Institute of Medicinal Plant Development

Lipid nanoparticles (LNPs) have shown high therapeutic utility as delivery vehicles for RNA, however, the way in which LNP chemical composition affects functional mRNA delivery is not completely understood.Surface and interface molecular analysis of LNPs can provide improved structure-function relationship information relevant to potency and tissue tropism in vivo, but there are limited techniques available to achieve the required interfacial characterisation.Secondary ion mass spectrometry (SIMS) has shown great potential for stratifying the lipid component of LNPs, with a prior study using qualitative depth profiles to confirm the basic structural arrangement of polyethylene glycol (PEG) lipids and RNA.This paper describes the first application of Quantitative cryogenic orbitrap secondary ion mass spectrometry (Q-Cryo-OrbiSIMS) to model the four canonical lipid components of LNPs, using quantified ion intensity profiles to plot the composition with depth.A combinatorial spot-in-spot mixing of the four lipid components generated 337 spots as a micro array on a glass slide.OrbiSIMS data is acquired from each spot.A variety of data pre-processing approaches are compared to scale the secondary ion intensity to minimise the effects of noise bias before construction of a calibration model.An artificial neural network model used to predict the four lipid compositions from the ion intensities produces a mean absolute error (MAE) of 14 %.The model is then used to predict quantitative molecular distributions from the OrbiSIMS profiles of cryogenically preserved LNPs.This demonstrates the feasibility of Q-Cryo-OrbiSIMS for LNPs, as well as the potential of this method for wider application to organic systems when suitable calibrant samples are available.

Mogrosides are natural, zero-calorie sweeteners derived from Siraitia grosvenorii. However, conventional production based on plant extraction is constrained by low yields and long cultivation cycles, limiting large-scale application. To address this, we reconstructed the mogroside biosynthetic pathway in Nicotiana tabacum through stable heterologous expression. A multigene construct (pMogroside), containing seven key S. grosvenorii genes involved in triterpene cyclization, hydroxylation, and glycosylation (SgSQE1, SgCS, SgEPH2, SgP450, AtCPR, SgUGT269-1, SgUGT289-3), was assembled using a modular strategy with P2A peptides and introduced via Agrobacterium-mediated transformation. Transgenic lines were confirmed by PCR and qRT-PCR, and HPLC-MS/MS analysis of leaves detected mogroside accumulation. Mogroside III was the dominant product (119.48 ± 25.17 ng/g FW), with lower levels of siamenoside I (21.74 ± 10.57 ng/g) and mogroside V (5.99 ± 0.61 ng/g). This work provides a sustainable plant-based platform for mogroside production and highlights the potential of N. tabacum as a versatile chassis in plant synthetic biology. Further enzyme engineering and pathway optimization will support future commercial applications in natural sweetener manufacturing.

The present study evaluated the effect of different foliar-applied biostimulants on growth, yield and fruit quality of organically cultivated strawberry cv. Festival under protected conditions in the Darjeeling Himalayan region during a single growing season (2021-22). Four biostimulants seaweed extract, humic acid, fulvic acid and amino acid were applied either once (30 days after transplanting, DAP) or twice (30 and 60 DAP), along with a water sprayed control in randomized block design with three replications. Foliar application of humic acid at 4 gL-¹ twice (30 and 60 DAP) significantly enhanced vegetative growth, flowering duration, fruit set, yield and biochemical quality. The highest fruit yield (650.10 g plant-¹), total soluble solids (9.73 °Brix) and ascorbic acid content (50.48 mg 100 g-¹) were recorded under this treatment, along with reduced titratable acidity. The findings indicate that humic acid applied twice during early growth stages can be effectively integrated into organic strawberry production systems in the Darjeeling Himalayan region for improving productivity and fruit quality.

N-Heterocyclic carbenes (NHCs) have emerged as promising nanoscale surface modifiers, enabling the formation of self-assembled monolayers (SAMs) on both flat surfaces and nanoparticles. However, most NHC-based adsorbates are derived from benzimidazole, with a few examples incorporating secondary metal coordination. Herein, we present the first example of a porphyrin-fused NHC monolayer on a gold surface. To prevent iodide contamination from conventional methylation reagents, dimethyl sulfate was used as an alternative. The resulting monolayer was thoroughly characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS), and surface-enhanced Raman spectroscopy (SERS). Furthermore, coordination with Ni(II) or Co(II) at the porphyrin core demonstrated its potential for nitric oxide (NO) detection. The monolayer exhibited a moderate thermal stability. Additionally, porphyrin-fused NHCs were incorporated into gold nanoparticles by using both top-down and bottom-up strategies. The resulting nanoparticles were characterized by transmission electron microscopy (TEM), energy-dispersive X-ray spectroscopy (EDX), and UV–vis spectroscopy. This work introduces a strategy for the nanoscale modification of porphyrin-fused NHCs on surfaces and nanoparticles, paving the way for future molecular complex-modified metal surface applications in sensing and catalysis.

