High-Throughput Surface Plasmon Resonance Biosensors for Identifying Diverse Therapeutic Monoclonal Antibodies.

Identification of antibodies targeting diverse functional epitopes on an antigen is highly crucial for discovering effective therapeutic candidates. Employing a traditional stepwise antibody "screening funnel" as well as prioritizing affinity-based selections over epitope-based selections, result in lead antibody panels lacking epitope diversity. In the present study, we employed an array-based surface plasmon resonance (SPR) platform to perform high-throughput epitope binning analysis on a large number of monoclonal antibodies (mAbs) generated in the early drug discovery process. The mAb panel contained clones from different antibody generation techniques and diverse transgenic mouse strains. The epitope binning results were analyzed in unique ways using various visualizations in the form of dendrograms and network plots, which assisted in determining diversity and redundancy in the mAb sample set. The binning data were further integrated with affinity information to evaluate the performance of seven different transgenic mouse strains. The combination of epitope binning results with binding kinetics and sequence analysis provided an effective and efficient way of selecting high affinity antibodies representing a diverse set of sequence families and epitopes.

Asymmetric excitation of left- and right-tail extreme events probed using a Hawkes model: Application to financial returns.

We construct a two-tailed peaks-over-threshold Hawkes model that captures asymmetric self- and cross-excitation in and between left- and right-tail extreme values within a time series. We demonstrate its applicability by investigating extreme gains and losses within the daily log-returns of the S&P 500 equity index. We find that the arrivals of extreme losses and gains are described by a common conditional intensity to which losses contribute twice as much as gains. However, the contribution of the former decays almost five times more quickly than that of the latter. We attribute these asymmetries to the different reactions of market traders to extreme upward and downward movements of asset prices: an example of negativity bias, wherein trauma is more salient than euphoria.

Discovery of Tirasemtiv, the First Direct Fast Skeletal Muscle Troponin Activator.

The identification and optimization of the first activators of fast skeletal muscle are reported. Compound 1 was identified from high-throughput screening (HTS) and subsequently found to improve muscle function via interaction with the troponin complex. Optimization of 1 for potency, metabolic stability, and physical properties led to the discovery of tirasemtiv (25), which has been extensively characterized in clinical trials for the treatment of amyotrophic lateral sclerosis.

Rapid cell separation with minimal manipulation for autologous cell therapies.

The ability to isolate specific, viable cell populations from mixed ensembles with minimal manipulation and within intra-operative time would provide significant advantages for autologous, cell-based therapies in regenerative medicine. Current cell-enrichment technologies are either slow, lack specificity and/or require labelling. Thus a rapid, label-free separation technology that does not affect cell functionality, viability or phenotype is highly desirable. Here, we demonstrate separation of viable from non-viable human stromal cells using remote dielectrophoresis, in which an electric field is coupled into a microfluidic channel using shear-horizontal surface acoustic waves, producing an array of virtual electrodes within the channel. This allows high-throughput dielectrophoretic cell separation in high conductivity, physiological-like fluids, overcoming the limitations of conventional dielectrophoresis. We demonstrate viable/non-viable separation efficacy of >98% in pre-purified mesenchymal stromal cells, extracted from human dental pulp, with no adverse effects on cell viability, or on their subsequent osteogenic capabilities.

The Role of Percutaneous Reduction and Fixation of Lisfranc Injuries.

To be able to perform percutaneous fixation of Lisfranc injuries, this article emphasizes that an anatomic reduction must be mandatory. When uncertainty remains as to whether closed reduction is anatomic, formal open reduction is recommended because accuracy of reduction is correlated with long-term outcome. Closed injuries with minimal displacement, bony avulsions, and skeletally immature individuals seem the most appropriate indications for percutaneous fixation. Not all injuries are ideal for this method of treatment, and this is an area that needs to be more clearly defined in the future.

Flavonoids from Engineered Tomatoes Inhibit Gut Barrier Pro-inflammatory Cytokines and Chemokines, via SAPK/JNK and p38 MAPK Pathways.

