An Electrochemical Approach to Directed Fluorination.

Electrosynthesis has made a revival in the field of organic chemistry and, in particular, radical-mediated reactions. Herein, we report a simple directed, electrochemical C-H fluorination method. Employing a dabconium mediator, commercially available Selectfluor, and RVC electrodes, we provide a range of steroid-based substrates with competent regioselective directing groups, including enones, ketones, and hydroxy groups, as well as never reported before lactams, imides, lactones, and esters.

Collision-free emergency planning and control methods for CAVs considering intentions of surrounding vehicles.

Autonomous emergency braking (AEB) systems are able to control vehicles as needed to avoid vehicle rear-end collisions. However, these systems are ineffective in scenarios with laterally cut-in vehicles and rapidly-changing dangerous scenes. This paper proposes a novel collision-free emergency braking system (CFEBS) that can enable intelligent connected vehicles (CAVs) to plan and execute a more conservative safety trajectory for the braking process in dangerous scenes by considering the longitudinal and lateral motion intentions of the surrounding vehicles. An intention identification model for surrounding vehicles is proposed based on long-short term memory (LSTM) networks and conditional random fields (CRFs). By considering the surrounding vehicles as risk sources and quantifying the risk with the speed of the risk flow, a potential risk flow model is built to calculate the potential risk map (PRM) around the ego vehicle. The global safest trajectory is generated via the PRM using the discrete method. The output trajectory profile is regarded as the reference for a model predictive controller (MPC). Simulation results show that the proposed CFEBS can predict vehicle intention with 91.6% accuracy and control the ego vehicle to perform effective collision-free braking operations in emergency traffic environments.

Crash mitigation controller for unavoidable T-bone collisions using reinforcement learning.

T-bone collision constitutes an emergency crash scenario that results in casualties and heavy losses; it is an excessively complicated scenario that cannot be handled by conventional control systems. This paper presents an innovative crash mitigation controller for application during unavoidable T-bone collisions to expand the vehicle-maneuverability envelope and minimize crash severity; this controller combines prior knowledge using an optimum expert-behavior policy and drift-operation mechanism based on an improved reinforcement learning algorithm, TD3. Vehicle and tire modeling are performed considering the nonlinear and coupled dynamics characteristics to improve control accuracy. Unlike conventional control systems and other reinforcement learning algorithms, the proposed controller realizes the optimum crash mitigation effect under different scenarios. It is expected to afford autonomous driving technologies with enhanced operating capabilities under extreme conditions.

Autonomous drift controller for distributed drive electric vehicle with input coupling and uncertain disturbance.

Exploring the drift maneuver could extend the dynamic control envelope and application range of autonomous vehicles. This paper presents a novel autonomous drift controller for a distributed drive electric vehicle, whose configuration provides more possibilities for drift. In the upper-level controller, a control channel recombination method transforms the over-actuated system into a standard square system, which is compatible with the proposed fuzzy-integral sliding-mode controller considering the input coupling and uncertain disturbance of the system to bring the vehicle into a marginally stable condition. The operation of the lower-level controller considers the dynamic characteristics of actuators and tires, and distributes the "virtual control input" among each physical actuator. This controller's performance with fast response and strong robustness was proved through the bench test.

Microfluidic technology for multiple single-cell capture.

Microfluidic devices are widely used in single-cell capture and for pairing single cells or groups of cells for cell-cell interaction analysis; these advances have improved drug screening and cell signal transduction analysis. The complex in vivo environment involves interactions between two cells and among multiple cells of the same or different phenotypes. This study reviewed the core principles and performance of several microfluidic multiple- and single-cell capture methods, namely, the microwell, valve, trap, and droplet methods. The advantages and disadvantages of the methods were compared, and suggestions regarding their application to multiple-cell capture were provided. The results may serve as a reference for research on microfluidic multiple single-cell coculture technology.

New strategy for precise cancer therapy: tumor-specific delivery of mitochondria-targeting photodynamic therapy agents and in situ O2-generation in hypoxic tumors.

