Genomic analysis reveals phylogeny of Zygophyllales and mechanism for water retention of a succulent xerophyte.

Revealing the genetic basis for stress-resistant traits in extremophile plants will yield important information for crop improvement. Zygophyllum xanthoxylum, an extant species of the ancient Mediterranean, is a succulent xerophyte that can maintain a favorable water status under desert habitats; however, the genetic basis of this adaptive trait is poorly understood. Furthermore, the phylogenetic position of Zygophyllales, to which Z. xanthoxylum belongs, remains controversial. In this study, we sequenced and assembled the chromosome-level genome of Z. xanthoxylum. Phylogenetic analysis showed that Zygophyllales and Myrtales form a separated taxon as a sister to the clade comprising fabids and malvids, clarifying the phylogenetic position of Zygophyllales at whole-genome scale. Analysis of genomic and transcriptomic data revealed multiple critical mechanisms underlying the efficient osmotic adjustment using Na+ and K+ as "cheap" osmolytes that Z. xanthoxylum has evolved through the expansion and synchronized expression of genes encoding key transporters/channels and their regulators involved in Na+/K+ uptake, transport, and compartmentation. It is worth noting that ZxCNGC1;1 (cyclic nucleotide-gated channels) and ZxCNGC1;2 constituted a previously undiscovered energy-saving pathway for Na+ uptake. Meanwhile, the core genes involved in biosynthesis of cuticular wax also featured an expansion and upregulated expression, contributing to the water retention capacity of Z. xanthoxylum under desert environments. Overall, these findings boost the understanding of evolutionary relationships of eudicots, illustrate the unique water retention mechanism in the succulent xerophyte that is distinct from glycophyte, and thus provide valuable genetic resources for the improvement of stress tolerance in crops and insights into the remediation of sodic lands.

Anti-Carbonic Anhydrase Goldbodies by Conformational Reconstruction of the Complementary-Determining Regions of Phage-Displayed Antibodies.

It has been demonstrated that the main complementary-determining region (CDR) fragments of antibodies could be grafted onto gold nanoparticles (AuNPs) to produce artificial antibody, dubbed Goldbody. Goldbody maintains the same binding specificity with the corresponding antigen as the original antibody, but has better stability than the antibody. However, the current design of Goldbodies is mainly based on the structures of antibody-antigen complexes. To extend this promising technique to the majority of antibodies whose complexes with the corresponding antigens are not structurally solved, herein, two anti-carbonic anhydrase (CA) antibodies screened by phage display were chosen to create anti-CA Goldbodies. One of the anti-CA antibodies, cAb-CA05, has a known complex structure with CA; but the other, cAb-CA06, does not. By conformational reconstruction of the CDR3 of cAb-CA06, which is identified by sequence alignment, as well as the CDR3 of cAb-CA05, two anti-CA Goldbodies have been created. Interestingly, our results show the two Goldbodies can bind to CA simultaneously, unambiguously indicating their binding sites on CA are far away. As the CDR3 is the major binding unit for many antibodies, which can be reliably predicted by sequence alignment, it could be used as a general strategy to develop artificial antibodies by directly grafting and conformationally reconstructing the predicted CDR3 of antibodies.

PEGylation of Goldbody: PEG-aided conformational engineering of peptides on gold nanoparticles.

It is still a great challenge to engineer flexible non-functional molecules into special conformations to carry out novel functions. Previously, we successfully restored the native conformations and functions of the flexible complementary-determining regions (CDRs) of antibodies on the surface of gold nanoparticles (AuNPs), and created a class of AuNP-based artificial antibodies, denoted as Goldbodies. Yet, in these Goldbodies, there are dozens of CDRs on one Goldbody. Herein, we show that the number of CDRs per Goldbody could be reduced by more than one order of magnitude, by replacing the majority of the CDRs with polyethylene glycol (PEG) with a molecular weight around 600 Da, while the native conformations and functions of the CDRs could still be restored on AuNPs. Also, we find that the PEG with two terminal -SH groups is much better than the PEG with a single -SH group for aiding the restoration of the native conformation of the CDRs on AuNPs. To demonstrate the potential generic applicability of the PEGylation in aiding conformational engineering of peptides, two PEGylated Goldbodies have been created, which can specifically recognize lysozyme and epidermal growth factor receptor, respectively. The PEGylated Goldbodies further prove the mechanism of conformational engineering and the "Confined Lowest Energy Fragments" (CLEFs) hypothesis, and pave the way for future applications of Goldbodies.

