CRISPR/Cas9-Engineered Large Fragment Deletion Mutations in Arabidopsis CEP Peptide-Encoding Genes Reveal Their Role in Primary and Lateral Root Formation.

C-TERMINALLY ENCODED PEPTIDEs (CEPs) are post-translationally modified peptides that play essential roles in root and shoot development, nitrogen absorption, nodule formation and stress resilience. However, it has proven challenging to determine biological activities of CEPs because of difficulties in obtaining loss-of-function mutants for these small genes. To overcome this challenge, we thus assembled a collection of easily detectable large fragment deletion mutants of Arabidopsis CEP genes through the clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9-engineered genome editing. This collection was then evaluated for the usability by functionally analyzing the Arabidopsis growth and development with a focus on the root. Most cep mutants displayed developmental defects in primary and lateral roots showing an increased primary root length and an enhanced lateral root number, demonstrating that the genetic resource provides a useful tool for further investigations into the roles of CEPs.

Infection Microenvironment-Sensitive Photothermal Nanotherapeutic Platform to Inhibit Methicillin-Resistant Staphylococcus aureus Infection.

Methicillin-resistant Staphylococcus aureus (MRSA) can induce multiple inflammations. The biofilm formed by MRSA is resistant to a variety of antibiotics and is extremely difficult to cure, which seriously threatens human health. Herein, a nanoparticle encapsulating berberine with polypyrrole core and pH-sensitive shell to provide chemo-photothermal dual therapy for MRSA infection is reported. By integrating photothermal agent polypyrrole, berberine, acid-degradable crosslinker, and acid-induced charge reversal polymer, the nanoparticle exhibited highly efficient MRSA infection treatment. In normal uninfected areas and bloodstream, nanoparticles showed negatively charged, demonstrating high biocompatibility and excellent hemocompatibility. However, once arriving at the MRSA infection site, the nanoparticle can penetrate and accumulate in the biofilm within 2 h. Simultaneously, berberine can be released into biofilm rapidly. Under the combined effect of photothermal response and berberine inhibition, 88.7% of the biofilm is removed at 1000 µg mL-1 . Moreover, the nanoparticles have an excellent inhibitory effect on biofilm formation, the biofilm inhibition capacity can reach up to 90.3%. Taken together, this pH-tunable nanoparticle can be employed as a new generation treatment strategy to fight against the fast-growing MRSA infection.

A group of CLE peptides regulates de novo shoot regeneration in Arabidopsis thaliana.

Known for their regulatory roles in stem cell homeostasis, CLAVATA3/ESR-RELATED (CLE) peptides also function as mediators of external stimuli such as hormones. De novo shoot regeneration, representing the remarkable plant cellular plasticity, involves reconstitution of stem cells under control of stem-cell regulators. Yet whether and how stem cell-regulating CLE peptides are implicated in plant regeneration remains unknown. By CRISPR/Cas9-induced loss-of-function studies, peptide application, precursor overexpression, and expression analyses, the role of CLE1-CLE7 peptides and their receptors in de novo shoot regeneration was studied in Arabidopsis thaliana. CLE1-CLE7 are induced by callus-induction medium and dynamically expressed in pluripotent callus. Exogenously-applied CLE1-CLE7 peptides or precursor overexpression effectively leads to shoot regeneration suppression, whereas their simultaneous mutation results in enhanced regenerative capacity, demonstrating that CLE1-CLE7 peptides redundantly function as negative regulators of de novo shoot regeneration. CLE1-CLE7-mediated shoot regeneration suppression is impaired in loss-of-function mutants of callus-expressed CLAVATA1 (CLV1) and BARELY ANY MERISTEM1 (BAM1) genes, indicating that CLV1/BAM1 are required for CLE1-CLE7-mediated shoot regeneration signaling. CLE1-CLE7 signaling resulted in transcriptional repression of WUSCHEL (WUS), a stem cell-promoting transcription factor known as a principal regulator of plant regeneration. Our results indicate that functionally-redundant CLE1-CLE7 peptides genetically act through CLV1/BAM1 receptors and repress WUS expression to modulate shoot-regeneration capacity, establishing the mechanistic basis for CLE1-CLE7-mediated shoot regeneration and a novel role for CLE peptides in hormone-dependent developmental plasticity.

The Alleviation of LPS-Induced Murine Acute Lung Injury by GSH-Mediated PEGylated Artesunate Prodrugs.

Acute lung injury (ALI) or its aggravated stage acute respiratory distress syndrome (ARDS) is a common severe clinical syndrome in intensive care unit, may lead to a life-threatening form of respiratory failure, resulting in high mortality up to 30-40% in most studies. Nanotechnology-mediated anti-inflammatory therapy is an emerging novel strategy for the treatment of ALI, has been demonstrated with unique advantages in solving the dilemma of ALI drug therapy. Artesunate (ART), a derivative of artemisinin, has been reported to have anti-inflammatory effects. Therefore, in the present study, we designed and synthesized PEGylated ART prodrugs and assessed whether ART prodrugs could attenuate lipopolysaccharide (LPS) induced ALI in vitro and in vivo. All treatment groups were conditioned with ART prodrugs 1 h before challenge with LPS. Significant increased inflammatory cytokines production and decreased GSH levels were observed in the LPS stimulated mouse macrophage cell line RAW264.7. Lung histopathological changes, lung W/D ratio, MPO activity and total neutrophil counts were increased in the LPS-induced murine model of ALI via nasal administration. However, these results can be reversed to some extent by treatment of ART prodrugs. The effectiveness of mPEG2k-SS-ART in inhibition of ALI induced by LPS was confirmed. In conclusion, our results demonstrated that the ART prodrugs could attenuate LPS-induced ALI effectively, and mPEG2k-SS-ART may serve as a novel strategy for treatment of inflammation induced lung injury.

