Novel Insights into the Wattle and Daub Model of Entamoeba Cyst Wall Formation and the Importance of Actin Cytoskeleton.

The "Wattle and Daub" model of cyst wall formation in Entamoeba invadens has been used to explain encystment in Entamoeba histolytica, the causal agent of amoebiasis, and this process could be a potential target for new antiamoebic drugs. In this study, we studied the morphological stages of chitin wall formation in E. invadens in more detail using fluorescent chitin-binding dyes and the immunolocalization of cyst wall proteins. It was found that chitin deposition was mainly initiated on the cell surface at a specific point or at different points at the same time. The cystic wall grew outward and gradually covered the entire surface of the cyst over time, following the model of Wattle and Daub. The onset of chitin deposition was guided by the localization of chitin synthase 1 to the plasma membrane, occurring on the basis of the Jacob lectin in the cell membrane. During encystation, F-actin was reorganized into the cortical region within the early stages of encystation and remained intact until the completion of the chitin wall. The disruption of actin polymerization in the cortical region inhibited proper wall formation, producing wall-less cysts or cysts with defective chitin walls, indicating the importance of the cortical actin cytoskeleton for proper cyst wall formation.

Nucleotide sugar transporters of Entamoeba histolytica and Entamoeba invadens involved in chitin synthesis.

Chitin, a homopolymer of ß-(1,4) linked N-acetylglucosamine (GlcNAc), is a major component of cyst wall in the protozoan parasites Entamoeba histolytica (Eh) and Entamoeba invadens (Ei). The Entamoeba chitin synthase makes chitin at the vesicular membrane rather than the plasma membrane in fungi, even though the chemistry of chitin synthesis is most likely the same. However, the role of nucleotide sugar transporter(s) (NSTs) that are involved in chitin synthesis in Entamoeba are not yet established. In this study, we have identified the putative UDP-GlcNAc transporter (EiNst5) of Ei by BLASTP analysis using the amino acid sequence of EhNst3, the UDP-GlcNAc transporter of Eh. Heterologous expression of both EhNst3 and EiNst5 was found to complement the function of Yea4p (UDP-GlcNAc transporter of S. cerevisiae) in YEA4 null mutant and increased the cell wall chitin content. Like Yea4p in S. cerevisiae, Myc-epitope tagged EhNst3 and EiNst5 were localized to the endoplasmic reticulum in Δyea4 cells. The EiNST5 transcript was up-regulated during the in vitro encystation and oxidative stress in E. invadens. Similar up-regulation was also seen for EhNST3 under oxidative stress in E. histolytica. Down-regulation of EiNst5 expression using gene-specific dsRNA significantly reduced cyst formation during in vitro encystation in E. invadens. Our observations suggest for the first time the involvement of EhNst3 and EiNst5 in chitin synthesis and so in encystation of Entamoeba.

Targeting NFE2L2, a transcription factor upstream of MMP-2: A potential therapeutic strategy for temozolomide resistant glioblastoma.

Glioblastoma (GBM) is the most malignant form of brain tumor posing a major threat to cancer amelioration. Temozolomide (TMZ) resistance is one of the major hurdles towards GBM prognosis. Oxidative stress and ECM remodeling are the two important processes involved in gaining chemo-resistance. Here, we established NFE2L2, an important member of oxidative stress regulation elevated in resistant cells, to be playing a transcriptional regulatory role on MMP-2, an ECM remodeling marker. This link led us to further explore targeted molecules to inhibit NFE2L2, thus affecting MMP-2, an important member promoting chemo-resistance. Thus, diosgenin was proposed as a novel NFE2L2 inhibitor acting as an alternative strategy to prevent the high dose administration of TMZ. Combinatorial therapy of diosgenin and TMZ significantly reduced the dosage regimen of TMZ and also showed affectivity in hitherto TMZ resistant GBM cells. GBM cells underwent apoptosis and early cell cycle arrest with significant reduction in MMP-2 levels. Thus preclinical validation of molecular interaction between diosgenin and NFE2L2 down-regulating MMP-2, EMT markers and promoting apoptosis, offers rationale for new therapeutic horizons in the field of glioblastoma management.

Highly Hydrophilic Luminescent Magnetic Mesoporous Carbon Nanospheres for Controlled Release of Anticancer Drug and Multimodal Imaging.

Judicious combination of fluorescence and magnetic properties along with ample drug loading capacity and control release property remains a key challenge in the design of nanotheranostic agents. This paper reports the synthesis of highly hydrophilic optically traceable mesoporous carbon nanospheres which can sustain payloads of the anticancer drug doxorubicin and T2 contrast agent such as cobalt ferrite nanoparticles. The luminescent magnetic hybrid system has been prepared on a mesoporous silica template using a resorcinol-formaldehyde precursor. The mesoporous matrix shows controlled release of the aromatic drug doxorubicin due to disruption of supramolecular π-π interaction at acidic pH. The particles show MR contrast behavior by affecting the proton relaxation with transverse relaxivity (r2) 380 mM(-1) S(-1). The multicolored emission and upconversion luminescence property of our sample are advantageous in bioimaging. In vitro cell experiments shows that the hybrid nanoparticles are endocyted by the tumor cells through passive targeting. The pH-responsive release of doxorubicin presents chemotherapeutic inhibition of cell growth through induction of apoptosis.

Design of Fe3O4@SiO2@Carbon Quantum Dot Based Nanostructure for Fluorescence Sensing, Magnetic Separation, and Live Cell Imaging of Fluoride Ion.

A robust reusable fluoride sensor comprised of a receptor in charge of the chemical recognition and a fluorophore responsible for signal recognition has been designed. Highly fluorescent carbon quantum dot (CD) and magnetically separable nickel ethylenediaminetetraacetic acid (EDTA) complex bound-silica coated magnetite nanoparticle (Fe3O4@SiO2-EDTA-Ni) have been used as fluorophore and fluoride ion receptor, respectively. The assay is based on the exchange reaction between the CD and F(-), which persuades the binding of fluoride to magnetic receptor. This method is highly sensitive, fast, and selective for fluoride ion in aqueous solution. The linear response range of fluoride (R(2) = 0.992) was found to be 1-20 µM with a minimum detection limit of 0.06 µM. Excellent magnetic property and superparamagnetic nature of the receptor are advantageous for the removal and well quantification of fluoride ion. The practical utility of the method is well tested with tap water. Because of high sensitivity, reusability, effectivity, and biocompatibility, it exhibits great promise as a fluorescent probe for intracellular detection of fluoride.