End-binding protein 1 controls signal propagation from the T cell receptor

The role of microtubules (MTs) in the control and dynamics of the immune synapse (IS) remains unresolved. Here, we show that T cell activation requires the growth of MTs mediated by the plus-end specific protein end-binding 1 (EB1). A direct interaction of the T cell receptor (TCR) complex with EB1 provides the molecular basis for EB1 activity promoting TCR encounter with signalling vesicles at the IS. EB1 knockdown alters TCR dynamics at the IS and prevents propagation of the TCR activation signal to LAT, thus inhibiting activation of PLCγ1 and its localization to the IS. These results identify a role for EB1 interaction with the TCR in controlling TCR sorting and its connection with the LAT/PLCγ1 signalosome.     

Disentangling signaling gradients generated by equivalent sources

Yeast cells approach a mating partner by polarizing along a gradient of mating pheromones that are secreted by cells of the opposite mating type. The Bar1 protease is secreted by a-cells and, paradoxically, degrades the α-factor pheromones which are produced by cells of the opposite mating type and trigger mating in a-cells. This degradation may assist in the recovery from pheromone signaling but has also been shown to play a positive role in mating. Previous studies suggested that widely diffusing protease can bias the pheromone gradient towards the closest secreting cell. Here, we show that restricting the Bar1 protease to the secreting cell itself, preventing its wide diffusion, facilitates discrimination between equivalent mating partners. This may be mostly relevant during spore germination, where most mating events occur in nature.     

Genome analysis of bovine-mastitis-associated Escherichia coli O32:H37 strain P4

Escherichia coli is a major pathogen of bovine intramammary infections. Here we report the first draft of the genome sequence of the E. coli O32:H37 P4 strain, which is widely used in experimental bovine mastitis studies.     

Draft Genome Sequence of Flavobacterium sp. Strain F52, Isolated from the Rhizosphere of Bell Pepper (Capsicum annuum L. cv. Maccabi)

Here we report the draft genome sequence of Flavobacterium sp. strain F52, isolated from the rhizosphere of bell pepper (Capsicum annuum L. cv. Maccabi). Flavobacterium spp. are ubiquitous in the rhizospheres of agricultural crops; however, little is known about their physiology. To our knowledge, this is the first published genome of a root-associated Flavobacterium strain.     

Selection of antibodies from synthetic antibody libraries

More than 2 dozen years had passed since the field of antibody engineering was established, with the first reports of bacterial [1-3] and mammalian cells [4] expression of recombinant antibody fragments, and in that time a lot of effort was dedicated to the development of efficient technological means, intended to assist in the creation of therapeutic monoclonal antibodies (mAbs). Research focus was given to two intertwined technological aspects: the selection platform and the recombinant antibody repertoires. In accordance with these areas of interest, it is the goal of this chapter to describe the various selection tools and antibody libraries existing, with emphasis on the later, and their applications. This chapter gives a far from exhaustive, subjective "historic account" of the field, describing the selection platforms, the different formats of antibody repertoires and the applications of both for selecting recombinant antibodies. Several excellent books provide detailed protocols for constructing antibody libraries and selecting antibodies from those libraries [5-13]. Such books may guide a newcomer to the field in the fine details of antibody engineering. We would like to offer advice to the novice: although seemingly simple, effective library construction and antibody isolation provide best benefits in the hands of professionals. It is an art as much as it is science.     

Studies of Mg2+/Ca2+ complexes of naturally occurring dinucleotides: potentiometric titrations, NMR, and molecular dynamics

Dinucleotides (Np(n)N'; N and N' are A, U, G, or C, n = 2-7) are naturally occurring physiologically active compounds. Despite the interest in dinucleotides, the composition of their complexes with metal ions as well as their conformations and species distribution in living systems are understudied. Therefore, we investigated a series of Mg(2+) and Ca(2+) complexes of Np(n)N's. Potentiometric titrations indicated that a longer dinucleotide polyphosphate (N is A or G, n = 3-5) linker yields more stable complexes (e.g., log K of 2.70, 3.27, and 3.73 for Ap(n)A-Mg(2+), n = 3, 4, 5, respectively). The base (A or G) or ion (Mg(2+) or Ca(2+)) has a minor effect on K(M)(ML) values. In a physiological medium, the longer Ap(n)As (n = 4, 5) are predicted to occur mostly as the Mg(2+)/Ca(2+) complexes. (31)P NMR monitored titrations of Np(n)N's with Mg(2+)/Ca(2+) ions showed that the middle phosphates of the dinucleotides coordinate with Mg(2+)/Ca(2+). Multidimensional potential of mean force (PMF) molecular dynamics (MD) simulations suggest that Ap(2)A and Ap(4)A coordinate Mg(2+) and Ca(2+) ions in both inner-sphere and outer-sphere modes. The PMF MD simulations additionally provide a detailed picture of the possible coordination sites, as well as the cation binding process. Moreover, both NMR and MD simulations showed that the conformation of the nucleoside moieties in Np(n)N'-Mg(2+)/Ca(2+) complexes remains the same as that of free mononucleotides.     

