Protective Properties of Copper-Loaded Chitosan Nanoparticles against Soybean Pathogens Pseudomonas savastanoi pv. glycinea and Curtobacterium flaccumfaciens pv. flaccumfaciens.

Soybeans are a valuable food product, containing 40% protein and a large percentage of unsaturated fatty acids ranging from 17 to 23%. Pseudomonas savastanoi pv. glycinea (Psg) and Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff) are harmful bacterial pathogens of soybean. The bacterial resistance of soybean pathogens to existing pesticides and environmental concerns requires new approaches to control bacterial diseases. Chitosan is a biodegradable, biocompatible and low-toxicity biopolymer with antimicrobial activity that is promising for use in agriculture. In this work, a chitosan hydrolysate and its nanoparticles with copper were obtained and characterized. The antimicrobial activity of the samples against Psg and Cff was studied using the agar diffusion method, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. The samples of chitosan and copper-loaded chitosan nanoparticles (Cu2+ChiNPs) significantly inhibited bacterial growth and were not phytotoxic at the concentrations of the MIC and MBC values. The protective properties of chitosan hydrolysate and copper-loaded chitosan nanoparticles against soybean bacterial diseases were tested on plants in an artificial infection. It was demonstrated that the Cu2+ChiNPs were the most effective against Psg and Cff. Treatment of pre-infected leaves and seeds demonstrated that the biological efficiencies of (Cu2+ChiNPs) were 71% and 51% for Psg and Cff, respectively. Copper-loaded chitosan nanoparticles are promising as an alternative treatment for bacterial blight and bacterial tan spot and wilt in soybean.

Effect of Seed Priming with Chitosan Hydrolysate on Lettuce (Lactuca sativa) Growth Parameters.

Seed priming increases germination, yield, and resistance to abiotic factors and phytopathogens. Chitosan is considered an ecofriendly growth stimulant and crop protection agent. Chitosan hydrolysate (CH) is an unfractionated product of hydrolysis of high-molecular-weight crab shell chitosan with a molecular weight of 1040 kDa and a degree of deacetylation of 85% with nitric acid. The average molecular weight of the main fraction in CH was 39 kDa. Lettuce seeds were soaked in 0.01-1 mg/mL CH for 6 h before sowing. The effects of CH on seed germination, plant morphology, and biochemical indicators at different growth stages were evaluated. Under the 0.1 mg/mL CH treatment, earlier seed germination was detected compared to the control. Increased root branching was observed, along with 100% and 67% increases in fresh weight (FW) at the 24th and 38th days after sowing (DAS), respectively. An increase in the shoot FW was found in CH-treated plants (33% and 4% at the 24th and 38th DAS, respectively). Significant increases in chlorophyll and carotenoid content compared to the control were observed at the 10th DAS. There were no significant differences in the activity of phenylalanine ammonia-lyase, polyphenol oxidase, ß-1,3-glucanase, and chitinase at the 24th and 38th DAS. Seed priming with CH could increase the yield and uniformity of plants within the group. This effect is important for commercial vegetable production.

Simple method for ultrasound assisted «click¼ modification of azido-chitosan derivatives by CuAAC.

New quaternized chitosan derivatives HT-TMC were synthesized as a result of copper catalyzed azide-alkyne [3 + 2] cycloaddition (CuAAC). The structure of the HT-TMC was verified by 2D NMR. The synthesis was carried out as a result of the formation of Cu(I) in situ, under the action of ultrasound in aerobic conditions in the presence of acetic acid and metallic copper (copper turnings). The new derivatives were characterized by increased pH range of solubility (DS range 18-76%) and the presence of antibacterial and fungicidal activities. The proposed catalytic system makes it possible to easily and efficiently obtain new derivatives of chitosan as a result of ultrasound-promoted CuAAC.

Optically Active Hybrid Materials Based on Natural Spider Silk.

Spider silk is a natural material possessing unique properties such as biocompatibility, regenerative and antimicrobial activity, and biodegradability. It is broadly considered an attractive matrix for tissue regeneration applications. Optical monitoring and potential control over tissue regrowth are attractive tools for monitoring of this process. In this work, we show upconversion modification of natural spider silk fibers with inorganic nanoparticles. To achieve upconversion, metal oxide nanoparticles were doped with low concentrations of rare-earth elements, producing potentially biocompatible luminescent nanomaterials. The suggested approach to spider silk modification is efficient and easy to perform, opening up sensing and imaging possibilities of biomaterials in a noninvasive and real-time manner in bio-integration approaches.

