Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera

Background: Camellia oleifera (C. oleifera) is a woody edible oil crop of great economic importance. Because of the lack of modern biotechnology research, C. oleifera faces huge challenges in both breeding and basic research. The protoplast and transient transformation system plays an important role in biological breeding, plant regeneration and somatic cell fusion. The objective of this present study was to develop a highly efficient protocol for isolating and purifying mesophyll protoplasts and transient transformation of C. oleifera. Several critical factors for mesophyll protoplast isolation from C. oleifera, including starting material (leaf age), pretreatment, enzymatic treatment (type of enzyme, concentration and digestion time), osmotic pressure and purification were optimized. Then the factors affecting the transient transformation rate of mesophyll protoplasts such as PEG molecular weights, PEG4000 concentration, plasmid concentration and incubation time were explored.Results: The in vitro grown seedlings of C. oleifera 'Huashuo' were treated in the dark for 24 h, then the 1st to 2nd true leaves were picked and vacuumed at - 0.07 MPa for 20 min. The maximum yield (3.5 x 10(7)/g.W) and viability (90.9%) of protoplast were reached when the 1st to 2nd true leaves were digested in the enzymatic solution containing1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10 and 0.25% (w/v) Snailase and 0.4 M mannitol for 10 h. Moreover, the protoplast isolation method was also applicable to the other two cultivars, the protoplast yield for 'TXP14' and 'DP47' was 1.1 x 10(7)/g.FW and 2.6 x 10(7)/g. FW, the protoplast viability for 'TXP14' and 'DP47' was 90.0% and 88.2%. The purification effect was the best when using W buffer as a cleaning agent by centrifugal precipitation. The maximum transfection efficiency (70.6%) was obtained with the incubation of the protoplasts with 15 mu g plasmid and 40% PEG4000 for 20 min.Conclusion: In summary, a simple and efficient system for isolation and transient transformation of C. oleifera mesophyll protoplast is proposed, which is of great significance in various aspects of C. oleifera research, including the study of somatic cell fusion, genome editing, protein function, signal transduction, transcriptional regulation and multi-omics analyses

Multi-classifier fusion based on belief-value for the diagnosis of autism spectrum disorder

IntroductionAutism Spectrum Disorder (ASD) has a significant impact on the health of patients, and early diagnosis and treatment are essential to improve their quality of life. Machine learning methods, including multi-classifier fusion, have been widely used for disease diagnosis and prediction with remarkable results. However, current multi-classifier fusion methods lack the ability to measure the belief level of different samples and effectively fuse them jointly.MethodsTo address these issues, a multi-classifier fusion classification framework based on belief-value for ASD diagnosis is proposed in this paper. The belief-value measures the belief level of different samples based on distance information (the output distance of the classifier) and local density information (the weight of the nearest neighbor samples on the test samples), which is more representative than using a single type of information. Then, the complementary relationships between belief-values are captured via a multilayer perceptron (MLP) network for effective fusion of belief-values.ResultsThe experimental results demonstrate that the proposed classification framework achieves better performance than a single classifier and confirm that the fusion method used can effectively fuse complementary relationships to achieve accurate diagnosis.DiscussionFurthermore, the effectiveness of our method has only been validated in the diagnosis of ASD. For future work, we plan to extend this method to the diagnosis of other neuropsychiatric disorders

Design, characterization of amphiphilic proteins and potential engineering applications

Orienting molecules as an ensemble to generate macroscopic response is the key step in realizing material applications. Based on hydrophobic concept, de novo designed maquettes have been rendered to be protein amphiphiles to create an ensemble at the air-water interface. Initially, we seek to construct a monolayer composed of four-α-helical bundles formed by association of di-α-helical peptides which are attached with hydrocarbon chains at the N-termini of peptide. Then, membrane proteins that mimic natural heme proteins, and vectorially-orient at the air-water interface as a well-ordered ensemble have been developed. A significant progress has been achieved in the area of development of transmembrane proteins that are capable of incorporating pigment cofactors required to promote light activated electron transfer. These designed membrane proteins have been demonstrated to insert into host phospholipids monolayers and to vectorially orient in the membrane. This new finding has been successfully applied to the design of model membrane proteins to understand the anesthetic binding mechanism