Besides tumor hypoxia and limitation of superficial lesions, the short lifetime of photoinduced reactive oxygen species (ROS) is another factor repressing photodynamic therapy (PDT) efficacy. To overcome these problems, this study developed newly designed mitochondria-specific, H2O2-activatable, and O2-producing nanoparticles to achieve highly selective and efficient PDT and self-sufficiency of O2 in hypoxic tumors. The newly designed nanoparticles (BDPP NPs) are composed of a mitochondria-targeting photosensitizer and catalase in the aqueous core and a black hole quencher and fluorescent tracker in the polymeric shell, and modified with the tumor-targeting cyclic pentapeptide c(RGDfK). Once taken up by αvß3 integrin-rich tumor cells, intracellular H2O2 easily penetrated the lipophilic shells into the aqueous cores of BDPP NPs, and it was catalyzed by catalase to quickly generate O2 gas, causing the rupture of the NPs to release the photosensitizer. Therefore in vivo tumor cell mitochondria targeting by BDPP can be realized together with the favorable hypoxia relief. In vitro and in vivo experiments demonstrate that the therapeutic efficiency was significantly improved by the mitochondria-specific feature and H2O2-controllable generation of 1O2. More importantly, BDPP NPs continuously generate O2 in the PDT process, which can be helpful for resolving the overconsumption of oxygen in PDT and enhancing the PDT efficiency of cancer chemotherapy. We anticipate that this work may provide new insight into the design of smart PDT systems to achieve highly selective in vivo PDT via targeting subcellular organelles and realize oxygen therapy in O2-deprived tumors.

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells' behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

The Effect of the Repression of Oxidative Stress on Tenocyte Differentiation: A Preliminary Study of a Rat Cell Model Using a Novel Differential Tensile Strain Bioreactor.

Because of limitations in the current understanding of the exact pathogenesis of tendinopathy, and the lack of an optimal experimental model, effective therapy for the disease is currently unavailable. This study aims to prove that repression of oxidative stress modulates the differentiation of tendon-derived cells (TDCs) sustaining excessive tensile strains, and proposes a novel bioreactor capable of applying differential tensile strains to cultured cells simultaneously. TDCs, including tendon-derived stem cells, tenoblasts, tenocytes, and fibroblasts, were isolated from the patellar tendons of Sprague‒Dawley rats. Cyclic uniaxial stretching with 4% or 8% strain at 0.5 Hz for 8 h was applied to TDCs. TDCs subjected to 8% strain were treated with epigallocatechin gallate (EGCG), piracetam, or no medication. Genes representing non-tenocyte lineage (Pparg, Sox9, and Runx2) and type I and type III collagen were analyzed by quantitative polymerase chain reaction. The 8% strain group showed increased expression of non-tenocyte lineage genes and type III/type I collagen ratios compared with the control and 4% strain groups, and the increased expression was ameliorated with addition of EGCG and piracetam. The model developed in this work could be applied to future research on the pathophysiology of tendinopathy and development of treatment options for the disease. Repression of oxidative stress diminishes the expression of genes indicating aberrant differentiation in a rat cell model, which indicates potential therapeutic intervention of tendinopathy, the often relentlessly degenerate condition.

Hydrodynamic shuttling for deterministic high-efficiency multiple single-cell capture in a microfluidic chip.

Studies on cellular heterogeneity have emerged as a powerful approach for developing new strategies to treat diseases including cancer. However, it is difficult to set up an in vitro co-culture experiment to study the interaction of individual live cells. In this paper, we report a hydrodynamic shuttling chip (HSC) which can deterministically capture single cells into microfluidic chambers to set up multiple single-cell co-culture experiments in which individual live cells can be microscopically observed. Using this chip device, we demonstrated a triple single-cell culture of oral squamous cell carcinoma and lymphatic endothelial cells to observe the effect of cell-cell interaction on the cell motility. Triple, single-cell pairing efficiency by our HSC device was eightfold higher than that of the probabilistic method. Using this HSC device, we were able to perform triple-culture experiments to show the cell type-dependent motility of oral squamous cell carcinoma and lymphatic endothelial cells, which was not observed in co-culture experiments.

Long-term T-DNA insert stability and transgene expression consistency in field propagated sugarcane.

