Microbial production of trans-aconitic acid.

Trans-aconitic acid (TAA) is a promising bio-based chemical with the structure of unsaturated tricarboxylic acid, and also has the potential to be a non-toxic nematicide as a potent inhibitor of aconitase. However, TAA has not been commercialized because the traditional production processes of plant extraction and chemical synthesis cannot achieve large-scale production at a low cost. The availability of TAA is a serious obstacle to its widespread application. In this study, we developed an efficient microbial synthesis and fermentation production process for TAA. An engineered Aspergillus terreus strain producing cis-aconitic acid and TAA was constructed by blocking itaconic acid biosynthesis in the industrial itaconic acid-producing strain. Through heterologous expression of exogenous aconitate isomerase, we further designed a more efficient cell factory to specifically produce TAA. Subsequently, the fermentation process was developed and scaled up step-by-step, achieving a TAA titer of 60 g L-1 at the demonstration scale of a 20 m3 fermenter. Finally, the field evaluation of the produced TAA for control of the root-knot nematodes was performed in a field trial, effectively reducing the damage of the root-knot nematode. Our work provides a commercially viable solution for the green manufacturing of TAA, which will significantly facilitate biopesticide development and promote its widespread application as a bio-based chemical.

Aconitate isomerase from maize leaves: Light-dependent expression and kinetic properties.

Aconitate isomerase (EC 5.3.3.7) interconverts cis- and trans-isomers of aconitic acid. Expression of the gene encoding this enzyme was studied in maize (Zea mays L.) leaves depending on light regime. Aconitate isomerase was induced by white and by red light indicating the involvement of phytochrome in the regulation of gene expression. The enzyme was partially purified from maize leaves. The value of Km was 0.75 mM with cis-aconitate and 0.92 mM with trans-aconitate, pH optimum was 8.0-8.2 with both substrates, citrate and malate suppressed its activity. It is concluded that aconitate isomerase actively participates in the interconversion of cis- and trans-aconitate in the light providing a possibility of using the pool of trans-aconitate for the regulation of the tricarboxylic acid cycle activity and mediating citrate/isocitrate supply for the biosynthetic and signaling purposes in photosynthetic cells.

The crystal structure of mouse IRG1 suggests that cis-aconitate decarboxylase has an open and closed conformation.

PROTEIN DATA BANK ACCESSION CODES: Coordinate and structural factors were deposited with the Protein Data Bank under PDB ID: 7BR9.

Construction of multilayer films with bactericidal and long-term antitumor drug release properties as a non-vascular stent coating for therapy in obstruction.

The construction of antibacterial and antitumor coatings could offer effective routes to improve the therapeutic effects of non-vascular stents for unresectable obstructions caused by malignant tumours. Herein, polyelectrolyte multilayers have been explored as bactericidal coatings with controlled antitumor drug release. To solve the challenges of loading and controlled release of small-molecule chemotherapeutic drugs in polyelectrolyte multilayers, the antitumor drug doxorubicin (DOX) was chemically conjugated onto polyethylenimine via cis aconitic anhydride (pH-sensitive linker), thus obtaining the polycation prodrug PEI-CA-DOX. Alginate sodium was oxidized (O-Alg) and mixed with DOX to prepare the O-Alg-DOX complex as a polyanion. QCM-D and contact angle tests were used to monitor and verify the progressive build-up of the PEI-CA-DOX/O-Alg-DOX multilayer films, which show a linear growth. The in vitro antibacterial tests indicated that the PEI-CA-DOX-terminated PEI-CA-DOX/O-Alg-DOX multilayers could kill the bacteria effectively. As-such multilayers also presented a long-term sustained DOX release behaviour in PBS due to the combination of slow release in PEI-CA-DOX and fast release in the O-Alg-DOX complex. The as-designed PEI-CA-DOX/O-Alg-DOX multilayers with combined antibacterial and antitumor properties may have great potential for applications in non-vascular stent coatings for palliative treatment of obstruction caused by malignant tumours.

pH and redox dual responsive carrier-free anticancer drug nanoparticles for targeted delivery and synergistic therapy.

