Single-Shot Analytical Assay Based on Graphene-Oxide-Modified Surface Acoustic Wave Biosensor for Detection of Single-Nucleotide Polymorphisms

The combination of a surface acoustic wave (SAW) biosensor with graphene oxide (GO) provides a promising perspective for detecting DNA mutation. The GO-modified SAW biosensor was prepared by conjugating GO onto the SAW chip surface via electrostatic interaction. Afterward, the probe was immobilized on the GO surface, and detection of DNA mutation was realized by hybridization. The hybridization with a variety of targets would yield different mass and conformational changes on the chip surface, causing the different SAW signals in real time. A total of 137 clinical samples were detected by a single-shot analytical assay based on GO-modified SAW biosensor and direct sequencing in parallel. The diagnostic performance (both sensitivity and specificity) of the assay was evaluated with the direct sequencing as a reference testing method. The phase-shift value of three genotypes in 137 clinical samples was significantly different (<i>p</i> < 0.001). Furthermore, testing of diagnostic performance yielded diagnostic sensitivity and specificity of 100% and 88.6% for identifying CT and CC genotype, 98.0% and 96.2% for identifying CT and TT genotype, respectively. The single-shot analytical assay based on the GO-modified SAW biosensor could be exploited as a potential useful tool to identify CYP2D6*10 polymorphisms in clinical practice of personalized medicine

Isolation and characterisation of tropomyosin from shrimp (<i>Penaeus vannamei</i> Boone) and its association property at high ionic strength

<div><p>Shrimps are important and highly demanded seafood, but they have been reported as a cause of food hypersensitive reaction. The major allergen of shrimp is tropomyosin (TM). However, so far, there has been few report on such purification procedure. In this study, we developed a strategy for the purification of TM from shrimp (<i>Penaeus vannamei</i> Boone). Subsequently, we demonstrated that the apparent MW of this protein is about 66 kDa, and this protein naturally contains two subunits (38.5 and 36.6 kDa) with a ratio of 1 to 1. Interestingly, different from other known TMs from vertebrates, shrimp TM can self-assemble into nanofibres at high ionic strength induced by ATP. These findings help to understand the structure and polymerisation property of TM from shrimps.</p></div

Label-Free Detection of Carbohydrate–Protein Interactions Using Nanoscale Field-Effect Transistor Biosensors

Carbohydrate–protein interactions play a significant role in cell communication, cell adhesion, cell trafficking, and immune responses. Many efforts have been made to demonstrate detection of carbohydrate–protein interactions. However, the existing methods are still tedious and expensive. Therefore, the detection of carbohydrate–protein interactions is of great significance, and new, efficient methods are required for fast and sensitive recognition testing. In this report, we, for the first time, developed the silicon nanowire (SiNW)-based biosensor capable of label-free electrical detection of carbohydrate–protein interactions with high selectivity and sensitivity by covalently immobilizing unmodified carbohydrates on the sensor surface. We fabricated new SiNW sensor chips with more SiNW arrays for potential detection of multiple analytes. In order to realize the immobilization of the unmodified carbohydrates on the SiNW surface, we used X-ray photoelectron spectra and fluorescence microscopy to verify the successful surface functionalization on the silicon surface. Furthermore, we demonstrated real-time detection of carbohydrate–protein interactions using the carbohydrate-modified SiNW sensor chips. The results show good specificity between galactose–lectin EC and mannose–Con A, which is in good agreement with that reported previously. Finally, the results also show that we are able to use the galactose-modified SiNW biosensor to detect lectin EC as low as 100 fg/mL, which is 4 orders of magnitude lower than that reported by other technologies. We believe that the developed SiNW biosensor paves a novel way for studying carbohydrate–protein interactions

TGF-β1-induced glutaminolysis accelerates endothelial migration and PP2A-Raf-MEK-ERK pathway is involved in this process.

<p>The cells were pretreated with U0126 (10μM) and OA (10 nM) for 30 minutes before TGF-β1 (10 ng/ml) stimulation and then cultured for 48 hours. (a) Cell migration was promoted by TGF-β1, and inhibition of PP2A-Raf-MEK-ERK pathway inhibited cells sprouting. (b) Bar graphs present the gap distance calculated by Image Pro Plus Software.**P<0.01 versus the control group, and *P<0.05 versus the TGF-β1 group. Bars represented means ±SEM.</p

Glutaminolysis Was Induced by TGF-β1 through PP2Ac Regulated Raf-MEK-ERK Signaling in Endothelial Cells

<div><p>Vascular endothelial cells can survive under hypoxic and inflammatory conditions by alterations of the cellular energy metabolism. In addition to high rates of glycolysis, glutaminolysis is another important way of providing the required energy to support cellular sprouting in such situations. However, the exact mechanism in which endothelial cells upregulate glutaminolysis remains unclear. Here we demonstrated that protein phosphatase 2A (PP2A)-mediated Raf-MEK-ERK signaling was involved in glutaminolysis in endothelial cells. Using models of human umbilical vein endothelial cells (HUVECs) treated with transforming growth factor-β1 (TGF-β1), we observed a dramatic induction in cellular glutamate levels accompanied by Raf-MEK-ERK activation. By addition of U0126, the specific inhibitor of MEK1/2, the expression of kidney-type glutaminase (KGA, a critical glutaminase in glutaminolysis) was significantly decreased. Moreover, inhibition of PP2A by okadaic acid (OA), a specific inhibitor of PP2A phosphatase activity or by depletion of its catalytic subunit (PP2Ac), led to a significant inactivation of Raf-MEK-ERK signaling and reduced glutaminolysis in endothelial cells. Taken together, these results indicated that PP2A-dependent Raf-MEK-ERK activation was involved in glutaminolysis and inhibition of PP2A signals was sufficient to block Raf-MEK-ERK pathway and reduced glutamine metabolism in endothelial cells.</p></div

