Anti-Fouling Magnetic Beads Combined with Signal Amplification Strategies for Ultra-Sensitive and Selective Electrochemiluminescence Detection of MicroRNAs in Complex Biological Media.

Herein, an electrochemiluminescence (ECL) microRNA biosensor based on anti-fouling magnetic beads (MBs) and two signal amplification strategies was developed. The newly designed anti-fouling dendritic peptide was wrapped on the surfaces of MBs to make them resistant to nonspecific adsorption of biomolecules in complex biological samples so as to realize accurate and selective target recognition. One of the amplification strategies was achieved through nucleic acid cycle amplification based on the DNAzyme on the surfaces of MBs. Then, the output DNA generated by the nucleic acid cycle amplification program stimulated the hybrid chain reaction (HCR) process on the modified electrode surface to generate the other amplification of the ECL response. Titanium dioxide nanoneedles (TiO2 NNs), as a co-reaction accelerator of the Ru(bpy)2(cpaphen)2+ and tripropylamine (TPrA) system, were wrapped with the electrodeposited polyaniline (PANI) on the electrode surface to enhance the ECL intensity of Ru(bpy)2(cpaphen)2+. The conducting polymer PANI can not only immobilize the TiO2 NNs but also improve the conductivity of the modified electrodes. The biosensor exhibited ultra-high sensitivity and excellent selectivity toward the detection of miRNA 21, with a detection limit of 0.13 fM. More importantly, with the anti-fouling MBs as a unique separation tool, this ECL biosensor was capable of assaying targets in complex biological media such as serum and cell lysate.

Overexpression of RING Domain E3 Ligase ZmXerico1 Confers Drought Tolerance through Regulation of ABA Homeostasis.

Drought stress is one of the main environmental problems encountered by crop growers. Reduction in arable land area and reduced water availability make it paramount to identify and develop strategies to allow crops to be more resilient in water-limiting environments. The plant hormone abscisic acid (ABA) plays an important role in the plants' response to drought stress through its control of stomatal aperture and water transpiration, and transgenic modulation of ABA levels therefore represents an attractive avenue to improve the drought tolerance of crops. Several steps in the ABA-signaling pathway are controlled by ubiquitination involving really interesting new genes (RING) domain-containing proteins. We characterized the maize (Zea mays) RING protein family and identified two novel RING-H2 genes called ZmXerico1 and ZmXerico2 Expression of ZmXerico genes is induced by drought stress, and we show that overexpression of ZmXerico1 and ZmXerico2 in Arabidopsis and maize confers ABA hypersensitivity and improved water use efficiency, which can lead to enhanced maize yield performance in a controlled drought-stress environment. Overexpression of ZmXerico1 and ZmXerico2 in maize results in increased ABA levels and decreased levels of ABA degradation products diphaseic acid and phaseic acid. We show that ZmXerico1 is localized in the endoplasmic reticulum, where ABA 8'-hydroxylases have been shown to be localized, and that it functions as an E3 ubiquitin ligase. We demonstrate that ZmXerico1 plays a role in the control of ABA homeostasis through regulation of ABA 8'-hydroxylase protein stability, representing a novel control point in the regulation of the ABA pathway.

Magnetic antifouling material based ratiometric electrochemical biosensor for the accurate detection of CEA in clinical serum.

A novel ratio electrochemical biosensor based on multi-functional nanocomposite was developed. Fe3O4 was synthesized in situ on carboxyl functionalized 2D nanomaterial MXene, and then covalently bonded with [Ru(NH3)6]3+ to obtain nanocomposites MXC-Fe3O4-Ru. Fe3O4 and [Ru(NH3)6]3+ can neutralize the electronegativity of the MXene to make the nanocomposites electrically neutral. Combine with the good hydrophilicity and conductivity of MXene, the nanocomposites can be utilized to construct antifouling electrochemical biosensors without modifying with specific antifouling materials. Moreover, Fe3O4 can endow the nanocomposites with magnetism, and [Ru(NH3)6]3+ is used as an internal standard molecule. The strong magnetic MXC-Fe3O4-Ru can be easily separated and firmly modified on the magnetic gold electrode (MGE). DNA double-stranded (dsDNA) containing an ferrocene (Fc)-modified carcinoembryonic antigen (CEA) aptamer can be specifically captured to the surface of the electrode by amido bond. In the presence of CEA, CEA binds to the aptamer and leaves the electrode surface, the electrochemical signal of Fc decreases, while the electrochemical signal of [Ru(NH3)6]3+ is fixed on the electrode surface remains basically unchanged. The ratio of the electrochemical signals of Fc and [Ru(NH3)6]3+ is proportional to the CEA concentration. The linear range of the sensor is 1 pg/mL to 1 µg/mL with a detection limit of 0.62 pg/mL. With the excellent antifouling performance, good conductivity of the nanocomposite, and the application of the ratiometric strategy, the biosensor can achieve high selectivity, accuracy, and sensitivity for the detection of targets even in complex samples, such as FBS and clinical serum.