Advanced drug delivery systems often employ nanomaterials as carriers to deliver drugs to desirable disease sites for enhanced efficacy. However, most systems have low drug loading capacity and cause safety concerns. Therefore, many anticancer therapeutics have recently been assembled to NPs form without using any additional nanocarrier to achieve high drug loading. However, carrier-free nanomedicines are often constrained by limitations such as inadequate stability and lack of control in drug release. Therefore, we synthesize carrier-free drug NPs containing cis-aconitic anhydride-modified doxorubicin and paclitaxel (CAD-PTX) and coating with crosslinked (CL) surfactant based on hyaluronic acid (HA) segment. With this design, the pure drug NPs possess pH and redox dual responsive release characteristic and could target CD44 overexpressed cancer cells. Our studies demonstrate that these CAD-PTX-CLHA NPs display high stability, excellent active targeting effect and controllable intracellular drug release, and ultimately achieve significantly better anti-cancer efficiency than individual doxorubicin and paclitaxel.

Integrative comparative analyses of metabolite and transcript profiles uncovers complex regulatory network in tomato (Solanum lycopersicum L.) fruit undergoing chilling injury.

Tomato fruit are especially susceptible to chilling injury (CI) when continuously exposed to temperatures below 12 °C. In this study, integrative comparative analyses of transcriptomics and metabolomics data were performed to uncover the regulatory network in CI tomato fruit. Metabolite profiling analysis found that 7 amino acids, 27 organic acids, 16 of sugars and 22 other compounds had a significantly different content while transcriptomics data showed 1735 differentially expressed genes (DEGs) were down-regulated and 1369 were up-regulated in cold-stored fruit. We found that the contents of citrate, cis-aconitate and succinate were increased, which were consistent with the expression of ATP-citrate synthase (ACS) and isocitrate dehydrogenase (IDH) genes in cold-treated tomato fruit. Cold stress promotes the expression of ACS and IDH which may increase the synthesis of citrate, cis-aconitate and succinate. Alanine and leucine had increased contents, which may result from alanine aminotransferase (ALT) and branched-chain amino acid aminotransferase (BcAT)'s high expression levels, respectively. Overall the transcriptomics and metabolomics data in our study explain the molecular mechanisms of the chilling injury and expands our understanding of the complex regulatory mechanisms of a metabolic network in response to chilling injury in tomato fruit.

Microenvironment-Induced In Situ Self-Assembly of Polymer-Peptide Conjugates That Attack Solid Tumors Deeply.

In cancer treatment, the unsatisfactory solid-tumor penetration of nanomaterials limits their therapeutic efficacy. We employed an in vivo self-assembly strategy and designed polymer-peptide conjugates (PPCs) that underwent an acid-induced hydrophobicity increase with a narrow pH-response range (from 7.4 to 6.5). In situ self-assembly in the tumor microenvironment at appropriate molecular concentrations (around the IC50 values of PPCs) enabled drug delivery deeper into the tumor. A cytotoxic peptide KLAK, decorated with the pH-sensitive moiety cis-aconitic anhydride (CAA), and a cell-penetrating peptide TAT were conjugated onto poly(ß-thioester) backbones to produce PT-K-CAA, which can penetrate deeply into solid tumors owing to its small size as a single chain. During penetration in vivo, CAA responds to the weak acid, leading to the self-assembly of PPCs and the recovery of therapeutic activity. Therefore, a deep-penetration ability for enhanced cancer therapy is provided by this in vivo assembly strategy.

Bromocatechol conjugates from a Chinese marine red alga, Symphyocladia latiuscula.

This study describes an investigation into polybromocatechol conjugates isolated from a marine red alga, Symphyocladia latiuscula (Harvey) Yamada, collected from coastal waters off Qingdao, China. We report on the isolation and characterisation of eight undescribed aconitic acid conjugates, symphyocladins R-X, including a likely solvolysis artifact of symphyocladin S, and an undescribed furanoyl conjugate, symphyocladin Y. Structure elucidation was achieved by detailed spectroscopic analysis. A plausible biosynthetic pathway linking all these co-metabolites through a cascade of quinone methide additions is proposed.

Polyaconitic acid/functional amine/azo dye composite as a novel hyper-branched polymer for cotton fabric functionalization.

