Microwave-assisted Nile red method for in vivo quantification of neutral lipids in microalgae

In vivo determination of neutral lipids with Nile red fluorescence has been used as a rapid screening method for certain types of microalgae, but has been unsuccessful in others, particularly those with thick, rigid cell walls that prevent penetration of the fluorescence dye into the cell. To solve the problem, a microwave-assisted Nile red staining method for microalgal lipid determination was developed. In a two-step staining protocol, 50 and 60 s were selected as the optimal microwave times for the pretreatment and staining process, respectively. Moreover, several calibration methods for quantitative analysis of neutral lipids in microalgae were investigated and compared with conventional gravimetric methods. Factors that affected the in vivo quantification of cellular neutral lipids were also investigated. Application of the new method for detection and quantification of neutral lipids in a number of green microalgae was demonstrated.     

Bistability versus bimodal distributions in gene regulatory processes from population balance

In recent times, stochastic treatments of gene regulatory processes have appeared in the literature in which a cell exposed to a signaling molecule in its environment triggers the synthesis of a specific protein through a network of intracellular reactions. The stochastic nature of this process leads to a distribution of protein levels in a population of cells as determined by a Fokker-Planck equation. Often instability occurs as a consequence of two (stable) steady state protein levels, one at the low end representing the "off" state, and the other at the high end representing the "on" state for a given concentration of the signaling molecule within a suitable range. A consequence of such bistability has been the appearance of bimodal distributions indicating two different populations, one in the "off" state and the other in the "on" state. The bimodal distribution can come about from stochastic analysis of a single cell. However, the concerted action of the population altering the extracellular concentration in the environment of individual cells and hence their behavior can only be accomplished by an appropriate population balance model which accounts for the reciprocal effects of interaction between the population and its environment. In this study, we show how to formulate a population balance model in which stochastic gene expression in individual cells is incorporated. Interestingly, the simulation of the model shows that bistability is neither sufficient nor necessary for bimodal distributions in a population. The original notion of linking bistability with bimodal distribution from single cell stochastic model is therefore only a special consequence of a population balance model.     

Structure-function relationships of the human bitter taste receptor hTAS2R1: insights from molecular modeling studies

Bitter taste receptors (T2Rs) belong to G-protein-coupled receptors (GPCRs). Despite extensive studies, the precise mechanisms of GPCR activation are still poorly understood. In this study, the models of the human bitter taste receptor hTAS2R1 alone and in complex with various ligands were constructed on the basis of template-based modeling and molecular docking. Then these models were subjected to all-atom molecular dynamics (MD) simulations in explicit lipid bilayers. The binding pocket of hTAS2R1 is mainly formed by transmembrane helix (TM) III, TM V, TM VI, and TM VII. Most of the residues contributing to ligand binding are positionally conserved comparing with other hTAS2Rs. By comparing the final conformations obtained by extensive MD simulations, we identified the changes in the transmembrane helices and the intra- and extracellular loops, which were expected to initiate the activation of the receptor. The intracellular loop II (ICL2) and TM III were found to play prominent roles in the process of activation. We proposed that a set of interactions between the aromatic Phe115 in the middle of ICL2 and three residues (Tyr103, Lys106, and Val107) at the cytoplasmic end of TM III may serve as a conformational switch of hTAS2R1 activation. All of the residues involved in the switch are highly conserved among T2Rs. This indicates that the control switch we proposed may be universal in T2Rs. Besides, our results also suggest that the formation of a short helical segment in ICL2 may be necessary for the activation of hTAS2R1.     

Fertilization and cytogenetic examination of interspecific reciprocal hybridization between the scallops, Chlamys farreri and Mimachlamys nobilis

Crossbreeding is a powerful tool for improving productivity and profitability in aquaculture. We conducted a pilot study of an artificial cross between two important cultivated scallops in China, Chlamys farreri and Mimachlamys nobilis, to test the feasibility of interspecific hybridization. Reciprocal hybridization experiments were performed using a single-pair mating strategy (M. nobilis ♀ × C. farreri ♂ and C. farreri ♀ × M. nobilis ♂). The fertilization of each pair was tracked using fluorescence staining of the gametes, and the chromosomes of the F1 hybrid larvae were examined via conventional karyotyping and genomic in situ hybridization (GISH). We observed moderate fertilization success in both interspecific crosses, although the overall fertilization was generally less rapid than that of intraspecific crosses. Conventional karyotyping showed that 70.4% of the viable F1 larvae in M. nobilis ♀ × C. farreri ♂ and 55.4% in C. farreri ♀ × M. nobilis ♂ comprised hybrid karyotypes (2n = 35 = 6m+5sm+11st+13t), and the results were further confirmed by GISH. Interestingly, we detected a few F1 from the M. nobilis ♀ × C. farreri ♂ cross that appeared to have developed gynogenetically. In addition, chromosome fragmentations, aneuploids and allopolyploids were observed in some F1 individuals. Our study presents evidence that the artificial cross between M. nobilis and C. farreri is experimentally possible. Further investigations of the potential heterosis of the viable F1 offspring at various developmental stages should be conducted to obtain a comprehensive evaluation of the feasibility of crossbreeding between these two scallop species.     