Flavonoids are a diverse group of plant secondary metabolites, known to reduce inflammatory bowel disease symptoms. How they achieve this is largely unknown. Our study focuses on the gut epithelium as it receives high topological doses of dietary constituents, maintains gut homeostasis, and orchestrates gut immunity. Dysregulation leads to chronic gut inflammation, via dendritic cell (DC)-driven immune responses. Tomatoes engineered for enriched sets of flavonoids (anthocyanins or flavonols) provided a unique and complex naturally consumed food matrix to study the effect of diet on chronic inflammation. Primary murine colonic epithelial cell-based inflammation assays consist of chemokine induction, apoptosis and proliferation, and effects on kinase pathways. Primary murine leukocytes and DCs were used to assay effects on transmigration. A murine intestinal cell line was used to assay wound healing. Engineered tomato extracts (enriched in anthocyanins or flavonols) showed strong and specific inhibitory effects on a set of key epithelial pro-inflammatory cytokines and chemokines. Chemotaxis assays showed a resulting reduction in the migration of primary leukocytes and DCs. Activation of epithelial cell SAPK/JNK and p38 MAPK signaling pathways were specifically inhibited. The epithelial wound healing-associated STAT3 pathway was unaffected. Cellular migration, proliferation, and apoptosis assays confirmed that wound healing processes were not affected by flavonoids. We show flavonoids target epithelial pro-inflammatory kinase pathways, inhibiting chemotactic signals resulting in reduced leukocyte and DC chemotaxis. Thus, both anthocyanins and flavonols modulate epithelial cells to become hyporesponsive to bacterial stimulation. Our results identify a viable mechanism to explain the in vivo anti-inflammatory effects of flavonoids.

The positive transcriptional elongation factor (P-TEFb) is required for neural crest specification.

Regulation of gene expression at the level of transcriptional elongation has been shown to be important in stem cells and tumour cells, but its role in the whole animal is only now being fully explored. Neural crest cells (NCCs) are a multipotent population of cells that migrate during early development from the dorsal neural tube throughout the embryo where they differentiate into a variety of cell types including pigment cells, cranio-facial skeleton and sensory neurons. Specification of NCCs is both spatially and temporally regulated during embryonic development. Here we show that components of the transcriptional elongation regulatory machinery, CDK9 and CYCLINT1 of the P-TEFb complex, are required to regulate neural crest specification. In particular, we show that expression of the proto-oncogene c-Myc and c-Myc responsive genes are affected. Our data suggest that P-TEFb is crucial to drive expression of c-Myc, which acts as a 'gate-keeper' for the correct temporal and spatial development of the neural crest.

A Database of microRNA Expression Patterns in Xenopus laevis.

MicroRNAs (miRNAs) are short, non-coding RNAs around 22 nucleotides long. They inhibit gene expression either by translational repression or by causing the degradation of the mRNAs they bind to. Many are highly conserved amongst diverse organisms and have restricted spatio-temporal expression patterns during embryonic development where they are thought to be involved in generating accuracy of developmental timing and in supporting cell fate decisions and tissue identity. We determined the expression patterns of 180 miRNAs in Xenopus laevis embryos using LNA oligonucleotides. In addition we carried out small RNA-seq on different stages of early Xenopus development, identified 44 miRNAs belonging to 29 new families and characterized the expression of 5 of these. Our analyses identified miRNA expression in many organs of the developing embryo. In particular a large number were expressed in neural tissue and in the somites. Surprisingly none of the miRNAs we have looked at show expression in the heart. Our results have been made freely available as a resource in both XenMARK and Xenbase.

Aseptic Raman spectroscopy can detect changes associated with the culture of human dental pulp stromal cells in osteoinductive culture.

There is an unmet need for the non-invasive characterisation of stem cells to facilitate the translation of cell-based therapies. Raman spectroscopy has proven utility in stem cell characterisation but as yet no method has been reported capable of taking repeated Raman measurements of living cells aseptically over time. The aim of this study was to determine if Raman spectroscopy could be used to monitor changes in a well characterised cell population (human dental pulp stromal cells (DPSCs)) by taking repeated Raman measurements from the same cell populations in osteoinductive culture over time and under aseptic conditions. DPSCs were isolated from extracted premolar teeth from 3 consenting donors. Following in vitro expansion, DPSCs were maintained for 28 days in osteo-inductive medium. Raman spectra were acquired from the cells at days 0, 3, 7, 10, 14 and 28. Principal component analysis (PCA) was carried out to assess if there was any temporal spectral variation. At day 28, osteoinduction was confirmed using alizarin red staining and qRT-PCR for alkaline phosphatase and osteocalcin. Alizarin red staining was positive in all samples at day 28 and significant increases in alkaline phosphatase (p < 0.001) and osteocalcin (p < 0.05) gene expression were also observed compared with day 0. PCA of the Raman data demonstrated trends in PC1 from days 0-10, influenced by protein associated features and PC2 from days 10-28, influenced by DNA/RNA associated features. We conclude that spectroscopy can be used to monitor changes in Raman signature with time associated with the osteoinduction of DPSCs using repeated measurements via an aseptic methodology.

