Stage progression and neurological symptoms in Trypanosoma brucei rhodesiense sleeping sickness: role of the CNS inflammatory response

Background: Human African trypanosomiasis progresses from an early (hemolymphatic) stage, through CNS invasion to the late (meningoencephalitic) stage. In experimental infections disease progression is associated with neuroinflammatory responses and neurological symptoms, but this concept requires evaluation in African trypanosomiasis patients, where correct diagnosis of the disease stage is of critical therapeutic importance. Methodology/Principal Findings: This was a retrospective study on a cohort of 115 T.b.rhodesiense HAT patients recruited in Eastern Uganda. Paired plasma and CSF samples allowed the measurement of peripheral and CNS immunoglobulin and of CSF cytokine synthesis. Cytokine and immunoglobulin expression were evaluated in relation to disease duration, stage progression and neurological symptoms. Neurological symptoms were not related to stage progression (with the exception of moderate coma). Increases in CNS immunoglobulin, IL-10 and TNF-α synthesis were associated with stage progression and were mirrored by a reduction in TGF-β levels in the CSF. There were no significant associations between CNS immunoglobulin and cytokine production and neurological signs of disease with the exception of moderate coma cases. Within the study group we identified diagnostically early stage cases with no CSF pleocytosis but intrathecal immunoglobulin synthesis and diagnostically late stage cases with marginal CSF pleocytosis and no detectable trypanosomes in the CSF. Conclusions: Our results demonstrate that there is not a direct linkage between stage progression, neurological signs of infection and neuroinflammatory responses in rhodesiense HAT. Neurological signs are observed in both early and late stages, and while intrathecal immunoglobulin synthesis is associated with neurological signs, these are also observed in cases lacking a CNS inflammatory response. While there is an increase in inflammatory cytokine production with stage progression, this is paralleled by increases in CSF IL-10. As stage diagnostics, the CSF immunoglobulins and cytokines studied do not have sufficient sensitivity to be of clinical value

Interrogating and Predicting Tolerated Sequence Diversity in Protein Folds: Application to E. elaterium Trypsin Inhibitor-II Cystine-Knot Miniprotein

Cystine-knot miniproteins (knottins) are promising molecular scaffolds for protein engineering applications. Members of the knottin family have multiple loops capable of displaying conformationally constrained polypeptides for molecular recognition. While previous studies have illustrated the potential of engineering knottins with modified loop sequences, a thorough exploration into the tolerated loop lengths and sequence space of a knottin scaffold has not been performed. In this work, we used the Ecballium elaterium trypsin inhibitor II (EETI) as a model member of the knottin family and constructed libraries of EETI loop-substituted variants with diversity in both amino acid sequence and loop length. Using yeast surface display, we isolated properly folded EETI loop-substituted clones and applied sequence analysis tools to assess the tolerated diversity of both amino acid sequence and loop length. In addition, we used covariance analysis to study the relationships between individual positions in the substituted loops, based on the expectation that correlated amino acid substitutions will occur between interacting residue pairs. We then used the results of our sequence and covariance analyses to successfully predict loop sequences that facilitated proper folding of the knottin when substituted into EETI loop 3. The sequence trends we observed in properly folded EETI loop-substituted clones will be useful for guiding future protein engineering efforts with this knottin scaffold. Furthermore, our findings demonstrate that the combination of directed evolution with sequence and covariance analyses can be a powerful tool for rational protein engineering

Structural model for the interaction of a designed Ankyrin Repeat Protein with the human epidermal growth factor receptor 2

Designed Ankyrin Repeat Proteins are a class of novel binding proteins that can be selected and evolved to bind to targets with high affinity and specificity. We are interested in the DARPin H10-2-G3, which has been evolved to bind with very high affinity to the human epidermal growth factor receptor 2 (HER2). HER2 is found to be over-expressed in 30% of breast cancers, and is the target for the FDA-approved therapeutic monoclonal antibodies trastuzumab and pertuzumab and small molecule tyrosine kinase inhibitors. Here, we use computational macromolecular docking, coupled with several interface metrics such as shape complementarity, interaction energy, and electrostatic complementarity, to model the structure of the complex between the DARPin H10-2-G3 and HER2. We analyzed the interface between the two proteins and then validated the structural model by showing that selected HER2 point mutations at the putative interface with H10-2-G3 reduce the affinity of binding up to 100-fold without affecting the binding of trastuzumab. Comparisons made with a subsequently solved X-ray crystal structure of the complex yielded a backbone atom root mean square deviation of 0.84-1.14 Ångstroms. The study presented here demonstrates the capability of the computational techniques of structural bioinformatics in generating useful structural models of protein-protein interactions

Understanding the Impact of Transparency on Algorithmic Decision Making Legitimacy

