De Novo Transcriptome Assembly and Gene Expression Profiling of the Copepod Calanus helgolandicus Feeding on the PUA-Producing Diatom Skeletonema marinoi.

Diatoms are the dominant component of the marine phytoplankton. Several diatoms produce secondary metabolites, namely oxylipins, with teratogenic effects on their main predators, crustacean copepods. Our study reports the de novo assembled transcriptome of the calanoid copepod Calanus helgolandicus feeding on the oxylipin-producing diatom Skeletonema marinoi. Differential expression analysis was also performed between copepod females exposed to the diatom and the control flagellate Prorocentrum minimum, which does not produce oxylipins. Our results showed that transcripts involved in carbohydrate, amino acid, folate and methionine metabolism, embryogenesis, and response to stimulus were differentially expressed in the two conditions. Expression of 27 selected genes belonging to these functional categories was also analyzed by RT-qPCR in C. helgolandicus females exposed to a mixed solution of the oxylipins heptadienal and octadienal at the concentration of 10 µM, 15 µM, and 20 µM. The results confirmed differential expression analysis, with up-regulation of genes involved in stress response and down-regulation of genes associated with folate and methionine metabolism, embryogenesis, and signaling. Overall, we offer new insights on the mechanism of action of oxylipins on maternally-induced embryo abnormality. Our results may also help identify biomarker genes associated with diatom-related reproductive failure in the natural copepod population at sea.

High-quality RNA extraction from copepods for Next Generation Sequencing: A comparative study.

Despite the ecological importance of copepods, few Next Generation Sequencing studies (NGS) have been performed on small crustaceans, and a standard method for RNA extraction is lacking. In this study, we compared three commonly-used methods: TRIzol®, Aurum Total RNA Mini Kit and Qiagen RNeasy Micro Kit, in combination with preservation reagents TRIzol® or RNAlater®, to obtain high-quality and quantity of RNA from copepods for NGS. Total RNA was extracted from the copepods Calanus helgolandicus, Centropages typicus and Temora stylifera and its quantity and quality were evaluated using NanoDrop, agarose gel electrophoresis and Agilent Bioanalyzer. Our results demonstrate that preservation of copepods in RNAlater® and extraction with Qiagen RNeasy Micro Kit were the optimal isolation method for high-quality and quantity of RNA for NGS studies of C. helgolandicus. Intriguingly, C. helgolandicus 28S rRNA is formed by two subunits that separate after heat-denaturation and migrate along with 18S rRNA. This unique property of protostome RNA has never been reported in copepods. Overall, our comparative study on RNA extraction protocols will help increase gene expression studies on copepods using high-throughput applications, such as RNA-Seq and microarrays.

Structure effect of carbon nanovectors in regulation of cellular responses.

Carbon nanostructures such as multiwalled carbon nanotubes (CNT) and graphene (G) are potential candidates in a large number of biomedical applications. However, there is limited understanding and connection between the physicochemical properties of diverse carbon nanostructures and biological systems, particularly with regard to cellular responses. It is also crucial to understand how the structure and surface composition of carbon nanostructures affect the cellular internalization process. Here, through in vitro cellular entry kinetics and cytotoxicity studies using MCF-7 breast cancer cells and H460 human lung cancer cells, we show that the structure and surface composition of CNT and G conjugates with various molecules such as PAMAM dendrimers (G4) and G4-poly(ethylene glycol) (PEG) are directly related to their cellular internalization ability and toxicity. Interestingly, the cellular association of CNT and G nanoconjugates was observed to be structure and surface composition dependent. We found that CNT conjugates internalized more compared to G conjugates. Furthermore, G4 conjugated CNT internalized more compared to G4-PEG conjugated CNT, whereas, higher internalization was found for G4-PEG conjugated G than G4 conjugated G. We have also correlated the cytotoxicity and cellular uptake mechanisms of CNT, G, and their conjugates through zeta potential measurements, fluorescence quenching studies and by fluorescence-activated cell sorting. Altogether these studies suggest different biological activities of the carbon nanostructures, with the shape and surface composition playing a primary role.

Poly(ethylene glycol) versus dendrimer prodrug conjugates: influence of prodrug architecture in cellular uptake and transferrin mediated targeting.

Many polymer based drug delivery nanosystems are currently being explored for delivering cytotoxic agents to the tumors. However, very few strategies delineate the comparative carrier ability of nanosystems, in similar experimental settings. As a result, it remains unclear how to optimally design polymer based multicomponent prodrug systems for delivery applications. The present study is aimed to design polymeric prodrug conjugate carriers for the comparative cellular delivery ability of anticancer drug doxorubicin hydrochloride (DOX) using linear poly(ethylene glycol) (PEG), hyperbranched poly(amido amine) (PAMAM) G4 dendrimer, and PAMAM G4 dendrimer-PEG conjugate using MCF-7 cells. Furthermore, the cellular targetability and in vitro anticancer activity of DOX conjugates is evaluated using transferrin (Tf) as a targeting ligand. Interestingly, conjugation of DOX to PAMAM G4-OH dendrimer significantly influences the cytotoxicity of DOX leading to -4 fold decrease in the IC50 dose when compared to pegylated DOX. This study establishes the rational and comparative structural activity relationship of polymeric prodrug carriers for delivery of anticancer drugs. The schematic representation of design of prodrug conjugates with varied polymeric architecures is as shown below (Fig. 1).