A robust spectroscopic method for the determination of protein conformational composition - application to the annealing of silk

The physical and mechanical properties of structural proteins such as silk fibroin can be modified by controlled conformational change, which is regularly monitored by Fourier transform infrared spectroscopy by peak fitting of the amide I band envelope. Although many variables affecting peak shape are well established, there is no fixed methodology to compare and follow secondary structural differences without significant operator input especially where low frequency spectral noise is a problem. The aim of this contribution is to establish a method for such analyses to be carried at high levels of autonomy to prevent subjective or erroneous fitting. A range of approaches was trialled with optimal peak parameters selected based on overall goodness of fit and reproducibility of fit of replicate sample spectra. The method was successfully tested against reference proteins having contrasting β content and the rationale for parameter selection is presented. Further, we applied this method to measure the effect of conformational change on the energy of the amide I band of silk fibroin during annealing. Energy changes were ca. 400 kJ mol−1 of fibroin. To confirm that this energy change was a consequence of increased hydrogen bonding we used a Thioflavin T staining method typically used to identify β aggregate type structures in amyloid plaques. We propose that the approach described herein can aid in the development of silk based materials for biomedical applications where tuning of the physical and mechanical properties of the silk are needed to guarantee optimum activity

Surgery combined with controlled-release doxorubicin silk films as a treatment strategy in an orthotopic neuroblastoma mouse model

Background: Neuroblastoma tumour resection goal is maximal tumour removal. We hypothesise that combining surgery with sustained, local doxorubicin application can control tumour growth.methods: We injected human neuroblastoma cells into immunocompromised mouse adrenal gland. When KELLY cell-induced tumour volume was >300 mm3, 80–90% of tumour was resected and treated as follows: instantaneous-release silk film with 100 μg doxorubicin (100IR), controlled-release film with 200 μg (200CR) over residual tumour bed; and 100 and 200 μg intravenous doxorubicin (100IV and 200IV). Tumour volume was measured and histology analysed.results: Orthotopic tumours formed with KELLY, SK-N-AS, IMR-32, SH-SY5Y cells. Tumours reached 1800±180 mm3 after 28 days, 2200±290 mm3 after 35 days, 1280±260 mm3 after 63 days, and 1700±360 mm3 after 84 days, respectively. At 3 days post KELLY tumour resection, tumour volumes were similar across all groups (P=0.6210). Tumour growth rate was similar in untreated vs control film, 100IV vs 100IR, and 100IV vs 200IV. There was significant difference in 100IR vs 200CR (P=0.0004) and 200IV vs 200CR (P=0.0003). Tumour growth with all doxorubicin groups was slower than that of control (P: <0.0001–0.0069). At the interface of the 200CR film and tumour, there was cellular necrosis, surrounded by apoptotic cells before reaching viable tumour cells.conclusions: Combining surgical resection and sustained local doxorubicin treatment is effective in tumour control. Administering doxorubicin in a local, controlled manner is superior to giving an equivalent intravenous dose in tumour control

SEARCHING FOR DEBRIS DISKS AROUND SEVEN RADIO PULSARS

We report on our searches for debris disks around seven relatively nearby radio pulsars, which are isolated sources that were carefully selected as targets on the basis of our deep Ks-band imaging survey. The Ks images obtained with the 6.5m Baade Magellan Telescope at Las Campanas Observatory are analyzed together with the Spitzer/IRAC images at 4.5 and 8.0μm and the WISE images at 3.4, 4.6, 12, and 22μm. No infrared counterparts of these pulsars are found, with flux upper limits of ∼μJy at near-infrared (λ < 10μm) and ∼10–1000μJy at mid-infrared wavelengths (λ > 10 μm). The results of this search are discussed in terms of the efficiency of converting the pulsar spin-down energy to thermal energy and X-ray heating of debris disks, with a comparison made of the two magnetars 4U 0142+61 and 1E 2259+586, which are suggested to harbor a debris disk.published_or_final_versio

Influence of silk–silica fusion protein design on silica condensation in vitro and cellular calcification

