Computationally driven deletion of broadly distributed T cell epitopes in a biotherapeutic candidate

Biotherapeutics are subject to immune surveillance within the body, and anti-biotherapeutic immune responses can compromise drug efficacy and patient safety. Initial development of targeted antidrug immune memory is coordinated by T cell recognition of immunogenic subsequences, termed “T cell epitopes.” Biotherapeutics may therefore be deimmunized by mutating key residues within cognate epitopes, but there exist complex trade-offs between immunogenicity, mutational load, and protein structure–function. Here, a protein deimmunization algorithm has been applied to P99 beta-lactamase, a component of antibody-directed enzyme prodrug therapies. The algorithm, integer programming for immunogenic proteins, seamlessly integrates computational prediction of T cell epitopes with both 1- and 2-body sequence potentials that assess protein tolerance to epitope-deleting mutations. Compared to previously deimmunized P99 variants, which bore only one or two mutations, the enzymes designed here contain 4–5 widely distributed substitutions. As a result, they exhibit broad reductions in major histocompatibility complex recognition. Despite their high mutational loads and markedly reduced immunoreactivity, all eight engineered variants possessed wild-type or better catalytic activity. Thus, the protein design algorithm is able to disrupt broadly distributed epitopes while maintaining protein function. As a result, this computational tool may prove useful in expanding the repertoire of next-generation biotherapeutics.     

HAX1 deficiency causes autosomal recessive severe congenital neutropenia (Kostmann disease)

Autosomal recessive severe congenital neutropenia (SCN) constitutes a primary immunodeficiency syndrome associated with increased apoptosis in myeloid cells, yet the underlying genetic defect remains unknown. Using a positional cloning approach and candidate gene evaluation, we identified a recurrent homozygous germline mutation in HAX1 in three pedigrees. After further molecular screening of individuals with SCN, we identified 19 additional affected individuals with homozygous HAX1 mutations, including three belonging to the original pedigree described by Kostmann. HAX1 encodes the mitochondrial protein HAX1, which has been assigned functions in signal transduction and cytoskeletal control. Here, we show that HAX1 is critical for maintaining the inner mitochondrial membrane potential and protecting against apoptosis in myeloid cells. Our findings suggest that HAX1 is a major regulator of myeloid homeostasis and underline the significance of genetic control of apoptosis in neutrophil development.     

Hep27, a member of the short-chain dehydrogenase/reductase family, is an NADPH-dependent dicarbonyl reductase expressed in vascular endothelial tissue

Human Hep27 was originally isolated from growth-arrested HepG2 cells and identified as a member of the superfamily of short-chain dehydrogenases/reductases (SDR). Its substrate specificity has not been determined, but a cross-species comparison suggests that it occurs in widely divergent species, such as human, Cenorhabditis elegans, Drosophila and Arabidopsis thaliana. In this study, Hep27 was expressed as a His₆ fusion protein, and subjected to a substrate screen, using a compound library of SDR substrates, comprising steroids, retinoids, sugars and carbonyl compounds. Whereas no steroid dehydrogenase or retinoid activity was detected, it was found that Hep27 catalyzed the NADPH-dependent reduction of dicarbonyl compounds, like 3,4-hexanedione and 1-phenyl-1,2-propanedione with similar turnover numbers as DCXR (a mitochondrial dicarbonyl reductase/xylulose reductase). In contrast, Hep27 does not convert sugar substrates like xylulose or threose. Based on its substrate specificity and expression in endothelial tissues, it is suggested that Hep27 functions as a dicarbonyl reductase in enzymatic inactivation of reactive carbonyls, involved in covalent modification of cellular components. Received 16 January 2006; received after revision 28 February 2006; accepted 31 March 2006     

Neuronal filtering of multiplexed odour representations

Neuronal activity patterns contain information in their temporal structure, indicating that information transfer between neurons may be optimized by temporal filtering. In the zebrafish olfactory bulb, subsets of output neurons (mitral cells) engage in synchronized oscillations during odour responses, but information about odour identity is contained mostly in non-oscillatory firing rate patterns. Using optogenetic manipulations and odour stimulation, we found that firing rate responses of neurons in the posterior zone of the dorsal telencephalon (Dp), a target area homologous to olfactory cortex, were largely insensitive to oscillatory synchrony of mitral cells because passive membrane properties and synaptic currents act as low-pass filters. Nevertheless, synchrony influenced spike timing. Moreover, Dp neurons responded primarily during the decorrelated steady state of mitral cell activity patterns. Temporal filtering therefore tunes Dp neurons to components of mitral cell activity patterns that are particularly informative about precise odour identity. These results demonstrate how temporal filtering can extract specific information from multiplexed neuronal codes.     

