Branched polymer grafted graphene oxide (GO) as a 2D template for calcium phosphate growth.

HYPOTHESIS: Graphene Oxide (GO)-templated deposition of inorganic materials through synthesis on dispersed single sheets of GO is often complicated by the loss of the desired 2D morphology owing to the coagulation of GO sheets at high salt concentrations and non-templated homogenous nucleation. Modifying GO with anionic polymer is expected to solve both problems by i) enhancing electrostatic(steric) stabilization upon exposure to high concentrations of the ionic precursors, and ii) offering additional nucleation sites at the grafted anionic moieties to avoid homogeneous secondary nucleation. EXPERIMENTS: GO was grafted with branched copolymers of poly(ethylene glycol) methacrylate (PEGMA 500) and diethylene glycol dimethacrylate (DEGDMA) and ω-vinyl terminated methacrylic acid macromonomer (P(MAA)), the latter serving as an addition-fragmentation chain transfer agent. The colloidal stability of GO dispersions in water toward salt was evaluated before and after modification. Precipitation of calcium phosphate (CaP) was performed by incubating modified GO in the precursor solutions. The conditions were optimized to maximize the nucleation selectively onto GO without homogeneous CaP nucleation and coagulation of the GO-sheets. FINDINGS: The copolymer grafted GO-sheets shows superior colloidal stability when dispersed in water. No aggregation occurs in the incubating ionic CaP precursor solutions. The optimum templated deposition of CaP onto the GO sheets by precipitation is to add a second shot of precursors after the nucleation stage to obtain GO sheets fully decorated with calcium phosphate nanorods without self-nucleation. Via the careful design on the GO modification and incubation process, the growth of calcium phosphate nanorods were confined in the desired 2D order exclusively, hereby achieving the goal of an efficient GO-templated synthesis.

Regional comparison on ciguatoxicity, hemolytic activity, and toxin profile of the dinoflagellate Gambierdiscus from Kiribati and Malaysia.

The dinoflagellates Gambierdiscus and Fukuyoa can produce Ciguatoxins (CTXs) and Maitotoxins (MTXs) that lead to ciguatera poisoning (CP). The CP hotspots, however, do not directly relate to the occurrence of the ciguatoxic Gambierdiscus and Fukuyoa. Species-wide investigations often showed no association between CTX level and the molecular identity of the dinoflagellates. In the Pacific region, Kiribati is known as a CP hotspot, while Malaysia has only three CP outbreaks reported thus far. Although ciguatoxic strains of Gambierdiscus were isolated from both Kiribati and Malaysia, no solid evidence on the contribution of ciguatoxic strains to the incidence of CP outbreak was recorded. The present study aims to investigate the regional differences in CP risks through region-specific toxicological assessment of Gambierdiscus and Fukuyoa. A total of 19 strains of Gambierdiscus and a strain of Fukuyoa were analyzed by cytotoxicity assay of the neuro-2a cell line, hemolytic assay of fish erythrocytes, and high-resolution mass spectrometry. Gambierdiscus from both Kiribati and Malaysia showed detectable ciguatoxicity; however, the Kiribati strains were more hemolytic. Putative 44-methylgambierone was identified as part of the contributors to the hemolytic activity, and other unknown hydrophilic toxins produced can be potentially linked to higher CP incidence in Kiribati.

The Occurrence, Distribution, and Toxicity of High-Risk Ciguatera Fish Species (Grouper and Snapper) in Kiritimati Island and Marakei Island of the Republic of Kiribati.

Ciguatera is one of the most widespread food poisonings caused by the ingestion of fish contaminated by ciguatoxins (CTXs). Snapper and grouper with high palatable and economic value are the primary food source and fish species for exportation in the Republic of Kiribati, but they are highly suspected CTX-contaminated species due to their top predatory characteristics. In this study, 60 fish specimens from 17 species of snappers and groupers collected from the Kiritimati Island and Marakei Island of the Republic of Kiribati were analyzed using mouse neuroblastoma (N2a) assay and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine Pacific CTX-1, -2 and -3 (P-CTX-1, -2 and -3). The LC-MS/MS results show that CTXs were detected in 74.5% of specimens from Marakei Island and 61.5% of specimens from Kiritimati Island. The most toxic fish Epinephelus coeruleopunctatus from Marakei Island and Cephalopholis miniata from Kiritimati Island were detected as 53-fold and 28-fold P-CTX-1 equivalents higher than the safety level of 10 pg/g P-CTX-1 equivalents, respectively. CTX levels and composition profiles varied with species and location. The N2a results suggested that fish specimens also contain high levels of other CTX-like toxins or sodium channel activators. The distribution patterns for ciguatoxic fish of the two islands were similar, with fish sampled from the northwest being more toxic than the southwest. This study shows that groupers and snappers are high-risk species for ciguatera in the Republic of Kiribati, and these species can further be used as indicator species in ciguatera endemic areas for risk assessment.

