Association of synovial fluid and urinary C2C-HUSA levels with surgical outcomes post-total knee arthroplasty.

OBJECTIVES: After total knee arthroplasty (TKA), ∼30% of knee osteoarthritis (KOA) patients show little symptomatic improvement. Earlier studies have correlated urinary (u) type 2 collagen C terminal cleavage peptide assay (C2C-HUSA), which detects a fragment of cartilage collagen breakdown, with KOA progression. This study determines whether C2C levels in urine, synovial fluid, or their ratio, are associated with post-surgical outcomes. METHODS: From a large sample of 489 subjects, diagnosed with primary KOA undergoing TKA, Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain and function scores were collected at baseline (time of surgery) and one-year post-TKA. Baseline urine (u) and synovial fluid (sf) were analysed using the IBEX-C2C-HUSA assay, with higher values indicating higher amounts of cartilage degradation. For urine, results were normalised to creatinine. Furthermore, subjects' changes in WOMAC scores were categorised based on percent reduction in pain or improvement in function, compared to baseline, such that >66.7%, >33.3 to ≤66.7%, and ≤33.3% denoted "strong", "moderate" and "mild/worse" responses, respectively. Associations of individual biofluid C2C-HUSA levels, or their ratio, with change in WOMAC pain and function scores up to one-year post-TKA, or category of change, were analysed by linear, logistic, or cumulative odds models. RESULTS: Higher baseline uC2C-HUSA levels or a lower ratio of baseline sfC2C-HUSA to uC2C-HUSA were associated with improvements in WOMAC pain by linear multivariable modelling [odds ratio -0.40 (95% confidence interval -0.76, -0.05) p = 0.03; 0.36 (0.01, 0.71), p = 0.04, respectively], while sfC2C-HUSA alone was not. However, lower ratios of sfC2C-HUSA to uC2C-HUSA were associated with improvements in WOMAC function [1.37 (0.18, 2.55), p = 0.02], while sfC2C-HUSA and uC2C-HUSA alone were not. Lower ratios of sfC2C-HUSA to uC2C-HUSA were also associated with an increased likelihood of a subject being categorised in a group where TKA was beneficial in both univariable [pain, 0.81 (0.68, 0.96), p = 0.02; function, 0.92 (0.85, 0.99), p = 0.035] and multivariable [pain, 0.81 (0.68, 0.97) p = 0.02; function, 0.92 (0.85, 1.00), p = 0.043] ordinal modelling, while sfC2C-HUSA and uC2C-HUSA alone were not. CONCLUSIONS: Overall, ratios of baseline sfC2C-HUSA to uC2C-HUSA, and baseline uC2C-HUSA, may play an important role in studying post-TKA surgical outcomes.

A Small Change With a Twist Ending: A Single Residue in EGF-CFC Drives Bilaterian Asymmetry.

Asymmetries are essential for proper organization and function of organ systems. Genetic studies in bilaterians have shown signaling through the Nodal/Smad2 pathway plays a key, conserved role in the establishment of body asymmetries. Although the main molecular players in the network for the establishment of left-right asymmetry (LRA) have been deeply described in deuterostomes, little is known about the regulation of Nodal signaling in spiralians. Here, we identified orthologs of the egf-cfc gene, a master regulator of the Nodal pathway in vertebrates, in several invertebrate species, which includes the first evidence of its presence in non-deuterostomes. Our functional experiments indicate that despite being present, egf-cfc does not play a role in the establishment of LRA in gastropods. However, experiments in zebrafish suggest that a single amino acid mutation in the egf-cfc gene in at least the common ancestor of chordates was the necessary step to induce a gain of function in LRA regulation. This study shows that the egf-cfc gene likely appeared in the ancestors of deuterostomes and "protostomes", before being adopted as a mechanism to regulate the Nodal pathway and the establishment of LRA in some lineages of deuterostomes.

Cellular and molecular profiles of larval and adult Xenopus corneal epithelia resolved at the single-cell level.