Tissue non-specific alkaline phosphatase production by human dental pulp stromal cells is enhanced by high density cell culture.

The cell surface hydrolase tissue non-specific alkaline phosphatase (TNAP) (also known as MSCA-1) is used to identify a sub-population of bone marrow stromal cells (BMSCs) with high mineralising potential and is found on subsets of cells within the dental pulp. We aim to determine whether TNAP is co-expressed by human dental pulp stromal cells (hDPSCs) alongside a range of BMSC markers, whether this is an active form of the enzyme and the effects of culture duration and cell density on its expression. Cells from primary dental pulp and culture expanded hDPSCs expressed TNAP. Subsequent analyses revealed persistent TNAP expression and co-expression with BMSC markers such as CD73 and CD90. Flow cytometry and biochemical assays showed that increased culture durations and cell densities enhanced TNAP expression by hDPSCs. Arresting the hDPSC cell cycle also increased TNAP expression. These data confirm that TNAP is co-expressed by hDPSCs together with other BMSC markers and show that cell density affects TNAP expression levels. We conclude that TNAP is a potentially useful marker for hDPSC selection especially for uses in mineralised tissue regenerative therapies.

Pain after cheilectomy of the first metatarsophalangeal joint: diagnosis and management.

Cheilectomy is commonly performed for osteoarthritis of the first metatarsophalangeal joint and generally has a successful outcome and high rate of patient satisfaction over the short to medium term. Despite the relatively good results achieved in most cases, a proportion of patients have ongoing pain after cheilectomy. This article outlines the potential causes of ongoing pain, including progression of osteoarthritis, neuralgic symptoms, and transfer metatarsalgia. Management strategies for treating the ongoing symptoms are discussed.

Weight-Bearing in the Nonoperative Treatment of Acute Achilles Tendon Ruptures: A Randomized Controlled Trial.

BACKGROUND: The rate of Achilles tendon ruptures is increasing, but there is a lack of consensus on treatment of acute injuries. The purpose of this trial was to compare outcomes of weight-bearing casts with those of traditional casts in the treatment of acute Achilles tendon ruptures. METHODS: Eighty-four patients with an acute Achilles tendon rupture were recruited over a two-year period. Patients were randomized to be treated with either a weight-bearing cast with a Böhler iron or a non-weight-bearing cast for eight weeks. Patients underwent muscle dynamometry testing at six months, with additional follow-up at one and two years. The primary outcomes that were assessed were the rerupture rate and the time taken to return to work. Secondary outcomes included return to sports, ankle pain and stiffness, footwear restrictions, and patient satisfaction. RESULTS: There were no significant differences between groups with regard to patient demographics or activity levels prior to treatment. At the time of follow-up at two years, one (3%) of the thirty-seven patients in the weight-bearing group and two (5%) of the thirty-seven in the non-weight-bearing group had sustained a rerupture (p = 0.62). The patients in the weight-bearing group experienced less subjective stiffness at one year. There were no significant differences in time taken to return to work, Leppilahti scores, patient satisfaction, pain, or return to sports between the groups. CONCLUSIONS: Use of weight-bearing casts for the nonoperative treatment of Achilles tendon ruptures appears to offer outcomes that are at least equivalent to those of non-weight-bearing casts. The overall rerupture rate in this study was low, supporting the continued use of initial nonoperative management for the treatment of acute Achilles tendon ruptures. LEVELS OF EVIDENCE: Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

Correlation of Pirani and Dimeglio scores with number of Ponseti casts required for clubfoot correction.