KEY MESSAGE: This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. T-DNA inserts are stable; no transgene rearrangements were observed. AmCYAN1 and PMI protein accumulation levels were maintained. There was no evidence that production of either protein declined across generations and no transgene silencing was observed in three commercial sugarcane varieties through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes over 4 years of field testing. Long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized. This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. These data are critical supporting information needed for successful commercialization of GM sugarcane. Here seventeen transgenic events, containing the AmCYAN1 gene driven by a CMP promoter and the E. coli PMI gene driven by either a CMP or Ubi promoter, were used to monitor T-DNA insert stability and consistency of transgene encoded protein accumulation through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes. The experiments were conducted in three commercial sugarcane varieties over 4 years of field testing. DNA gel blot analysis showed that the T-DNA inserts are stable; no transgene rearrangements were observed. Quantitative ELISA showed no evidence of decreasing AmCYAN1 and PMI protein levels across generations and no transgene silencing was observed. These results indicate that long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized.

A microfluidic dual-well device for high-throughput single-cell capture and culture.

In vitro culture of single cells facilitates biological studies by deconvoluting complications from cell population heterogeneity. However, there is still a lack of simple yet high-throughput methods to perform single cell culture experiments. In this paper, we report the development and application of a microfluidic device with a dual-well (DW) design concept for high-yield single-cell loading (~77%) in large microwells (285 and 485 µm in diameter) which allowed for cell spreading, proliferation and differentiation. The increased single-cell loading yield is achieved by using sets of small microwells termed "capture-wells" and big microwells termed "culture-wells" according to their utilities for single-cell capture and culture, respectively. This novel device architecture allows the size of the "culture" microwells to be flexibly adjusted without affecting the single-cell loading efficiency making it useful for cell culture applications as demonstrated by our experiments of KT98 mouse neural stem cell differentiation, A549 and MDA-MB-435 cancer cell proliferation, and single-cell colony formation assay with A549 cells in this paper.

Trait stacking in transgenic crops: challenges and opportunities.

In recent years, there has been a rapid increase in the planting of transgenic crops with stacked traits. Most of these products have been formed by conventional breeding, i.e. the crossing of transgenic plant (event) containing individual transgenes with other event(s) containing single or double transgenic traits. Many biotech companies are developing stacked trait products with increasing numbers of insect and herbicide tolerance genes for controlling a broad range of insect pests and weeds. There has also been an increase in development of technologies for molecular stacking of multiple traits in a single transgene locus. In this review we look at the status of stacked trait products, crop trait stacking technologies and the technical challenges we are facing. We also review recent progress in developing technology for assembling large transgene arrays in vitro (molecular stacks), their delivery to crop plants and issues they pose for transgene expression.

Comparative functional analysis of three abiotic stress-inducible promoters in transgenic rice.

To identify minimal effective promoters for driving abiotic stress-inducible transgene expression in rice, we selected promoter elements of three stress-responsive genes, viz. rab16A coding for dehydrin, OsABA2 coding for zeaxanthin epoxidase, and a gene coding for a hypothetical protein (HP1) based on the presence of ABA-, salt- and drought-responsive cis-acting elements. These were translationally fused to the gusA reporter gene and introduced into rice to study their effect on heterologous gene expression. The OsABA2 promoter was found to be the most effective and desirable promoter among the three in terms of driving a low constitutive transgene expression under normal conditions and high induction in response to ABA, salt and drought stress, the highest being a 12-fold induction in response to ABA. The rab16A and HP1 promoters resulted in high levels of constitutive expression. While induction of GUS activity was generally two- to threefold for all the treatments in roots for both the promoters, induction in leaves was generally insignificant, the exceptions being rab16A in response to continuous salt stress and HP1 in response to water deficit. It was also observed that the three promoters, in general, resulted in lower constitutive expression, but higher induction in roots as compared to leaves.

Identification of a rice actin2 gene regulatory region for high-level expression of transgenes in monocots.

