Plant-based production of an orally active cyclotide for the treatment of multiple sclerosis.

Multiple sclerosis (MS) is a debilitating disease that requires prolonged treatment with often severe side effects. One experimental MS therapeutic currently under development is a single amino acid mutant of a plant peptide termed kalata B1, of the cyclotide family. Like all cyclotides, the therapeutic candidate [T20K]kB1 is highly stable as it contains a cyclic backbone that is cross-linked by three disulfide bonds in a knot-like structure. This stability is much sought after for peptide drugs, which despite exquisite selectivity for their targets, are prone to rapid degradation in human serum. In preliminary investigations, it was found that [T20K]kB1 retains oral activity in experimental autoimmune encephalomyelitis, a model of MS in mice, thus opening up opportunities for oral dosing of the peptide. Although [T20K]kB1 can be synthetically produced, a recombinant production system provides advantages, specifically for reduced scale-up costs and reductions in chemical waste. In this study, we demonstrate the capacity of the Australian native Nicotiana benthamiana plant to produce a structurally identical [T20K]kB1 to that of the synthetic peptide. By optimizing the co-expressed cyclizing enzyme, precursor peptide arrangements, and transgene regulatory regions, we demonstrate a [T20K]kB1 yield in crude peptide extracts of ~ 0.3 mg/g dry mass) in whole plants and close to 1.0 mg/g dry mass in isolated infiltrated leaves. With large-scale plant production facilities coming on-line across the world, the sustainable and cost-effective production of cyclotide-based therapeutics is now within reach.

Insights into the synthesis strategies of plant-derived cyclotides.

Cyclotides are plant peptides characterized with a head-to-tail cyclized backbone and three interlocking disulfide bonds, known as a cyclic cysteine knot. Despite the variations in cyclotides peptide sequences, this core structure is conserved, underlying their most useful feature: stability against thermal and chemical breakdown. Cyclotides are the only natural peptides known to date that are orally bioavailable and able to cross cell membranes. Cyclotides also display bioactivities that have been exploited and expanded to develop as potential therapeutic reagents for a wide range of conditions (e.g., HIV, inflammatory conditions, multiple sclerosis, etc.). As such, in vitro production of cyclotides is of the utmost importance since it could assist further research on this peptide class, specifically the structure-activity relationship and its mechanism of action. The information obtained could be utilized to assist drug development and optimization. Here, we discuss several strategies for the synthesis of cyclotides using both chemical and biological routes.

Bibliometric Review of the Literature on Cone Snail Peptide Toxins from 2000 to 2022.

The venom of marine cone snails is mainly composed of peptide toxins called conopeptides, among which conotoxins represent those that are disulfide-rich. Publications on conopeptides frequently state that conopeptides attract considerable interest for their potent and selective activity, but there has been no analysis yet that formally quantifies the popularity of the field. We fill this gap here by providing a bibliometric analysis of the literature on cone snail toxins from 2000 to 2022. Our analysis of 3028 research articles and 393 reviews revealed that research in the conopeptide field is indeed prolific, with an average of 130 research articles per year. The data show that the research is typically carried out collaboratively and worldwide, and that discoveries are truly a community-based effort. An analysis of the keywords provided with each article revealed research trends, their evolution over the studied period, and important milestones. The most employed keywords are related to pharmacology and medicinal chemistry. In 2004, the trend in keywords changed, with the pivotal event of that year being the approval by the FDA of the first peptide toxin drug, ziconotide, a conopeptide, for the treatment of intractable pain. The corresponding research article is among the top ten most cited articles in the conopeptide literature. From the time of that article, medicinal chemistry aiming at engineering conopeptides to treat neuropathic pain ramped up, as seen by an increased focus on topological modifications (e.g., cyclization), electrophysiology, and structural biology.

Exploring the Sequence Diversity of Cyclotides from Vietnamese Viola Species.

Viola is the largest genus in the Violaceae plant family and is known for its ubiquitous natural production of cyclotides. Many Viola species are used as medicinal herbs across Asia and are often consumed by humans in teas for the treatment of diseases, including ulcers and asthma. Previous studies reported the isolation of cyclotides from Viola species in many countries in the hope of discovering novel compounds with anti-cancer activities; however, Viola species from Vietnam have not been investigated to date. Here, the discovery of cyclotides from three Viola species (V. arcuata, V. tonkinensis, and V. austrosinensis) collected in the northern mountainous region of Vietnam is reported. Ten cyclotides were isolated from these three Viola species: four are novel and six were previously reported to be expressed in other plants. The structures of three of the new bracelet cyclotides are similar to that of cycloviolacin O2. Because cycloviolacin O2 has previously been shown to have potent activity against a wide range of cancer cell lines including HeLa (human cervical cancer cells) and PC-3 (human prostate cancer cells), the cancer cytotoxicity of the cyclotides isolated from V. arcuata was assessed. All tested cyclotides were cytotoxic against cancer cells, albeit to varying degrees. The sequences discovered in this study significantly expand the understanding of cyclotide diversity, especially in comparison with other cyclotides found in plants from the Asian region.

