovarian tumor domain protein

Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of approximately 130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys(48) linkages, having little or no activity on Lys(63)- and Lys(29)-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.     

Functional Implication of Dp71 in Osmoregulation and Vascular Permeability of the Retina

Functional alterations of Müller cells, the principal glia of the retina, are an early hallmark of most retina diseases and contribute to their further progression. The molecular mechanisms of these reactive Müller cell alterations, resulting in disturbed retinal homeostasis, remain largely unknown. Here we show that experimental detachment of mouse retina induces mislocation of the inwardly rectifying potassium channels (Kir4.1) and a downregulation of the water channel protein (AQP4) in Müller cells. These alterations are associated with a strong decrease of Dp71, a cytoskeleton protein responsible for the localization and the clustering of Kir4.1 and AQP4. Partial (in detached retinas) or total depletion of Dp71 in Müller cells (in Dp71-null mice) impairs the capability of volume regulation of Müller cells under osmotic stress. The abnormal swelling of Müller cells In Dp71-null mice involves the action of inflammatory mediators. Moreover, we investigated whether the alterations in Müller cells of Dp71-null mice may interfere with their regulatory effect on the blood-retina barrier. In the absence of Dp71, the retinal vascular permeability was increased as compared to the controls. Our results reveal that Dp71 is crucially implicated in the maintenance of potassium homeostasis, in transmembraneous water transport, and in the Müller cell-mediated regulation of retinal vascular permeability. Furthermore, our data provide novel insights into the mechanisms of retinal homeostasis provided by Müller cells under normal and pathological conditions.     

Discovery of Klotho peptide antagonists against Wnt3 and Wnt3a target proteins using combination of protein engineering,protein–protein docking,peptide docking and molecular dynamics simulations

The Klotho is known as lifespan enhancing protein involved in antagonizing the effect of Wnt proteins. Wnt proteins are stem cell regulators, and uninterrupted exposure of Wnt proteins to the cell can cause stem and progenitor cell senescence, which may lead to aging. Keeping in mind the importance of Klotho in Wnt signaling, in silico approaches have been applied to study the important interactions between Klotho and Wnt3 and Wnt3a (wingless-type mouse mammary tumor virus (MMTV) integration site family members 3 and 3a). The main aim of the study is to identify important residues of the Klotho that help in designing peptides which can act as Wnt antagonists. For this aim, a protein engineering study is performed for Klotho, Wnt3 and Wnt3a. During the theoretical analysis of homology models, unexpected role of number of disulfide bonds and secondary structure elements has been witnessed in case of Wnt3 and Wnt3a proteins. Different in silico experiments were carried out to observe the effect of correct number of disulfide bonds on 3D protein models. For this aim, total of 10 molecular dynamics (MD) simulations were carried out for each system. Based on the protein–protein docking simulations of selected protein models of Klotho with Wnt3 and Wnt3a, different peptides derived from Klotho have been designed. Wnt3 and Wnt3a proteins have three important domains: Index finger, N-terminal domain and a patch of ～10 residues on the solvent exposed surface of palm domain. Protein–peptide docking of designed peptides of Klotho against three important domains of palmitoylated Wnt3 and Wnt3a yields encouraging results and leads better understanding of the Wnt protein inhibition by proposed Klotho peptides. Further in vitro studies can be carried out to verify effects of novel designed peptides as Wnt antagonists.