Ciguatoxin-Producing Dinoflagellate Gambierdiscus in the Beibu Gulf: First Report of Toxic Gambierdiscus in Chinese Waters.

Ciguatera poisoning is mainly caused by the consumption of reef fish that have accumulated ciguatoxins (CTXs) produced by the benthic dinoflagellates Gambierdiscus and Fukuyoa. China has a long history of problems with ciguatera, but research on ciguatera causative organisms is very limited, especially in the Beibu Gulf, where coral reefs have been degraded significantly and CTXs in reef fish have exceeded food safety guidelines. Here, five strains of Gambierdiscus spp. were collected from Weizhou Island, a ciguatera hotspot in the Beibu Gulf, and identified by light and scanning electron microscopy and phylogenetic analyses based on large and small subunit rDNA sequences. Strains showed typical morphological characteristics of Gambierdiscus caribaeus, exhibiting a smooth thecal surface, rectangular-shaped 2', almost symmetric 4″, and a large and broad posterior intercalary plate. They clustered in the phylogenetic tree with G. caribaeus from other locations. Therefore, these five strains belonged to G. caribaeus, a globally distributed Gambierdiscus species. Toxicity was determined through the mouse neuroblastoma assay and ranged from 0 to 5.40 fg CTX3C eq cell-1. The low level of toxicity of G. caribaeus in Weizhou Island, with CTX-contaminated fish above the regulatory level in the previous study, suggests that the long-term presence of low toxicity G. caribaeus might lead to the bioaccumulation of CTXs in fish, which can reach dangerous CTX levels. Alternatively, other highly-toxic, non-sampled strains could be present in these waters. This is the first report on toxic Gambierdiscus from the Beibu Gulf and Chinese waters and will provide a basis for further research determining effective strategies for ciguatera management in the area.

A Rhizobium bacterium and its population dynamics under different culture conditions of its associated toxic dinoflagellate Gambierdiscus balechii.

Rhizobium bacteria are known as symbionts of legumes for developing nodules on plant roots and fixing N2 for the host plants but unknown for associations with dinoflagellates. Here, we detected, isolated, and characterized a Rhizobium species from the marine toxic dinoflagellate Gambierdiscus culture. Its 16S rRNA gene (rDNA) is 99% identical to that of Rhizobium rosettiformans, and the affiliation is supported by the phylogenetic placement of its cell wall hydrolase -encoding gene (cwh). Using quantitative PCR of 16S rDNA and cwh, we found that the abundance of this bacterium increased during the late exponential growth phase of Gambierdiscus and under nitrogen limitation, suggesting potential physiological interactions between the dinoflagellate and the bacterium. This is the first report of dinoflagellate-associated Rhizobium bacterium, and its prevalence and ecological roles in dinoflagellate-Rhizobium relationships remain to be investigated in the future. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-021-00102-1.

On Particle Size Distributions in Catalytic Chain Transfer Emulsion Polymerization: Chain-Extension and the Use of Derived Macromonomers as Reactive Surfactants in Emulsion Polymerization.

Catalytic chain transfer emulsion polymerization (CCTP) and subsequent chain extension via reversible addition-fragmentation chain transfer (RAFT) were used to synthesize amphiphilic macromonomers (MM), in the form of polymer latexes. The macromonomers consisted of two blocks whose first was a random copolymer of methacrylic acid and methyl methacrylate, at 35:65 mol:mol, while the second block was n-butyl methacrylate P[(MAA-co-MMA)-block-PBMA]. The block copolymer colloids were disintegrated and micellized upon addition of ammonia. The resulting nanosized polymer dispersions were used as reactive surfactants in the emulsion polymerization of n-butyl methacrylate. For this, a dual stage slow-fast monomer feed profile was used. The final polymer latexes were in the sub-100 nm range for the particle diameter at 30% w/w total polymer content. The emphasis of the work is to discuss and find an explanation for the observed particle size distributions in the three consecutive emulsion polymerization steps. The particle size distribution of the ω-unsaturated macromonomer latex synthesized by CCTP emulsion polymerization was found to be much broader than expected. This discrepancy is attributed to an extended particle nucleation period. The chain extension step in the macromonomer latex preparation showed considerable secondary nucleation. The presence of water-soluble macromonomer species from the CCTP emulsion polymerization step assured that control of chain growth persisted. The use of the amphiphilic macromonomers as reactive surfactants in the form of a nanosized aggregate seed dispersion showed that the average particle diameter could be tuned and that the molecular weight distributions could be regulated, when monomer starved conditions were used in the emulsion polymerizations.

