The secreted, multidomain protein follistatin binds activins with high affinity, inhibiting their receptor interaction. We have dissected follistatin's domain structure and shown that the minimal activin-inhibiting fragment of follistatin is comprised of the first and second Fs domains (Fs12). This protein can bind to activin dimer and form a stable complex containing two Fs12 molecules and one activin dimer. We have solved crystal structures of activin A alone and its complex with Fs12 fragment to 2 Å resolution. The complex structure shows how Fs12 molecules wrap around the back of the 'wings' of activin, blocking the type II receptor-binding site on activin A. Arginine 192 in Fs2 is a key residue in this interaction, inserting itself in between activin's fingers. Complex formation imposes a novel orientation for the EGF-and Kazal-like subdomains in the Fs2 domain and activin A shows further variation from the canonical TGF-b family fold. The structure provides a detailed description of the inhibitory mechanism and gives insights into interactions of follistatin with other TGF-b family proteins.

As part of an effort to develop nanoelectronic sensors for biological targets, we tested the potential to incorporate coiled coils as metallized, self-assembling, site-specific molecular linkers on carbon nanotubes (CNTs). Based on a previously conceived modular anchor-probe approach, a system was designed in which hydrophobic residues (valines and leucines) form the interface between the two helical peptide components. Charged residues (glutamates and arginines) on the borders of the hydrophobic interface increase peptide solubility, and provide stability and specificity for anchor-probe assembly. Two histidine residues oriented on the exposed hydrophilic exterior of each peptide were included as chelating sites for metal ions such as cobalt. Cysteines were incorporated at the peptide termini for oriented, thiol-mediated coupling to surface plasmon resonance (SPR) biosensor surfaces, gold nanoparticles or CNT substrates. The two peptides were produced by solid phase peptide synthesis using Fmoc chemistry: an acidic 42-residue peptide E42C, and its counterpart in the heterodimer, a basic 39-residue peptide R39C. The ability of E42C and R39C to bind cobalt was demonstrated by immobilized metal affinity chromatography and isothermal titration calorimetry. SPR biosensor kinetic analysis of dimer assembly revealed apparent sub-nanomolar affinities in buffers with and without 1 mM CoCl2 using two different reference surfaces. For device-oriented CNT immobilization, R39C was covalently anchored to CNT tips via a C-terminal cysteine residue. Scanning electron microscopy was used to visualize the assembly of probe peptide (E42C) N-terminally labeled with 15 nm gold nanoparticles, when added to the R39C-CNT surface. The results obtained open the way to develop CNT tip-directed recognition surfaces, using recombinant and chemically synthesized chimeras containing binding epitopes fused to the E42C sequence domain.

Public expenditure on healthcare in India is low by international comparison, and access to essential treatment pushes many uninsured citizens below the poverty line. In many countries, policymakers utilize health technology assessment (HTA) methodologies to direct investments in healthcare, to obtain the maximum benefit for the population as a whole. With rising incomes and a commitment from the Government of India to increase the proportion of gross domestic product spent on health, this is an opportune moment to consider how HTA might help to allocate healthcare spending in India, in an equitable and efficient manner. Despite the predominance of out-of-pocket payments in the Indian healthcare sector, payers of all types are increasingly demanding value for money from expenditure on healthcare. In this review we demonstrate how HTA can be used to inform several aspects of healthcare provision. Areas in which HTA could be applied in the Indian context include, drug pricing, development of clinical practice guidelines, and prioritizing interventions that represent the greatest value within a limited budget. To illustrate the potential benefits of using the HTA approach, we present an example from a mature HTA market (Canada) that demonstrates how a new treatment for patients with atrial fibrillation — although more expensive than the current standard of care — improves clinical outcomes and represents a cost-effective use of public health resources. If aligned with the prevailing cultural and ethical considerations, and with the necessary investment in expert staff and resources, HTA promises to be a valuable tool for development of the Indian healthcare sector.

Interleukin (IL)-5 exerts hematopoietic functions through binding to the IL-5 receptor subunits, ␣ and ␤c. Specific assembly steps of full-length subunits as they occur in cell membranes, ultimately leading to receptor activation, are not well understood. We tracked the oligomerization of IL-5 receptor subunits using fluorescence resonance energy transfer (FRET) imaging. Full-length IL-5R␣ and ␤c were expressed in Phoenix cells as chimeric proteins fused to enhanced cyan or yellow fluorescent protein (CFP or YFP, respectively). A time-and dose-dependent increase in FRET signal between IL-5R␣-CFP and ␤c-YFP was observed in response to IL-5, indicative of heteromeric receptor ␣-␤c subunit interaction. This response was inhibited by AF17121, a peptide antagonist of IL-5R␣. Substantial FRET signals with ␤c-CFP and ␤c-YFP co-expressed in the absence of IL-5R␣ demonstrated that ␤c subunits exist as preformed homo-oligomers. IL-5 had no effect on this ␤c-alone FRET signal. Interestingly, the addition of IL-5 to cells co-expressing ␤c-CFP, ␤c-YFP, and nontagged IL-5R␣ led to further increase in FRET efficiency. Observation of preformed ␤c oligomers fits with the view that this form can lead to rapid cellular responses upon IL-5 stimulation. The IL-5-induced effects on ␤c assembly in the presence of nontagged IL-5R␣ provide direct evidence that IL-5 can cause higher order rearrangements of ␤c homooligomers. These results suggest that IL-5 and perhaps other ␤c cytokines (IL-3 and granulocyte/macrophage colony-stimulating factor) trigger cellular responses by the sequential binding of cytokine ligand to the specificity receptor (subunit ␣), followed by binding of the ligand-subunit ␣ complex to, and consequent rearrangement of, a ground state form of ␤c oligomers.It is generally thought that most cytokines trigger cellular signal transduction by inducing the association of receptor subunits that leads to increased proximity of associated intracellular kinases and initiation of phosphorylation cascades. Recent studies of human growth hormone and erythropoietin have shown that the assembly of their receptor subunits leads to conformational rearrangement and that this process plays a role in receptor activation (1-3). However, evidence for such cytokine-induced receptor rearrangement for other cytokinereceptor complexes remains limited. In the case of interleukin (IL) 2 -5 and the other ␤c cytokines IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF), the states of receptor subunit assembly in membranes that lead to activation have been proposed, but experimental evidence for these has remained largely indirect.Human IL-5 is a T H 2 cell-derived cytokine that regulates hematopoiesis and inflammation. It is implicated in the pathogenesis of allergic disorders, asthma, and other forms of hypereosinophilic syndromes, through its influence on eosinophil maturation, proliferation, activation, expansion, and tissue distribution (4 -7). IL-5 exerts its biological functions by binding to a heteromeric cell surface rece...

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