Incidence of Cancer in ANCA-Associated Vasculitis: A Meta-Analysis of Observational Studies

<div><p>Objective</p><p>The purpose of this paper is to examine cancer incidence in patients with ANCA-associated vasculitis (AASV) derived from population-based cohort studies by means of meta-analysis.</p><p>Methods</p><p>Relevant electronic databases were searched for studies characterizing the associated risk of overall malignancy in patients with AASV. Standardized incidence rates (SIRs) with 95% confidence intervals (CIs) were used to evaluate the strength of association. We tested for publication bias and heterogeneity and stratified for site-specific cancers.</p><p>Results</p><p>Six studies (n = 2,578) were eventually identified, of which six provided the SIR for overall malignancy, five reported the SIR for non-melanoma skin cancer (NMSC), four for leukemia, five for bladder cancer, three for lymphoma, three for liver cancer, four for lung cancer, three for kidney cancer, four for prostate cancer, four for colon cancer and four for breast cancer. Overall, the pooled SIR of cancer in AASV patients was 1.74 (95%CI = 1.37–2.21), with moderate heterogeneity among these studies (I² = 65.8%, P = 0.012). In sub-analyses for site-specific cancers, NMSC, leukemia and bladder cancer were more frequently observed in patients with AASV with SIR of 5.18 (95%CI = 3.47–7.73), 4.89 (95%CI = 2.93–8.16) and 3.84 (95%CI = 2.72–5.42) respectively. There was no significant increase in the risk of kidney cancer (SIR = 2.12, 95%CI = 0.66–6.85), prostate cancer (SIR = 1.45, 95%CI = 0.87–2.42), colon cancer (SIR = 1.26, 95%CI = 0.70–2.27), and breast cancer (SIR = 0.95, 95%CI = 0.50–1.79). Among these site-specific cancers, only NMSC showed moderate heterogeneity (I² = 55.8%, P = 0.06). No publication bias was found by using the Begg’s test and Egger's test.</p><p>Conclusions</p><p>This meta-analysis shows that AASV patients treatment with cyclophosphamide (CYC) are at increased risk of late-occurring malignancies, particularly of the NMSC, leukemia and bladder cancer. However, there is no significant association between AASV and kidney cancer, prostate cancer, colon cancer and breast cancer. These findings emphasize monitoring and preventative management in AASV patients after cessation of CYC therapy is momentous.</p></div

Synthesis of co-crystals of meloxicam with carboxylic acids by grinding

Search strategy. (DOCX 13 kb

Knockdown of PP2Ac by siRNA prevents the activation of Raf/MEK/ERK pathway in response to TGF-β1.

<p>(a) HUVECs were transiently transfected with 100 nmol/L of PP2Ac siRNA or control siRNA for 12 h. PP2Ac protein expression was detected to confirm the knockdown effect. A representative western blot was shown. (b) Densitometry analysis of PP2Ac protein expression was plotted (n≥3). (c, d, e, f) After transfection of PP2Ac siRNA, cells were incubated with TGF-β1 for 15 min and 60 min, respectively. A representative western blot of p-c-Raf (Ser 259), T-c-Raf, p-ERK 1/2, T-ERK 1/2 in each group were shown. The histograms showed the ratio of p-c-Raf/T-c-Raf and p-ERK/T-ERK (n≥3).*P<0.05 compared to control group. Bars represented means ±SEM.</p

Raf-MEK-ERK signaling pathway is involved in KGA activation in HUVECs.

<p>The cells were treated with 10ng/ml of TGF-β1 for different indicated periods, from 15 minutes to 90 minutes. (a, c) Representative western blot of p-ERK1/2, T-ERK1/2, p-c-Raf (Ser 259) and T-c-Raf in each group was shown. (b, d, e, f) The histogram are normalized to a GAPDH control and showed the ratio of p-ERK1/2 to T-ERK1/2 and p-c-Raf to T-c-Raf (n≥3). (g, h) HUVECs were pretreated with MEK 1/2 inhibitor (U0126, 10μM) for 30 min. KGA protein expressions were detected by western blot with densitometry analysis (n≥3). *P<0.05 compared to control group. Bars represented means ±SEM.</p

Glutaminolysis is induced by TGF-β1 in HUVECs.

<p>The cells were treated with 10ng/ml of TGF-β1 for different indicated periods, from 15 minutes to 48 hours. (a) KGA protein expression was up-regulated by TGF-β1. Representative western blot of KGA in each group is shown. (b) Densitometry analysis of KGA protein expression was plotted (n≥3). (c) Intracellular glutamate level in the medium was analyzed using theAmplex^®Red Glutamic Acid / Glutamate Oxidase Assay Kit (n≥3). *P<0.05 compared to control group. Bars represented means ± SEM.</p