A new hyperbranched polymer based on aconitic acid and two different amine (triethnaol amine and diethylenetriamine) with different functional groups; hydroxyl and amine groups respectively was successfully synthesised by A2B3 polymerization technique and characterised using Fourier Transform Infrared (FT-IR), Nuclear Magnetic Resonance (NMR), rheological properties, antimicrobial and cytotoxicity activity. In addition, a new heterocyclic azo dye was synthesised and characterised using FT-IR, NMR, mass spectra and antimicrobial activity. Characterisation provides that both composites and azo dye have been well prepared. A mixture from both hyberbranched polymer and synthesised azo dye have been applied to cotton fabrics. Evaluation of treated fabrics shows that, the surface of treated fabrics has a thin film from applied composite which coated the whole fibre surface. Treated fabrics have good antimicrobial activity against gram positive, gram negative bacteria and fungi. Fastness properties, physical and mechanical properties for treated fabrics were also evaluated.

Rod-like cellulose nanocrystal/cis-aconityl-doxorubicin prodrug: A fluorescence-visible drug delivery system with enhanced cellular uptake and intracellular drug controlled release.

Rod-like nanomedicines facilitate cellular uptake. This research is aimed to develop fluorescence-visible rod-like nanomedicines with enhanced cellular uptake and intracellular drug controlled release based on cis-aconityl-doxorubicin (CAD) labeled cellulose nanocrystal rods (CNR). Particularly, CAD was synthesized by the ring-opening reaction between cis-aconitic anhydride (CAA) and the amino group of Doxorubicin (DOX). Amidation reaction occurred between the 6-carboxylic groups of CAD and the amino groups of aminated CNR to give CAD labeled CNR (CAD@CNR). Compared with CNR, CAD@CNR showed similar morphology and crystal structure. The mean length of CAD@CNR was ca. 118 nm with aspect ratio ranging from 12 to 15, facilitating their endocytosis. CAD@CNR prodrug was rather stable in pH 7.4 phosphate buffer solution but tended to be hydrolyzed to release DOX under acidic condition, due to the rapid degradation of amide bonds between DOX and cis-aconitic acid via an intramolecular acid-catalyzed mechanism. CAD@CNR prodrug showed sustained drug release profiles over 40 h, and the cumulative drug release showed a tendency to increase from 36 to 80% with the pH value decreasing from 7.4 to 5.0. The half maximal inhibitory concentration (IC50) of CAD@CNR prodrug against NCI H 460 cells without NH4Cl (lysosomotropic weak bases) pretreatment was 1.75 times higher than that with 40 mM NH4Cl pretreatment, further confirmed that the DOX release from the CAD@CNR prodrug was triggered by the low pH value of lysosome (pH 5.0). Compared with DOX·HCl, CAD@CNR prodrug showed enhanced cellular uptake ability during 12 or 24 h of incubation due to the endocytosis mechanism of CAD@CNR prodrug. After incubation with cells, CAD@CNR prodrug could be observed by using fluorescence microscope due to the red fluorescence of DOX. In a word, CAD@CNR showed great potential as fluorescence-visible drug delivery system with enhanced cellular uptake and intracellular drug release due to its rod-like morphology, suitable aspect ratio, and acid-triggered drug release.

Esterification of trans-aconitic acid improves its anti-inflammatory activity in LPS-induced acute arthritis.

trans-Aconitic acid (TAA) is an abundant constituent in the leaves of Echinodorus grandiflorus, a medicinal plant used to treat rheumatoid arthritis in Brazil. Esterification was explored as a strategy to increase lipophilicity and biopharmaceutical properties of TAA, a highly polar tricarboxylic acid. We herein report the synthesis of TAA esters via Fischer esterification with ethanol, n-butanol and n-octanol. The reaction kinetics was investigated to produce mono-, di- and tri- derivatives. Mono- and diesters of TAA were obtained as a mixture of positional isomers, whereas the triesters were recovered as pure compounds. The obtained esters were screened in a model of acute arthritis induced by the injection of LPS in the knee joint of Swiss mice. The diesters were the most active compounds, regardless of the alcohol employed in the reaction, whereas bioactivity of the derivatives improved by increasing the length of the aliphatic chain of the alcohol employed in esterification. In general, the esters showed higher potency than TAA. When administered orally to mice at doses of 0.017-172.3 µmol/Kg, the diethyl, di-n-butyl and di-n-octyl esters of TAA reduced the cellular infiltration into the knee joint, especially of neutrophils. The study identified diesters of TAA as potential useful derivatives for the management of rheumatoid arthritis and other inflammatory diseases.