BACKGROUND: A number of grading systems for severity of clubfoot have been reported in the literature, but none are universally accepted. The aim of this study was to find the correlation between 2 of the most widely utilized classification systems (the Pirani score and the Dimeglio score) with number of Ponseti casts required to achieve initial clubfeet correction. METHODS: A retrospective study of prospectively collected data was performed. All clubfeet assessed at our dedicated clubfoot clinic from January 2007 to December 2011 were included. Clubfoot severity was assessed using both the Pirani score and the Dimeglio score. The total number of casts was calculated from the first cast to the time of initiation of the foot abduction orthosis. RESULTS: The mean number of Ponseti casts required to achieve initial correction was 5.8 (range, 2 to 10 casts). A low correlation (rs 0.21) was identified when the total Dimeglio score was compared with the number of casts. No correlation (rs 0.12) was identified between the Pirani score and the number of casts. CONCLUSIONS: The Dimeglio and Pirani scores remain the most widely accepted clubfoot severity grading systems. However, their prognostic value remains questionable, at least in the early treatment stages. LEVEL OF EVIDENCE: Prognostic study level II.

Cell separation: Terminology and practical considerations.

Cell separation is a powerful tool in biological research. Increasing usage, particularly within the tissue engineering and regenerative medicine communities, means that researchers from a diverse range of backgrounds are utilising cell separation technologies. This review aims to offer potential solutions to cell sorting problems and to clarify common ambiguities in terminology and experimental design. The frequently used cell separation terms of 'purity', 'recovery' and 'viability' are discussed, and attempts are made to reach a consensus view of their sometimes ambiguous meanings. The importance of appropriate experimental design is considered, with aspects such as marker expression, tissue isolation and original cell population analysis discussed. Finally, specific technical issues such as cell clustering, dead cell removal and non-specific antibody binding are considered and potential solutions offered. The solutions offered may provide a starting point to improve the quality of cell separations achieved by both the novice and experienced researcher alike.

The New Zealand Joint Registry: report of 11-year data for ankle arthroplasty.

This article describes the results of the first 11 years of ankle arthroplasty data for the New Zealand Joint Registry. The main purpose is to collect accurate outcome information regarding these procedures and to guide orthopedic surgeons in the care of their patients. Trends can often be identified early, and implants with higher revision rates can be identified. In addition, individual surgeons can be given data that compare their performance with the collective data, providing invaluable feedback. Patient-based questionnaires are highly important for gauging the results of surgery. Patient response rates have been less than optimal, particularly after revision surgery.

Chemical genetics and drug discovery in Xenopus.

Chemical genetics uses small molecules to modulate protein function and has the potential to perturb any biochemical event in a complex cellular context. The application of chemical genetics to dissect biological processes has become an attractive alternative to mutagenesis screens due to its technical simplicity, inexpensive reagents, and low-startup costs. Xenopus embryos are particularly amenable to whole organism chemical genetic screens. Here we describe the basic protocols we have developed to screen small compound libraries on Xenopus laevis embryos. We score embryos either by observing phenotypic changes in the whole tadpole or by changes in gene expression pattern using automated wholemount in situ hybridization.

Office-based deep sedation for pediatric ophthalmologic procedures using a sedation service model.

Aims. (1) To assess the efficacy and safety of pediatric office-based sedation for ophthalmologic procedures using a pediatric sedation service model. (2) To assess the reduction in hospital charges of this model of care delivery compared to the operating room (OR) setting for similar procedures. Background. Sedation is used to facilitate pediatric procedures and to immobilize patients for imaging and examination. We believe that the pediatric sedation service model can be used to facilitate office-based deep sedation for brief ophthalmologic procedures and examinations. Methods. After IRB approval, all children who underwent office-based ophthalmologic procedures at our institution between January 1, 2000 and July 31, 2008 were identified using the sedation service database and the electronic health record. A comparison of hospital charges between similar procedures in the operating room was performed. Results. A total of 855 procedures were reviewed. Procedure completion rate was 100% (C.I. 99.62-100). There were no serious complications or unanticipated admissions. Our analysis showed a significant reduction in hospital charges (average of $1287 per patient) as a result of absent OR and recovery unit charges. Conclusions. Pediatric ophthalmologic minor procedures can be performed using a sedation service model with significant reductions in hospital charges.