Folding of Flexible Protein Fragments and Design of Nanoparticle-Based Artificial Antibody Targeting Lysozyme.

It is generally believed that a protein's sequence solely determines its native structure, but how the long- and short-range interactions jointly determine the native structure/conformation of the protein or every local fragment of the protein is still not fully understood. Since most protein fragments are unstructured on their own, direct observation of the folding of flexible protein fragments is very difficult. Interestingly, we show that it is possible to graft the complementary-determining regions (CDRs) of antibodies onto the surface of a gold nanoparticle (AuNP) to create AuNP-based artificial antibodies (denoted as Goldbodies), such as an antilysozyme Goldbody. Goldbodies can specifically recognize the corresponding antigens like the original natural antibodies do, but direct structural evidence for the refolding or restoration of native conformation of the grafted CDRs on AuNPs is still missing and in high demand. Herein we design a new Goldbody that targets an epitope on the lysozyme different from that of the previous antilysozyme Goldbody, and the one circle of helix in the CDR makes it possible to distinguish the unfolded conformation of the free CDR and its folded conformation on AuNPs by circular dichroism (CD) spectroscopy. The refolding of flexible protein fragments on NPs provides unique evidence and inspiration for understanding the fundamental principles of protein folding.

Conformationally engineering flexible peptides on silver nanoparticles.

Molecular conformational engineering is to engineer flexible non-functional molecules into unique conformations to create novel functions just like natural proteins fold. Obviously, it is a grand challenge with tremendous opportunities. Based on the facts that natural proteins are only marginally stable with a net stabilizing energy roughly equivalent to the energy of two hydrogen bonds, and the energy barriers for the adatom diffusion of some metals are within a similar range, we propose that metal nanoparticles can serve as a general replacement of protein scaffolds to conformationally engineer protein fragments on the surface of nanoparticles. To prove this hypothesis, herein, we successfully restore the antigen-recognizing function of the flexible peptide fragment of a natural anti-lysozyme antibody on the surface of silver nanoparticles, creating a silver nanoparticle-base artificial antibody (Silverbody). A plausible mechanism is proposed, and some general principles for conformational engineering are summarized to guide future studies in this area.

Reduced Graphene Oxide (rGO)-Supported and Pyrolytic Carbon (PC)-Coated γ-Fe2 O3 /PC-rGO Composite Anode Material with Enhanced Li Storage Performance.

As a high-capacity anode material for lithium ion batteries, γ-Fe2 O3 is a promising alternative to conventional graphite among multifarious transition metal oxides owing to its high theoretical specific capacity (1007 mAh g-1 ), abundant reserves, good safety and low cost. However, improving the electrical conductivity and overcoming the morphological damage caused by the severe volume expansion during cycling are still the tricky problems to be solved. Herein, a three-dimensional heterostructure composite (γ-Fe2 O3 /PC-rGO60 ) was prepared by a facile solvothermal reaction followed by heat treatment in inert atmosphere. This composite material exhibits a reversible charge specific capacity of 1035 mAh g-1 at the current density of 0.1 A g-1 . After 100 cycles at 0.2 A g-1 , the capacity is increased from 966.2 to 1091.1 mAh g-1 . Even cycled for 200 cycles at 1 A g-1 , the capacity is only decreased from 751.4 to 670.6 mAh g-1 , giving capacity retention of 89.3%. The rGO network supported flexible composite architecture is beneficial for accommodating the volume expansion of the γ-Fe2 O3 active material during the lithiation/delithiation process. Besides, the conductive rGO network and the in-situ formed pyrolytic carbon (PC) can provide a smooth electron transmission path and a favorable lithium ion transport channel.

A Potential MDM2 Inhibitor Formed by Restoring the Native Conformation of the p53 α-Helical Peptide on Gold Nanoparticles.

