Advances in carbon nanotube based electrochemical sensors for bioanalytical applications

Electrochemical (EC) sensing approaches have exploited the use of carbon nanotubes (CNTs) as electrode materials owing to their unique structures and properties to provide strong electrocatalytic activity with minimal surface fouling. Nanofabrication and device integration technologies have emerged along with significant advances in the synthesis, purification, conjugation and biofunctionalization of CNTs. Such combined efforts have contributed towards the rapid development of CNT-based sensors for a plethora of important analytes with improved detection sensitivity and selectivity. The use of CNTs opens an opportunity for the direct electron transfer between the enzyme and the active electrode area. Of particular interest are also excellent electrocatalytic activities of CNTs on the redox reaction of hydrogen peroxide and nicotinamide adenine dinucleotide, two major by-products of enzymatic reactions. This excellent electrocatalysis holds a promising future for the simple design and implementation of on-site biosensors for oxidases and dehydrogenases with enhanced selectivity. To date, the use of an anti-interference layer or an artificial electron mediator is critically needed to circumvent unwanted endogenous electroactive species. Such interfering species are effectively suppressed by using CNT based electrodes since the oxidation of NADH, thiols, hydrogen peroxide, etc. by CNTs can be performed at low potentials. Nevertheless, the major future challenges for the development of CNT-EC sensors include miniaturization, optimization and simplification of the procedure for fabricating CNT based electrodes with minimal non-specific binding, high sensitivity and rapid response followed by their extensive validation using “real world” samples. A high resistance to electrode fouling and selectivity are the two key pending issues for the application of CNT-based biosensors in clinical chemistry, food quality and control, waste water treatment and bioprocessing.     

PMC,a potent hydrophilic α‐tocopherol derivative,inhibits NF‐κB activation via PP2A but not IκBα‐dependent signals in vascular smooth muscle cells

The hydrophilic α‐tocopherol derivative, 2,2,5,7,8‐pentamethyl‐6‐hydroxychromane (PMC), is a promising alternative to vitamin E in clinical applications. Critical vascular inflammation leads to vascular dysfunction and vascular diseases, including atherosclerosis, hypertension and abdominal aortic aneurysms. In this study, we investigated the mechanisms of the inhibitory effects of PMC in vascular smooth muscle cells (VSMCs) exposed to pro‐inflammatory stimuli, lipopolysaccharide (LPS) combined with interferon (IFN)‐γ. Treatment of LPS/IFN‐γ‐stimulated VSMCs with PMC suppressed the expression of inducible nitric oxide synthase (iNOS) and matrix metalloproteinase‐9 in a concentration‐dependent manner. A reduction in LPS/IFN‐γ‐induced nuclear factor (NF)‐κB activation was also observed in PMC‐treated VSMCs. The translocation and phosphorylation of p65, protein phosphatase 2A (PP2A) inactivation and the formation of reactive oxygen species (ROS) were significantly inhibited by PMC in LPS/IFN‐γ‐activated VSMCs. However, neither IκBα degradation nor IκB kinase (IKK) or ribosomal s6 kinase‐1 phosphorylation was affected by PMC under these conditions. Both treatments with okadaic acid, a PP2A‐selective inhibitor, and transfection with PP2A siRNA markedly reversed the PMC‐mediated inhibition of iNOS expression, NF‐κB‐promoter activity and p65 phosphorylation. Immunoprecipitation analysis of the cellular extracts of LPS/IFN‐γ‐stimulated VSMCs revealed that p65 colocalizes with PP2A. In addition, p65 phosphorylation and PP2A inactivation were induced in VSMCs by treatment with H₂O₂, but neither IκBα degradation nor IKK phosphorylation was observed. These results collectively indicate that the PMC‐mediated inhibition of NF‐κB activity in LPS/IFN‐γ‐stimulated VSMCs occurs through the ROS‐PP2A‐p65 signalling cascade, an IKK‐IκBα‐independent mechanism. Therapeutic interventions using PMC may therefore be beneficial for the treatment of vascular inflammatory diseases.     

The Transforming Growth Factor-β Type III Receptor Mediates Distinct Subcellular Trafficking and Downstream Signaling of Activin-like Kinase (ALK)3 and ALK6 Receptors

Bone morphogenetic proteins (BMPs) signal through the BMP type I and type II receptors to regulate cellular processes, including embryonic development. The type I BMP receptors activin-like kinase (ALK)3 and ALK6 share a high degree of homology, yet possess distinct signaling roles. Here, we report that although the transforming growth factor (TGF)-β type III receptor (TβRIII) enhanced both ALK3 and ALK6 signaling, TβRIII more potently enhanced ALK6-mediated stimulation of the BMP-responsive promoters XVent2 and 3GC2, and up-regulation of the early response gene Smad6. In contrast, TβRIII specifically enhanced ALK3-mediated up-regulation of the early response gene ID-1. TβRIII associated with ALK3 primarily through their extracellular domains, whereas its interaction with ALK6 required both the extracellular and cytoplasmic domains. TβRIII, along with its interacting scaffolding protein β-arrestin2, induced the internalization of ALK6. In contrast, TβRIII colocalized with and resulted in the cell surface retention of ALK3, independently of β-arrestin2. Although complex formation between TβRIII, ALK6, and β-arrestin2 and TβRIII/ALK6 internalization resulted in maximal BMP signaling, the TβRIII mutant unable to interact with β-arrestin2, TβRIII-T841A, was unable to do so. These studies support a novel role for TβRIII in mediating differential ALK3 and ALK6 subcellular trafficking resulting in distinct signaling downstream of ALK3 and ALK6.     

WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma

Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12–Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.     

