Site-specific sonoporation of human melanoma cells at the cellular level using high lateral-resolution ultrasonic micro-transducer arrays

We developed a new instrumental method by which human melanoma cells (LU1205) are sonoporated via radiation pressures exerted by highly-confined ultrasonic waves produced by high lateral-resolution ultrasonic micro-transducer arrays (UMTAs). The method enables cellular-level site-specific sonoporation within the cell monolayer due to UMTAs and can be applicable in the delivery of drugs and gene products in cellular assays. In this method, cells are seeded on the biochip that employs UMTAs for high spatial resolution and specificity. UMTAs are driven by 30-MHz sinusoidal signals and the resulting radiation pressures induce sonoporation in the targeted cells. The sonoporation degree and the effective lateral resolution of UMTAs are determined by performing fluorescent microscopy and analysis of carboxylic-acid-derivatized CdSe/ZnS quantum dots passively transported into the cells. Models representing the transducer-generated ultrasound radiation pressure, the ultrasound-inflicted cell membrane wound, and the transmembrane transport through the wound are developed to determine the ultrasound-pressure-dependent wound size and enhanced cellular uptake of nanoparticles. Model-based calculations show that the effective wound size and cellular uptake of nanoparticles increase linearly with increasing ultrasound pressure (i.e., at applied radiation pressures of 0.21, 0.29, and 0.40 MPa, the ultrasound-induced initial effective wound radii are 150, 460, and 650 nm, respectively, and the post-sonoporation intracellular quantum-dot concentrations are 7.8, 22.8, and 29.9 nM, respectively) and the threshold pressure required to induce sonoporation in LU1205 cells is ～0.12 MPa.     

Preparation of cross-linked lipase-coated micro-crystals for biodiesel production from waste cooking oil

A dual modification procedure composed of cross-linking and protein coating with K₂SO₄ was employed to modify Geotrichum sp. lipase for catalyzing biodiesel production from waste cooking oil. Compared to single modification of protein coating with K₂SO₄, the dual modification of cross-linking and lipase coating improved catalytic properties in terms of thermostable stability, organic solvent tolerance, pH stability and operational stability in biodiesel production process, although biodiesel yield and initial reaction rate for CLPCMCs were not improved. After five successive batch reactions, CLPCMCs could still maintain 80% of relative biodiesel yield. CLPCMCs retained 64% of relative biodiesel yield after incubation in a pH range of 4-6 for 4 h, and 85% of relative biodiesel yield after incubation in a range of 45-50 °C for 4 h. CLPCMCs still maintained 83% of relative biodiesel yield after both treated in polar organic solvent and non-polar organic solvent for 4 h.     

Molecular characterization and mRNA expression of catalase from pearl oyster Pinctada fucata

Catalase (EC 1.11.1.6) is an important antioxidant enzyme that protects aerobic organisms against oxidative damage by degrading hydrogen peroxide to water and oxygen. In the present study, a catalase cDNA of peal oyster Pincatada fucata (designated as PoCAT) is cloned and characterized by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) methods. PoCAT is 2428 bp long and consists of a 5′-UTR of 140 bp, an unusually long 3′-UTR of 749 bp, and an open reading frame (ORF) of 1539 bp. The ORF of PoCAT encodes a polypeptide of 512 amino acids with molecular weight of 58.1 kDa and the theoretical isoelectric point of 8.4. PoCAT shares 62.3–82.2% identity and 73.0–92.0% similarity to other catalase amino acid sequences. Sequence alignment indicates that PoCAT contains the proximal heme-ligand signature sequence (R³⁵¹LFSYSDT³⁵⁸), the proximal active site signature (F⁶¹NRERIPERVVHAKGGGA⁷⁸), and the three catalytic amino acid residues (His⁷², Asn¹⁴⁵, and Tyr ³⁵⁵). PoCAT has two potential glycosylation sites (N⁴³⁶YS⁴³⁸ and N⁴⁷⁸FS⁴⁸⁰) and a peroxisome targeting signal (ASL). PoCAT mRNA was ubiquitously expressed in all detected tissues, and the expression level of PoCAT mRNA was higher in intestine and mantle. The expression profile analysis showed that the expression level of PoCAT mRNA in intestine was significantly up-regulated at 2, 4 and 12 h after Vibrio alginolyticus stimulation. These results demonstrated that PoCAT is a typical member of catalase family and might be involved in innate immune responses of pearl oyster.     