Caspase-1 causes truncation and aggregation of the Parkinson's disease-associated protein α-synuclein.

The aggregation of α-synuclein (aSyn) leading to the formation of Lewy bodies is the defining pathological hallmark of Parkinson's disease (PD). Rare familial PD-associated mutations in aSyn render it aggregation-prone; however, PD patients carrying wild type (WT) aSyn also have aggregated aSyn in Lewy bodies. The mechanisms by which WT aSyn aggregates are unclear. Here, we report that inflammation can play a role in causing the aggregation of WT aSyn. We show that activation of the inflammasome with known stimuli results in the aggregation of aSyn in a neuronal cell model of PD. The insoluble aggregates are enriched with truncated aSyn as found in Lewy bodies of the PD brain. Inhibition of the inflammasome enzyme caspase-1 by chemical inhibition or genetic knockdown with shRNA abated aSyn truncation. In vitro characterization confirmed that caspase-1 directly cleaves aSyn, generating a highly aggregation-prone species. The truncation-induced aggregation of aSyn is toxic to neuronal culture, and inhibition of caspase-1 by shRNA or a specific chemical inhibitor improved the survival of a neuronal PD cell model. This study provides a molecular link for the role of inflammation in aSyn aggregation, and perhaps in the pathogenesis of sporadic PD as well.

A soluble α-synuclein construct forms a dynamic tetramer.

A heterologously expressed form of the human Parkinson disease-associated protein α-synuclein with a 10-residue N-terminal extension is shown to form a stable tetramer in the absence of lipid bilayers or micelles. Sequential NMR assignments, intramonomer nuclear Overhauser effects, and circular dichroism spectra are consistent with transient formation of α-helices in the first 100 N-terminal residues of the 140-residue α-synuclein sequence. Total phosphorus analysis indicates that phospholipids are not associated with the tetramer as isolated, and chemical cross-linking experiments confirm that the tetramer is the highest-order oligomer present at NMR sample concentrations. Image reconstruction from electron micrographs indicates that a symmetric oligomer is present, with three- or fourfold symmetry. Thermal unfolding experiments indicate that a hydrophobic core is present in the tetramer. A dynamic model for the tetramer structure is proposed, based on expected close association of the amphipathic central helices observed in the previously described micelle-associated "hairpin" structure of α-synuclein.

Development of a shape-based algorithm for identification of asymptomatic vertebral compression fractures: A proof-of-principle study.

Objectives: Vertebral fracture is both common and serious among adults, yet it often goes undiagnosed. This study aimed to develop a shape-based algorithm (SBA) for the automatic identification of vertebral fractures. Methods: The study included 144 participants (50 individuals with a fracture and 94 without a fracture) whose plain thoracolumbar spine X-rays were taken. Clinical diagnosis of vertebral fracture (grade 0 to 3) was made by rheumatologists using Genant's semiquantitative method. The SBA algorithm was developed to determine the ratio of vertebral body height loss. Based on the ratio, SBA classifies a vertebra into 4 classes: 0 = normal, 1 = mild fracture, 2 = moderate fracture, 3 = severe fracture). The concordance between clinical diagnosis and SBA-based classification was assessed at both person and vertebra levels. Results: At the person level, the SBA achieved a sensitivity of 100% and specificity of 62% (95% CI, 51%-72%). At the vertebra level, the SBA achieved a sensitivity of 84% (95% CI, 72%-93%), and a specificity of 88% (95% CI, 85%-90%). On average, the SBA took 0.3 s to assess each X-ray. Conclusions: The SBA developed here is a fast and efficient tool that can be used to systematically screen for asymptomatic vertebral fractures and reduce the workload of healthcare professionals.

Biochar from Cyperus alternifolius Linn.: from a waste of phytoremediation processing to efficient depolluting agent.

Phytoremediation is one of the most powerful and viable solutions for developing countries to clean the soil and water bodies from metallic pollutants. Cyperus alternifolius Linn. (CAL), a tropical wetland plant, has been widely researched for removing harmful contaminants due to its hyperaccumulation ability. However, the waste biomass of phytoremediation processing may risk secondary environmental pollution. Thus, the preparation and application of biochar from metal-contaminated plants can be considered a new approach. In a 60-day experiment, CAL plants were irrigated with different concentrations of Zn(II) (200, 700, 1200, 1700, and 2200 mg·L-1), and then the plants were converted into biochar via the pyrolysis process. The characteristics of biochar including of surface composition and morphology, phase formation, and optical property were analyzed. The biochar enriched with Zn(II) at 1200 mg·L-1 had a bandgap value of 3.17 eV and consisted of carbon microparticles intermingled with ZnO and SiO2 nanoparticles. Furthermore, the adsorption and photocatalysis of the biochar were studied in the discolouration of methylene blue (MB), as a test reaction, with the maximum MB removal capacities of 55.2 mg·g-1. Such results will serve as the basis for new research aiming at the potential for reusing metal-contaminated plants to produce efficient depolluting biochar.

Cysteine-rich peptides: From bioactivity to bioinsecticide applications.