Synergistic radiochemotherapy for breast cancer is often hindered by critical challenges, including tumor hypoxia and inherent radioresistance, which limit treatment efficacy. Overcoming these barriers requires advanced nanoplatforms capable of modulating the tumor microenvironment while precisely codelivering therapeutics. Here, we address these challenges by designing and developing a multifunctional bovine serum albumin (BSA)-stabilized MnO2–Bi2S3-Methotrexate (MTX) nanoplatform. Synthesized through a biomineralization approach, the nanoplatform exhibits a hydrodynamic diameter of 67 nm and a zeta potential of −20.9 mV, ensuring excellent colloidal stability. Experimental investigations revealed a pH-responsive MTX release profile and enhanced radiosensitization, which is attributed to the high-Z properties of Bi2S3 and the hypoxia-mitigating, oxygen-generating function of MnO2. To gain deeper mechanistic insights, computational analyses were employed: molecular docking studies confirmed MTX’s stable binding to BSA subdomains IIA and IIIA with a binding free energy (ΔG) of −14.79 kcal/mol, while density functional theory (DFT) calculations revealed a robust interaction energy of −154 kcal/mol for the MTX-α-MnO2–Bi2S3 complex, featuring a HOMO–LUMO gap of 2.56 eV and a Fermi energy of −3.74 eV (Bi2S3: −3.98 eV, MnO2: −4.41 eV), underscoring its electronic suitability for radiosensitization. Additionally, MD simulations over 10,000 ps at 310 K demonstrated structural stability with a root-mean-square deviation (RMSD) of ∼1.8–2.0 nm, dynamic flexibility with a root-mean-square fluctuation (RMSF) of ∼0.15–0.3 nm, stable hydrogen bonding (∼2–3 bonds for MTX-MnO2–Bi2S3, radial distribution function peak at ∼2.0 Å), and compactness with a radius of gyration (Rg) of ∼0.5 nm for MTX and ∼1.5 nm for MnO2–Bi2S3. These combined experimental and computational findings highlight the nanoplatform’s potential to overcome tumor hypoxia, deliver MTX effectively, and enhance radiotherapy outcomes, offering a robust and innovative framework for synergistic cancer therapy and paving the way for future preclinical development.

This study unravels the function of SmMYC2 regulating resistance to B. cinerea in S. miltiorrhiza. Salvia miltiorrhiza is a traditional medicinal herb and commonly used to treat cardiovascular and cerebrovascular diseases. Gray mold caused by Botrytis cinerea poses a significant threat to production of many medicinal plants. The key transcription factor SmMYC2 has been found to contribute to promoting the biosynthesis of tanshinones or phenolic acids, and also mediated salt resistance in S. miltiorrhiza. However, limited information underlying its role of resistance to B. cinerea is available. In this study, simultaneous improvement of tanshinone and phenolic acid biosynthesis was observed in transgenic S. miltiorrhiza plants overexpressing SmMYC2 (OE-SmMYC2). Besides, expression pattern analysis revealed that SmMYC2 transcript increased significantly after B. cinerea infection. OE-SmMYC2 plants significantly improved their resistance to B. cinerea, and enhanced the expression level of the defense marker genes, such as SmPR1, SmPR10, SmSam a1, and SmPR-STH2. Besides, three antioxidant enzymes including catalase, peroxidase and superoxide dismutase were increased in OE-SmMYC2 lines, and SmMYC2 reduced the H2O2 accumulation. Overall, our results revealed that SmMYC2 played positive regulatory roles in resistance to B. cinerea in S. miltiorrhiza.

Assembling and upscaling biomolecular activity to perform work in man-made devices is a challenge in synthetic biology. Here we report the step-by-step process to construct fully protein-based micro-three-dimensional (3D) printed robotic structures, which are coated with and actuated by a minimal actomyosin cortex. This approach can be used to program self-powered soft robots assembled from multiple biomolecular modules, devising biophysical assays to quantify active forces produced in 3D and engineering smart 3D microchips for synthetic cell assembly. The procedure covers the establishment of 3D printing microstructures from protein materials, the assembly of actomyosin-based active coatings and the robotic structure design and characterization. The detailed step-by-step instructions will guide scientists in replicating the preparation procedures, facilitating the adoption of biomolecular microrobots and the development of 3D protein-based robotic technology and their applications. The procedure is suited for users with expertise in biomaterials and requires 15 d to complete.

