Proteomic Landscape Has Revealed Small Rubber Particles Are Crucial Rubber Biosynthetic Machines for Ethylene-Stimulation in Natural Rubber Production.

Rubber particles are a specific organelle for natural rubber biosynthesis (NRB) and storage. Ethylene can significantly improve rubber latex production by increasing the generation of small rubber particles (SRPs), regulating protein accumulation, and activating many enzyme activities. We conducted a quantitative proteomics study of different SRPs upon ethylene stimulation by differential in-gel electrophoresis (DIGE) and using isobaric tags for relative and absolute quantification (iTRAQ) methods. In DIGE, 79 differentially accumulated proteins (DAPs) were determined as ethylene responsive proteins. Our results show that the abundance of many NRB-related proteins has been sharply induced upon ethylene stimulation. Among them, 23 proteins were identified as rubber elongation factor (REF) and small rubber particle protein (SRPP) family members, including 16 REF and 7 SRPP isoforms. Then, 138 unique phosphorylated peptides, containing 129 phosphorylated amino acids from the 64 REF/SRPP family members, were identified, and most serine and threonine were phosphorylated. Furthermore, we identified 226 DAPs from more than 2000 SRP proteins by iTRAQ. Integrative analysis revealed that almost all NRB-related proteins can be detected in SRPs, and many proteins are positively responsive to ethylene stimulation. These results indicate that ethylene may stimulate latex production by regulating the accumulation of some key proteins. The phosphorylation modification of REF and SRPP isoforms might be crucial for NRB, and SRP may act as a complex natural rubber biosynthetic machine.

A protein extraction method for low protein concentration solutions compatible with the proteomic analysis of rubber particles.

The extraction of high-purity proteins from the washing solution (WS) of rubber particles (also termed latex-producing organelles) from laticifer cells in rubber tree for proteomic analysis is challenging due to the low concentration of proteins in the WS. Recent studies have revealed that proteins in the WS might play crucial roles in natural rubber biosynthesis. To further examine the involvement of these proteins in natural rubber biosynthesis, we designed an efficiency method to extract high-purity WS proteins. We improved our current borax and phenol-based method by adding reextraction steps with phenol (REP) to improve the yield from low protein concentration samples. With this new method, we extracted WS proteins that were suitable for proteomics. Indeed, compared to the original borax and phenol-based method, the REP method improved both the quality and quantity of isolated proteins. By repeatedly extracting from low protein concentration solutions using the same small amount of phenol, the REP method yielded enough protein of sufficiently high-quality from starting samples containing less than 0.02 mg of proteins per milliliter. This method was successfully applied to extract the rubber particle proteins from the WS of natural rubber latex samples. The REP-extracted WS proteins were resolved by 2DE, and 28 proteins were positively identified by MS. This method has the potential to become widely used for the extraction of proteins from low protein concentration solutions for proteomic analysis.

Prevention of bacterial biofilm formation on orthodontic brackets by non-crosslinked chitosan coating.

During orthodontic treatment, the patients are susceptible to dental caries as a result of the bacterial adhesion and biofilm formation around the orthodontic brackets. Prevention of the caries-related biofilm formation is of significance for maintaining both aesthetics and health of the teeth. Herein, the brackets were functionalized with antibacterial activity via coating a layer of non-crosslinked chitosan (CS). We firstly demonstrated the ability of free CS scaffolds (not coated on brackets) to inhibit the formation of Streptococcus mutans biofilms (inhibition rate 94.3 % for CS-0.3 mg) and to eradicate the mature biofilms (biofilm loss rate 99.8 % for CS-1.2 mg). Further, the inhibition of S. mutans biofilm formation on brackets by CS coating was investigated for the first time. As a result, the CS-coated brackets (Br-CS) kept the great biofilm inhibition capacity of free CS scaffolds. In detail, the Br-CS, prepared by immersing brackets in CS solutions (containing 1.0, 2.5, 5.0 and 10 mg/mL CS) and freeze-drying, showed the biofilm inhibition rate of 48.5 %, 88.6 %, 96.4 % and 99.6 %, respectively. In conclusion, coating orthodontic brackets with the non-crosslinked CS is a potential approach for inhibiting biofilm formation and protecting patients from dental caries.

Efficient Removal of Dental Plaque Biofilm from Training Typodont Teeth via Water Flosser.

Plaque biofilms play critical roles in the development of dental caries. Mechanical plaque control methods are considered to be most effective for plaque removal, such as brushing teeth or using flosser. Recently, water flosser has been paid much attention. Here, we tested the ability of a water flosser to remove the adhered sucrose and the dental plaque biofilms formed by Streptococcus mutans, Streptococcus sanguinis, and Actinobacillus viscosus. We found that the residual sucrose concentration was 3.54 mg/mL in the control group, 1.75 mg/mL in the syringe group (simulating the ordinary mouthwash), and 0 mg/mL in water flosser group. In addition, the residual bacterial concentration was 3.6 × 108 CFU/mL in the control group, 1.6 × 107 CFU/mL in the syringe group, and only 5.5 × 105 CFU/mL in the water flosser group. In summary, water flosser is effective for cleaning the teeth, which may have significant potential in preventing dental caries and maintaining oral health.

Temperature sensitive liposome based cancer nanomedicine enables tumour lymph node immune microenvironment remodelling.

