AU4S: A novel synthetic peptide to measure the activity of ATG4 in living cells

ATG4 plays a key role in autophagy induction, but the methods for monitoring ATG4 activity in living cells are limited. Here we designed a novel fluorescent peptide named AU4S for noninvasive detection of ATG4 activity in living cells, which consists of the cell-penetrating peptide (CPP), ATG4-recognized sequence “GTFG,” and the fluorophore FITC. Additionally, an ATG4-resistant peptide AG4R was used as a control. CPP can help AU4S or AG4R to penetrate cell membrane efficiently. AU4S but not AG4R can be recognized and cleaved by ATG4, leading to the change of fluorescence intensity. Therefore, the difference between AU4S- and AG4R-measured fluorescence values in the same sample, defined as “F-D value,” can reflect ATG4 activity. By detecting the F-D values, we found that ATG4 activity paralleled LC3B-II levels in rapamycin-treated cells, but neither paralleled LC3B-II levels in starved cells nor presented a correlation with LC3B-II accumulation in WBCs from healthy donors or leukemia patients. However, when DTT was added to the system, ATG4 activity not only paralleled LC3B-II levels in starved cells in the presence or absence of autophagy inhibitors, but also presented a positive correlation with LC3B-II accumulation in WBCs from leukemia patients (R² = 0.5288). In conclusion, this study provides a convenient, rapid, and quantitative method to monitor ATG4 activity in living cells, which may be beneficial to basic and clinical research on autophagy.     

Noncovalent assembly of anti-dendritic cell antibodies and antigens for evoking immune responses in vitro and in vivo

Targeting of Ags directly to dendritic cells (DCs) through anti-DC receptor Ab fused to Ag proteins is a promising approach to vaccine development. However, not all Ags can be expressed as a rAb directly fused to a protein Ag. In this study, we show that noncovalent assembly of Ab-Ag complexes, mediated by interaction between dockerin and cohesin domains from cellulose-degrading bacteria, can greatly expand the range of Ags for this DC-targeting vaccine technology. rAbs with a dockerin domain fused to the rAb H chain C terminus are efficiently secreted by mammalian cells, and many Ags not secreted as rAb fusion proteins are readily expressed as cohesin directly fused to Ag either via secretion from mammalian cells or as soluble cytoplasmic Escherichia coli products. These form very stable and homogeneous complexes with rAb fused to dockerin. In vitro, these complexes can efficiently bind to human DC receptors followed by presentation to Ag-specific CD4(+) and CD8(+) T cells. Low doses of the HA1 subunit of influenza hemagglutinin conjugated through this means to anti-Langerin rAbs elicited Flu HA1-specific Ab and T cell responses in mice. Thus, the noncovalent assembly of rAb and Ag through dockerin and cohesin interaction provides a useful modular strategy for development and testing of prototype vaccines for elicitation of Ag-specific T and B cell responses, particularly when direct rAb fusions to Ag cannot be expressed.     

Complete genome sequence of a highly prevalent porcine circovirus 2 isolated from piglet stool samples in China

Porcine circovirus type 2 (PCV2) is the etiologic agent of porcine circovirus-associated disease. Here, we first report the complete genome sequence of PCV2 strain JSTZ, which was isolated from piglet stool samples and is highly prevalent in China. It will help in understanding the epidemiology and molecular characteristics of PCV2.     

Complete genome sequence of a novel species of Porcine Bocavirus, PBoV5

Porcine bocavirus 5 is a novel porcine bocavirus species found in a pig with clinical diarrhea from a farm in China. Here, we report the complete genome sequence of strain PBoV5/JS677, which will help toward understanding the molecular and evolutionary characteristics of the porcine bocavirus.     

Prostaglandin E2 affects T cell responses through modulation of CD46 expression

The ubiquitous protein CD46, a regulator of complement activity, promotes T cell activation and differentiation toward a regulatory Tr1-like phenotype. The CD46-mediated differentiation pathway is defective in several chronic inflammatory diseases, underlying the importance of CD46 in controlling T cell function and the need to understand its regulatory mechanisms. Using an RNA interference-based screening approach in primary T cells, we have identified that two members of the G protein-coupled receptor kinases were involved in regulating CD46 expression at the surface of activated cells. We have investigated the role of PGE(2), which binds to the E-prostanoid family of G protein-coupled receptors through four subtypes of receptors called EP 1-4, in the regulation of CD46 expression and function. Conflicting roles of PGE(2) in T cell functions have been reported, and the reasons for these apparent discrepancies are not well understood. We show that addition of PGE(2) strongly downregulates CD46 expression in activated T cells. Moreover, PGE(2) differentially affects T cell activation, cytokine production, and phenotype depending on the activation signals received by the T cells. This was correlated with a distinct pattern of the PGE(2) receptors expressed, with EP4 being preferentially induced by CD46 activation. Indeed, addition of an EP4 antagonist could reverse the effects observed on cytokine production after CD46 costimulation. These data demonstrate a novel role of the PGE(2)-EP4 axis in CD46 functions, which might at least partly explain the diverse roles of PGE(2) in T cell functions.     

Effects of honey bee (Apis mellifera L.) queen insemination volume on worker behavior and physiology

Honey bee colonies consist of tens of thousands of workers and a single reproductive queen that produces a pheromone blend which maintains colony organization. Previous studies indicated that the insemination quantity and volume alter queen mandibular pheromone profiles. In our 11-month long field study we show that workers are more attracted to high-volume versus low-volume inseminated queens, however, there were no significant differences between treatments in the number of queen cells built by workers in preparation for supersedure. Workers exposed to low-volume inseminated queens initiated production of queen-like esters in their Dufour's glands, but there were no significant difference in the amount of methyl farnesoate and juvenile hormone in worker hemolymph. Lastly, queen overwintering survival was unexpectedly lower in high-volume inseminated queens. Our results suggest that the queen insemination volume could ultimately affect colony health and productivity.     

Genome sequences of three species in the family Planctomycetaceae

Most of the species in the family Planctomycetaceae are of interest for their eukaryotic-like cell structures and characteristics of resistance to extreme environments. Here, we report draft genome sequences of three aquatic parasitic species of this family, Singulisphaera acidiphila (DSM 18658T), Schlesneria paludicola (DSM 18645T), and Zavarzinella formosa (DSM 19928T).