[A mini-review on anti-tumor effect of cannabidiol].

Cannabidiol is the main non-psychoactive component of Cannabis sativa, which has multiple medicinal activities, such as antiepileptic, immunomodulation, analgesic, antioxidant, anticonvulsant, anti-anxiety and other functions. In recent years, it has been found that cannabidiol can inhibit the proliferation of various tumor cells, induce apoptosis and autophagy of tumor cells, arrest cell cycle, interrupt invasion and metastasis of tumor cells, regulate tumor microenvironment, exert synergistic therapy with other chemotherapeutic drugs, and reduce the toxicity of chemotherapeutic drugs. However, its anti-tumor effect remains controversial and its application is limited. The study of microspheres, nano liposomes and other new drug delivery systems can improve the anti-tumor effect of cannabidiol. In this study, the anti-tumor mechanism and application of cannabidiol were summarized and discussed in order to provide inspirations for its further investigation and application.

Probing the interactions of GlcNH2 with boric acid via NMR spectroscopy.

The essential role of boronic esters in controlling both the direction and selectivity of chemical reactions as well as their significant function in catalytic activity have been demonstrated for industrially important processes. The specific interaction analyses of the monosaccharide GlcNH2 with boric acid are of interest since monosaccharides serve as model systems for the more sophisticated carbohydrate molecules. The interaction of GlcNH2 with boric acid was systematically investigated by numerous NMR techniques. A 1 : 1 chelate boron complex coordinated at the cis-1,2 position of GlcNH2 was identified as the major species in DMSO-d6 solution via1H and 13C INEPT DOSY NMR spectroscopy. This specific boron nitrogen coordination mechanism was further supported by the 1H-15N HSQC spectra. Variations in the spin-lattice relaxation times (T1) of the 13C1 nucleus also provided quantitative data regarding this non-covalent interactions. This is an application of 1H, 13C INEPT DOSY, 1H-15N HSQC, and relaxation methods to study such aggregations in solutions. These methods have potential applications in the characterization of reactive intermediates in biomass conversions.

Generation of Oligodendrocyte Progenitor Cells From Mouse Bone Marrow Cells.

Oligodendrocyte progenitor cells (OPCs) are a subtype of glial cells responsible for myelin regeneration. Oligodendrocytes (OLGs) originate from OPCs and are the myelinating cells in the central nervous system (CNS). OLGs play an important role in the context of lesions in which myelin loss occurs. Even though many protocols for isolating OPCs have been published, their cellular yield remains a limit for clinical application. The protocol proposed here is novel and has practical value; in fact, OPCs can be generated from a source of autologous cells without gene manipulation. Our method represents a rapid, and high-efficiency differentiation protocol for generating mouse OLGs from bone marrow-derived cells using growth-factor defined media. With this protocol, it is possible to obtain mature OLGs in 7-8 weeks. Within 2-3 weeks from bone marrow (BM) isolation, after neurospheres formed, the cells differentiate into Nestin+ Sox2+ neural stem cells (NSCs), around 30 days. OPCs specific markers start to be expressed around day 38, followed by RIP+O4+ around day 42. CNPase+ mature OLGs are finally obtained around 7-8 weeks. Further, bone marrow-derived OPCs exhibited therapeutic effect in shiverer (Shi) mice, promoting myelin regeneration and reducing the tremor. Here, we propose a method by which OLGs can be generated starting from BM cells and have similar abilities to subventricular zone (SVZ)-derived cells. This protocol significantly decreases the timing and costs of the OLGs differentiation within 2 months of culture.

Glycosylation intermediates studied using low temperature 1H- and 19F-DOSY NMR: new insight into the activation of trichloroacetimidates.

Low temperature 1H- and 19F-DOSY have been used for analyzing reactive intermediates in glycosylation reactions, where a glycosyl trichloroacetimidate donor has been activated using different catalysts. The DOSY protocols have been optimized for low temperature experiments and provided new insight into acid catalyzed glycosylation chemistry. From the study a new glycosylation intermediate was characterized.

Glucosamine condensation catalyzed by 1-ethyl-3-methylimidazolium acetate: mechanistic insight from NMR spectroscopy.

The basic ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) could efficiently catalyze the conversion of 2-amino-2-deoxy-d-glucose (GlcNH2) into deoxyfructosazine (DOF) and fructosazine (FZ). Mechanistic investigation by NMR studies disclosed that [C2C1Im][OAc], exhibiting strong hydrogen bonding basicity, could coordinate with the hydroxyl and amino groups of GlcNH2via the promotion of hydrogen bonding in bifunctional activation of substrates and further catalyzing product formation, based on which a plausible reaction pathway involved in this homogeneous base-catalyzed reaction was proposed. Hydrogen bonding as an activation force, therefore, is responsible for the remarkable selectivity and rate enhancement observed.

OVCA1 inhibits the proliferation of epithelial ovarian cancer cells by decreasing cyclin D1 and increasing p16.

OVCA1, a tumor suppressor gene, is deleted or lower expressed in about 80% of ovarian cancer. Over expression of OVCA1 in human ovarian cancer A2780 cells inhibits cell proliferation and arrests cells in G1 stage. However, the fact that the molecular mechanism of OVCA1 inhibits cell growth is presently elusive. Here we investigated the potential signaling pathway induced by over-expression of OVCA1. Our results show that over-expression of human OVCA1 in ovarian cancer cells A2780 leads to down-regulation of cyclin D1, and up-regulation of p16, but no effect on the expression of NF-κB. It indicates that OVCA1 could inhibit the proliferation of ovarian cancer cell A2780 by p16/cyclin D1 pathway, but not by NF-κB.