Corneal Epithelial Stem Cells (CESCs) and their proliferative progeny, the Transit Amplifying Cells (TACs), are responsible for homeostasis and maintaining corneal transparency. Owing to our limited knowledge of cell fates and gene activity within the cornea, the search for unique markers to identify and isolate these cells remains crucial for ocular surface reconstruction. We performed single-cell RNA sequencing of corneal cells from larval and adult stages of Xenopus. Our results indicate that as the cornea develops and matures, there is an increase in cellular diversity, which is accompanied by a substantial shift in transcriptional profile, gene regulatory network and cell-cell communication dynamics. Our data also reveals several novel genes expressed in corneal cells and changes in gene expression during corneal differentiation at both developmental time-points. Importantly, we identify specific basal cell clusters in both the larval and adult cornea that comprise a relatively undifferentiated cell type and express distinct stem cell markers, which we propose are the putative larval and adult CESCs, respectively. This study offers a detailed atlas of single-cell transcriptomes in the frog cornea. In the future, this work will be useful to elucidate the function of novel genes in corneal epithelial homeostasis, wound healing and regeneration.

Mercapto-functionalized polyhedral oligomeric silsesquioxane as a soluble support for the synthesis of peptide thioesters.

The revival of peptide-based drugs has led to the increasing demand for the development of large-scale synthesis of these complex molecules. To meet this demand, the use of mercapto-functionalized polyhedral oligomeric silsesquioxane (POSS-SH) as a soluble support for the synthesis of a model pentapeptide POSS-thioester is reported. The synthetic process provided a total yield of 62% for the pentapeptide POSS-thioester and the 1H NMR spectra validated the high purity of the products. The successful synthesis of the pentapeptide POSS-thioester with high yield and purity provides a promising way to the scale-up chemical synthesis of peptide thioesters, peptides, peptide amides, cyclic peptides, and even proteins.

BMP signaling plays a role in anterior-neural/head development, but not organizer activity, in the gastropod Crepidula fornicata.

BMP signaling is involved in many aspects of metazoan development, with two of the most conserved functions being to pattern the dorsal-ventral axis and to specify neural versus epidermal fates. An active area of research within developmental biology asks how BMP signaling was modified over evolution to build disparate body plans. Animals belonging to the superclade Spiralia/Lophotrochozoa are excellent experimental subjects for studying the evolution of BMP signaling because a highly conserved, stereotyped early cleavage program precedes the emergence of distinct body plans. In this study we examine the role of BMP signaling in one representative, the slipper snail Crepidula fornicata. We find that mRNAs encoding BMP pathway components (including the BMP ligand decapentaplegic, and BMP antagonists chordin and noggin-like proteins) are not asymmetrically localized along the dorsal-ventral axis in the early embryo, as they are in other species. Furthermore, when BMP signaling is perturbed by adding ectopic recombinant BMP4 protein, or by treating embryos with the selective Activin receptor-like kinase-2 (ALK-2) inhibitor Dorsomorphin Homolog 1 (DMH1), we observe no obvious effects on dorsal-ventral patterning within the posterior (post-trochal) region of the embryo. Instead, we see effects on head development and the balance between neural and epidermal fates specifically within the anterior, pre-trochal tissue derived from the 1q1 lineage. Our experiments define a window of BMP signaling sensitivity that ends at approximately 44-48 â€‹hours post fertilization, which occurs well after organizer activity has ended and after the dorsal-ventral axis has been determined. When embryos were exposed to BMP4 protein during this window, we observed morphogenetic defects leading to the separation of the anterior, 1q lineage from the rest of the embryo. The 1q-derived organoid remained largely undifferentiated and was radialized, while the post-trochal portion of the embryo developed relatively normally and exhibited clear signs of dorsal-ventral patterning. When embryos were exposed to DMH1 during the same time interval, we observed defects in the head, including protrusion of the apical plate, enlarged cerebral ganglia and ectopic ocelli, but otherwise the larvae appeared normal. No defects in shell development were noted following DMH1 treatments. The varied roles of BMP signaling in the development of several other spiralians have recently been examined. We discuss our results in this context, and highlight the diversity of developmental mechanisms within spiral-cleaving animals.