IRE1α induces thioredoxin-interacting protein to activate the NLRP3 inflammasome and promote programmed cell death under irremediable ER stress

When unfolded proteins accumulate to irremediably high levels within the endoplasmic reticulum (ER), intracellular signaling pathways called the unfolded protein response (UPR) become hyperactivated to?cause programmed cell death. We discovered that?thioredoxin-interacting protein (TXNIP) is?a critical node in this "terminal UPR." TXNIP becomes rapidly induced by IRE1α, an ER bifunctional kinase/endoribonuclease (RNase). Hyperactivated IRE1α increases TXNIP mRNA stability by reducing levels of a TXNIP destabilizing microRNA, miR-17. In turn, elevated TXNIP protein activates the NLRP3 inflammasome, causing procaspase-1 cleavage and interleukin 1β (IL-1β) secretion. Txnip gene deletion reduces pancreatic β cell death during ER stress and suppresses diabetes caused by proinsulin misfolding in the Akita mouse. Finally, small molecule?IRE1α RNase inhibitors suppress TXNIP production to block IL-1β secretion. In summary, the IRE1α-TXNIP pathway is used in the terminal UPR to promote sterile inflammation and programmed cell death and may be targeted to develop effective treatments for cell degenerative diseases.     

Influenza A H5N1 clade 2.3.4 virus with a different antiviral susceptibility profile replaced clade 1 virus in humans in northern Vietnam

Background

Prior to 2007, highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry and humans in Vietnam were consistently reported to be clade 1 viruses, susceptible to oseltamivir but resistant to amantadine. Here we describe the re-emergence of human HPAI H5N1 virus infections in Vietnam in 2007 and the characteristics of the isolated viruses.

Methods and Findings

Respiratory specimens from patients suspected to be infected with avian influenza in 2007 were screened by influenza and H5 subtype specific polymerase chain reaction. Isolated H5N1 strains were further characterized by genome sequencing and drug susceptibility testing. Eleven poultry outbreak isolates from 2007 were included in the sequence analysis. Eight patients, all of them from northern Vietnam, were diagnosed with H5N1 in 2007 and five of them died. Phylogenetic analysis of H5N1 viruses isolated from humans and poultry in 2007 showed that clade 2.3.4 H5N1 viruses replaced clade 1 viruses in northern Vietnam. Four human H5N1 strains had eight-fold reduced in-vitro susceptibility to oseltamivir as compared to clade 1 viruses. In two poultry isolates the I117V mutation was found in the neuraminidase gene, which is associated with reduced susceptibility to oseltamivir. No mutations in the M2 gene conferring amantadine resistance were found.

Conclusion

In 2007, H5N1 clade 2.3.4 viruses replaced clade 1 viruses in northern Vietnam and were susceptible to amantadine but showed reduced susceptibility to oseltamivir. Combination antiviral therapy with oseltamivir and amantadine for human cases in Vietnam is recommended.     

Mechanisms of ligand transfer by the hepatic tocopherol transfer protein

alpha-Tocopherol is a member of the vitamin E family that functions as the principal fat-soluble antioxidant in vertebrates. Body-wide distribution of tocopherol is regulated by the hepatic alpha-tocopherol transfer protein (alphaTTP), which stimulates secretion of the vitamin from hepatocytes to circulating lipoproteins. This biological activity of alphaTTP is thought to stem from its ability to facilitate the transfer of vitamin E between membranes, but the mechanism by which the protein exerts this activity remains poorly understood. Using a fluorescence energy transfer methodology, we found that the rate of tocopherol transfer from lipid vesicles to alphaTTP increases with increasing alphaTTP concentration. This concentration dependence indicates that ligand transfer by alphaTTP involves direct protein-membrane interaction. In support of this notion, equilibrium analyses employing filtration, dual polarization interferometry, and tryptophan fluorescence demonstrated the presence of a stable alphaTTP-bilayer complex. The physical association of alphaTTP with membranes is markedly sensitive to the presence of vitamin E in the bilayer. Some naturally occurring mutations in alphaTTP that cause the hereditary disorder ataxia with vitamin E deficiency diminish the effect of tocopherol on the protein-membrane association, suggesting a possible mechanism for the accompanying pathology.     

Delivery of retinoid-based therapies to target tissues

Through its various metabolites, vitamin A controls essential physiological functions. Both naturally occurring metabolites and novel retinoid analogues have shown effectiveness in many clinical settings that include skin diseases and cancer, and in animal models of human conditions affecting vision. In this review, we analyze several potential retinoid-based therapies from the point of view of drug metabolism and transport to target tissues. We focus on the endogenous factors that affect the absorption, transport, and metabolism of retinoids by taking into account data obtained from the analysis of animal models that lack the enzymes or proteins involved in the storage and absorption of retinoids. We also discuss findings of toxicity associated with retinoids in an effort to improve the outcome of retinoid-based therapies. In this context, we review evidence that esterification of retinol and retinol-based drugs within target tissues provides one of the most efficient means to improve the absorption and to reduce the toxicity associated with pharmacological doses of retinoids. Future retinoid-based therapeutic strategies could involve targeted delivery mechanisms leading to lower toxicity and improved effectiveness of retinoids.