Biomaterial design via genetic engineering can be utilized for the rational functionalization of proteins to promote biomaterials integration and tissue regeneration. Spider silk has been extensively studied for its biocompatibility, biodegradability and extraordinary material properties. As a protein-based biomaterial, recombinant DNA derived derivatives of spider silks have been modified with biomineralization domains which lead to silica deposition and potentially accelerated bone regeneration. However, the influence of the location of the R5 (SSKKSGSYSGSKGSKRRIL) silicifying domain fused with the spider silk protein sequence on biosilicification process remains to be determined. Here we designed two silk-R5 fusion proteins that differed in the location of the R5 peptide, C- vs. N-terminus, where the spider silk domain consisted of a 15mer repeat of a 33 amino acid consensus sequence of the major ampullate dragline Spidroin 1 from Nephila clavipes (SGRGGLGG QG AGAAAAAGGA GQGGYGGLGSQGT). The chemical, physical and silica deposition properties of these recombinant proteins were assessed and compared to a silk 15mer control without the R5 present. The location of the R5 peptide did not have significant effect on wettability and surface energies, while the C - terminal location of the R5 promoted more controlled silica precipitation, suggesting differences in protein folding and possibly different access to charged amino acids that drive the silicification process. Further, cell compatibility in vitro, as well as the ability to promote human bone marrow derived mesenchymal stem cells (hMSC) differentiation were demonstrated for both variants of the fusion proteins

Expandable and Rapidly Differentiating Human Induced Neural Stem Cell Lines for Multiple Tissue Engineering Applications

Limited availability of human neurons poses a significant barrier to progress in biological and preclinical studies of the human nervous system. Current stem cell-based approaches of neuron generation are still hindered by prolonged culture requirements, protocol complexity, and variability in neuronal differentiation. Here we establish stable human induced neural stem cell (hiNSC) lines through the direct reprogramming of neonatal fibroblasts and adult adipose-derived stem cells. These hiNSCs can be passaged indefinitely and cryopreserved as colonies. Independently of media composition, hiNSCs robustly differentiate into TUJ1-positive neurons within 4 days, making them ideal for innervated co-cultures. In vivo, hiNSCs migrate, engraft, and contribute to both central and peripheral nervous systems. Lastly, we demonstrate utility of hiNSCs in a 3D human brain model. This method provides a valuable interdisciplinary tool that could be used to develop drug screening applications as well as patient-specific disease models related to disorders of innervation and the brain

The JAK inhibitor tofacitinib suppresses synovial JAK1-STAT signalling in rheumatoid arthritis.

ObjectiveTofacitinib is an oral Janus kinase (JAK) inhibitor for the treatment of rheumatoid arthritis (RA). The pathways affected by tofacitinib and the effects on gene expression in situ are unknown. Therefore, tofacitinib effects on synovial pathobiology were investigated.MethodsA randomised, double-blind, phase II serial synovial biopsy study (A3921073; NCT00976599) in patients with RA with an inadequate methotrexate response. Patients on background methotrexate received tofacitinib 10 mg twice daily or placebo for 28 days. Synovial biopsies were performed on Days -7 and 28 and analysed by immunoassay or quantitative PCR. Clinical response was determined by disease activity score and European League Against Rheumatism (EULAR) response on Day 28 in A3921073, and at Month 3 in a long-term extension study (A3921024; NCT00413699).ResultsTofacitinib exposure led to EULAR moderate to good responses (11/14 patients), while placebo was ineffective (1/14 patients) on Day 28. Tofacitinib treatment significantly reduced synovial mRNA expression of matrix metalloproteinase (MMP)-1 and MMP-3 (p&lt;0.05) and chemokines CCL2, CXCL10 and CXCL13 (p&lt;0.05). No overall changes were observed in synovial inflammation score or the presence of T cells, B cells or macrophages. Changes in synovial phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT3 strongly correlated with 4-month clinical responses (p&lt;0.002). Tofacitinib significantly decreased plasma CXCL10 (p&lt;0.005) at Day 28 compared with placebo.ConclusionsTofacitinib reduces metalloproteinase and interferon-regulated gene expression in rheumatoid synovium, and clinical improvement correlates with reductions in STAT1 and STAT3 phosphorylation. JAK1-mediated interferon and interleukin-6 signalling likely play a key role in the synovial response.Trial registration numberNCT00976599

