Newly synthesized peptide,Ara-27, exhibits significant improvement in cell-penetrating ability compared to conventional peptides

Cell-penetrating peptides (CPPs) are short amino acid sequences known to act as a vehicle for enhancing the intracellular translocating efficiency of extracellular molecules. Although many groups have attempted to develop peptides with high cell-penetrating efficiencies, very few have demonstrated efficient cellular uptake of CPPs at low concentrations. Here, we describe a newly synthesized peptide derived from Arabidopsis, Ara-27, which exhibits significant improvement in cell-penetrating efficiency compared to existing CPPs. The cell-penetrating efficiency of Ara-27 was compared with the commonly used Tat-protein transduction domain (Tat-PTD) and membrane translocating sequence (MTS) in human dermal fibroblast (HDF) and human dental pulp stem cells (hDPSC). Cell-penetrating efficiency of fluorescein isothiocyanate (FITC)-labeled CPPs were assessed by flow cytometry and visualized by confocal microscopy. Flow cytometric analysis revealed >99% cell-penetrating efficiency for 2 μM Ara-27 in both HDF and hDPSC. In contrast, 2 μM Tat-PTD and MTS showed <10% cell-penetrating efficiency in both cells. In support, relative fluorescence intensities of FITC-labeled Ara-27 were around 8 to 22 times higher than those of Tat-PTD and MTS in both cells. Confocal analysis revealed internalization of 0.2 and 2 μM Ara-27 in both human cells, which was not observed for Tat-PTD and MTS at either concentration. In conclusion, this study describes a novel CPP, Ara-27, which exhibit significant improvement in intracellular uptake compared to conventional CPPs, without affecting cell viability. Thus, development of Ara-27 based peptides may lead to improved delivery of functional cargo such as small molecules, siRNA, and drugs for in vivo studies.     

Ca2+ Sensitivity of Anoctamin 6/TMEM16F Is Regulated by the Putative Ca2+-Binding Reservoir at the N-Terminal Domain

Anoctamin 6/TMEM16F (ANO6) is a dual-function protein with Ca²⁺-activated ion channel and Ca²⁺-activated phospholipid scramblase activities, requiring a high intracellular Ca²⁺ concentration (e.g., half-maximal effective Ca²⁺ concentration [EC₅₀] of [Ca²⁺]_i > 10 μM), and strong and sustained depolarization above 0 mV. Structural comparison with Anoctamin 1/TMEM16A (ANO1), a canonical Ca²⁺-activated chloride channel exhibiting higher Ca²⁺ sensitivity (EC₅₀ of 1 μM) than ANO6, suggested that a homologous Ca²⁺-transferring site in the N-terminal domain (Nt) might be responsible for the differential Ca²⁺ sensitivity and kinetics of activation between ANO6 and ANO1. To elucidate the role of the putative Ca²⁺-transferring reservoir in the Nt (Nt-CaRes), we constructed an ANO6-1-6 chimera in which Nt-CaRes was replaced with the corresponding domain of ANO1. ANO6-1-6 showed higher sensitivity to Ca²⁺ than ANO6. However, neither the speed of activation nor the voltage-dependence differed between ANO6 and ANO6-1-6. Molecular dynamics simulation revealed a reduced Ca²⁺ interaction with Nt-CaRes in ANO6 than ANO6-1-6. Moreover, mutations on potentially Ca²⁺-interacting acidic amino acids in ANO6 Nt-CaRes resulted in reduced Ca²⁺ sensitivity, implying direct interactions of Ca²⁺ with these residues. Based on these results, we cautiously suggest that the net charge of Nt-CaRes is responsible for the difference in Ca²⁺ sensitivity between ANO1 and ANO6.     

Small intestinal immune-environmental changes induced by oral tolerance inhibit experimental atopic dermatitis

Atopic dermatitis is a chronic skin inflammatory disease mediated by Th2-type immune responses. Although intestinal immune responses have been shown to play a critical role in the development or prevention of atopic dermatitis, the precise influence of intestinal immunity on atopic dermatitis is incompletely understood. We show here that orally tolerized mice are protected from experimental atopic dermatitis induced by sensitization and epicutaneous (EC) challenge to ovalbumin. Although the expression of Th2-type cytokines in the small intestine of orally tolerized and EC-challenged mice did not change significantly, these mice showed decreased inflammatory responses in the small intestine with restoration of microbial change elicited by the EC challenge. Interestingly, an increase in small intestinal eosinophils was observed with the EC challenge, which was also inhibited by oral tolerance. The role of small intestinal eosinophils and microbiota in the pathogenesis of experimental atopic dermatitis was further substantiated by decreased inflammatory mediators in the small intestine and attenuated Th2-type inflammation in the skin of eosinophil-deficient and microbiota-ablated mice with EC challenges. Based on these data, we propose that the bidirectional interaction between the skin and the intestine has a role in the pathogenesis of atopic dermatitis and that modulation of the intestinal microenvironments could be a therapeutic approach to atopic dermatitis.Subject terms: Allergy, Mucosal immunology     

