Molecular Engineered Carbon-Based Sensor for Ultrafast and Specific Detection of Neurotransmitters.

In the quest for designing affordable diagnostic devices with high performance, precisely functionalized carbon-based materials with high accuracy and selectivity are required. Every material has its own unique ability to interact with the analyte, and its performance can be enhanced by probing the interaction mechanism. Herein, p-aminophenol (PAP)-functionalized reduced graphene oxide (rGO) nanoscale material is developed by a one-step synthetic route as an all-organic-based sensor. As the PAP molecules are precisely covalently interacted with the rGO at the basal plane and form a wrinkled-paper-like structure, the functionalized material exhibits an outstanding sensing ability (7.5 nM neurotransmitter dopamine (DA) at a wide linear range, 0.01-100 µM) with fast electrical transduction (<3 s) and good recyclability (∼10 cycles) in a real sample. Combining various analytical and density functional theory (DFT) calculation methods, physicochemical properties and the interaction mechanism of analyte-materials transduction are discussed exclusively. Besides, the potential application of the well-dispersed rGO-PAP gravure ink in flexible-printed electronics fields is explored. This study not only provides new insights into the surface/interface chemistry and working principle of this unique anchoring of PAP on rGO but also offers a new pathway for developing other forms of metal-free/organic functionalized biosensors with high efficiency.

Transcriptional Repressor HIC1 Contributes to Suppressive Function of Human Induced Regulatory T Cells.

Regulatory T (Treg) cells are critical in regulating the immune response. In vitro induced Treg (iTreg) cells have significant potential in clinical medicine. However, applying iTreg cells as therapeutics is complicated by the poor stability of human iTreg cells and their variable suppressive activity. Therefore, it is important to understand the molecular mechanisms of human iTreg cell specification. We identified hypermethylated in cancer 1 (HIC1) as a transcription factor upregulated early during the differentiation of human iTreg cells. Although FOXP3 expression was unaffected, HIC1 deficiency led to a considerable loss of suppression by iTreg cells with a concomitant increase in the expression of effector T cell associated genes. SNPs linked to several immune-mediated disorders were enriched around HIC1 binding sites, and in vitro binding assays indicated that these SNPs may alter the binding of HIC1. Our results suggest that HIC1 is an important contributor to iTreg cell development and function.

Interaction of glycated protein and DFO mimicked hypoxia in cellular responses of HUVECs.

The accelerated non-enzymatic modification of proteins by Maillard reaction during prolonged hyperglycemia is a key player in the diabetes associated pathology. In addition, hypoxia has been implicated in the recent past as a modulating factor. Therefore we have examined the interaction of glycation modified human serum albumin (AGE-HSA) and deferoxamine (DFO) mimicked hypoxia on the expression of hypoxia inducible factor 1α (HIF-1α), and the role of RAGE (receptor for AGE) signaling in up-regulation of HIF-1α. Expression of VEGF (a downstream target of HIF-1α) and sICAM-1 (inflammatory marker) was also detected. When HUVEC were subjected to hypoxia, highest expression of HIF-1α was observed. When treated with AGE-HSA at two concentrations, higher expression was found vis-a-vis control, with 0.2 mg ml(-1) than 2.0 mg ml(-1) which was mediated in part by RAGE as determined by RAGE silencing. However, when the cells were exposed to a combination treatment of hypoxia and AGE-HSA, a biphasic effect at the two different concentrations was observed as compared to the individual treatments. VEGF was synergistically up-regulated by hypoxia and AGE-HSA. On the other hand sICAM-1 was up-regulated by AGE-HSA but down-regulated by hypoxia. These results show that AGE-HSA functions as a non-hypoxic factor which modulates the expression of HIF-1α in a concentration dependent manner in the range studied. It can be concluded that glycated serum proteins may activate HIF-1α independently in diabetes. Further, when both glycated proteins and hypoxic conditions are present, they act in opposition in regulation of HIF-1α.

Interaction of glycated human serum albumin with endothelial cells in a hemodynamic environment: structural and functional correlates.

Advanced glycation end products (AGEs) are known to be involved in the pathogenesis of several diseases, in particular diabetes, via signaling through their receptor. Numerous studies have been carried out on protein-sugar interactions at very high concentrations of the latter. The objective of this investigation was to determine the effects of nonenzymatic glycation induced by reducing sugars on the secondary structure of human serum albumin (HSA) under different physiological conditions and to correlate that with expression of RAGE (receptor for advanced glycation end products) on HUVECs (human umbilical vein endothelial cells) in a controlled hemodynamic environment. Our results indicate that RAGE expression is shear stress modulated and that glycated HSA enhances the expression further. The secondary structure of AGE-HSA derived from glucose at 20 mM contains higher α-helical content and elicits maximum expression of the receptor. The effect of shear stress at 10 dynes cm(-2) is independent of AGE-HSA.

Effect of different mitogens and serum concentration on HUVEC morphology and characteristics: implication on use of higher passage cells.

Human umbilical vein endothelial cells (HUVEC) were cultured in two different media, viz. the commonly used M199 containing 20% fetal bovine serum (FBS) and endothelial cell growth factor and a defined media EGM-2 containing 2% FBS along with growth supplements in known concentrations. The purpose of this study was to determine the effect of different media on the growth potential and cell morphology in subsequent passages. We have established that a dual coating of gelatin and human fibronectin extracellular matrix provides optimal cell attachment. Growth rate for primary culture was almost double in defined media. For secondary culture a two fold higher proliferation rate was observed in defined EGM-2 media. Histological studies were done using phase contrast, confocal and scanning electron microscopy which showed that cells cultured in M199 started losing their morphological characteristic from 3rd passage and after 6th passage appeared to come in senescent stage, while in case of defined media there was no change observed in the cells up to 10th passage. A significant difference was found in the expression of soluble intracellular adhesion molecule-1 (sICAM-1) which is an endothelial cell marker on cells cultured in different media. Additionally it was observed that exposure duration to trypsin-EDTA during cell detachment also plays an important role in maintaining cell morphological characteristics. These results show that significant morphological changes appear in higher order passages if cells are grown in routine medium for a long time and therefore may not be suitable for cell signaling experiments.