In this study, we evaluated some conditions affecting the extraction of pluthiophenol rich in pluthiophenol from the leaves of Pluchea indica collected in Hanoi. The investigated factors included extraction method, extraction solvent, medicinal plant size, material mass/solvent volume ratio, extraction time, and number of extraction times. Additionally, several methods of refining and enriching pluthiophenol in the Pluchea indica extract were also investigated. The criteria used to evaluate and select extraction conditions was the pluthiophenol content by the HPLC method. The best results included using hot extraction at 80 oC, extraction solvent of 60% ethanol, material size of 1 mm, extraction three times and 2 hours each time, with material/solvent ratios of 1/8, 1/6, and 1/6 (g/mL). Refining and enriching the pluthiophenol component was done using a liquid-liquid distribution and column chromatography (Amberlite XAD4 polymer adsorbent). The obtained extract after refining had a pluthiophenol content of 6.12%, and the pluthiophenol recovery efficiency of the whole process was 79.74% on a laboratory scale (50 g). In the 1 kg scale extraction procedure, the obtained extract had a pluthiophenol content of 6.29%.

The dynamics of chemical reactions in solution are of paramount importance in fields ranging from biology to materials science. Because the hydrogen-bond network and proton dynamics govern the behavior of aqueous solutions, they have been the subject of numerous studies over the years. Here, we report the observation of a previously unknown associative state in the hydroxide ion that forms when a proton from a neighboring water molecule approaches the hydroxide ion, utilizing resonant inelastic soft X-ray scattering (RIXS) and quantum dynamical simulations. State-of-the-art theoretical analysis reveals state mixing in the electronically excited states between aqueous hydroxide ions and the solvent. Our results give new insights into chemical bonding and excited-state dynamics in the aqueous environment. This investigation of associative states opens up new pathways for spectroscopic studies of chemical reaction dynamics and lays the foundation for directly accessing dynamic proton exchange in solution.

Mitochondria are essential organelles that provide energy for plants. They are semi-autonomous, maternally inherited, and closely linked to cytoplasmic male sterility (CMS) in plants. Lonicera macranthoides, a widely used medicinal plant from the Caprifoliaceae family, is rich in chlorogenic acid (CGA) and its analogues, which are known for their antiviral and anticancer properties. However, studies on the mitogenome of L. macranthoides still remain limited. The mitochondrial DNA contained in the whole genome DNA was extracted from a male sterile cultivar of L. macranthoides, named 'Yulei 1'. Next-generation sequencing (NGS) and third-generation sequencing (TGS) technologies were combined to obtain the mitogenome. RNA editing events were identified by integrating the mitogenome data with RNA sequencing data from leaf, stem, and flower tissues. The potential causes of male sterility in 'Yulei 1' were analyzed based on the loss of functional genes, mitogenome rearrangements, RNA editing events, and open reading frames (ORFs). The complete mitogenome of L. macranthoides 'Yulei 1' was obtained for the first time, with a length of 1,002,202 bp. It contains 48 protein-coding genes (PCGs), 26 tRNA genes, and 3 rRNA genes. Additionally, 79 simple sequence repeats (SSRs), 39 tandem repeats, and 99 dispersed repeats were identified. Among these, two direct repeats (RP1a/1b, RP2a/2b) and two inverse repeats (RP3a/b, RP4a/b) may facilitate mitogenome recombination. Gene transfer analysis revealed that 4.36% and 21.98% of mitogenomic sequences mapped to the chloroplast and nuclear genomes, respectively. Phylogenetic analysis indicated that L. macranthoides is closest to L. japonica at the mitogenome level. Notably, RNA editing events varied across different plant tissues, with 357 editing sites in 30 PCGs in leaves, 138 sites in 24 PCGs in flowers, and 68 sites in 13 PCGs in stems. Finally, all indications of CMS in the mitogenome were screened, including the detection of ORFs, and the findings showed no mutations in the mitogenome that would explain the sterility of 'Yulei 1'. Overall, our study provides a complete mitogenome of L. macranthoides, which will aid in its genetic marker exploration, evolutionary relationship analysis, and breeding programs.