Producing amyloid fibrils in vitro: A tool for studying AL amyloidosis.

Amyloid light-chain (AL) amyloidosis is the second most common form of systemic amyloidosis which is characterized by a high level of mortality and no effective treatment to remove fibril deposition. This disorder is caused by malfunctioning of B-cells resulting in production of abnormal protein fibrils composed of immunoglobulin light chain fragments that tend to deposit on various organs and tissues. AL amyloidosis is set apart from other forms of amyloidosis in that no specific sequences have been identified in the immunoglobulin light chains that are amyloid fibril formation causative and patient specific. This unusual feature hinders the therapeutic progress and requires either direct access to patient samples (which is not always possible) or a source of in vitro produced fibrils. While isolated reports of successful AL amyloid fibril formation from various patient-specific protein sequences can be found in literature, no systematic research on this topic was performed since 1999. In the present study we have developed a generalized approach to in vitro fibril production from various types of previously reported [[1], [2], [3]] amyloidogenic immunoglobulin light chains and their fragments. We describe the procedure from selection and generation of starting material, through finding of optimal assay conditions, to applying a panel of methods to confirm successful fibril formation. Procedure details are discussed in the light of the most recent findings and theories on amyloid fibril formation. The reported protocol produces high quality AL amyloid fibrils that can subsequently be used in the development of the much-needed amyloid-targeting diagnostic and therapeutic approaches.

Structural basis for ligand reception by anaplastic lymphoma kinase.

The proto-oncogene ALK encodes anaplastic lymphoma kinase, a receptor tyrosine kinase that is expressed primarily in the developing nervous system. After development, ALK activity is associated with learning and memory1 and controls energy expenditure, and inhibition of ALK can prevent diet-induced obesity2. Aberrant ALK signalling causes numerous cancers3. In particular, full-length ALK is an important driver in paediatric neuroblastoma4,5, in which it is either mutated6 or activated by ligand7. Here we report crystal structures of the extracellular glycine-rich domain (GRD) of ALK, which regulates receptor activity by binding to activating peptides8,9. Fusing the ALK GRD to its ligand enabled us to capture a dimeric receptor complex that reveals how ALK responds to its regulatory ligands. We show that repetitive glycines in the GRD form rigid helices that separate the major ligand-binding site from a distal polyglycine extension loop (PXL) that mediates ALK dimerization. The PXL of one receptor acts as a sensor for the complex by interacting with a ligand-bound second receptor. ALK activation can be abolished through PXL mutation or with PXL-targeting antibodies. Together, these results explain how ALK uses its atypical architecture for its regulation, and suggest new therapeutic opportunities for ALK-expressing cancers such as paediatric neuroblastoma.

Impact of benzyl butyl phthalate on shoaling behavior in Fundulus heteroclitus (mummichog) populations.

Fundulus heteroclitus preference for association with familiar conspecifics of similar body length was impacted by benzyl butyl phthalate (BBP); this was found to be a statically significant result with a p < 0.0001. When presented with equally sized shoals consisting of either large or small fish, the majority of unexposed (84%) and acetone exposed control (82%) fish selected the shoal of large fish. A small number of control fish chose either the shoal of small fish (6% and 10%) or the neutral zone (10% and 8%) where they were clear morphological outliers. Fish exposed to 0.1 mg/L BBP exposure daily for four weeks selected the shoal of small fish more often than unexposed or acetone controls (7.5- and 4.5-fold respectively). They also remained in the neutral zone and displayed agitation at levels more than twice that of control. Agitation and shoal choice disruption are quantifiable behavioral responses that support the use of F. heteroclitus as a model for detecting sub-lethal BBP exposure.