Fatty acid synthesis is critical for stem cell pluripotency via promoting mitochondrial fission

Pluripotent stem cells are known to display distinct metabolic phenotypes than their somatic counterparts. While accumulating studies are focused on the roles of glucose and amino acid metabolism in facilitating pluripotency, little is known regarding the role of lipid metabolism in regulation of stem cell activities. Here, we show that fatty acid (FA) synthesis activation is critical for stem cell pluripotency. Our initial observations demonstrated enhanced lipogenesis in pluripotent cells and during cellular reprogramming. Further analysis indicated that de novo FA synthesis controls cellular reprogramming and embryonic stem cell pluripotency through mitochondrial fission. Mechanistically, we found that de novo FA synthesis regulated by the lipogenic enzyme ACC1 leads to the enhanced mitochondrial fission via (i) consumption of AcCoA which affects acetylation‐mediated FIS1 ubiquitin–proteasome degradation and (ii) generation of lipid products that drive the mitochondrial dynamic equilibrium toward fission. Moreover, we demonstrated that the effect of Acc1 on cellular reprogramming via mitochondrial fission also exists in human iPSC induction. In summary, our study reveals a critical involvement of the FA synthesis pathway in promoting ESC pluripotency and iPSC formation via regulating mitochondrial fission.     

Core–shell structured CdTe/CdS@SiO2@CdTe@SiO2 composite fluorescent spheres: Synthesis and application for Cd2+ detection

Core–shell structured quantum dot (QD)–silica fluorescent nanoparticles have attracted a great deal of attention due to the excellent optical properties of QDs and the stability of silica. In this study, core–shell structured CdTe/CdS@SiO₂@CdTe@SiO₂ fluorescent nanospheres were synthesized based on the Stöber method using multistep silica encapsulation. The second silica layer on the CdTe QDs maintained the optical stability of nanospheres and decreased adverse influences on the probe during subsequent processing. Red‐emissive CdTe/CdS QDs (630 nm) were used as a built‐in reference signal and green‐emissive CdTe QDs (550 nm) were used as a responding probe. The fluorescence of CdTe QDs was greatly quenched by added S^2?, owing to a S^2?‐induced change in the CdTe QDs surface state in the shell. Upon addition of Cd²⁺ to the S^2?‐quenched CdTe/CdS@SiO₂@CdTe@SiO₂ system, the responding signal at 550 nm was dramatically restored, whereas the emission at 630 nm remained almost unchanged; this response could be used as a ratiometric ‘off–on’ fluorescent probe for the detection of Cd²⁺. The sensing mechanism was suggested to be: the newly formed CdS‐like cluster with a higher band gap facilitated exciton/hole recombination and effectively enhanced the fluorescence of the CdTe QDs. The proposed probe shows a highly sensitive and selective response to Cd²⁺ and has potential application in the detection of Cd²⁺ in environmental or biological samples.     

Morphological description of three marine ciliates (Ciliophora, Scuticociliatia), with establishment of a new genus and two new species

Three marine scuticociliates, Falcicyclidium fangi nov. gen., nov. spec., Falcicyclidium atractodes nov. spec., and Cristigera media Kahl, 1928 were investigated using live observation and silver impregnation methods. The genus Falcicyclidium is distinguished by the combination of: (i) dorsoventrally flattened body, (ii) hook-like (falciform) paroral membrane, (iii) anterior end of paroral membrane posterior to anterior end of membranelle 1, and (iv) multiple caudal cilia. Falcicyclidium fangi nov. spec., the type of the new genus, can be recognized by the combination of its large size, extremely dorsoventrally flattened (3:1) body, consistently 10 somatic kineties, and the broad, elongate buccal area occupying 60% of the body length. Falcicyclidium atractodes nov. spec. is mainly characterized by a unique spine projecting from both the anterior and posterior end. The uncommon form, Cristigera media is redescribed based on the population from Qingdao, the statistic data and additional features, especially the morphology of the living cells, are documented.     

Regulation of endothelial permeability by Src kinase signaling: vascular leakage versus transcellular transport of drugs and macromolecules

An important function of the endothelium is to regulate the transport of liquid and solutes across the semi-permeable vascular endothelial barrier. Two cellular pathways have been identified controlling endothelial barrier function. The normally restrictive paracellular pathway, which can become "leaky" during inflammation when gaps are induced between endothelial cells at the level of adherens and tight junctional complexes, and the transcellular pathway, which transports plasma proteins the size of albumin via transcytosis in vesicle carriers originating from cell surface caveolae. During non-inflammatory conditions, caveolae-mediated transport may be the primary mechanism of vascular permeability regulation of fluid phase molecules as well as lipids, hormones, and peptides that bind avidly to albumin. Src family protein tyrosine kinases have been implicated in the upstream signaling pathways that lead to endothelial hyperpermeability through both the paracellular and transcellular pathways. Endothelial barrier dysfunction not only affects vascular homeostasis and cell metabolism, but also governs drug delivery to underlying cells and tissues. In this review of the field, we discuss the current understanding of Src signaling in regulating paracellular and transcellular endothelial permeability pathways and effects on endogenous macromolecule and drug delivery.