An automated aquatic rack system for rearing marine invertebrates.

BACKGROUND: One hundred years ago, marine organisms were the dominant systems for the study of developmental biology. The challenges in rearing these organisms outside of a marine setting ultimately contributed to a shift towards work on a smaller number of so-called model systems. Those animals are typically non-marine organisms with advantages afforded by short life cycles, high fecundity, and relative ease in laboratory culture. However, a full understanding of biodiversity, evolution, and anthropogenic effects on biological systems requires a broader survey of development in the animal kingdom. To this day, marine organisms remain relatively understudied, particularly the members of the Lophotrochozoa (Spiralia), which include well over one third of the metazoan phyla (such as the annelids, mollusks, flatworms) and exhibit a tremendous diversity of body plans and developmental modes. To facilitate studies of this group, we have previously described the development and culture of one lophotrochozoan representative, the slipper snail Crepidula atrasolea, which is easy to rear in recirculating marine aquaria. Lab-based culture and rearing of larger populations of animals remain a general challenge for many marine organisms, particularly for inland laboratories. RESULTS: Here, we describe the development of an automated marine aquatic rack system for the high-density culture of marine species, which is particularly well suited for rearing filter-feeding animals. Based on existing freshwater recirculating aquatic rack systems, our system is specific to the needs of marine organisms and incorporates robust filtration measures to eliminate wastes, reducing the need for regular water changes. In addition, this system incorporates sensors and associated equipment for automated assessment and adjustment of water quality. An automated feeding system permits precise delivery of liquid food (e.g., phytoplankton) throughout the day, mimicking real-life feeding conditions that contribute to increased growth rates and fecundity. CONCLUSION: This automated system makes laboratory culture of marine animals feasible for both large and small research groups, significantly reducing the time, labor, and overall costs needed to rear these organisms.

The role of sensory innervation in cornea-lens regeneration.

BACKGROUND: Numerous sensory nerves in the cornea contribute to normal tissue homeostasis. Interestingly, cells within the basal corneal epithelium can regenerate new lenses in the frog, Xenopus. In this study, we investigated whether cornea sensory nerves or their neuropeptides are important for supporting cornea-lens regeneration. RESULTS: Attempts to sever the trigeminal nerve trunk, which provides sensory nerve branches to the cornea, did not inhibit lens regeneration. However, using this approach we found that it was not possible to completely disrupt sensory innervation, as these nerves are able to quickly regenerate back to the cornea. On the other hand, attenuation of neuropeptide levels with capsaicin was found to significantly inhibit lens regeneration, as visualized by a reduction of Substance P. These treatments also led to a reduction of cornea sensory innervation. Interestingly, inhibition of the Substance P-preferred receptor NK-1 with Spantide II did not affect lens-regeneration rates. CONCLUSIONS: This study provides evidence that cornea nerves support cornea-lens regeneration, which could occur through the release of various neurotrophic factors. Substance P, however, does not appear to be the critical component of this signaling pathway. Further studies are needed to investigate what role other known neurotrophic factors may play in this process.

Molecular markers for corneal epithelial cells in larval vs. adult Xenopus frogs.