Accelerated maximum likelihood parameter estimation for stochastic biochemical systems

ABSTRACT: BACKGROUND: A prerequisite for the mechanistic simulation of a biochemical system is detailed knowledge of its kinetic parameters. Despite recent experimental advances, the estimation of unknown parameter values from observed data is still a bottleneck for obtaining accurate simulation results. Many methods exist for parameter estimation in deterministic biochemical systems; methods for discrete stochastic systems are less well developed. Given the probabilistic nature of stochastic biochemical models, a natural approach is to choose parameter values that maximize the probability of the observed data with respect to the unknown parameters, a.k.a. the maximum likelihood parameter estimates (MLEs). MLE computation for all but the simplest models requires the simulation of many system trajectories that are consistent with experimental data. For models with unknown parameters, this presents a computational challenge, as the generation of consistent trajectories can be an extremely rare occurrence. RESULTS: We have developed Monte Carlo Expectation-Maximization with Modified Cross-Entropy Method (MCEM2): an accelerated method for calculating MLEs that combines advances in rare event simulation with a computationally efficient version of the Monte Carlo expectation-maximization (MCEM) algorithm. Our method requires no prior knowledge regarding parameter values, and it automatically provides a multivariate parameter uncertainty estimate. We applied the method to five stochastic systems of increasing complexity, progressing from an analytically tractable pure-birth model to a computationally demanding model of yeast-polarization. Our results demonstrate that MCEM2 substantially accelerates MLE computation on all tested models when compared to a stand-alone version of MCEM. Additionally, we show how our method identifies parameter values for certain classes of models more accurately than two recently proposed computationally efficient methods. CONCLUSIONS: This work provides a novel, accelerated version of a likelihood-based parameter estimation method that can be readily applied to stochastic biochemical systems. In addition, our results suggest opportunities for added efficiency improvements that will further enhance our ability to mechanistically simulate biological processes.     

Molecular and phylogenetic characterization of Spodoptera litura granulovirus

Baculovirus, Spodoptera litura granulovirus (SlGV) was isolated from the infected S. litura larvae, and was characterized. The granule of SlGV was ovoidal shape with an approximate size of 240∼340 nm× 140∼180 nm. Each granule contained one single rod-shape virion with a mean size of 180∼200 nm×20∼40 nm. Restriction endonuclease fragment analysis estimated that the total genome size of SlGV is about 115 kb. Necleotide sequence analysis of the granulin gene showed that the gene encodes 249 amino acids with a predicted molecular mass of 29 kDa. When the phylogenic relationship was analyzed using the nucleotide sequence of the granulin gene, SlGV was most closely related to Trichoplusia ni granulovirus (TnGV) and Xestia c-nigrum granulovirus (XcGV) which belong to Type I granulovirus.     

Isolation of a strain of Bacillus thuringiensis ssp. kurstaki HD-1 encoding δ-endotoxin Cry1E

A strain of Bacillus thuringiensis, STB-1, toxic against Spodoptera exigua , was isolated. Bacillus thuringiensis STB-1 produced bipyramidal inclusions and reacted with the H antiserum of B. thuringiensis ssp. kurstaki . The plasmid and protein profiles of B. thuringiensis STB-1 were compared with those of its reference strains, ssp. kurstaki and ssp. kenyae . To verifiy the gene type of B. thuringiensis STB-1, PCR analysis was performedwith Spodoptera -specific cry gene primers. The result showed that B. thuringiensis STB-1, unlike its reference strains, had cry1Aa , cry1Ab , cry1Ac and cry1E , suggesting that B. thuringiensis STB-1 was a unique strain with respect to gene type. In addition, B. thuringiensis STB-1 showed a high level of toxicity against both S. exigua and Bombyx mori , whereas B. thuringiensis ssp. kurstaki HD-1 or ssp. kenyae showed a high level of toxicity against only Bombyx mori or S. exigua , respectively.