Arabidopsis Carboxylesterase 20 Binds Strigolactone and Increases Branches and Tillers When Ectopically Expressed in Arabidopsis and Maize.

Severe drought stress can delay maize silk emergence relative to the pollen shedding period, resulting in poor fertilization and reduced grain yield. Methods to minimize the delay in silking could thus improve yield stability. An Arabidopsis enhancer-tagged carboxylesterase 20 (AtCXE20) line was identified in a drought tolerance screen. Ectopic expression of AtCXE20 in Arabidopsis and maize resulted in phenotypes characteristic of strigolactone (SL)-deficient mutants, including increased branching and tillering, decreased plant height, delayed senescence, hyposensitivity to ethylene, and reduced flavonols. Maize silk growth was increased by AtCXE20 overexpression, and this phenotype was partially complemented by exogenous SL treatments. In drought conditions, the transgenic maize plants silked earlier than controls and had decreased anthesis-silking intervals. The purified recombinant AtCXE20 protein bound SL in vitro, as indicated by SL inhibiting AtCXE20 esterase activity and altering AtCXE20 intrinsic fluorescence. Homology modeling of the AtCXE20 three-dimensional (3D) protein structure revealed a large hydrophobic binding pocket capable of accommodating, but not hydrolyzing SLs. The AtCXE20 protein concentration in transgenic maize tissues was determined by mass spectrometry to be in the micromolar range, well-above known endogenous SL concentrations. These results best support a mechanism where ectopic expression of AtCXE20 with a strong promoter effectively lowers the concentration of free SL by sequestration. This study revealed an agriculturally important role for SL in maize silk growth and provided a new approach for altering SL levels in plants.

Expression of the Arabidopsis thaliana BBX32 gene in soybean increases grain yield.

Crop yield is a highly complex quantitative trait. Historically, successful breeding for improved grain yield has led to crop plants with improved source capacity, altered plant architecture, and increased resistance to abiotic and biotic stresses. To date, transgenic approaches towards improving crop grain yield have primarily focused on protecting plants from herbicide, insects, or disease. In contrast, we have focused on identifying genes that, when expressed in soybean, improve the intrinsic ability of the plant to yield more. Through the large scale screening of candidate genes in transgenic soybean, we identified an Arabidopsis thaliana B-box domain gene (AtBBX32) that significantly increases soybean grain yield year after year in multiple transgenic events in multi-location field trials. In order to understand the underlying physiological changes that are associated with increased yield in transgenic soybean, we examined phenotypic differences in two AtBBX32-expressing lines and found increases in plant height and node, flower, pod, and seed number. We propose that these phenotypic changes are likely the result of changes in the timing of reproductive development in transgenic soybean that lead to the increased duration of the pod and seed development period. Consistent with the role of BBX32 in A. thaliana in regulating light signaling, we show that the constitutive expression of AtBBX32 in soybean alters the abundance of a subset of gene transcripts in the early morning hours. In particular, AtBBX32 alters transcript levels of the soybean clock genes GmTOC1 and LHY-CCA1-like2 (GmLCL2). We propose that through the expression of AtBBX32 and modulation of the abundance of circadian clock genes during the transition from dark to light, the timing of critical phases of reproductive development are altered. These findings demonstrate a specific role for AtBBX32 in modulating soybean development, and demonstrate the validity of expressing single genes in crops to deliver increased agricultural productivity.