A DNA minimachine for selective and sensitive detection of DNA.

Synthetic molecular machines have been explored to manipulate matter at the molecular level. Here, we designed a multifunctional DNA nano-construct, dubbed a 'DNA minimachine' (DMM), which (i) tightly binds complementary DNA; (ii) recognizes specific fragments with high selectivity and (iii) amplifies output signals. DMM1 detects lower concentrations of both single-stranded DNA and double-stranded DNA compared to a conventional probe. This study sets a direction towards the development of molecular machines for selective, sensitive and cost-efficient DNA analysis.

Broccoli Fluorets: Split Aptamers as a User-Friendly Fluorescent Toolkit for Dynamic RNA Nanotechnology.

RNA aptamers selected to bind fluorophores and activate their fluorescence offer a simple and modular way to visualize native RNAs in cells. Split aptamers which are inactive until the halves are brought within close proximity can become useful for visualizing the dynamic actions of RNA assemblies and their interactions in real time with low background noise and eliminated necessity for covalently attached dyes. Here, we design and test several sets of F30 Broccoli aptamer splits, that we call fluorets, to compare their relative fluorescence and physicochemical stabilities. We show that the splits can be simply assembled either through one-pot thermal annealing or co-transcriptionally, thus allowing for direct tracking of transcription reactions via the fluorescent response. We suggest a set of rules that enable for the construction of responsive biomaterials that readily change their fluorescent behavior when various stimuli such as the presence of divalent ions, exposure to various nucleases, or changes in temperature are applied. We also show that the strand displacement approach can be used to program the controllable fluorescent responses in isothermal conditions. Overall, this work lays a foundation for the future development of dynamic systems for molecular computing which can be used to monitor real-time processes in cells and construct biocompatible logic gates.

N-[4-(N,N,N-trimethylammonium)benzyl]chitosan chloride: Synthesis, interaction with DNA and evaluation of transfection efficiency.

А novel cationic chitosan derivative, N-[4-(N,N,N-trimethylammonium)benzyl]chitosan chloride (TMAB-CS), with different degrees of substitution (DS) was synthesized by a chemoselective interaction of 4-formyl-N,N,N-trimethylanilinium iodide with chitosan amino groups using a reductive amination method. Several factors (pH, reactant ratio, reaction time, and chitosan structure) were studied for their effects on the DS of the resulting TMAB-CS. The obtained derivatives were characterized by 1H NMR and FTIR spectroscopy. Turbidimetric titration showed enhanced solubility over a wide pH range even for low-substituted TMAB-CS. TMAB-CS provided strong DS-dependent binding of plasmid DNA. Dynamic light scattering measurements revealed the formation of stable polyplexes with hydrodynamic diameters of 200-300nm and ζ-potential of 20-30mV. TMAB-CS with relatively low DS (25%) demonstrated more pronounced transfection efficiency (up to 2000 cell/cm2) of plasmid DNA into the HEK293 cell line promoted by free TMAB-CS. The positive effects of lower DS can be related to a better polyplex dissociation within the cell. The cytotoxicity of TMAB-CS was comparable to that of the initial chitosan at concentrations up to 300ng/µL, even at high DS.

Cross-presentation of lactoferrin encapsulated into chitosan-based nanoparticles.

Induction of CD8+ cytotoxic T-cell response is essential for the protection from intracellular pathogens. It requires major histocompatibility complex class I processing of newly synthesized proteins transported from the cytosolic pathway. Presentation of mature soluble proteins occurs via a cross-presentation (CP) pathway that is much less efficient in the activation of cytotoxic response. Encapsulation of proteins into polymeric nanoparticles (NPs) can modulate the efficacy of antigen CP. In this article, a model antigen lactoferrin (L) was encapsulated into polysaccharide NPs with different physicochemical properties (size, charge, and hydrophobicity) and used as an immunogen. CD8+ or CD4+ associated IgG2a or IgG1 subclasses of L-specific antibodies, respectively, served as a measure of CD8+ versus CD4+ T-cell activation. Among five types of NPs produced, only succinylchitosan-galactomannan (LSG) and succinylchitosan-PEG-chitosan (LSPC) NPs induced a significant IgG2a response. IgG1 production was comparable in all but hydrophobic succinyl-dodecyl-chitosan (LSD) NPs, where it was only marginal. Confocal studies demonstrated that galactomannan-equipped LSG-NPs induced vacuolar type of CP, while positively charged LSPC-NPs were transported mostly via the cytosolic CP pathway.