Direct Macromolecular Drug Delivery to Cerebral Ischemia Area using Neutrophil-Mediated Nanoparticles.

Delivery of macromolecular drugs to the brain is impeded by the blood brain barrier. The recruitment of leukocytes to lesions in the brain, a typical feature of neuroinflammation response which occurs in cerebral ischemia, offers a unique opportunity to deliver drugs to inflammation sites in the brain. In the present study, cross-linked dendrigraft poly-L-lysine (DGL) nanoparticles containing cis-aconitic anhydride-modified catalase and modified with PGP, an endogenous tripeptide that acts as a ligand with high affinity to neutrophils, were developed to form the cl PGP-PEG-DGL/CAT-Aco system. Significant binding efficiency to neutrophils, efficient protection of catalase enzymatic activity from degradation and effective transport to receiver cells were revealed in the delivery system. Delivery of catalase to ischemic subregions and cerebral neurocytes in MCAO mice was significantly enhanced, which obviously reducing infarct volume in MCAO mice. Thus, the therapeutic outcome of cerebral ischemia was greatly improved. The underlying mechanism was found to be related to the inhibition of ROS-mediated apoptosis. Considering that neuroinflammation occurs in many neurological disorders, the strategy developed here is not only promising for treatment of cerebral ischemia but also an effective approach for various CNS diseases related to inflammation.

Co-delivery of docetaxel and curcumin prodrug via dual-targeted nanoparticles with synergistic antitumor activity against prostate cancer.

PURPOSE: Combination therapy is increasingly used as a primary cancer treatment regimen. In this report, we designed EGFR peptide decorated nanoparticles (NPs) to co-deliver docetaxel (DTX) and pH sensitive curcumin (CUR) prodrug for the treatment of prostate cancer. RESULTS: EGFR peptide (GE11) targeted, pH sensitive, DTX and CUR prodrug NPs (GE11-DTX-CUR NPs) had an average diameter of 167nm and a zeta potential of -37.5mV. The particle size of the NPs was adequately maintained in serum and a sustained drug release pattern was observed. Improved inhibition of cancer cell and tumor tissue growth was shown in the GE11-DTX-CUR NPs group compared to the other groups. CONCLUSION: It can be summarized that DTX and CUR prodrug could be delivered into tumor cells simultaneously by the GE 11 targeting and the EPR effect of NPs. The resulting GE11-DTX-CUR NPs is a promising system for the synergistic antitumor treatment of prostate cancer.

Contribution of trans-aconitic acid to DPPH scavenging ability in different media.

The antioxidant properties of trans-aconitic acid (TAA) alone or in the presence of usual antioxidants were assessed by DPPH assay. The IC50 value equal to 70mM was very high compared to usual antioxidants (vitamin C and trolox). A joint experimental/theoretical study suggested that hydrogen atom abstraction in TAA by DPPH was located on -CH2- methylene bridge because the corresponding radical was more stabilized than COO(·) and CC(·) radicals. In combination with antioxidants (vitamin C, gallic acid, caffeic acid, trolox), synergy or additivity effects were noticed. The magnitude of the synergistic effect varied between 1.06 and 1.24 depending on the type and concentration of antioxidant for a concentration of TAA equal to 22.3mM. Especially, the addition of TAA at a concentration below 32mM to a solution containing 20µM of vitamin C had a synergy effect. Beyond this concentration, TAA showed an additive effect.

Preparation and characterization of pH-sensitive camptothecin-cis-aconityl grafted chitosan oligosaccharide nanomicelles.

Camptothecin (CPT) was introduced to water-soluble chitosan oligosaccharide (CHO) using cis-aconityl (CA), as a pH-sensitive linker, to develop a new hydrophobic structure, i.e. CPTCACHO. The triple conjugates were synthesized in three ratios (5%, 7.5%, and 10%) and characterized by Fourier transform infrared (FT-IR) and proton nuclear magnetic resonance (1HNMR). Thermo gravimetric analysis and critical micelle concentration (CMC) assessments were performed. Prepared nano-micelles were analyzed for particle size, polydispersity index (PDI), drug release and in vitro cytotoxicity. CPTCACHO 7.5% micelles as optimum micelles had a mean diameter of 50nm (observed by transmission electron microscopy), a zeta potential of +45.9mV, and a CMC of about 9.97×10-5g/L. The release results showed that CPTCACHO 7.5% has the burst release at acidic pH, and cytotoxicity study indicated that IC50 of CPTCACHO 7.5% for MCF-7 cell line was 0.8µg/mL. These properties altogether make CPTCACHO micelles, as a pH sensitive cargo with inherent cytotoxicity, a potential candidate for hydrophobic anticancer drugs.