Corneal Epithelial Stem Cells (CESCs) and their proliferative progeny, the Transit Amplifying Cells (TACs), are responsible for maintaining the integrity and transparency of the cornea. These stem cells (SCs) are widely used in corneal transplants and ocular surface reconstruction. Molecular markers are essential to identify, isolate and enrich for these cells, yet no definitive CESC marker has been established. An extensive literature survey shows variability in the expression of putative CESC markers among vertebrates; being attributed to species-specific variations, or other differences in developmental stages of these animals, approaches used in these studies and marker specificity. Here, we expanded the search for CESC markers using the amphibian model Xenopus laevis. In previous studies we found that long-term label retaining cells (suggestive of CESCs and TACs) are present throughout the larval basal corneal epithelium. In adult frogs, these cells become concentrated in the peripheral cornea (limbal region). Here, we used immunofluorescence to characterize the expression of nine proteins in the corneas of both Xenopus larvae and adults (post-metamorphic). We found that localization of some markers change between larval and adult stages. Markers such as p63, Keratin 19, and ß1-integrin are restricted to basal corneal epithelial cells of the larvae. After metamorphosis their expression is found in basal and intermediate layer cells of the adult frog corneal epithelium. Another protein, Pax6 was expressed in the larval corneas, but surprisingly it was not detected in the adult corneal epithelium. For the first time we report that Tcf7l2 can be used as a marker to differentiate cornea vs. skin in frogs. Tcf7l2 is present only in the frog skin, which differs from reports indicating that the protein is expressed in the human cornea. Furthermore, we identified the transition between the inner, and the outer surface of the adult frog eyelid as a key boundary in terms of marker expression. Although these markers are useful to identify different regions and cellular layers of the frog corneal epithelium, none is unique to CESCs or TACs. Our results confirm that there is no single conserved CESC marker in vertebrates. This molecular characterization of the Xenopus cornea facilitates its use as a vertebrate model to understand the functions of key proteins in corneal homeostasis and wound repair.

Ex Vivo Eye Tissue Culture Methods for Xenopus.

Lens regeneration can be studied in whole animals following removal of the original lens (lentectomy). However, culturing a whole animal can be impractical for assays involving small molecule inhibitors or proteins. Ex vivo eye tissue culture is an alternative approach for examining lens regeneration. The ex vivo culture system offers certain advantages when compared to the in vivo regeneration assay, as the percentage of cases showing lens differentiation can exceed that seen in whole animals. This culture system also allows for the treatment of eye tissues in small volumes, which helps ensure reproducibility and reduces the amount (and cost) of small-molecule inhibitors or exogenous proteins, etc., necessary to conduct an experiment. Additionally, different eye tissues can be combined, such as nontransgenic and transgenic tissues (e.g., eyecup and cornea) that carry reporters or inducible transgenes. This approach represents a very useful tool in the analysis of lens regeneration or for simply culturing specific eye tissues, and can be used to culture either Xenopus laevis or Xenopus tropicalis eye tissues.

Methods for Examining Lens Regeneration in Xenopus.

Some vertebrates are able to regenerate the lens following its removal. This includes species in the genus Xenopus (i.e., X. laevis, X. tropicalis, and X. borealis), the only anurans known to undergo lens regeneration. In Xenopus the regenerated lens is derived de novo from cells located within the basal-most layer of the larval corneal epithelium, and is triggered by factors provided by the neural retina. In larval frogs the corneal epithelium is underlain by an endothelium separated from the corneal epithelium except for a small central attachment (i.e., the "stromal-attracting center"). This connection grows larger as the stroma forms and the frogs approach metamorphosis. Here we provide instructions for performing lentectomies (removal of the original lens) to study lens regeneration.

Ectomesoderm and epithelial-mesenchymal transition-related genes in spiralian development.