Amygdala circuitry mediating reversible and bidirectional control of anxiety

Anxiety--a sustained state of heightened apprehension in the absence of immediate threat--becomes severely debilitating in disease states. Anxiety disorders represent the most common of psychiatric diseases (28% lifetime prevalence) and contribute to the aetiology of major depression and substance abuse. Although it has been proposed that the amygdala, a brain region important for emotional processing, has a role in anxiety, the neural mechanisms that control anxiety remain unclear. Here we explore the neural circuits underlying anxiety-related behaviours by using optogenetics with two-photon microscopy, anxiety assays in freely moving mice, and electrophysiology. With the capability of optogenetics to control not only cell types but also specific connections between cells, we observed that temporally precise optogenetic stimulation of basolateral amygdala (BLA) terminals in the central nucleus of the amygdala (CeA)--achieved by viral transduction of the BLA with a codon-optimized channelrhodopsin followed by restricted illumination in the downstream CeA--exerted an acute, reversible anxiolytic effect. Conversely, selective optogenetic inhibition of the same projection with a third-generation halorhodopsin (eNpHR3.0) increased anxiety-related behaviours. Importantly, these effects were not observed with direct optogenetic control of BLA somata, possibly owing to recruitment of antagonistic downstream structures. Together, these results implicate specific BLA-CeA projections as critical circuit elements for acute anxiety control in the mammalian brain, and demonstrate the importance of optogenetically targeting defined projections, beyond simply targeting cell types, in the study of circuit function relevant to neuropsychiatric disease.     

Exact Efficient Handling of Interrupted Illumination in Helical Cone-Beam Computed Tomography with Arbitrary Pitch

We present a theoretically-exact and stable computed tomography(CT) reconstruction algorithm that is capable of handling interrupted illumination and therefore of using all measured data at arbitrary pitch.This algorithm is based on a differentiated backprojection(DBP) on M-lines.First,we discuss the problem of interrupted illumination and how it affects the DBP.Then we show that it is possible to take advantage of some properties of the DBP to compensate for the effects of interrupted illumination in a mat...     

The Moxee City (Washington) mammoth: morphostratigraphic, taphonomic, and taxonomic considerations

A nearly complete, but highly fractured, proboscidean tusk was unearthed during parking lot construction near Moxee City in central Washington in May 2001. Schreger angle analysis revealed that the tusk was from a mammoth. AMS radiocarbon dating of the tusk established that the mammoth died 14,570 14C yr BP. The age, combined with the biogeography of proboscidean finds in the Pacific Northwest, suggests the tusk is from a Columbian mammoth ( Mammuthus columbi ). The condition of the tusk and its association with basalt and crystalline erratics suggest that a locally derived tusk was swept up in the advancing flood and transported to ～320 m elevation, where it was deposited in the sediments of the 3rd of 3 Missoula Floods that are preserved in the area. The tusk's weathering indicates subaerial exposure prior to burial in the slackwater sediments. Slackwater deposits at the site are pale, ～30-100 cm thick, calcareous, fine-textured strata that include occasional coarse basalt and crystalline sand and gravel. They are intruded by numerous clastic dikes. The sediments encapsulating the tusk lack rhythmites because of their deposition in the nearshore zone of an ephemeral slackwater lake. The first 2 floods inundated the site between 15,300 14 C yr BP and 14,570 14 C yr BP, stripping the A horizon from a well-developed soil formed in alluvial fan sediments sitting above an Ellensburg Formation pediment. The last flood to reach the site occurred later than 14,570 14 C yr BP, as indicated by the presence of the dated tusk. Post-flood and post-MSH S tephra loess derived from the Yakima River floodplain mantles these slackwater deposits. The Warden soil is forming in the now-stable loess parent material.     

Supporting Consistency in Linked Specialized Engineering Models Through Bindings and Updating

Currently, some commercial software applications support users to work in an integrated environment. However, this is limited to the suite of models provided by the software vendor and consequently it forces all the parties to use the same software. In contrast, the research described in this paper investigates ways of using standard software applications, which may be specialized for different professional domains. These are linked for effective transfer of information and a binding mechanism is provided to support consistency. The proposed solution was implemented using a CAD application and an independent finite element application in order to verify the theoretical aspects of this work.