A first-in-human phase 0 clinical study of RNA interference-based spherical nucleic acids in patients with recurrent glioblastoma.

Glioblastoma (GBM) is one of the most difficult cancers to effectively treat, in part because of the lack of precision therapies and limited therapeutic access to intracranial tumor sites due to the presence of the blood-brain and blood-tumor barriers. We have developed a precision medicine approach for GBM treatment that involves the use of brain-penetrant RNA interference-based spherical nucleic acids (SNAs), which consist of gold nanoparticle cores covalently conjugated with radially oriented and densely packed small interfering RNA (siRNA) oligonucleotides. On the basis of previous preclinical evaluation, we conducted toxicology and toxicokinetic studies in nonhuman primates and a single-arm, open-label phase 0 first-in-human trial (NCT03020017) to determine safety, pharmacokinetics, intratumoral accumulation and gene-suppressive activity of systemically administered SNAs carrying siRNA specific for the GBM oncogene Bcl2Like12 (Bcl2L12). Patients with recurrent GBM were treated with intravenous administration of siBcl2L12-SNAs (drug moniker: NU-0129), at a dose corresponding to 1/50th of the no-observed-adverse-event level, followed by tumor resection. Safety assessment revealed no grade 4 or 5 treatment-related toxicities. Inductively coupled plasma mass spectrometry, x-ray fluorescence microscopy, and silver staining of resected GBM tissue demonstrated that intravenously administered SNAs reached patient tumors, with gold enrichment observed in the tumor-associated endothelium, macrophages, and tumor cells. NU-0129 uptake into glioma cells correlated with a reduction in tumor-associated Bcl2L12 protein expression, as indicated by comparison of matched primary tumor and NU-0129-treated recurrent tumor. Our results establish SNA nanoconjugates as a potential brain-penetrant precision medicine approach for the systemic treatment of GBM.

Regulation by fungal endophyte of Rhodiola crenulata from enzyme genes to metabolites based on combination of transcriptome and metabolome.

BACKGROUND: The contents of some its crucial metabolites tend to decrease when Rhodiola crenulata is cultured at low altitude. Interestingly, it was found that an endophyte, Phialocephala fortinii, could alleviate this problem. RESULTS: There were 16 151 differential genes including 14 706 up-regulated and 1445 down-regulated unigenes with significant differences (P < 0.05), and a total of 1432 metabolites exhibited statistically significant (P < 0.05) metabolic differences comprising 27 different marker metabolites which showed highly significant values of VIP > 5 and P < 0.01. Results highlight differential regulation of 20 enzymatic genes that are involved in the biosynthesis of five different marker metabolites including acetaldehyde, homocysteine, cyclopropylamine, 1-pyrrolinium and halistanol sulfate. CONCLUSIONS: The positive physiological effect of P. fortinii on R. crenulata encompasses differential regulation in carbohydrate metabolism, lipid metabolism and secondary metabolite synthesis. © 2020 Society of Chemical Industry.

Correlation in Chemical Metabolome and Endophytic Mycobiome in Cynomorium songaricum from Different Desert Locations in China.

Cynomorium songaricum Rupr. is a valuable food and medicinal plant with functions, such as an increase in sexual function, mainly attributed to its complex secondary metabolites. However, the effect of internal microbes on metabolite production in C. songaricum is still largely unclear. In this study, the relationship between endophytes and differential secondary metabolites in C. songaricum from seven major producing regions of China were explored based on established methods of metabolomics and high-throughput sequencing. The results showed that there were 13 different marker metabolites, seven shared fungal OTUs, and numerous unshared OTUs among C. songaricum distributed at different locations in China and identified significant correlations between metabolites and endophytic fungi. Our study revealed that endophytic fungi may be one possible factor that can affect the plant secondary metabolite composition.

Fungal adaptation to plant defences through convergent assembly of metabolic modules.