Many efforts have been made to develop inhibitors of MDM2 as potential drugs for cancer therapy. In this work, we use our previous developed conformational engineering technique to stabilize the binding conformation of the p53 transcription activation domain (TAD) peptide on gold nanoparticles (AuNPs), and create an AuNP-based anti-MDM2 artificial antibody, denoted as anti-MDM2 Goldbody, that specifically binds MDM2. Though the free TAD peptide is unstructured, circular dichroism (CD) spectra confirm that its α-helical conformation in the original p53 protein is restored on the anti-MDM2 Goldbody, and surface plasmon resonance (SPR) experiments confirm that there is strong specific interaction between the anti-MDM2 Goldbody and MDM2, demonstrating the anti-MDM2 Goldbody as a potential inhibitor of MDM2. This work demonstrates that the conformational engineering technique is not limited to the antigen-antibody systems, but can also be applied more widely in other protein-protein interfaces to create increasingly more artificial proteins for various biomedical applications.

Clinical Randomized Controlled Study of Acupuncture Treatment on Children with Autism Spectrum Disorder (ASD): A Systematic Review and Meta-Analysis.

This study aimed to summarize the effectiveness and safety of acupuncture in the treatment of autism spectrum disorder (ASD) through literature analysis and evaluation. All studies were retrieved from various databases as follows: English databases, such as PubMed, Cochrane Library, Ovid, and Web of Science, and Chinese databases, such as China National Knowledge Infrastructure (CNKI), WanFang Data (WF), and Technology Periodical Database (VIP). The Cochrane Collaboration's Bias Risk Assessment Scale was used to assess the studies' risk of bias. The effects of acupuncture treatment for ASD were determined using the following indicators: childhood autism rating scale (CARS), autism behavior check list (ABC), Reynell developmental language scale (RDLS), and functional independence measure of children (WeeFIM). The risk map of bias of these studies' quality and the meta-analysis results of the indicators was prepared with RevMan 5.2 software. Finally, 16 studies were included, five of which were in English and 11 were in Chinese. The 16 studies included 1332 patients. The CARS results for subgroup analysis were as follows: acupuncture subgroup (MD = -2.65, 95% CI (-3.22, -2.07)) and acupuncture plus massage subgroup (MD = -10.35, 95% CI (-11.34, -9.36)). The ABC results were as follows: (MD = -6.70, 95% CI (-9.10, -4.29)). The analysis results of sensory, relating, language, body and object use, and social/self-help in the subitems of ABC were as follows: sensory (MD = -2.67, 95% CI (-2.90, -2.44)), relating (MD = -3.28, 95% CI (-3.55, -3.02)), language (MD = -2.45, 95% CI (-2.73, -2.16)), body and object use (MD = -1.19, 95% CI (-1.38, -1.00)), and social/self-help (MD = -2.09, 95% CI (-2.30, -1.89)). For the analysis results of comprehension and expression ages in the subitems of RDLS, the comprehension age results were as follows: (MD = 0.08, 95% CI (-0.06, 0.22), P = 0.27). Those of expression age were as follows: (MD = 0.15, 95% CI (0.04, 0.26), P=0.009). The WeeFIM results were as follows: (MD = 3.70, 95% CI (2.38, 5.02)). This study suggested that acupuncture could effectively treat ASD. However, acupuncture methods and prescriptions at this stage remain heterogeneous, and acupuncture treatment operations require standardization. Studies using rigorous and standard research designs are needed to draw stronger conclusions about the advantages of using acupuncture to treat children and adolescents with ASD.

ZxNHX1 indirectly participates in controlling K+ homeostasis in the xerophyte Zygophyllum xanthoxylum.

