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Ensuring biotech companies are sufficiently capitalized to propel innovation and development remains a central focus for management. In this article, we draw on our broad perspective interacting with venture capitalists to offer thoughts on investor feedback. Understanding venture capitalists' mindsets and investment theses will increase the probability of securing needed capital.

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Serum amyloid A (SAA) is a plasma protein that transports lipids during inflammation. To explore SAA solution conformations and lipid-binding mechanism, we used hydrogen-deuterium exchange mass spectrometry, lipoprotein reconstitution, amino acid sequence analysis, and molecular dynamics simulations. Solution conformations of lipid-bound and lipid-free mSAA1 at pH~7.4 agreed in details with the crystal structures but also showed important differences. The results revealed that amphipathic α-helices h1 and h3 comprise a lipid-binding site that is partially pre-formed in solution, is stabilized upon binding lipids, and shows lipid-induced folding of h3. This site sequesters apolar ligands via a concave hydrophobic surface in SAA oligomers. The largely disordered/dynamic C-terminal region is conjectured to mediate the promiscuous binding of other ligands. The h1-h2 linker region is predicted to form an unexpected ß-hairpin that may represent an early amyloidogenic intermediate. The results help establish structural underpinnings for understanding SAA interactions with its key functional ligands, its evolutional conservation, and its transition to amyloid.

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Transthyretin (TTR) is a globular tetrameric transport protein in plasma. Nearly 140 single amino acid substitutions in TTR cause life-threatening amyloid disease. We report a one-of-a-kind pathological variant featuring a Glu51, Ser52 duplication mutation (Glu51_Ser52dup). The proband, heterozygous for the mutation, exhibited an unusually aggressive amyloidosis that was refractory to treatment with the small-molecule drug diflunisal. To understand the poor treatment response and expand therapeutic options, we explored the structure and stability of recombinant Glu51_Ser52dup. The duplication did not alter the protein secondary or tertiary structure but decreased the stability of the TTR monomer and tetramer. Diflunisal, which bound with near-micromolar affinity, partially restored tetramer stability. The duplication had no significant effect on the free energy and enthalpy of diflunisal binding, and hence on the drug-protein interactions. However, the duplication induced tryptic digestion of TTR at near-physiological conditions, releasing a C-terminal fragment 49-129 that formed amyloid fibrils under conditions in which the full-length protein did not. Such C-terminal fragments, along with the full-length TTR, comprise amyloid deposits in vivo. Bioinformatics and structural analyses suggested that increased disorder in the surface loop, which contains the Glu51_Ser52dup duplication, not only helped generate amyloid-forming fragments but also decreased structural protection in the amyloidogenic residue segment 25-34, promoting misfolding of the full-length protein. Our studies of a unique duplication mutation explain its diflunisal-resistant nature, identify misfolding pathways for amyloidogenic TTR variants, and provide therapeutic targets to inhibit amyloid fibril formation by variant TTR.

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Serum amyloid A (SAA) is an acute-phase protein whose action in innate immunity and lipid homeostasis is unclear. Most circulating SAA binds plasma high-density lipoproteins (HDL) and reroutes lipid transport. In vivo SAA binds existing lipoproteins or generates them de novo upon lipid uptake from cells. We explored the products of SAA-lipid interactions and lipoprotein remodeling in vitro. SAA complexes with palmitoyl-oleoyl phosphocholine (POPC) were analyzed for structure and stability using circular dichroism and fluorescence spectroscopy, electron microscopy, gel electrophoresis and gel filtration. The results revealed the formation of 8-11nm lipoproteins that were∼50% α-helical and stable at near-physiological conditions but were irreversibly remodeled at Tm∼52°C. Similar HDL-size nanoparticles formed spontaneously at ambient conditions or upon thermal remodeling of parent lipoproteins containing various amounts of proteins and lipids, including POPC and cholesterol. Therefore, such HDL-size particles formed stable kinetically accessible structures in a wide range of conditions. Based on their size and stoichiometry, each particle contained about 12 SAA and 72 POPC molecules, with a protein:lipid weight ratio circa 2.5:1, suggesting a structure distinct from HDL. High stability of these nanoparticles and their HDL-like size suggest that similar lipoproteins may form in vivo during inflammation or injury when SAA concentration is high and membranes from dead cells require rapid removal. We speculate that solubilization of membranes by SAA to generate lipoproteins in a spontaneous energy-independent process constitutes the primordial function of this ancient protein, providing the first line of defense in clearing cell debris from the injured sites.

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Serum amyloid A is a major acute-phase plasma protein that modulates innate immunity and cholesterol homeostasis. We combine sequence analysis with x-ray crystal structures to postulate that SAA acts as an intrinsically disordered hub mediating interactions among proteins, lipids and proteoglycans. A structural model of lipoprotein-bound SAA monomer is proposed wherein two α-helices from the N-domain form a concave hydrophobic surface that binds lipoproteins. A C-domain, connected to the N-domain via a flexible linker, binds polar/charged ligands including cell receptors, bridging them with lipoproteins and rerouting cholesterol transport. Our model is supported by the SAA cleavage in the interdomain linker to generate the 1-76 fragment deposited in reactive amyloidosis. This model sheds new light on functions of this enigmatic protein.

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An international nurse shortage, tightening fiscal constraints, and increased service demands have seen health systems increasingly turn to employing assistants in nursing (AIN) as a cost-effective means to meet demand. This paper describes social positioning from the perspective of 11 AIN who were employed to work in specialist mental health settings in a metropolitan health service in Sydney. Data was collected by means of semistructured interviews. Interview questions encouraged AIN to explore their experience with reference to positioning within the service, role perception, role development, staff relationship, and role satisfaction. Thematic analysis was utilized to generate themes and explore meaning within the data. The following themes emerged: role definition and clarity; socialization and adaptation; and enhancing education. Analysis suggests that whilst AIN were integrated into mainstream service, the scope of activities or role remains geographically variable and inconsistent. Encouragingly, as AIN became familiar with their work environments and teams, they considered themselves to be of value and were able to play a meaningful role. A desire for learning and a need for continuing education also emerged as a primary theme. Findings from the data suggest that AIN in the mental health setting remain a novel and, to some extent, poorly utilized resource.

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PURPOSE: To determine a suitable animal model for further characterization of the nasolacrimal drainage system. METHODS: A comprehensive Pubmed literature search was performed to locate articles pertaining to the histology or anatomy of the nasolacrimal drainage system in research animals. The histology of 2 nasolacrimal drainage systems of a cynomolgus monkey was also evaluated using hematoxylin-eosin stain. RESULTS: Sixteen articles were identified that describe the anatomy and/or histology of the nasolacrimal drainage system in rabbits, rats, pigs, sheep, goats, horses, deer, llamas, camels, apes, dogs, and cats, with significant homology between these models. Notable exceptions were found in rat histology and rabbit anatomy. In the few experiments using animal models to investigate the nasolacrimal drainage system, the rabbit model was the most commonly used system. Light microscopy of the cynomolgus monkey nasolacrimal drainage system revealed markedly similar anatomy and histology to that of humans. CONCLUSIONS: Literature review demonstrates a great deal of similarity in the anatomy and histology of the nasolacrimal drainage systems of 12 mammalian species. Although a common ophthalmic research animal, the rat model has significant histologic differences that prevent its use for comparison with the human lacrimal excretory system. Current literature suggests that despite anatomical variation, the rabbit animal model could potentially be used for further characterization of the nasolacrimal drainage system because it pertains to clinical applications in human patients. Preliminary light microscopy suggests that the cynomolgus monkey may be a superior model for nasolacrimal drainage research, but further studies are required.

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