Graphene oxide-incorporated pH-responsive folate-albumin-photosensitizer nanocomplex as image-guided dual therapeutics.

The objective of this study was to develop an active-targeted, pH-responsive albumin-photosensitizer-incorporated graphene oxide nanocomplex as an image-guided theranostic agent for dual therapies. Herein, bovine serum albumin (BSA)-cis-aconityl pheophorbide-a (c-PheoA) conjugate was complexed with graphene oxide (GO) at ratios of 1:1, 1:0.5, and 1:0.1 with the mean hydrodynamic diameter of the resulting complex being 100-200nm. Further, with the 1:0.5 ratio, we developed a folate-BSA-c-PheoA conjugate:GO complex incorporated free PheoA (PheoA+GO:FA-BSA-c-PheoA NC) with a mean hydrodynamic diameter of 182.0±33.2nm. The release study showed that the photosensitizer from the nanocomplex was released rapidly at pH5.5 compared to that at pH7.4 when incubated for 24h. Cellular uptake results showed that the PheoA+GO:FA-BSA-c-PheoA NCs was readily taken up by B16F10 and MCF7 cancer cells. In vitro phototoxicity results showed that PheoA+GO:FA-BSA-c-PheoA NC has a higher efficacy against cancer cells than free PheoA, thereby demonstrating the synergistic effect of PS and GO in response to a single laser of 670nm. In vivo and ex vivo bioimaging results showed that fluorescence signals of higher intensity were observed in the tumor area of mice treated with PheoA+GO:FA-BSA-c-PheoA NC than those in the tumor of mice treated with free PheoA, thereby suggesting that the targeted nanocomplex selectively accumulated in the tumor area compared to free PheoA. Through antitumor study, PheoA+GO:FA-BSA-c-PheoA NC showed a synergistic effect in tumor-bearing mice by a single 671nm laser treatment. These results demonstrate that our prepared PheoA+GO:FA-BSA-c-PheoA NC can be used as a theranostic agent in phototherapies and for the photodiagnosis of cancer.

Ustilago maydis produces itaconic acid via the unusual intermediate trans-aconitate.

Itaconic acid is an important biomass-derived chemical building block but has also recently been identified as a metabolite produced in mammals, which has antimicrobial activity. The biosynthetic pathway of itaconic acid has been elucidated in the ascomycetous fungus Aspergillus terreus and in human macrophages. In both organisms itaconic acid is generated by decarboxylation of the tricarboxylic acid (TCA) cycle intermediate cis-aconitate. Here, we show that the basidiomycetous fungus Ustilago maydis uses an alternative pathway and produces itaconic acid via trans-aconitate, the thermodynamically favoured isomer of cis-aconitate. We have identified a gene cluster that contains all genes involved in itaconic acid formation. Trans-aconitate is generated from cis-aconitate by a cytosolic aconitate-Δ-isomerase (Adi1) that belongs to the PrpF family of proteins involved in bacterial propionate degradation. Decarboxylation of trans-aconitate is catalyzed by a novel enzyme, trans-aconitate decarboxylase (Tad1). Tad1 displays significant sequence similarity with bacterial 3-carboxy-cis,cis-muconate lactonizing enzymes (CMLE). This suggests that U. maydis has evolved an alternative biosynthetic pathway for itaconate production using the toxic intermediate trans-aconitate. Overexpression of a pathway-specific transcription factor (Ria1) or a mitochondrial tricarboxylic acid transporter (Mtt1) resulted in a twofold increase in itaconate yield. Therefore, our findings offer new strategies for biotechnological production of this valuable biomass-derived chemical.

Enzymatic characterization and gene identification of aconitate isomerase, an enzyme involved in assimilation of trans-aconitic acid, from Pseudomonas sp. WU-0701.