Intracellular pathways involved in bone regeneration triggered by recombinant silk-silica chimeras

Biomineralization at the organic-inorganic interface is critical to many biology material functions in vitro and in vivo. Recombinant silk-silica fusion peptides are organic-inorganic hybrid material systems that can be effectively used to study and control biologically-mediated mineralization due to the genetic basis of sequence control. However, to date, the mechanisms by which these functionalized silk-silica proteins trigger the differentiation of human mesenchymal stem cells (hMSCs) to osteoblasts remain unknown. To address this challenge, we analyzed silk-silica surfaces for silica-hMSC receptor binding and activation, and the intracellular pathways involved in the induction of osteogenesis on these bioengineered biomaterials. The induction of gene expression of αVβ3 integrin, all three Mitogen-activated Protein Kinsases (MAPKs) as well as c-Jun, Runt-related Transcription Factor 2 (Runx2) and osteoblast marker genes was demonstrated upon growth of the hMSCs on the silk-silica materials. This induction of key markers of osteogenesis correlated with the content of silica on the materials. Moreover, computational simulations were performed for silk/silica-integrin binding which showed activation of αVβ3 integrin in contact with silica. This integrated computational and experimental approach provides insight into interactions that regulate osteogenesis towards more efficient biomaterial designs

Sustained release silk fibroin discs: Antibody and protein delivery for HIV prevention

With almost 2 million new HIV infections worldwide each year, the prevention of HIV infection is critical for stopping the pandemic. The only approved form of pre-exposure prophylaxis is a costly daily pill, and it is recognized that several options will be needed to provide protection to the various affected communities around the world. In particular, many at-risk people would benefit from a prevention method that is simple to use and does not require medical intervention or a strict daily regimen. We show that silk fibroin protein can be formulated into insertable discs that encapsulate either an antibody (IgG) or the potent HIV inhibitor 5P12-RANTES. Several formulations were studied, including silk layering, water vapor annealing and methanol treatment to stabilize the protein cargo and impact the release kinetics over weeks. In the case of IgG, high concentrations were released over a short time using methanol treatment, with more sustained results with the use of water vapor annealing and layering during device fabrication. For 5P12-RANTES, sustained release was obtained for 31 days using water vapor annealing. Further, we show that the released inhibitor 5P12-RANTES was functional both in vitro and in ex vivo colorectal tissue. This work shows that silk fibroin discs can be developed into formidable tools to prevent HIV infection

Balancing influence between actors in healthcare decision making

<p>Abstract</p> <p>Background</p> <p>Healthcare costs in most developed countries are not clearly linked to better patient and public health outcomes, but are rather associated with service delivery orientation. In the U.S. this has resulted in large variation in healthcare availability and use, increased cost, reduced employer participation in health insurance programs, and reduced overall population health outcomes. Recent U.S. healthcare reform legislation addresses only some of these issues. Other countries face similar healthcare issues.</p> <p>Discussion</p> <p>A major goal of healthcare is to enhance patient health outcomes. This objective is not realized in many countries because incentives and structures are currently not aligned for maximizing population health. The misalignment occurs because of the competing interests between "actors" in healthcare. In a simplified model these are individuals motivated to enhance their own health; enterprises (including a mix of nonprofit, for profit and government providers, payers, and suppliers, etc.) motivated by profit, political, organizational and other forces; and government which often acts in the conflicting roles of a healthcare payer and provider in addition to its role as the representative and protector of the people. An imbalance exists between the actors, due to the resources and information control of the enterprise and government actors relative to the individual and the public. Failure to use effective preventive interventions is perhaps the best example of the misalignment of incentives. We consider the current Pareto efficient balance between the actors in relation to the Pareto frontier, and show that a significant change in the healthcare market requires major changes in the utilities of the enterprise and government actors.</p> <p>Summary</p> <p>A variety of actions are necessary for maximizing population health within the constraints of available resources and the current balance between the actors. These actions include improved transparency of all aspects of medical decision making, greater involvement of patients in shared medical decision making, greater oversight of guideline development and coverage decisions, limitations on direct to consumer advertising, and the need for an enhanced role of the government as the public advocate.</p