DNAzyme-based highly sensitive electronic detection of lead via quantum dot-assembled amplification labels

An electronic DNAzyme sensor for highly sensitive detection of Pb(2+) is demonstrated by coupling the significant signal enhancement of the layer-by-layer (LBL) assembled quantum dots (QDs) with Pb(2+) specific DNAzymes. The presence of Pb(2+) cleaves the DNAzymes and releases the biotin-modified fragments, which further hybridize with the complementary strands immobilized on the gold substrate. The streptavidin-coated, QD LBL assembled nanocomposites were captured on the gold substrate through biotin-streptavidin interactions. Subsequent electrochemical signals of the captured QDs upon acid dissolution provide quantitative information on the concentrations of Pb(2+) with a dynamic range from 1 to 1000 nM. Due to the dramatic signal amplification by the numerous QDs, subnanomolar level (0.6 nM) of Pb(2+) can be detected. The proposed sensor also shows good selectivity against other divalent metal ions and thus holds great potential for the construction of general DNAzyme-based sensing platform for the monitoring of other heavy metal ions.     

Detection of membrane protein two-dimensional crystals in living cells

It is notoriously difficult to grow membrane protein crystals and solve membrane protein structures. Improved detection and screening of membrane protein crystals are needed. We have shown here that second-order nonlinear optical imaging of chiral crystals based on second harmonic generation can provide sensitive and selective detection of two-dimensional protein crystalline arrays with sufficiently low background to enable crystal detection within the membranes of live cells. The method was validated using bacteriorhodopsin crystals generated in live Halobacterium halobium bacteria and confirmed by electron microscopy from the isolated crystals. Additional studies of alphavirus glycoproteins indicated the presence of localized crystalline domains associated with virus budding from mammalian cells. These results suggest that in vivo crystallization may provide a means for expediting membrane protein structure determination for proteins exhibiting propensities for two-dimensional crystal formation.     

93-kDa twin-domain serine protease inhibitor (Serpin) has a regulatory function on the beetle Toll proteolytic signaling cascade

Serpins are protease inhibitors that play essential roles in the down-regulation of extracellular proteolytic cascades. The core serpin domain is highly conserved, and typical serpins are encoded with a molecular size of 35–50 kDa. Here, we describe a novel 93-kDa protein that contains two complete, tandemly arrayed serpin domains. This twin serpin, SPN93, was isolated from the larval hemolymph of the large beetle Tenebrio molitor. The N-terminal serpin domain of SPN93 forms a covalent complex with the Spätzle-processing enzyme, a terminal serine protease of the Toll signaling cascade, whereas the C-terminal serpin domain of SPN93 forms complexes with a modular serine protease and the Spätzle-processing enzyme-activating enzyme, which are two different enzymes of the cascade. Consequently, SPN93 inhibited β-1,3-glucan-mediated Toll proteolytic cascade activation in an in vitro system. Site-specific proteolysis of SPN93 at the N-terminal serpin domain was observed after activation of the Toll proteolytic cascade in vivo, and down-regulation of SPN93 by RNAi sensitized β-1,3-glucan-mediated larval death. Therefore, SPN93 is the first serpin that contains twin tandemly arrayed and functionally active serpin domains that have a regulatory role in the larval Toll proteolytic signaling cascade.     

Phytane and neoclerodane diterpenes from the aerial parts of Inula nervosa Wall.

Three new aliphatic diterpenes (1–3), together with three known neoclerodane-type diterpenes (4–6) were isolated from the aerial parts of Inula nervosa Wall. The structures of 1-3 were elucidated on the basis of 1D and 2D spectroscopic analysis. Additionally, phytane-type and neoclerodane-type diterpenes have not been reported in any species of the genus Inula yet. The phytane-type and neoclerodane-type diterpenes obtained from I. nervosa Wall. suggest this plant maybe have remote genetic relations with other Inula species.     

Methane emission and soil microbial communities in early rice paddy as influenced by urea-N fertilization

Plant and Soil - Moso bamboo (Phyllostachys edulis) invasions into adjacent forests are becoming increasingly common. Moso bamboo invasions affect litter quality, soil nutrients, and microbial...     

Expression profile of circular RNA in rat intimal hyperplasia and target gene prediction

Intimal hyperplasia is an important cause of stenosis or occlusion after vascular injury. Circular RNAs (circRNAs) are known to be related to various cardiovascular diseases. However, the expression profile of circRNAs in the neointima has not been reported in detail. In this study, we established a rat common carotid artery (CCA) injury model. A microarray detection showed significant differences in circRNA expression between the normal and injured CCA. Real-time quantitative polymerase chain reaction verified the differences. We used bioinformatics to predict the microRNAs that possibly interact with the differentially expressed (DE) circRNAs and linked the potential functions of circRNAs to the target genes of the microRNAs. We believe that the DE circRNA in neointima may affect the differentiation, proliferation, and migration of vascular cells through a variety of target genes. The intervention or utilization of certain circRNAs should be a new method for preventing and treating intimal hyperplasia.