BACKGROUND: Spiralians (e.g., annelids, molluscs, and flatworms) possess two sources of mesoderm. One is from endodermal precursors (endomesoderm), which is considered to be the ancestral source in metazoans. The second is from ectoderm (ectomesoderm) and may represent a novel cell type in the Spiralia. In the mollusc Crepidula fornicata, ectomesoderm is derived from micromere daughters within the A and B cell quadrants. Their progeny lie along the anterolateral edges of the blastopore. There they undergo epithelial-mesenchymal transition (EMT), become rounded and undergo delamination/ingression. Subsequently, they assume the mesenchymal phenotype, and migrate beneath the surface ectoderm to differentiate various cell types, including muscles and pigment cells. RESULTS: We examined expression of several genes whose homologs are known to regulate Type 1 EMT in other metazoans. Most of these genes were expressed within spiralian ectomesoderm during EMT. CONCLUSIONS: We propose that spiralian ectomesoderm, which exhibits analogous cellular behaviors to other populations of mesenchymal cells, may be controlled by the same genes that drive EMT in other metazoans. Perhaps these genes comprise a conserved metazoan EMT gene regulatory network (GRN). This study represents the first step in elucidating the GRN controlling the development of a novel spiralian cell type (ectomesoderm). Developmental Dynamics 247:1097-1120, 2018. © 2018 Wiley Periodicals, Inc.

Molecular, phylogenetic and developmental analyses of Sall proteins in bilaterians.

BACKGROUND: Sall (Spalt-like) proteins are zinc-finger transcription factors involved in a number of biological processes. They have only been studied in a few model organisms, such as Drosophila melanogaster, Caenorhabditis elegans, Schmidtea mediterranea and some vertebrates. Further taxon sampling is critical to understand the evolution and diversification of this protein and its functional roles in animals. RESULTS: Using genome and transcriptome mining, we confirmed the presence of sall genes in a range of additional animal taxa, for which their presence had not yet been described. We show that sall genes are broadly conserved across the Bilateria, and likely appeared in the bilaterian stem lineage. Our analysis of the protein domains shows that the characteristic arrangement of the multiple zinc-finger domains is conserved in bilaterians and may represent the ancient arrangement of this family of transcription factors. We also show the existence of a previously unknown zinc-finger domain. In situ hybridization was used to describe the gene expression patterns in embryonic and larval stages in two species of snails: Crepidula fornicata and Lottia gigantea. In L. gigantea, sall presents maternal expression, although later on the expression is restricted to the A and B quadrants during gastrulation and larval stage. In C. fornicata, sall has no maternal expression and it is expressed mainly in the A, C and D quadrants during blastula stages and in an asymmetric fashion during the larval stage. DISCUSSION: Our results suggest that the bilaterian common ancestor had a Sall protein with at least six zinc-finger domains. The evolution of Sall proteins in bilaterians might have occurred mostly as a result of the loss of protein domains and gene duplications leading to diversification. The new evidence complements previous studies in highlighting an important role of Sall proteins in bilaterian development. Our results show maternal expression of sall in the snail L. gigantea, but not C. fornicata. The asymmetric expression shown in the ectoderm of the trochophore larva of snails is probably related to shell/mantle development. The observed sall expression in cephalic tissue in snails and some other bilaterians suggests a possible ancestral role of sall in neural development in bilaterians.

RNA helicase Mov10 is essential for gastrulation and central nervous system development.

BACKGROUND: Mov10 is an RNA helicase that modulates access of Argonaute 2 to microRNA recognition elements in mRNAs. We examined the role of Mov10 in Xenopus laevis development and show a critical role for Mov10 in gastrulation and in the development of the central nervous system (CNS). RESULTS: Knockdown of maternal Mov10 in Xenopus embryos using a translation blocking morpholino led to defects in gastrulation and the development of notochord and paraxial mesoderm, and a failure to neurulate. RNA sequencing of the Mov10 knockdown embryos showed significant upregulation of many mRNAs when compared with controls at stage 10.5 (including those related to the cytoskeleton, adhesion, and extracellular matrix, which are involved in those morphogenetic processes). Additionally, the degradation of the miR-427 target mRNA, cyclin A1, was delayed in the Mov10 knockdowns. These defects suggest that Mov10's role in miRNA-mediated regulation of the maternal to zygotic transition could lead to pleiotropic effects that cause the gastrulation defects. Additionally, the knockdown of zygotic Mov10 showed that it was necessary for normal head, eye, and brain development in Xenopus consistent with a recent study in the mouse. CONCLUSIONS: Mov10 is essential for gastrulation and normal CNS development. Developmental Dynamics 247:660-671, 2018. © 2017 Wiley Periodicals, Inc.