The succulent xerophyte Zygophyllum xanthoxylum (Bunge) Engl. can absorb Na+ from the soil as an osmoticum in order to resist osmotic stress. The tonoplast Na+/H+ antiporter ZxNHX1 is essential for maintaining the salt-accumulation characteristics of Z. xanthoxylum by compartmentalizing Na+ into vacuoles. Previous results revealed that the silencing of ZxNHX1 greatly decreased Na+ accumulation in Z. xanthoxylum under 50 mM NaCl due to the weakened compartmentalisation; in addition, K+ concentration also significantly reduced in ZxNHX1-RNAi lines. Yet, whether the reduction of K+ concentration was directly triggered by the silencing of ZxNHX1 remains unclear. In this study, the growth parameters and expression levels of ZxSOS1, ZxHKT1;1, ZxAKT1 and ZxSKOR were measured in wild-type and ZxNHX1-RNAi lines under control or -0.5 MPa osmotic stress. The results showed that the silencing of ZxNHX1 inhibited the plant growth, decreased Na+ concentration in leaves, reduced the transcript abundance of ZxSOS1 and dramatically increased that of ZxHKT1;1 in roots of Z. xanthoxylum under osmotic stress; whereas tissue K+ concentrations and the expression level of ZxSKOR displayed no significant variations, and the expression of ZxAKT1 were significantly reduced in ZxNHX1-RNAi lines under osmotic stress, compared with the wild type. These results suggest that in Z. xanthoxylum, ZxNHX1 can maintain the normal growth by compartmentalizing Na+ into vacuoles, and regulate the spatial distribution of Na+ indirectly by affecting the expressions of ZxSOS1 and ZxHKT1;1. Moreover, the silencing of ZxNHX1 is not the main reason that led to the reduction of K+ concentration in ZxNHX1-RNAi lines under 50 mM NaCl, and ZxNHX1 might be indirectly involved in regulating K+ homeostasis.

Characterization of the Specific Interactions between Nanoparticles and Proteins at Residue-Resolution by Alanine Scanning Mutagenesis.

The interaction between nanoparticles and proteins is a central problem in the nano-bio-fields. However, it is still a great challenge to characterize the specific interaction between nanoparticles and proteins in structural details. Using the Goldbodies, the artificial antibodies created by grafting complementary-determining regions (CDRs) of natural antibodies onto gold nanoparticles, as the models, we manage to identify the key residues of the CDR peptides on gold nanoparticles for the specific interactions by alanine scanning mutagenesis. Each and every residue of the CDR peptides on two Goldbodies (which specifically bind with hen egg white lysozyme and epidermal growth factor receptor, respectively) is mutated to alanine one by one, generating a total of 18 single-mutants of the two Goldbodies. Experimental results reveal that the key residues of the CDR peptides for the specific interactions between the two Goldbodies and the corresponding antigens are exactly the same as those in the natural antibodies, thus proving that the correct conformations of the CDRs of natural antibodies have been successfully reconstructed on AuNPs. This is the first residue-resolution structural illustration for the specific interaction between a designed nanoparticle and a protein.

Cloning and functional characterization of epidermis-specific promoter MtML1 from Medicago truncatula.

Epidermis-specific promoters are necessary for ectopic expression of specific functional genes such as the cuticle-related genes. Previous studies indicated that both ECERIFERUM 6 (AtCER6) and MERISTEM L1 LAYER (ATML1) promoters from Arabidopsis thaliana can drive gene expression specifically in the epidermis of shoot apical meristems (SAMs) and leaves. However, the epidermis-specific promoters from legume plants have not been reported. Here, we cloned a 5' flanking sequence from the upstream -2150 bp to the translational start ATG codon of MtML1 gene of legume model plant Medicago truncatula. PlantCARE analysis indicated that this sequence matches the characteristics of a promoter, having TATA box and CAAT box, as well as contains some conserved elements of epidermis-specific promoters like AtCER6 and ATML1 promoters. The ß-glucuronidase (GUS) histochemical analysis showed that MtML1 promoter can drive GUS gene expression in transiently transformed Nicotiana tabacum leaves under non-inducing condition. Furthermore, it can also control GUS expression in leaves and siliques rather than roots of the stably transformed Arabidopsis. More importantly, the leaf cross-section observations indicated that MtML1 exclusively expressed in the epidermis of leaves. These results suggested that MtML1 promoter performed the epidermis-specific in plant shoot. Our study establishes the foundation for driving the cuticle-related gene to express in epidermis, which may be very useful in genetic engineering of legume plants.