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The Antarctic nematode, Panagrolaimus sp. DAW1 (formerly called Panagrolaimus davidi) , is the best documented example of an organism able to survive intracellular ice formation in all of its compartments. Not only is it able to survive such extreme physiological disruption, but it is able to produce progeny once thawed from such a state. In addition, under slower rates, or less extreme degrees, of cooling, its body remains unfrozen and the vapour pressure difference between the supercooled body fluids and the surrounding ice leads to a process termed cryoprotective dehydration. In contrast to a fairly large body of work in building up our molecular understanding of cryoprotective dehydration, no comparable work has been undertaken on intracellular freezing. This paper describes an experiment subjecting cultures of Panagrolaimus sp. DAW1 to a range of temperatures including a rapid descent to −10 °C, in a medium just prior to, and after, freezing. Through deep sequencing of RNA libraries we have gained a snapshot of which genes are highly abundant when P. sp. DAW1 is undergoing an intracellular freezing event. The onset of freezing correlated with a high production of genes involved in cuticle formation and subsequently, after 24 h in a frozen state, protease production. In addition to the mapping of RNA sequencing, we have focused on a select set of genes arising both from the expression profiles, as well as implicated from other cold tolerance studies, to undertake qPCR. Among the most abundantly represented transcripts in the RNA mapping is the zinc-metalloenzyme, neprilysin, which also shows a particularly strong upregulated signal through qPCR once the nematodes have frozen. [ABSTRACT FROM AUTHOR]

The Antarctic nematode Panagrolaimus sp. DAW1 is one of the only organisms known to survive extensive intracellular freezing throughout its tissues. Although the physiological mechanisms of this extreme adaptation are partly understood, the molecular mechanisms remain largely unknown. RNAi is a method that allows the examination of gene function in a direct, targeted manner, by knocking out specific mRNAs and revealing the effects on the phenotype. In this study we have explored the viability of RNAi in Panagrolaimus sp. DAW1. In the first trial, nematodes were fed E. coli expressing Panagrolaimus sp. DAW1 dsRNA of the embryonic lethal genes rps-2 and dhc, and the blister gene duox. Pd-rps-2(RNAi)-treated nematodes showed a significant decrease in larval hatching. However, qPCR showed no significant decrease in the amount of rps-2 mRNA in Pd-rps-2(RNAi)-treated animals. Several soaking protocols for dsRNA uptake were investigated using the fluorescent dye FITC. Desiccation-enhanced soaking showed the strongest uptake of FITC and resulted in a significant and consistent decrease of mRNA levels of two of the four tested genes (rps-2 and tps-2a), suggesting effective uptake of dsRNA-containing solution by the nematode. These findings suggest that RNAi by desiccation-enhanced soaking is viable in Panagrolaimus sp. DAW1 and provide the first functional genomic approach to investigate freezing tolerance in this non-model organism. RNAi, in conjunction with qPCR, can be used to screen for candidate genes involved in intracellular freezing tolerance in Panagrolaimus sp. DAW1. [ABSTRACT FROM AUTHOR]

Apical-basal and planar cell polarities are hallmarks of metazoan epithelia required to separate internal and external environments and to regulate trans- and intracellular transport, cytoskeletal organization, and morphogenesis. Mechanisms of cell polarization have been intensively studied in bilaterian model organisms, particularly in early embryos and cultured cells, while cell polarity in pre-bilaterian tissues is poorly understood. Here, we have studied apical-basal and planar polarization in regenerating (aggregating) clusters of epitheliomuscular cells of Hydra , a simple representative of the ancestral, pre-bilaterian phylum Cnidaria. Immediately after dissociation, single epitheliomuscular cells do not exhibit cellular polarity, but they polarize de novo during aggregation. Reestablishment of the Hydra -specific epithelial bilayer is a result of short-range cell sorting. In the early phase of aggregation, apical-basal polarization starts with an enlargement of the epithelial apical-basal diameter and by the development of belt-like apical septate junctions. Specification of the basal pole of epithelial cells occurs shortly later and is linked to synthesis of mesoglea, development of hemidesmosome-like junctions, and formation of desmosome-like junctions connecting the basal myonemes of neighbouring cells. Planar polarization starts, while apical-basal polarization is already ongoing. It is executed gradually starting with cell-autonomous formation, parallelization, and condensation of myonemes at the basal end of each epithelial cell and continuing with a final planar alignment of epitheliomuscular cells at the tissue level. Our findings reveal that epithelial polarization in Hydra aggregates occurs in defined steps well accessible by histological and ultrastructural techniques and they will provide a basis for future molecular studies. [ABSTRACT FROM AUTHOR]