We have isolated and characterized the 5' region of the rice actin2 gene (OsAct2), which contains 793 bp of sequence upstream of the OsAct2 transcription initiation site, 58 bp of the first non-coding exon, 1736 bp of the 5' intron and the first 8 bp (non-coding sequence) of the second exon. It was found that the 5' region of OsAct2 is an efficient gene regulatory region for driving the constitutive expression of foreign genes in transgenic rice. In situ histochemical results indicated that OsAct2::GUS (GUS, beta-glucuronidase) gene expression in transgenic rice plants is high in sporophytic and gametophytic tissues. It was demonstrated that a 2.6-kb upstream sequence of the OsAct2 translation initiation codon contains all of the 5' regulatory elements necessary for high-level gus expression in transgenic rice tissues. OsAct2 promoter activity was significantly enhanced by the deletion of a 1590-bp segment from the central region of the first intron. The +96 to +274 region of the intron negatively regulates gus expression in leaves. To identify regulatory elements within the OsAct2 promoter, nested truncations of the promoter region were made and fused to gus. The results showed that the region from -1 to -376 was sufficient for promoter activity. In addition, two OsAct2-based expression vectors for use in monocot transformation were developed to promote the high-level expression of foreign genes.

An efficient field screening procedure for identifying transposants for constructing an Ac/Ds-based insertional-mutant library of rice.

An efficient system was developed, and several variables tested, for generating a large-scale insertional-mutagenesis population of rice. The most important feature in this improved Ac/Ds tagging system is that one can conveniently carry out large-scale screening in the field and select transposants at the seedling stage. Rice was transformed with a plasmid that includes a Basta-resistance gene (bar). After the Ds element is excised during transposition, bar becomes adjacent to the ubiquitin promoter, and the rice plant becomes resistant to the herbicide Basta. In principle, one can plant up to one million plants in the field and select those plants that survive after spraying with Basta. To test the utility of this system, 4 Ds starter lines were crossed with 14 different Ac plants, and many transposants were successfully identified after planting 134,285 F2 plants in the field. Over 2,800 of these transposants were randomly chosen for PCR analysis, and the results fully confirmed the reliability of the field screening procedure.

An efficient method for producing an indexed, insertional-mutant library in rice.

Generation of an indexed, saturated, insertional-mutant library is an aid to understanding the functions of genes in an organism. However, 10 years of work by many investigators have not yet yielded such a library in rice. The major reason is that determining the chromosomal locations of a very large number of random insertion mutants by flanking sequence analysis is highly labor intensive, and therefore, libraries that do exist have not been indexed. We report here an efficient procedure to construct an indexed, region-specific, insertional-mutant library of rice. The procedure makes use of efficient long-PCR-based high-throughput indexing, coupled with a random but anchored population of Ds transposants. Long-PCR indexing allows rapid and simultaneous determination of the chromosomal locations of a large number of mutants that surround a particular anchor line, thus converting a random library into an indexed one. Such a library can be used directly, without the need to screen a large random library for a desired mutant plant.

Evaluation of the stress-inducible production of choline oxidase in transgenic rice as a strategy for producing the stress-protectant glycine betaine.

Glycine betaine (GB) is a compatible solute that is also capable of stabilizing the structure and function of macromolecules. Several GB-producing transgenic rice lines were generated in which the Arthrobacter pascens choline oxidase (COX) gene, fused to a chloroplast targeting sequence (TP) was expressed under the control of an ABA-inducible promoter (SIP; stress-inducible promoter) or a ubiquitin (UBI) gene promoter that is considered to be constitutive. This comparison led to interesting observations that suggest complex regulation with respect to GB synthesis and plant growth response under stress. In spite of the use of the well-studied stress-inducible promoter, the highest level of GB accumulation (up to 2.60 micromol g(-1) DW) in the SIP lines grown under saline conditions was not as high as in the UBI lines (up to 3.12 micromol g(-1) DW). Therefore, the use of an ABA-inducible promoter was not more beneficial for de novo production of GB. Interestingly, saline growth conditions enhanced GB accumulation by up to 89% in the SIP lines, whereas up to 44% increase was seen in a UBI line. In all these cases the GB levels were many-fold below the range reported for plant species that produce GB naturally. In spite of lower GB concentrations, statistically greater levels of stress tolerance were found in SIP lines than in UBI lines, suggesting that the stress protection observed in SIP plants cannot be totally explained by the increase in the GB content.