A novel electrogenerated chemiluminescence peptide-based biosensor (ECL-PB) for the determination of prostate-specific antigen (PSA) was developed on the basis of target-induced cleavage of a specific peptide within Nafion film incorporated with gold nanoparticles (AuNPs) and ECL emitting species. A specific peptide (CHSSKLQK) was used as a molecular recognition element; tris(2,2'-ripyridine) dichlororuthenium(II) (Ru(bpy)3(2+)) was used as ECL emitting species, and ferrocene carboxylic acid (Fc) was employed as ECL quencher. The ECL-PB biosensor was fabricated by casting the mixture of Nafion and AuNPs onto the surface of glassy carbon electrode to form AuNPs/Nafion film, and then, Ru(bpy)3(2+) was electrostatically adsorbed into the AuNPs/Nafion film; finally, the peptide-tagged with ferrocene carboxylic acid (Fc-peptide) was self-assembled onto the surface of the AuNPs. When PSA was present, it specifically cleaved the Fc-peptide, leading the quencher to leave the electrode and resulting in the increase of the ECL intensity obtained from the resulted electrode in 0.1 M phosphate buffer saline (pH 7.4) containing tri-n-propylamine. The results showed that the increased ECL intensity was directly linear to the logarithm of the concentration of PSA in the range from 5.0 × 10(-12) to 5.0 × 10(-9) g/mL. An extremely low detection limit of 8 × 10(-13) g/mL was achieved because of the signal amplification through AuNPs and the ECL background suppression through Fc as ECL quencher. This work demonstrates that the combination of the direct transduction of peptide cleavage events with the highly sensitive ECL method is a promising strategy for the design of enzymatic cleavage-based ECL biosensors with high sensitivity and selectivity.

Haematococcus pluvialis is a commercial microalga, that produces abundant levels of astaxanthin under stress conditions. Acetate and Fe2+ are reported to be important for astaxanthin accumulation in H. pluvialis. In order to study the synergistic effects of high light stress and these two factors, we obtained transcriptomes for four groups: high light irradiation (HL), addition of 25 mM acetate under high light (HA), addition of 20 μM Fe2+ under high light (HF) and normal green growing cells (HG). Among the total clean reads of the four groups, 156,992 unigenes were found, of which 48.88% were annotated in at least one database (Nr, Nt, Pfam, KOG/COG, SwissProt, KEGG, GO). The statistics for DEGs (differentially expressed genes) showed that there were more than 10 thousand DEGs caused by high light and 1800–1900 DEGs caused by acetate or Fe2+. The results of DEG analysis by GO and KEGG enrichments showed that, under the high light condition, the expression of genes related to many pathways had changed, such as the pathway for carotenoid biosynthesis, fatty acid elongation, photosynthesis-antenna proteins, carbon fixation in photosynthetic organisms and so on. Addition of acetate under high light significantly promoted the expression of key genes related to the pathways for carotenoid biosynthesis and fatty acid elongation. Furthermore, acetate could obviously inhibit the expression of genes related to the pathway for photosynthesis-antenna proteins. For addition of Fe2+, the genes related to photosynthesis-antenna proteins were promoted significantly and there was no obvious change in the gene expressions related to carotenoid and fatty acid synthesis.

Inflammation plays a pivotal role in the pathogenesis of many diseases in the central nervous system. Caudate nucleus (CN), the largest nucleus in the brain, is also implicated in many neurological disorders. 2-Arachidonoylglycerol (2-AG), the most abundant endogenous cannabinoid and the true natural ligand for CB1 receptors, has been shown to exhibit neuroprotective effects through its anti-inflammatory action from proinflammatory stimuli in hippocampus. However, it is still not clear whether 2-AG is also able to protect CN neurons from proinflammation stimuli. In the present study, we discovered that 2-AG significantly protects CN neurons in culture against lipopolysaccharide (LPS)-induced inflammatory response. 2-AG is capable of suppressing elevation of LPS-induced cyclooxygenase-2 expression associated with ERK/p38MAPK/NF-κB signaling pathway in CB1 receptor-dependant manner in primary cultured CN neurons. Moreover, 2-AG inhibits LPS-induced increase in voltage-gated sodium channel currents and hyperpolarizing shift of activation curves through CB1 receptor-dependant pathway. Our study suggests the therapeutic potential of 2-AG for the treatment of some inflammation-induced neurological disorders and pain.

A sensitive electrogenerated chemiluminescence (ECL) bioassay was developed for the detection of two protein kinases incorporating the peptide phosphorylation and a versatile ECL probe. Cyclic adenosine monophosphate-dependent protein kinase (PKA) and casein kinase II (CK2) were used as proof-of-concept targets while a PKA-specific peptide (CLRRASLG) and a CK2-specific peptide (CRRRADDSDDDDD) were used as the recognition substrates. Taking advantage of the ability of protein A binding with the Fc region of a variety of antibodies with high affinity, a ruthenium derivative-labeled protein A was utilized as a versatile ECL probe for bioassay of multiple protein kinases. A specific peptide substrate toward target protein kinase was first self-assembled on the surface of gold electrode and then serine in the specific peptide on the electrode was phosphorylated by target protein kinase in the presence of adenosine-5′-triphosphate. After recognition of the phosphorylated peptide by monoclonal antiphosphoserine antibody, the versatile ECL probe was specifically bound to the antiphosphoserine antibody on the electrode surface. The ECL bioassay was developed successfully in the individual detection of PKA and CK2 with detection limit of 0.005 U/mL and 0.004 U/mL, respectively. In addition, the ECL bioassay was applied to quantitative analysis of the kinase inhibitors and monitoring drug-triggered kinase activation in cell lysates. Moreover, an ECL imaging bioassay using electron-multiplying charged coupled device as detector on the gold electrode array was developed for the simultaneous detection of PKA and CK2 activity from 0.01 U/mL to 0.4 U/mL, respectively, at one time. This work demonstrates that the ingenious design and use of a versatile ECL probe are promising to simultaneous detection of multiple protein kinases and screening of kinase inhibitor.

Homocysteine (Hcy) is a high risk factor for Alzheimer's disease (AD). Caudate nucleus (CN), the major component of basal ganglia in the brain, is also involved in many neurological disorders. 2-Arachidonoylglycerol (2-AG), the true natural ligand for cannabinoid type-1 (CB1) receptors and the most abundant endogenous cannabinoid, has been shown to exhibit neuroprotective effects through its anti-inflammatory action from proinflammatory stimuli in the hippocampus and CN. However, it is still not well understood whether that 2-AG is also able to protect CN neurons from Hcy harmful insults. In the present work, we explored that 2-AG significantly protects CN neurons in culture against Hcy-induced response. 2-AG is capable of inhibiting elevation of Hcy-induced cyclooxygenase-2 expression associated with nuclear factor-kappaB/p38MAPK/ERK1/2 signaling pathway through CB1 receptors-dependent way in primary cultured CN neurons. Our study reveals the therapeutic potential for 2-AG for the treatment of neurodegenerative diseases, such as AD.