Evaluation of inpatient postpartum recovery using the Obstetric Quality of Recovery-10 patient-reported outcome measure: a single-center observational study.

BACKGROUND: Few adequately validated patient-reported outcome measures are available, which can assess recovery profiles following childbirth. OBJECTIVE: We aimed to determine whether quantitative recovery (using the Obstetric Quality of Recovery-10 patient-reported outcome measure) was superior following vaginal delivery compared with cesarean delivery and evaluate validity, reliability, and responsiveness of this patient-reported outcome measure in the obstetrical setting in the United States. STUDY DESIGN: Women recruited into this single-center observational cohort study completed the Obstetric Quality of Recovery-10 and EuroQol 5-dimension 3L patient-reported outcome measures within 72 hours of childbirth. We assessed the validity with hypothesis testing and structural validity. In hypothesis testing, the primary outcome was Obstetric Quality of Recovery-10 scores after vaginal vs cesarean delivery. Secondary outcomes were differences in Obstetric Quality of Recovery-10 scores for vaginal delivery following induction of labor vs spontaneous labor and scheduled vs unplanned cesarean delivery, correlation with clinical parameters (American Society of Anesthesiologists classification grade, body mass index, length of hospital stay, estimated blood loss, transfusion requirement, antiemetic use, and neonatal intensive care unit admission), and qualitative ranking of Obstetric Quality of Recovery-10 items for each delivery mode. Structural validity was assessed by determining the correlation of the Obstetric Quality of Recovery-10 scores with the EuroQol 5-dimension 3L and global health visual analog scale scores. Reliability was assessed using Cronbach alpha and inter-item correlation of Obstetric Quality of Recovery-10 items. Responsiveness was assessed by evaluating the change in Obstetric Quality of Recovery-10 scores over the 72-hour postpartum period. RESULTS: Data from 215 women were analyzed. In hypothesis testing, the median (interquartile range) Obstetric Quality of Recovery-10 scores were higher following vaginal delivery than cesarean delivery (86 [77-94] vs 77 [64-86], respectively; P<.001). Multivariate model demonstrated that Obstetric Quality of Recovery-10 scores were significantly lower after cesarean delivery when adjusting for American Society of Anesthesiologists classification grade, age, body mass index, and ethnicity (R=-8.97; P<.001). Obstetric Quality of Recovery-10 scores were similar between induction of labor and spontaneous labor, and scheduled cesarean delivery and unplanned cesarean delivery. Obstetric Quality of Recovery-10 was correlated with length of hospital stay (R=-0.248; P<.001), estimated blood loss (R=-0.3429; P<.001), transfusion requirement (R=-0.140; P=.041), and antiemetic use (R=-0.280; P<.001). The highest ranked Obstetric Quality of Recovery-10 items were ability to hold baby, feeling in control, and ability to look after personal hygiene. The lowest ranked items were pain and shivering. In structural validity, correlation of Obstetric Quality of Recovery-10 score was moderate with the global health visual analog scale (r=0.511) and EuroQol 5-dimension 3L scores (r=-0.509). In reliability, Cronbach alpha was 0.72 and more than 80% of individual items correlated. In responsiveness, Obstetric Quality of Recovery-10 scores did not change significantly over the study period. CONCLUSION: Quantitative inpatient recovery following vaginal delivery is superior to cesarean delivery. The Obstetric Quality of Recovery-10 appears to be a valid and reliable patient-reported outcome measure following these delivery modes. Further studies are needed to determine how to improve recovery domains identified in this study, to evaluate Obstetric Quality of Recovery-10 in different languages and determine whether these domains impact outcomes beyond hospitalization.

Mga Modulates Bmpr1a Activity by Antagonizing Bs69 in Zebrafish.

MAX giant associated protein (MGA) is a dual transcriptional factor containing both T-box and bHLHzip DNA binding domains. In vitro studies have shown that MGA functions as a transcriptional repressor or activator to regulate transcription of promotors containing either E-box or T-box binding sites. BS69 (ZMYND11), a multidomain-containing (i.e., PHD, BROMO, PWWP, and MYND) protein, has been shown to selectively recognizes histone variant H3.3 lysine 36 trimethylation (H3.3K36me3), modulates RNA Polymerase II elongation, and functions as RNA splicing regulator. Mutations in MGA or BS69 have been linked to multiple cancers or neural developmental disorders. Here, by TALEN and CRISPR/Cas9-mediated loss of gene function assays, we show that zebrafish Mga and Bs69 are required to maintain proper Bmp signaling during early embryogenesis. We found that Mga protein localized in the cytoplasm modulates Bmpr1a activity by physical association with Zmynd11/Bs69. The Mynd domain of Bs69 specifically binds the kinase domain of Bmpr1a and interferes with its phosphorylation and activation of Smad1/5/8. Mga acts to antagonize Bs69 and facilitate the Bmp signaling pathway by disrupting the Bs69-Bmpr1a association. Functionally, Bmp signaling under control of Mga and Bs69 is required for properly specifying the ventral tailfin cell fate.

Establishment of the Vertebrate Germ Layers.