D Amino Acids Highlight the Catalytic Power of the Ribosome

The possible mechanism(s) by which ribosomes make peptide bonds during protein synthesis have been explored for decades. Yet, there is no agreement on how the catalytic site, the peptidyl transferase center (PTC), promotes this reaction. Here, we discuss the results of recent investigations of translation with D amino acids that provide fresh insights into that longstanding question

Allosteric regulation in NMDA receptors revealed by the genetically encoded photo-cross- linkers

International audienceAllostery is essential to neuronal receptor function, but its transient nature poses a challenge for characterization. The N-terminal domains (NTDs) distinct from ligand binding domains are a major locus for allosteric regulation of NMDA receptors (NMDARs), where different modulatory binding sites have been observed. The inhibitor ifenprodil, and related phenylethanoamine compounds specifically targeting GluN1/GluN2B NMDARs have neuroprotective activity. However, whether they use differential structural pathways than the endogenous inhibitor Zn2+ for regulation is unknown. We applied genetically encoded unnatural amino acids (Uaas) and monitored the functional changes in living cells with photo-cross-linkers specifically incorporated at the ifenprodil binding interface between GluN1 and GluN2B subunits. We report constraining the NTD domain movement, by a light induced crosslinking bond that introduces minimal perturbation to the ligand binding, specifically impedes the transduction of ifenprodil but not Zn2+ inhibition. Subtle distance changes reveal interfacial flexibility and NTD rearrangements in the presence of modulators. Our results present a much richer dynamic picture of allostery than conventional approaches targeting the same interface, and highlight key residues that determine functional and subtype specificity of NMDARs. The light-sensitive mutant neuronal receptors provide complementary tools to the photo-switchable ligands for opto-neuropharmacology

Development of a new method to identify aminoacylated RNA

A RT-PCR method is developed to isolate RNA aminoacylated on their 3’ end from large pools of RNA. The method is being applied in two separate projects. We are interested in isolating a new class of ribozymes that could successively catalyze the two chemical reactions leading to their own 3’ aminoacylation (ATP activation of an amino acid followed by 3' esterification of the RNA). The catalysis of each of the two reactions has independently been demonstrated for some RNA isolated with the SELEX methodology [1-2]. However, the coupling of both reactions on a same molecule has not been achieved yet. The identification of these still hypothetical ribozymes may help understand how the former translation system started in the absence of the aminoacyltRNA Synthetase, which catalyzes the above two reactions on tRNA in modern cells. In another project, we would like to identify the whole repertoire of aminoacylated RNA (the “aminoacylome”) in cells. There are strong indications that other RNA besides tRNA and tmRNA may be aminoacylated for biological purposes [3-4]

The operations and properties of reproducibility of the empty marking

Kurt Lautenbach presented sufficient and necessary conditions for reproducibility of the empty marking and proved that the empty marking is reproducible if and only if there is non-negative T-invariant, whose net representations have neither siphons nor traps, containing a positive entry for at least one fact and goal transition. This paper extends these results, we prove that composition, insertion, deletion and substitution do not influence reproducibility of the empty marking, and a net with reproducibility of the empty marking preserves the reproducibility in its backward net (N is a net, its backward net N-1 arises from N by reversing the direction of all arcs). We also show that the empty marking in acyclic P/T nets with a positive entry for at least one fact and goal transition is reproducible if and only if the net is covered by T-invariant

Fine-Scale Population Estimation by 3D Reconstruction of Urban Residential Buildings

Fine-scale population estimation is essential in emergency response and epidemiological applications as well as urban planning and management. However, representing populations in heterogeneous urban regions with a finer resolution is a challenge. This study aims to obtain fine-scale population distribution based on 3D reconstruction of urban residential buildings with morphological operations using optical high-resolution (HR) images from the Chinese No. 3 Resources Satellite (ZY-3). Specifically, the research area was first divided into three categories when dasymetric mapping was taken into consideration. The results demonstrate that the morphological building index (MBI) yielded better results than built-up presence index (PanTex) in building detection, and the morphological shadow index (MSI) outperformed color invariant indices (CIIT) in shadow extraction and height retrieval. Building extraction and height retrieval were then combined to reconstruct 3D models and to estimate population. Final results show that this approach is effective in fine-scale population estimation, with a mean relative error of 16.46% and an overall Relative Total Absolute Error (RATE) of 0.158. This study gives significant insights into fine-scale population estimation in complicated urban landscapes, when detailed 3D information of buildings is unavailable