Targeting tumour immunosuppressive microenvironment is a crucial strategy in immunotherapy. However, the critical role of the tumour lymph node (LN) immune microenvironment (TLIME) in the tumour immune homoeostasis is often ignored. Here, we present a nanoinducer, NIL-IM-Lip, that remodels the suppressed TLIME via simultaneously mobilizing T and NK cells. The temperature-sensitive NIL-IM-Lip is firstly delivered to tumours, then directed to the LNs following pH-sensitive shedding of NGR motif and MMP2-responsive release of IL-15. IR780 and 1-MT induces immunogenic cell death and suppress regulatory T cells simultaneously during photo-thermal stimulation. We demonstrate that combining NIL-IM-Lip with anti-PD-1 significantly enhances the effectiveness of T and NK cells, leading to greatly suppressed tumour growth in both hot and cold tumour models, with complete response in some instances. Our work thus highlights the critical role of TLIME in immunotherapy and provides proof of principle to combine LN targeting with immune checkpoint blockade in cancer immunotherapy.

Two-Wave Variable Nanotheranostic Agents for Dual-Mode Imaging-Guided Photo-Induced Triple-Therapy for Cancer.

Photothermal therapy (PTT) is a promising strategy for cancer treatment, but its clinical application relies heavily on accurate tumor positioning and effective combination. Nanotheranostics has shown superior application in precise tumor positioning and treatment, bringing potential opportunities for developing novel PTT-based therapies. Here, a nanotheranostic agent is proposed to enhance magnetic resonance imaging (MRI)/ near-infrared fluorescence imaging (NIRFI) imaging-guided photo-induced triple-therapy for cancer. Thermosensitive liposomes co-loaded with SPIONs/IR780 and Abemaciclib (SIA-TSLs), peptide ACKFRGD, and click group 2-cyano-6-amino-benzothiazole (CABT) are co-modified on the surface of SIA-TSLs to form SIA-αTSLs. ACKFRGD can be hydrolyzed to expose the 1, 2-thiolamino groups in the presence of cathepsin B in tumors, which click cycloaddition with the cyano group on CABT, resulting in the formation of SIA-αTSLs aggregates. The aggregation of SIA-αTSLs in tumors enhances the MRI/NIRFI imaging capability and enables precise PTT. Photo-induced triple-therapy enhances precision cancer therapy. First, PTT ablates specific tumors and induces ICD via localized photothermal. Second, local tumor heating promotes the rupture of SIA-αTSLs, which release Abemaciclib to block the tumor cell cycle and inhibit Tregs proliferation. Third, injecting GM-CSF into tumor tissue leads to recruitment of dendritic cells and initiation of antitumor immunity. Collectively, these results present a promising nanotheranostic strategy for future cancer therapy.

Subcellular proteome profiles of different latex fractions revealed washed solutions from rubber particles contain crucial enzymes for natural rubber biosynthesis.

Rubber particle (RP) is a specific organelle for natural rubber biosynthesis (NRB) and storage in rubber tree Hevea brasiliensis. NRB is processed by RP membrane-localized proteins, which were traditionally purified by repeated washing. However, we noticed many proteins in the discarded washing solutions (WS) from RP. Here, we compared the proteome profiles of WS, C-serum (CS) and RP by 2-DE, and identified 233 abundant proteins from WS by mass spectrometry. Many spots on 2-DE gels were identified as different protein species. We further performed shotgun analysis of CS, WS and RP and identified 1837, 1799 and 1020 unique proteins, respectively. Together with 2-DE, we finally identified 1825 proteins from WS, 246 were WS-specific. These WS-specific proteins were annotated in Gene Ontology, indicating most abundant pathways are organic substance metabolic process, protein degradation, primary metabolic process, and energy metabolism. Protein-protein interaction analysis revealed these WS-specific proteins are mainly involved in ribosomal metabolism, proteasome system, vacuolar protein sorting and endocytosis. Label free and Western blotting revealed many WS-specific proteins and protein complexes are crucial for NRB initiation. These findings not only deepen our understanding of WS proteome, but also provide new evidences on the roles of RP membrane proteins in NRB. SIGNIFICANCE: Natural rubber is stored in rubber particle from the rubber tree. Rubber particles were traditionally purified by repeated washing, but many proteins were identified from the washing solutions (WS). We obtained the first visualization proteome profiles with 1825 proteins from WS, including 246 WS-specific ones. These WS proteins contain almost all enzymes for polyisoprene initiation and may play important roles in rubber biosynthesis.

Comprehensive Proteomics Analysis of Laticifer Latex Reveals New Insights into Ethylene Stimulation of Natural Rubber Production.

Ethylene is a stimulant to increase natural rubber latex. After ethylene application, both fresh yield and dry matter of latex are substantially improved. Moreover, we found that ethylene improves the generation of small rubber particles. However, most genes involved in rubber biosynthesis are inhibited by exogenous ethylene. Therefore, we conducted a proteomics analysis of ethylene-stimulated rubber latex, and identified 287 abundant proteins as well as 143 ethylene responsive latex proteins (ERLPs) with mass spectrometry from the 2-DE and DIGE gels, respectively. In addition, more than 1,600 proteins, including 404 ERLPs, were identified by iTRAQ. Functional classification of ERLPs revealed that enzymes involved in post-translational modification, carbohydrate metabolism, hydrolase activity, and kinase activity were overrepresented. Some enzymes for rubber particle aggregation were inhibited to prolong latex flow, and thus finally improved latex production. Phosphoproteomics analysis identified 59 differential phosphoproteins; notably, specific isoforms of rubber elongation factor and small rubber particle protein that were phosphorylated mainly at serine residues. This post-translational modification and isoform-specific phosphorylation might be important for ethylene-stimulated latex production. These results not only deepen our understanding of the rubber latex proteome but also provide new insights into the use of ethylene to stimulate rubber latex production.