Human Factors and Simulation in Emergency Medicine.

This consensus group from the 2017 Academic Emergency Medicine Consensus Conference "Catalyzing System Change through Health Care Simulation: Systems, Competency, and Outcomes" held in Orlando, Florida, on May 16, 2017, focused on the use of human factors (HF) and simulation in the field of emergency medicine (EM). The HF discipline is often underutilized within EM but has significant potential in improving the interface between technologies and individuals in the field. The discussion explored the domain of HF, its benefits in medicine, how simulation can be a catalyst for HF work in EM, and how EM can collaborate with HF professionals to effect change. Implementing HF in EM through health care simulation will require a demonstration of clinical and safety outcomes, advocacy to stakeholders and administrators, and establishment of structured collaborations between HF professionals and EM, such as in this breakout group.

Beyond the sea: Crepidula atrasolea as a spiralian model system.

This paper introduces the black-footed slipper snail, Crepidula atrasolea, as a new model for biological studies in the Spiralia. C. atrasolea is a calyptraeid gastropod, and congener of the Atlantic slipper snail, C. fornicata. Like C. fornicata, C. atrasolea shares a sedentary, filter-feeding, protandrous lifestyle, but is preferable as a developmental model because of its short generation time, year-round reproduction, and direct development. In our lab, individuals go from egg to reproductive females in under six months, as compared to an estimated 1-2 years for C. fornicata. Here we provide details for collecting and transporting animals, setting up inland aquaria, and maintaining laboratory colonies of C. atrasolea. We also describe early development, which is similar to that in other calyptraeids. Females brood encapsulated embryos for three weeks, which hatch as "crawl-away" juveniles. We also present a developmental transcriptome for C. atrasolea, covering early cleavage through late organogenesis stages, as a useful tool for future studies of gene expression and function. We provide this information to the broader developmental community to facilitate widespread use of this system.

Establishment and activity of the D quadrant organizer in the marine gastropod Crepidula fornicata.

During development in metazoan embryos, the fundamental embryonic axes are established by organizing centers that influence the fates of nearby cells. Among the spiralians, a large and diverse branch of protostome metazoans, studies have shown that an organizer sets up the dorsal-ventral axis, which arises from one of the four basic cell quadrants during development (the dorsal, D quadrant). Studies in a few species have also revealed variation in terms of how and when the D quadrant and the organizer are established. In some species the D quadrant is specified conditionally, via cell-cell interactions, while in others it is specified autonomously, via asymmetric cell divisions (such as those involving the formation of polar lobes). The third quartet macromere (3D) typically serves as the spiralian organizer; however, other cells born earlier or later in the D quadrant lineage can serve as the organizer, such as the 2d micromere in the annelid Capitella teleta or the 4d micromere in the mollusc Crepidula fornicata. Here we present work carried out in the snail C. fornicata to show that establishment of a single D quadrant appears to rely on a combination of both autonomous (via inheritance of the polar lobe) and conditional mechanisms (involving induction via the progeny of the first quartet micromeres). Through systematic ablation of cells, we show that D quadrant identity is established between 5th and 6th cleavage stages, as it is in other spiralians that use conditional specification. Subsequently, following the next cell cycle, organizer activity takes place soon after the birth of the 4d micromere. Therefore, unlike the case in other spiralians that use conditional specification, the specification of the D quadrant and the activity of the dorso-ventral organizer are temporally and spatially uncoupled. We also present data on organizer function in naturally-occurring and experimentally-induced twin embryos, which possess multiple D quadrants. We show that supernumerary D quadrants can arise in C. fornicata (either spontaneously or following polar lobe removal); when multiple D quadrants are present these do not exhibit effective organizer activity. We conclude that the polar lobe is not required for D quadrant specification, though it could play a role in effective organizer activity. We also tested whether the inheritance of the small polar lobe by the D quadrant is associated with the ability to laterally inhibit neighboring quadrants by direct contact in order to normally prevent supernumerary organizers from arising. Finally, we discuss the variation of spiralian organizers in a phylogenetic context.