Part 2: Social Implications of Algorithmic PhenomenaInternational audienceIn recent years the volume, velocity and variety of the Big Data being produced has presented several opportunities to improve all our lives. It has also generated several challenges not the least of which is humanities ability to analyze, process and take decisions on that data. Algorithmic Decision Making (ADM) represents a solution to these challenge. Whilst ADM has been around for many years, it has come under increased scrutiny in recent years because of concerns related to the increasing breadth of application and the inherent lack of Transparency in these algorithms, how they operate and how they are created. This has impacted the perceived Legitimacy of this technology which has led to government legislation to limit and regulate its use. This paper begins the process of understanding the impact of Transparency on ADM Legitimacy by breaking down Transparency in Algorithmic Decision Making into the components of Validation, Visibility and Variability and by using legitimacy theory to theorize the impact of transparency on ADM Legitimacy. A useful first step in the development of a framework is achieved by developing a series of testable propositions to be used in further proposed research regarding the impact of Transparency on ADM Legitimacy

Targeting Extracellular Domains D4 and D7 of Vascular Endothelial Growth Factor Receptor 2 Reveals Allosteric Receptor Regulatory Sites

Vascular endothelial growth factors (VEGFs) activate three receptor tyrosine kinases, VEGFR-1, -2, and -3, which regulate angiogenic and lymphangiogenic signaling. VEGFR-2 is the most prominent receptor in angiogenic signaling by VEGF ligands. The extracellular part of VEGF receptors consists of seven immunoglobulin homology domains (Ig domains). Earlier studies showed that domains 2 and 3 (D23) mediate ligand binding, while structural analysis of dimeric ligand/receptor complexes by electron microscopy and small-angle solution scattering revealed additional homotypic contacts in membrane-proximal Ig domains D4 and D7. Here we show that D4 and D7 are indispensable for receptor signaling. To confirm the essential role of these domains in signaling, we isolated VEGFR-2-inhibitory “designed ankyrin repeat proteins” (DARPins) that interact with D23, D4, or D7. DARPins that interact with D23 inhibited ligand binding, receptor dimerization, and receptor kinase activation, while DARPins specific for D4 or D7 did not prevent ligand binding or receptor dimerization but effectively blocked receptor signaling and functional output. These data show that D4 and D7 allosterically regulate VEGFR-2 activity. We propose that these extracellular-domain-specific DARPins represent a novel generation of receptor-inhibitory drugs for in vivo applications such as targeting of VEGFRs in medical diagnostics and for treating vascular pathologies

Control of repeat-protein curvature by computational protein design

Shape complementarity is an important component of molecular recognition, and the ability to precisely adjust the shape of a binding scaffold to match a target of interest would greatly facilitate the creation of high affinity protein reagents and therapeutics. Here we describe a general approach to control the shape of the binding surface on repeat protein scaffolds, and apply it to leucine rich repeat proteins. First, a set of self-compatible building block modules are designed that when polymerized each generate surfaces with unique but constant curvatures. Second, a set of junction modules that connect the different building blocks are designed. Finally, new proteins with custom designed shapes are generated by appropriately combining building block and junction modules. Crystal structures of the designs illustrate the power of the approach in controlling repeat protein curvature. Repeat protein scaffolds have attracted much attention as alternative binding scaffolds to antibodies1-4 and also as building blocks of protein nanomaterials5-7 because of their intrinsic modularity and high stability. The leucine rich repeat (LRR) is a repeat protein scaffold with a horseshoe-like global structure in which the concave surface is often

The Scent of Danger: the Impact of Predator Chemical Cues on Emergence from Refuge and Willingness to Autotomize Limbs in the House Cricket (Acheta domesticus)

Prey can accurately assess predation risk via the detection of chemical cues and take appropriate measures to survive encounters with predators. Research on the chemical ecology of terrestrial invertebrate predator-prey interactions has repeatedly found that direct chemical cues can alter prey organisms’ antipredator behavior. However, much of this research has focused on the chemical mediation of avoidance and immobility by cues from lycosid spiders neglecting other prominent invertebrate predators and behavior such as autotomy. In our study, house crickets (Acheta domesticus) were exposed to cues from cricket-fed orange-footed centipedes (Cormocephalus aurantiipes), red-back spiders (Latrodectus hasselti), an odorous (cologne) control, and a non-odorous control to determine whether direct chemical cues had any influence on two types of anti-predatory behavior: the willingness (latency) to emerge from a refuge and to autotomize limbs. Exposure to C. aurantiipes cues resulted in a significantly slower emergence from a refuge, but exposure to L. hasselti cues did not. Direct chemical cues had no influence on initial autotomy, but exposure to L. hasselti cues did significantly decrease the latency to autotomize a second limb. That cues from L. hasselti had an influence on a second autotomy, but not initial autotomy may be because crickets that undergo autotomy for a second time may perceive themselves to be already at a higher risk of predation as they were already missing a limb. Variation in responses to cues from different predators demonstrates a need to examine the influence of chemical cues from a wider variety of invertebrate predators on anti-predator behavior