Long-term changes in river-floodplain dynamics: implications for salmonid habitat in the Interior Columbia Basin, USA.

Rivers and their associated floodplains are among the world's most highly altered ecosystems, resulting in billions of dollars in restoration expenditures. Successful restoration of these systems requires information at multiple spatial scales (from localized reaches to broader-scale watersheds), as well as information spanning long time frames. Here, we develop a suite of historical landscape indicators of riverine status, primarily from the perspective of salmonid management, using a case study in the Interior Columbia Basin, Washington, USA. We use a combination of historical and modern aerial photography to quantify changes in land cover and reach type, as well as potential fish habitat within channel and off-channel floodplain areas. As of 1949, 55% of the Wenatchee River floodplain had been converted to agriculture. By 2006, 62% had been modified by anthropogenic development, of which 20% was due to urban expansion. The historical percentage of agricultural land in the watershed and the contemporary percentage of urban area surpass thresholds in land cover associated with deleterious impacts on river systems. In addition, the abundance of reach types associated with the highest quality salmonid habitat (island braided and meandering reaches) has declined due to conversion to straight reach types. The area occupied by fish habitats associated with channel migration (slow/stagnant channels and dry channels) has declined approximately 25-30%. Along highly modified rivers, these habitats have also become increasingly fragmented. Caveats related to visual quality and seasonal timing of historical photographs were important considerations in the interpretation of changes witnessed for headwater island braided systems, as well as for floodplain ponds. Development of rigorous, long-term, multi-scale monitoring techniques is necessary to guide the management and restoration of river-floodplain systems for the diversity of ecosystem services they provide.

DHODH modulates transcriptional elongation in the neural crest and melanoma.

Melanoma is a tumour of transformed melanocytes, which are originally derived from the embryonic neural crest. It is unknown to what extent the programs that regulate neural crest development interact with mutations in the BRAF oncogene, which is the most commonly mutated gene in human melanoma. We have used zebrafish embryos to identify the initiating transcriptional events that occur on activation of human BRAF(V600E) (which encodes an amino acid substitution mutant of BRAF) in the neural crest lineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and lack p53 (also known as tp53) have a gene signature that is enriched for markers of multipotent neural crest cells, and neural crest progenitors from these embryos fail to terminally differentiate. To determine whether these early transcriptional events are important for melanoma pathogenesis, we performed a chemical genetic screen to identify small-molecule suppressors of the neural crest lineage, which were then tested for their effects on melanoma. One class of compound, inhibitors of dihydroorotate dehydrogenase (DHODH), for example leflunomide, led to an almost complete abrogation of neural crest development in zebrafish and to a reduction in the self-renewal of mammalian neural crest stem cells. Leflunomide exerts these effects by inhibiting the transcriptional elongation of genes that are required for neural crest development and melanoma growth. When used alone or in combination with a specific inhibitor of the BRAF(V600E) oncogene, DHODH inhibition led to a marked decrease in melanoma growth both in vitro and in mouse xenograft studies. Taken together, these studies highlight developmental pathways in neural crest cells that have a direct bearing on melanoma formation.

Determinants of R-loop formation at convergent bidirectionally transcribed trinucleotide repeats.

R-loops have been described at immunoglobulin class switch sequences, prokaryotic and mitochondrial replication origins, and disease-associated (CAG)n and (GAA)n trinucleotide repeats. The determinants of trinucleotide R-loop formation are unclear. Trinucleotide repeat expansions cause diseases including DM1 (CTG)n, SCA1 (CAG)n, FRAXA (CGG)n, FRAXE (CCG)n and FRDA (GAA)n. Bidirectional convergent transcription across these disease repeats can occur. We find R-loops formed when CTG or CGG and their complementary strands CAG or CCG were transcribed; GAA transcription, but not TTC, yielded R-loops. R-loop formation was sensitive to DNA supercoiling, repeat length, insensitive to repeat interruptions, and formed by extension of RNA:DNA hybrids in the RNA polymerase. R-loops arose by transcription in one direction followed by transcription in the opposite direction, and during simultaneous convergent bidirectional transcription of the same repeat forming double R-loop structures. Since each transcribed disease repeat formed R-loops suggests they may have biological functions.