Morphogenesis along the animal-vegetal axis: fates of primary quartet micromere daughters in the gastropod Crepidula fornicata.

BACKGROUND: The Spiralia are a large, morphologically diverse group of protostomes (e.g. molluscs, annelids, nemerteans) that share a homologous mode of early development called spiral cleavage. One of the most highly-conserved features of spiralian development is the contribution of the primary quartet cells, 1a-1d, to the anterior region of the embryo (including the brain, eyes, and the anterior ciliary band, called the prototroch). Yet, very few studies have analyzed the ultimate fates of primary quartet sub-lineages, or examined the morphogenetic events that take place in the anterior region of the embryo. RESULTS: This study focuses on the caenogastropod slipper snail, Crepidula fornicata, a model for molluscan developmental biology. Through direct lineage tracing of primary quartet daughter cells, and examination of these cells during gastrulation and organogenesis stages, we uncovered behaviors never described before in a spiralian. For the first time, we show that the 1a2-1d2 cells do not contribute to the prototroch (as they do in other species) and are ultimately lost before hatching. During gastrulation and anterior-posterior axial elongation stages, these cells cleavage-arrest and spread dramatically, contributing to a thin provisional epidermis on the dorsal side of the embryo. This spreading is coupled with the displacement of the animal pole, and other pretrochal cells, closer to the ventrally-positioned mouth, and the vegetal pole. CONCLUSIONS: This is the first study to document the behavior and fate of primary quartet sub-lineages among molluscs. We speculate that the function of 1a2-1d2 cells (in addition to two cells derived from 1d12, and the 2b lineage) is to serve as a provisional epithelium that allows for anterior displacement of the other progeny of the primary quartet towards the anterior-ventral side of the embryo. These data support a new and novel mechanism for axial bending, distinct from canonical models in which axial bending is suggested to be driven primarily by differential proliferation of posterior dorsal cells. These data suggest also that examining sub-lineages in other spiralians will reveal greater variation than previously assumed.

Effects of Participation in Sports Programs on Walking Ability and Endurance Over Time in Children With Cerebral Palsy.

OBJECTIVE: Children with cerebral palsy may benefit from maintaining a high level of physical fitness similar to typically developing children especially in terms of long-term physical performance, although in practice this is often difficult. The purpose of this study was to determine the effect of participation in sports programs on walking ability and endurance over time. DESIGN: A retrospective cohort study included participants with cerebral palsy, aged 6 to 20 yrs, who attended a summer sports program from 2004 to 2012. There were 256 participant sessions with pre/post data recorded. The participants consisted of a total of 97 children (mean age [SD] = 11.4 [3.1] yrs), many of whom attended multiple programs throughout the years. Programs were held 6 hrs/d, 5 d/wk for up to 4 wks. Outcome measures included the Timed Up and Go, modified 6-min walk, and 25-ft walk/run. RESULTS: The results showed significant improvements in the Timed Up and Go, modified 6-min walk distance and 25-ft walk/run over time. Children in Gross Motor Classification System level III made the largest gains. CONCLUSIONS: Walking ability and endurance seem to improve after participation in an intensive summer sports programs. Higher frequency of program attendance resulted in significant improvements in the Timed Up and Go. TO CLAIM CME CREDITS: Complete the self-assessment activity and evaluation online at http://www.physiatry.org/JournalCME CME OBJECTIVES: Upon completion of this article, the reader should be able to: (1) Discuss the importance of physical activity at the participation level (sports programs) for children with cerebral palsy; (2) Contrast the changes in walking ability and endurance for children in Gross Motor Function Classification System level I, II, and III after sports programs; and (3) Identify the impact of higher frequency of sports program attendance over time on walking ability. LEVEL: Advanced ACCREDITATION: The Association of Academic Physiatrists is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians.The Association of Academic Physiatrists designates this Journal-based CME activity for a maximum of 0.75 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.