The ongoing diversification of plant defence compounds exerts dynamic selection pressures on the microorganisms that colonize plant tissues. Evolutionary processes that generate resistance towards these compounds increase microbial fitness by giving access to plant resources and increasing pathogen virulence. These processes entail sequence-based mechanisms that result in adaptive gene functions, and combinatorial mechanisms that result in novel syntheses of existing gene functions. However, the priority and interactions among these processes in adaptive resistance remain poorly understood. Using a combination of molecular genetic and computational approaches, we investigated the contributions of sequence-based and combinatorial processes to the evolution of fungal metabolic gene clusters encoding stilbene cleavage oxygenases (SCOs), which catalyse the degradation of biphenolic plant defence compounds known as stilbenes into monophenolic molecules. We present phylogenetic evidence of convergent assembly among three distinct types of SCO gene clusters containing alternate combinations of phenolic catabolism. Multiple evolutionary transitions between different cluster types suggest recurrent selection for distinct gene assemblages. By comparison, we found that the substrate specificities of heterologously expressed SCO enzymes encoded in different clusters types were all limited to stilbenes and related molecules with a 4'-OH group, and differed modestly in substrate range and activity under the experimental conditions. Together, this work suggests a primary role for genome structural rearrangement, and the importance of enzyme modularity, in promoting fungal metabolic adaptation to plant defence chemistry.

Horizontal gene cluster transfer increased hallucinogenic mushroom diversity.

Secondary metabolites are a heterogeneous class of chemicals that often mediate interactions between species. The tryptophan-derived secondary metabolite, psilocin, is a serotonin receptor agonist that induces altered states of consciousness. A phylogenetically disjunct group of mushroom-forming fungi in the Agaricales produce the psilocin prodrug, psilocybin. Spotty phylogenetic distributions of fungal compounds are sometimes explained by horizontal transfer of metabolic gene clusters among unrelated fungi with overlapping niches. We report the discovery of a psilocybin gene cluster in three hallucinogenic mushroom genomes, and evidence for its horizontal transfer between fungal lineages. Patterns of gene distribution and transmission suggest that synthesis of psilocybin may have provided a fitness advantage in the dung and late wood-decay fungal niches, which may serve as reservoirs of fungal indole-based metabolites that alter behavior of mycophagous and wood-eating invertebrates. These hallucinogenic mushroom genomes will serve as models in neurochemical ecology, advancing the (bio)prospecting and synthetic biology of novel neuropharmaceuticals.

AP2 transcription factor CBX1 with a specific function in symbiotic exchange of nutrients in mycorrhizal Lotus japonicus.

The arbuscular mycorrhizal (AM) symbiosis, a widespread mutualistic association between land plants and fungi, depends on reciprocal exchange of phosphorus driven by proton-coupled phosphate uptake into host plants and carbon supplied to AM fungi by host-dependent sugar and lipid biosynthesis. The molecular mechanisms and cis-regulatory modules underlying the control of phosphate uptake and de novo fatty acid synthesis in AM symbiosis are poorly understood. Here, we show that the AP2 family transcription factor CTTC MOTIF-BINDING TRANSCRIPTION FACTOR1 (CBX1), a WRINKLED1 (WRI1) homolog, directly binds the evolutionary conserved CTTC motif that is enriched in mycorrhiza-regulated genes and activates Lotus japonicus phosphate transporter 4 (LjPT4) in vivo and in vitro. Moreover, the mycorrhiza-inducible gene encoding H+-ATPase (LjHA1), implicated in energizing nutrient uptake at the symbiotic interface across the periarbuscular membrane, is coregulated with LjPT4 by CBX1. Accordingly, CBX1-defective mutants show reduced mycorrhizal colonization. Furthermore, genome-wide-binding profiles, DNA-binding studies, and heterologous expression reveal additional binding of CBX1 to AW box, the consensus DNA-binding motif for WRI1, that is enriched in promoters of glycolysis and fatty acid biosynthesis genes. We show that CBX1 activates expression of lipid metabolic genes including glycerol-3-phosphate acyltransferase RAM2 implicated in acylglycerol biosynthesis. Our finding defines the role of CBX1 as a regulator of host genes involved in phosphate uptake and lipid synthesis through binding to the CTTC/AW molecular module, and supports a model underlying bidirectional exchange of phosphorus and carbon, a fundamental trait in the mutualistic AM symbiosis.