Characterizing ciguatoxin (CTX)- and Non-CTX-producing strains of Gambierdiscus balechii using comparative transcriptomics.

Gambierdiscus spp. can produce the polyketide compound, ciguatoxin (CTX), and are hence responsible for ciguatera fish poisoning (CFP). Studying the molecular mechanism that regulates CTX production is crucial for understanding the environmental trigger of CTX as well as for better informing fishery management. Commonly, polyketide synthases are important for polyketide synthesis; however, no gene has been confirmatively assigned to CTX production. Here, suppression subtractive hybridization (SSH) and transcriptome sequencing (RNA-Seq) were used to compare a CTX-producing strain with a non-CTX-producing strain. Using both methods, a total of 52 polyketide synthase (PKS) genes were identified to be up-regulated in the CTX-producing G. balechii, including transcripts encoding single-domain PKSs as well as transcripts encoding multi-domain PKSs. Using reverse transcription quantitative PCR, the expression of these genes in the CTX-producing strain and in nitrogen-limited cultures of the strain was further documented. These data suggest that PKSs are likely involved in polyketide synthesis and potentially in CTX synthesis in this dinoflagellate species. Our study provides the candidate biomarkers for the detection of CTXs or CFP in waters or any other organisms as well as a valuable genomic resource for the research on Gambierdiscus and other dinoflagellates.

Synthesis of Janus and Patchy Particles Using Nanogels as Stabilizers in Emulsion Polymerization.

Polymer nanogels are used as colloidal stabilizers in emulsion polymerization. The nanogels were made by the covalent cross-linking of block copolymer micelles, the macromolecular building blocks of which were synthesized using a combination of catalytic chain transfer emulsion polymerization and reversible addition-fragmentation chain transfer (RAFT) of methacrylate monomers. The use of the nanogels in an emulsion polymerization led to anisotropic Janus and patchy colloids, where a latex particle was decorated by a number of patches on its surface. Control of the particle size and patch density was achieved by tailoring of the reaction conditions, such as varying the amount of nanogels, pH, and salt concentration. Overall, the emulsion polymerization process in the presence of nanogels as stabilizers is shown to be a versatile and easily scalable route toward the fabrication of Janus and patchy polymer colloids.

Taxonomic assignment of the benthic toxigenic dinoflagellate Gambierdiscus sp. type 6 as Gambierdiscus balechii (Dinophyceae), including its distribution and ciguatoxicity.

Recent molecular phylogenetic studies of Gambierdiscus species flagged several new species and genotypes, thus leading to revitalizing its systematics. The inter-relationships of clades revealed by the primary sequence information of nuclear ribosomal genes (rDNA), however, can sometimes be equivocal, and therefore, in this study, the taxonomic status of a ribotype, Gambierdiscus sp. type 6, was evaluated using specimens collected from the original locality, Marakei Island, Republic of Kiribati; and specimens found in Rawa Island, Peninsular Malaysia, were further used for comparison. Morphologically, the ribotype cells resembled G. scabrosus, G. belizeanus, G. balechii, G. cheloniae and G. lapillus in thecal ornamentation, where the thecal surfaces are reticulate-foveated, but differed from G. scabrosus by its hatchet-shaped Plate 2', and G. belizeanus by the asymmetrical Plate 3'. To identify the phylogenetic relationship of this ribotype, a large dataset of the large subunit (LSU) and small subunit (SSU) rDNAs were compiled, and performed comprehensive analyses, using Bayesian-inference, maximum-parsimony, and maximum-likelihood, for the latter two incorporating the sequence-structure information of the SSU rDNA. Both the LSU and SSU rDNA phylogenetic trees displayed an identical topology and supported the hypothesis that the relationship between Gambierdiscus sp. type 6 and G. balechii was monophyletic. As a result, the taxonomic status of Gambierdiscus sp. type 6 was revised, and assigned as Gambierdiscus balechii. Toxicity analysis using neuroblastoma N2A assay confirmed that the Central Pacific strains were toxic, ranging from 1.1 to 19.9 fg P-CTX-1 eq cell-1, but no toxicity was detected in a Western Pacific strain. This suggested that the species might be one of the species contributing to the high incidence rate of ciguatera fish poisoning in Marakei Island.