Spiralian gastrulation: germ layer formation, morphogenesis, and fate of the blastopore in the slipper snail Crepidula fornicata.

BACKGROUND: Gastrulation is a critical step in bilaterian development, directly linked to the segregation of germ layers, establishment of axes, and emergence of the through-gut. Theories about the evolution of gastrulation often concern the fate of the blastopore (site of endomesoderm internalization), which varies widely in a major branch of bilaterians, the Spiralia. In this group, the blastopore has been said to become the mouth, the anus, both, or neither. Different developmental explanations for this variation exist, yet no modern lineage tracing study has ever correlated the position of cells surrounding the blastopore with their contribution to tissues of the mouth, foregut, and anus in a spiralian. This is the first study to do so, using the gastropod Crepidula fornicata. RESULTS: Crepidula gastrulation occurs by epiboly: the first through third quartet micromeres form an epithelial animal cap that expands to cover vegetal endomesodermal precursors. Initially, descendants of the second and third quartet micromeres (2a-2d, 3a-3d) occupy a portion of the blastopore lip. As the blastopore narrows, the micromeres' progeny exhibit lineage-specific behaviors that result in certain sublineages leaving the lip's edge. Anteriorly, cells derived from 3a(2) and 3b(2) undergo a unique epithelial-to-mesenchymal transition involving proliferation and a collective movement of cells into the archenteron. These cells make a novel spiralian germ layer, the ectomesoderm. Posteriorly, cells derived from 3c(2) and 3d(2) undergo a form of convergence and extension that involves zippering of cells and their intercalation across the ventral midline. During this process, several of these cells, as well as the 2d clone, become displaced posteriorly, away from the blastopore. Progeny of 2a-2c and 3a-3d make the mouth and foregut, and the blastopore becomes the opening to the mouth. The anus forms days later, as a secondary opening within the 2d(2) clone, and not from the classically described "anal cells", which we identify as the 3c(221) and 3d(221) cells. CONCLUSIONS: Our analysis of Crepidula gastrulation constitutes the first description of blastopore lip morphogenesis and fates using lineage tracing and live imaging. These data have profound implications for hypotheses about the evolution of the bilaterian gut and help explain observed variation in blastopore morphogenesis among spiralians.

A High Through-put Platform for Recombinant Antibodies to Folded Proteins.

Antibodies are key reagents in biology and medicine, but commercial sources are rarely recombinant and thus do not provide a permanent and renewable resource. Here, we describe an industrialized platform to generate antigens and validated recombinant antibodies for 346 transcription factors (TFs) and 211 epigenetic antigens. We describe an optimized automated phage display and antigen expression pipeline that in aggregate produced about 3000 sequenced Fragment antigen-binding domain that had high affinity (typically EC50<20 nm), high stability (Tm∼80 °C), good expression in E. coli (∼5 mg/L), and ability to bind antigen in complex cell lysates. We evaluated a subset of Fabs generated to homologous SCAN domains for binding specificities. These Fragment antigen-binding domains were monospecific to their target SCAN antigen except in rare cases where they cross-reacted with a few highly related antigens. Remarkably, immunofluorescence experiments in six cell lines for 270 of the TF antigens, each having multiple antibodies, show that ∼70% stain predominantly in the cytosol and ∼20% stain in the nucleus which reinforces the dominant role that translocation plays in TF biology. These cloned antibody reagents are being made available to the academic community through our web site recombinant-antibodies.org to allow a more system-wide analysis of TF and chromatin biology. We believe these platforms, infrastructure, and automated approaches will facilitate the next generation of renewable antibody reagents to the human proteome in the coming decade.