Secondary Metabolite Accumulation Associates with Ecological Succession of Endophytic Fungi in Cynomorium songaricum Rupr.

Cynomorium songaricum Rupr. is a rare root-parasitic plant distributed in the desert ecosystem. Little is known about the role of endophytes in accumulation of metabolites in C. songaricum. Here, the correlations between the seven active components (total sugars, flavonoids, protocatechuic acid, catechins, tannins, gallic acid, and ursolic acid) and the endophytic fungi of C. songaricum were investigated, and their causal relationships are discussed further. The results showed that the accumulation of these components and the assembly of endophytic fungi changed with different plant developmental stages. Diverse relationships including positive and negative correlation were found among chemicals and endophytic fungal operational taxonomic units based on correlation coefficient matrices, which demonstrated that the accumulation of secondary metabolites in C. songaricum is closely related to the endophytic fungal community composition. These results present new opportunities to deeply understand plant-fungal symbioses and secondary metabolite productions.

Partitioning of Fungal Endophyte Assemblages in Root-Parasitic Plant Cynomorium songaricum and Its Host Nitraria tangutorum.

Endophytic fungi are an integral part and even seen as host organs of plant, influencing physiology, ecology, and development of host plants. However, little is known about micro-ecosystems and functional interactions of endophytic fungi in root-parasitic interactions of Cynomorium songaricum and its host Nitraria tangutorum. Here, distribution and dynamics of endophytic fungi were objectively investigated in their associations with C. songaricum and N. tangutorum based on mycobiome studies using high-throughput sequencing. Results suggest that endophytic fungi may be exchanged between C. songaricum and its host N. tangutorum probably through haustorium, connection of xylem and phloem in the vascular system. The similarity of endophytic fungal composition between C. songaricum and parasitized N. tangutorum was 3.88% which was significantly higher than the fungal similarity of 0.10% observed between C. songaricum and non-parasitized N. tangutorum. The similarities of fungal community in parasitized N. tangutorum were much closer to C. songaricum than to the non-parasitized N. tangutorum. The composition of endophytic fungi in these associations increased in progressive developmental stages of C. songaricum from sprouting to above ground emergence, and decreased subsequently probably due to host recognition and response by fungi. However, the shared fungal operational taxonomic units (OTUs) increased among interactions of C. songaricum with parasitized and non-parasitized N. tangutorum. Studies of bioactivity on culturable endophytic fungi showed that isolates such as Fusarium spp. possess the ability to promote seed germination of C. songaricum. Our study reports for the first time the special ecological system of endophytic fungi in C. songaricum and its host N. tangutorum. Overall, we hypothesize that a deeper understanding of the sharing, movement, and role of endophytic fungi between root-parasitic plant and its host may lead to finding alternative approaches to help increase the output of ethno-pharmacologically important medicinal plants.

Integrated multi-omics analysis supports role of lysophosphatidylcholine and related glycerophospholipids in the Lotus japonicus-Glomus intraradices mycorrhizal symbiosis.

Interaction of plant roots with arbuscular mycorrhizal fungi (AMF) is a complex trait resulting in cooperative interactions among the two symbionts including bidirectional exchange of resources. To study arbuscular mycorrhizal symbiosis (AMS) trait variation in the model plant Lotus japonicus, we performed an integrated multi-omics analysis with a focus on plant and fungal phospholipid (PL) metabolism and biological significance of lysophosphatidylcholine (LPC). Our results support the role of LPC as a bioactive compound eliciting cellular and molecular response mechanisms in Lotus. Evidence is provided for large interspecific chemical diversity of LPC species among mycorrhizae with related AMF species. Lipid, gene expression and elemental profiling emphasize the Lotus-Glomus intraradices interaction as distinct from other arbuscular mycorrhizal (AM) interactions. In G. intraradices, genes involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs were enhanced, while in Lotus, FA synthesis genes were up-regulated during AMS. Furthermore, FAS protein localization to mitochondria suggests FA biosynthesis and elongation may also occur in AMF. Our results suggest the existence of interspecific partitioning of PL resources for generation of LPC and novel candidate bioactive PLs in the Lotus-G. intraradices symbiosis. Moreover, the data advocate research with phylogenetically diverse Glomeromycota species for a broader understanding of the molecular underpinnings of AMS.