CME‐1, a novel polysaccharide,suppresses iNOS expression in lipopolysaccharide‐stimulated macrophages through ceramide‐initiated protein phosphatase 2A activation

CME‐1, a novel water‐soluble polysaccharide purified from Ophiocordyceps sinensis mycelia, has anti‐oxidative, antithrombotic and antitumour properties. In this study, other major attributes of CME‐1, namely anti‐inflammatory and immunomodulatory properties, were investigated. Treating lipopolysaccharide (LPS)‐stimulated RAW 264.7 cells with CME‐1 concentration‐dependently suppressed nitric oxide formation and inducible nitric oxide synthase (iNOS) expression. In the CME‐1‐treated RAW 264.7 cells, LPS‐induced IκBα degradation and the phosphorylation of p65, Akt and mitogen‐activated protein kinases (MAPKs), including extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38, were reduced. Treatment with a protein phosphatase 2A (PP2A)‐specific inhibitor, significantly reversed the CME‐1‐suppressed iNOS expression; IκBα degradation; and p65, Akt and MAPK phosphorylation. PP2A activity up‐regulation and PP2A demethylation reduction were also observed in the cells. Moreover, CME‐1‐induced PP2A activation and its subsequent suppression of LPS‐activated RAW 264.7 cells were diminished by the inhibition of ceramide signals. LPS‐induced reactive oxygen species (ROS) and hydroxyl radical formation were eliminated by treating RAW 264.7 cells with CME‐1. Furthermore, the role of ceramide signalling pathway and anti‐oxidative property were also demonstrated in CME‐1‐mediated inhibition of LPS‐activated primary peritoneal macrophages. In conclusion, CME‐1 suppressed iNOS expression by up‐regulating ceramide‐induced PP2A activation and reducing ROS production in LPS‐stimulated macrophages. CME‐1 is a potential therapeutic agent for treating inflammatory diseases.     

The implementation of the consensus on the management of Helicobacter pylori and barriers to consensus

Background

A consensus on the management of Helicobacter pylori has been developed. We aimed to assess whether dissemination through continuing medical education (CME) could enhance the adoption of this consensus among clinicians and to explore potential barriers to acceptance.

Materials and methods

Four CME courses were held to disseminate the consensus. Adoption surveys were performed to evaluate participants’ behavior in the past and their commitment to adopt the consensus in future clinical practice after CME. The gaps and barriers to adoption were also surveyed.

Results

A total of 240 physicians had attended the CME courses and received surveys with the 22 statements/substatements of the consensus. Before CME, adoption was good in six, fair in ten, and poor in six. After CME, 21 statements had either an initial >90% adoption or improvement to good or fair (P < 0.001), but one still had poor even though it showed improvement (P = 0.02). Although commitment was good or fair after CME, there was a >20% gap between “commitment” and “no barrier” to adoption for 11 statements, ten of which had a main barrier of financial incentives. Among the statements with fair or poor commitment after CME, less commitment to adoption and more barriers related to financial incentives were pronounced in clinicians serving in regional/district hospitals or clinics compared to those serving in medical centers.

Conclusions

Continuing medical education may improve the adoption of the H. pylori consensus. The financial incentives were shown to be a main barrier to adoption of the consensus and should be improved.     

Heterogeneous Expression of Transketolase in Rat Brain

Abstract: Transketolase (TK; EC 2.2.1.1) is a key pentose phosphate shunt enzyme that plays an important role in the production of reducing equivalents and pentose sugars. TK activity declines in the brains of patients with Alzheimer's disease or Wernicke-Korsakoff syndrome, as well as in thiamine-deficient rats. Understanding the role of TK in the pathophysiology of these neurodegenerative conditions requires knowledge of its regional, cellular, and subcellular distribution within the brain. The current study employed in situ hybridization and immunocytochemistry to examine the distribution of TK mRNA and its encoded protein in adult rat brain. TK mRNA and protein were widely distributed throughout the brain. However, they were enriched in selective perikarya in the piriform cortex, nucleus of the diagonal band, red nucleus, dorsal raphe, pontine nucleus, locus coeruleus, trapezoid, inferior olive, and several cranial nerve nuclei. Lower expression of TK mRNA and protein occurred in layer V of cortex, olfactory tubercle, ventral pallidum, medial septal nucleus, hippocampus, thalamic and hypothalamic nuclei, mammillary body, central gray, and the substantia nigra. TK immunoreactivity also occurred in the nuclei of ubiquitously distributed glial cells, as well as ependymal cells. The heterogeneous distribution of TK may reflect a variety of metabolic activities among different brain regions but does not provide a simple molecular explanation for selective cell death in either thiamine deficiency or other conditions where TK is reduced.     

Zinc status in plasma of obese individuals during glucose administration

To know whether plasma zinc status is altered under acute hyperglycemic state, the interrelationships among plasma glucose, insulin, and zinc concentrations during oral glucose tolerance test (OGTT) in obese individuals and their lean controls were studied. Plasma glucose and insulin concentrations under fasting as well as those values in response to OGTT were significantly higher in obese individuals than those in lean controls. On the other hand, the obese had lower fasting plasma zinc concentrations compared to lean controls (13.5 vs 18.1 Μmol/L,p < 0.005). Under fasting, plasma zinc concentrations in overall individuals inversely correlated to their body mass index (BMI) (r = -0.516), plasma glucose (r = -0.620), and plasma insulin (r = -0.510). However, there were no significant changes in plasma zinc and copper values during OGTT in both obese individuals and lean controls. This study showed that plasma zinc values had no changes during OGTT in obese individuals. The results also indicated that lower fasting plasma zinc concentrations in obese individuals were not the short-term metabolic result.