Desmosomes are cell-cell junctions that link tissue cells experiencing intense mechanical stress. Although the structure of the desmosomal cadherins is known, the desmosome architecture--which is essential for mediating numerous functions--remains elusive. Here, we recorded cryo-electron tomograms (cryo-ET) in which individual cadherins can be discerned; they appear variable in shape, spacing, and tilt with respect to the membrane. The resulting subtomogram average reaches a resolution of ~26 Å, limited by the inherent flexibility of desmosomes. To address this challenge typical of dynamic biological assemblies, we combine sub-tomogram averaging with atomistic molecular dynamics (MD) simulations. We generate models of possible cadherin arrangements and perform an in silico screening according to biophysical and structural properties extracted from MD simulation trajectories. We find a truss-like arrangement of cadherins that resembles the characteristic footprint seen in the electron micrograph. The resulting model of the desmosomal architecture explains their unique biophysical properties and strength. [ABSTRACT FROM AUTHOR]

[Display omitted] Polyphosphate nanoparticles as phosphatase-degradable carriers for Penicillium chrysogenum antifungal protein (PAF) can enhance the antifungal activity of the protein against Candida albicans biofilm. PAF-polyphosphate (PP) nanoparticles (PAF-PP NPs) were obtained through ionic gelation. The resulting NPs were characterized in terms of their particle size, size distribution and zeta potential. Cell viability and hemolysis studies were carried out in vitro on human foreskin fibroblasts (Hs 68 cells) and human erythrocytes, respectively. Enzymatic degradation of NPs was investigated by monitoring release of free monophosphates in the presence of isolated as well as C. albicans -derived phosphatases. In parallel, shift in zeta potential of PAF-PP NPs as a response to phosphatase stimuli was determined. Diffusion of PAF and PAF-PP NPs through C. albicans biofilm matrix was analysed by fluorescence correlation spectroscopy (FCS). Antifungal synergy was evaluated on C. albicans biofilm by determining the colony forming units (CFU). PAF-PP NPs were obtained with a mean size of 300.9 ± 4.6 nm and a zeta potential of −11.2 ± 2.8 mV. In vitro toxicity assessments revealed that PAF-PP NPs were highly tolerable by Hs 68 cells and human erythrocytes similar to PAF. Within 24 h, 21.9 ± 0.4 μM of monophosphate was released upon incubation of PAF-PP NPs having final PAF concentration of 156 μg/ml with isolated phosphatase (2 U/ml) leading to a shift in zeta potential up to −0.7 ± 0.3 mV. This monophosphate release from PAF-PP NPs was also observed in the presence of C. albicans -derived extracellular phosphatases. The diffusivity of PAF-PP NPs within 48 h old C. albicans biofilm matrix was similar to that of PAF. PAF-PP NPs enhanced antifungal activity of PAF against C. albicans biofilm decreasing the survival of the pathogen up to 7-fold in comparison to naked PAF. In conclusion, phosphatase-degradable PAF-PP NPs hold promise as nanocarriers to augment the antifungal activity of PAF and enable its efficient delivery to C. albicans cells for the potential treatment of Candida infections. [ABSTRACT FROM AUTHOR]

[Display omitted] The aim of this study was to evaluate the uptake mechanism of thiolated nanostructured lipid carriers (NLCs). NLCs were decorated with a short-chain polyoxyethylene(10)stearyl ether with a terminal thiol group (NLCs-PEG 10 -SH) or without (NLCs-PEG 10 -OH) as well as with a long-chain polyoxyethylene(1 0 0)stearyl ether with thiolation (NLCs-PEG 100 -SH) or without (NLCs-PEG 100 -OH). NLCs were evaluated for size, polydispersity index (PDI), surface morphology, zeta potential and storage stability over six months. Cytotoxicity, adhesion to the cell surface and internalization of these NLCs in increasing concentrations were evaluated on Caco-2 cells. The influence of NLCs on the paracellular permeability of lucifer yellow was determined. Furthermore, cellular uptake was examined with and without various endocytosis inhibitors as well as reducing and oxidizing agents. NLCs were obtained in a size ranging from 164 to 190 nm, a PDI of 0.2, a negative zeta potential < –33 mV and stability over six months. Cytotoxicity was shown to be concentration dependent and to be lower for NLCs with shorter PEG chains. Permeation of lucifer yellow was 2-fold increased by NLCs-PEG 10 -SH. All NLCs displayed concentration dependent adhesion to the cell surface and internalization, which was in particular 9.5-fold higher for NLCs-PEG 10 -SH compared to NLCs-PEG 10 -OH. Short PEG chain NLCs and especially thiolated short PEG chain NLCs showed higher cellular uptake than NLCs with longer PEG chain. Cellular uptake of all NLCs was mainly clathrin-mediated endocytosis. Thiolated NLCs showed also caveolae-dependent and clathrin- and caveolae-independent uptake. Macropinocytosis was involved in NLCs with long PEG chains. NLCs-PEG 10 -SH indicated thiol-dependent uptake, which was influenced by reducing and oxidizing agents. Due to thiol groups on the surface of NLCs their cellular uptake and paracellular permeation enhancing properties can be substantially improved. [ABSTRACT FROM AUTHOR]