Greater levels of insect resistance and constraints on the use of current pesticides have recently led to increased crop losses in agricultural production. Further, the health and environmental impacts of pesticides now restrict their application. Biologics based on peptides are gaining popularity as efficient crop protection agents with low environmental toxicity. Cysteine-rich peptides (whether originated from venoms or plant defense substances) are chemically stable and effective as insecticides in agricultural applications. Cysteine-rich peptides fulfill the stability and efficacy requirements for commercial uses and provide an environmentally benign alternative to small-molecule insecticides. In this article, cysteine-rich insecticidal peptide classes identified from plants and venoms will be highlighted, focusing on their structural stability, bioactivity and production.

Cover crop influence on pore size distribution and biopore dynamics: Enumerating root and soil faunal effects.

Pore structure is a key determinant of soil functioning, and both root growth and activity of soil fauna are modified by and interact with pore structure in multiple ways. Cover cropping is a rapidly growing popular strategy for improving agricultural sustainability, including improvements in pore structure. However, since cover crop species encompass a variety of contrasting root architectures, they can have disparate effects on formation of soil pores and their characteristics, thus on the pore structure formation. Moreover, utilization of the existing pore systems and its modification by new root growth, in conjunction with soil fauna activity, can also vary by cover crop species, affecting the dynamics of biopores (creation and demolition). The objectives of this study were (i) to quantify the influence of 5 cover crop species on formation and size distribution of soil macropores (>36 µm Ø); (ii) to explore the changes in the originally developed pore architecture after an additional season of cover crop growth; and (iii) to assess the relative contributions of plant roots and soil fauna to fate and modifications of biopores. Intact soil cores were taken from 5 to 10 cm depth after one season of cover crop growth, followed by X-ray computed micro-tomography (CT) characterization, and then, the cores were reburied for a second root growing period of cover crops to explore subsequent changes in pore characteristics with the second CT scanning. Our data suggest that interactions of soil fauna and roots with pore structure changed over time. While in the first season, large biopores were created at the expense of small pores, in the second year these biopores were reused or destroyed by the creation of new ones through earthworm activities and large root growth. In addition, the creation of large biopores (>0.5 mm) increased total macroporosity. During the second root growing period, these large sized macropores, however, are reduced in size again through the action of soil fauna smaller than earthworms, suggesting a highly dynamic equilibrium. Different effects of cover crops on pore structure mainly arise from their differences in root volume, mean diameter as well as their reuse of existing macropores.

Make it or break it: Plant AEPs on stage in biotechnology.

Asparaginyl endopeptidases (AEPs) are cysteine proteases that control a myriad of cellular functions in plants, including maturation of seed storage proteins and programmed cell death. Recently, several noteworthy AEPs have been discovered that primarily function as transpeptidases rather than hydrolases, to instead catalyse the formation of new peptide bonds. These AEPs appear to have evolved for the cyclisation of a large class of plant defence peptides called cyclotides. Here we describe recent insights into the structural differences between AEPs that preference peptide ligation over hydrolysis. This knowledge is instrumental for the deployment of AEP ligases as biotechnological tools for in vitro applications such as protein labelling and or cyclization, and for plant molecular farming applications.

Effects of elevated temperature and elevated CO2 on soil nitrification and ammonia-oxidizing microbial communities in field-grown crop.

Rising global air temperature and atmospheric CO2 are expected to have considerable effects on soil nutrient cycling and plant productivity. Soil nitrification controlled by ammonia-oxidizing bacteria and archaea (AOB and AOA) communities plays a key role in contributing to plant nitrogen (N) availability; however, response of soil nitrification and functional microbial communities to climate change and subsequent consequences for crop yields remain largely unknown. Cotton productivity is a function of temperature and N availability under well-watered conditions. In general, cotton growth responds positively to elevated CO2, but simultaneous warming may offset benefits of rising CO2. In this study, cotton was used as a model system to elucidate the short-term response of soil nitrification and ammonia-oxidizing communities to elevated temperature and elevated CO2 using field-based environmentally-controlled chambers. Elevated temperature (ambient + 1.1 °C) altered the AOA community, while elevated temperature and elevated CO2 (ambient + 132 ppm) significantly increased soil nitrification rate and shifted AOB and AOA communities, but these effects depended on cotton developmental stages. Ammonia-oxidizing community abundance and structure were statistically correlated with nitrifying activity. Our findings suggest that climate change will positively affect soil nitrifying communities, leading to an increase in process rates and subsequent N availability, which is directly linked to crop productivity.

A new dimeric alkylresorcinol from the stem barks of Swintonia floribunda (Anacardiaceae).

From an EtOAc-soluble fraction of the stem barks of Swintonia floribunda (Anacardiaceae), one new dimeric alkylresorcinol named integracin E (1), together with 4 known compounds (2-5) were isolated. Their chemical structures were elucidated based on the spectroscopic data interpretation. The absolute configuration of 1 was determined by the specific rotation analysis of its acid-catalyzed hydrolysis product. Compound 1 showed potent tyrosinase inhibitory activity with an IC50 value of 48.2 µM.