UNLABELLED: trans-Aconitic acid is an unsaturated organic acid that is present in some plants such as soybean and wheat; however, it remains unclear how trans-aconitic acid is degraded and/or assimilated by living cells in nature. From soil, we isolated Pseudomonas sp. WU-0701 assimilating trans-aconitic acid as a sole carbon source. In the cell-free extract of Pseudomonas sp. WU-0701, aconitate isomerase (AI; EC 5.3.3.7) activity was detected. Therefore, it seems likely that strain Pseudomonas sp. WU-0701 converts trans-aconitic acid to cis-aconitic acid with AI, and assimilates this via the tricarboxylic acid cycle. For the characterization of AI from Pseudomonas sp. WU-0701, we performed purification, determination of enzymatic properties and gene identification of AI. The molecular mass of AI purified from cell-free extract was estimated to be ~ 25 kDa by both SDS/PAGE and gel filtration analyses, indicating that AI is a monomeric enzyme. The optimal pH and temperature of purified AI for the reaction were 6.0 °C and 37 °C, respectively. The gene ais encoding AI was cloned on the basis of the N-terminal amino acid sequence of the protein, and Southern blot analysis revealed that only one copy of ais is located on the bacterial genome. The gene ais contains an ORF of 786 bp, encoding a polypeptide of 262 amino acids, including the N-terminal 22 amino acids as a putative periplasm-targeting signal peptide. It is noteworthy that the amino acid sequence of AI shows 90% and 74% identity with molybdenum ABC transporter substrate-binding proteins of Pseudomonas psychrotolerans and Xanthomonas albilineans, respectively. This is the first report on purification to homogeneity, characterization and gene identification of AI. DATABASE: The nucleotide sequence of ais described in this article is available in the DDBJ/EMBL/GenBank nucleotide sequence databases under the Accession No. LC010980.

Preclinical evaluation of antitumor activity of acid-sensitive PEGylated doxorubicin.

The acid-sensitive PEGylated doxorubicin (DOX) with exact chemical structure was designed and prepared as a potential tumor intracellular microenvironment-responsive drug delivery system. First, the insensitive succinic anhydride-functionalized DOX (i.e., SAD) and acid-sensitive cis-aconitic anhydride-modified DOX (i.e., CAD) were synthesized through the ring-opening reaction. Subsequently, the insensitive and acid-sensitive PEGylated DOX (i.e., mPEG-SAD and mPEG-CAD) was prepared by the condensation reaction between the terminal hydroxyl group of mPEG and the carboxyl group in SAD and CAD, respectively. The obtained mPEG-SAD and mPEG-CAD could spontaneously self-assemble into micelles in phosphate-buffered saline at pH 7.4 with diameters of about 100 nm. The DOX release of mPEG-CAD micelle could be accelerated by the decrease of pH from 7.4, 6.8, to 5.5 in relation to that of mPEG-SAD micelle. On the other hand, the result of the cellular proliferation inhibition test indicated that mPEG-CAD micelle exhibited favorable antiproliferative activity in vitro. In addition, the selective intratumoral accumulation and antitumor efficacy of mPEG-CAD micelle were significantly better than those of free DOX and mPEG-SAD. More importantly, the prodrug micelles exhibited upregulated security in vivo as compared to free DOX. Overall, the mPEG-CAD micelle with enhanced antitumor efficacy and decreased side effects was a fascinating prospect for the clinical chemotherapy of malignancy.

Quantitation of trans-aconitic acid in different stages of the sugar-manufacturing process.

The sugar cane industry has seen how biomass production in sugar mills would be converted to a readily available source of molecules besides sugar. Properly managed, byproducts would be transformed into a sustainable source of renewable and environmentally friendly chemical products. As a principal and more abundant organic acid in sugar cane juice, trans-aconitic acid (TAA) has been studied for use as a plasticizer in the polymer industry. However, up to now no industrial-scale application has been reported. As a reasonable approach to recover TAA from a sugar mill, first, an analytical method to determine its presence in all stages of the sugar-manufacturing process is needed. A new modern method was developed to measure TAA in seven stages in a sugar mill located in Valle del Cauca, Colombia. The stages with higher content of TAA were syrup, with 3363.6 ± 589.3 mg/L, and honey (molasses), with 6110.05 ± 139.5 mg/L.