Network of GRAS transcription factors involved in the control of arbuscule development in Lotus japonicus.

Arbuscular mycorrhizal (AM) fungi, in symbiosis with plants, facilitate acquisition of nutrients from the soil to their host. After penetration, intracellular hyphae form fine-branched structures in cortical cells termed arbuscules, representing the major site where bidirectional nutrient exchange takes place between the host plant and fungus. Transcriptional mechanisms underlying this cellular reprogramming are still poorly understood. GRAS proteins are an important family of transcriptional regulators in plants, named after the first three members: GIBBERELLIC ACID-INSENSITIVE, REPRESSOR of GAI, and SCARECROW. Here, we show that among 45 transcription factors up-regulated in mycorrhizal roots of the legume Lotus japonicus, expression of a unique GRAS protein particularly increases in arbuscule-containing cells under low phosphate conditions and displays a phylogenetic pattern characteristic of symbiotic genes. Allelic rad1 mutants display a strongly reduced number of arbuscules, which undergo accelerated degeneration. In further studies, two RAD1-interacting proteins were identified. One of them is the closest homolog of Medicago truncatula, REDUCED ARBUSCULAR MYCORRHIZATION1 (RAM1), which was reported to regulate a glycerol-3-phosphate acyl transferase that promotes cutin biosynthesis to enhance hyphopodia formation. As in M. truncatula, the L. japonicus ram1 mutant lines show compromised AM colonization and stunted arbuscules. Our findings provide, to our knowledge, new insight into the transcriptional program underlying the host's response to AM colonization and propose a function of GRAS transcription factors including RAD1 and RAM1 during arbuscule development.

Phosphoenolpyruvate provision to plastids is essential for gametophyte and sporophyte development in Arabidopsis thaliana.

Restriction of phosphoenolpyruvate (PEP) supply to plastids causes lethality of female and male gametophytes in Arabidopsis thaliana defective in both a phosphoenolpyruvate/phosphate translocator (PPT) of the inner envelope membrane and the plastid-localized enolase (ENO1) involved in glycolytic PEP provision. Homozygous double mutants of cue1 (defective in PPT1) and eno1 could not be obtained, and homozygous cue1 heterozygous eno1 mutants [cue1/eno1(+/-)] exhibited retarded vegetative growth, disturbed flower development, and up to 80% seed abortion. The phenotypes of diminished oil in seeds, reduced flavonoids and aromatic amino acids in flowers, compromised lignin biosynthesis in stems, and aberrant exine formation in pollen indicate that cue1/eno1(+/-) disrupts multiple pathways. While diminished fatty acid biosynthesis from PEP via plastidial pyruvate kinase appears to affect seed abortion, a restriction in the shikimate pathway affects formation of sporopollonin in the tapetum and lignin in the stem. Vegetative parts of cue1/eno1(+/-) contained increased free amino acids and jasmonic acid but had normal wax biosynthesis. ENO1 overexpression in cue1 rescued the leaf and root phenotypes, restored photosynthetic capacity, and improved seed yield and oil contents. In chloroplasts, ENO1 might be the only enzyme missing for a complete plastidic glycolysis.

Biomechanical factors and injury risk in high-severity rollovers.

The number of rolls, as well as other factors, has been associated with increased injury risk in rollovers. Data from NASS-CDS from 1995-2003 were used to evaluate the biomechanical implications of vehicle kinematics during multiple rolls and to evaluate the risk of injuries to different body regions during rollovers. The data showed that the risk of injury increased with increasing number of rolls. The rate of increase in risk varied by the region of the body affected and injury severity. The increased risk was particularly great when a vehicle rolled more than two complete rolls.