The process of germ layer formation is a universal feature of animal development. The germ layers separate the cells that produce the internal organs and tissues from those that produce the nervous system and outer tissues. Their discovery in the early nineteenth century transformed embryology from a purely descriptive field into a rigorous scientific discipline, in which hypotheses could be tested by observation and experimentation. By systematically addressing the questions of how the germ layers are formed and how they generate overall body plan, scientists have made fundamental contributions to the fields of evolution, cell signaling, morphogenesis, and stem cell biology. At each step, this work was advanced by the development of innovative methods of observing cell behavior in vivo and in culture. Here, we take an historical approach to describe our current understanding of vertebrate germ layer formation as it relates to the long-standing questions of developmental biology. By comparing how germ layers form in distantly related vertebrate species, we find that highly conserved molecular pathways can be adapted to perform the same function in dramatically different embryonic environments.

Identification of a novel insertion mutation in FGFR3 that causes thanatophoric dysplasia type 1.

Thanatophoric dysplasia is a type of short-limbed neonatal dwarfism that is usually lethal in the perinatal period. It is characterized by short limbs, a narrow, bell-shaped thorax, macrocephaly with a prominent forehead, and flattened vertebral bodies. These malformations result from autosomal dominant mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. In this report, we describe a novel FGFR3 insertion mutation in a fetus with shortened limbs, curved femurs, and a narrow thorax. The diagnosis of thanatophoric dysplasia type 1 was suspected clinically, and FGFR3 sequencing showed a c.742_743insTGT variant, which predicts p.R248delinsLC. In vivo studies in zebrafish demonstrated that this mutation resulted in the overexpression of zebrafish Fgfr3, leading to the over-activation of downstream signaling and dorsalized embryos. To date, no insertions or deletions in FGFR3 have been reported to cause thanatophoric dysplasia types 1 or 2; therefore, this represents the first report to describe such a mutation. © 2016 Wiley Periodicals, Inc.

Extraembryonic signals under the control of MGA, Max, and Smad4 are required for dorsoventral patterning.

In vertebrates, extraembryonic tissues can act as signaling centers that impose a reproducible pattern of cell types upon the embryo. Here, we show that the zebrafish yolk syncytial layer (YSL) secretes a ventralizing signal during gastrulation. This activity is mediated by Bmp2b/Swirl (Swr) expressed under the control of Max's giant associated protein (MGA) and its binding partners, Max and Smad4. MGA coimmunoprecipitates with both Max and Smad4 in embryo extracts, and the three proteins form a complex in vitro. Furthermore, all three proteins bind to a DNA fragment upstream of the bmp2b transcription start site. Targeted depletion of MGA, its binding partners, or Bmp2b/Swr from the YSL reduces BMP signaling throughout the embryo, resulting in a mildly dorsalized phenotype. We conclude that MGA, Max, and Smad4 act in the extraembryonic YSL to initiate a positive feedback loop of Bmp signaling within the embryo.

Embryological manipulations in zebrafish.

Due to the powerful combination of genetic and embryological techniques, the teleost fish Danio rerio has emerged in the last decade as an important model organism for the study of embryonic development. It is relatively easy to inject material such as mRNA or synthetic oligonucleotides to reduce or increase the expression of a gene product. Changes in gene expression can be analyzed at the level of mRNA, by whole-mount in situ hybridization, or at the level of protein, by immunofluorescence. It is also possible to quantitatively analyze protein levels by Western and immunoprecipitation. Cell behavior can be analyzed in detail by cell transplantation and by fate mapping. Because a large number of mutations have been identified in recent years, these methods can be applied in a variety of contexts to provide a deep understanding of gene function that is often more difficult to achieve in other vertebrate model systems.

MEC-17 is an alpha-tubulin acetyltransferase.

In most eukaryotic cells, subsets of microtubules are adapted for specific functions by post-translational modifications (PTMs) of tubulin subunits. Acetylation of the epsilon-amino group of K40 on alpha-tubulin is a conserved PTM on the luminal side of microtubules that was discovered in the flagella of Chlamydomonas reinhardtii. Studies on the significance of microtubule acetylation have been limited by the undefined status of the alpha-tubulin acetyltransferase. Here we show that MEC-17, a protein related to the Gcn5 histone acetyltransferases and required for the function of touch receptor neurons in Caenorhabditis elegans, acts as a K40-specific acetyltransferase for alpha-tubulin. In vitro, MEC-17 exclusively acetylates K40 of alpha-tubulin. Disruption of the Tetrahymena MEC-17 gene phenocopies the K40R alpha-tubulin mutation and makes microtubules more labile. Depletion of MEC-17 in zebrafish produces phenotypes consistent with neuromuscular defects. In C. elegans, MEC-17 and its paralogue W06B11.1 are redundantly required for acetylation of MEC-12 alpha-tubulin, and contribute to the function of touch receptor neurons partly via MEC-12 acetylation and partly via another function, possibly by acetylating another protein. In summary, we identify MEC-17 as an enzyme that acetylates the K40 residue of alpha-tubulin, the only PTM known to occur on the luminal surface of microtubules.

TTLL3 Is a tubulin glycine ligase that regulates the assembly of cilia.

In most ciliated cell types, tubulin is modified by glycylation, a posttranslational modification of unknown function. We show that the TTLL3 proteins act as tubulin glycine ligases with chain-initiating activity. In Tetrahymena, deletion of TTLL3 shortened axonemes and increased their resistance to paclitaxel-mediated microtubule stabilization. In zebrafish, depletion of TTLL3 led to either shortening or loss of cilia in several organs, including the Kupffer's vesicle and olfactory placode. We also show that, in vivo, glutamic acid and glycine ligases oppose each other, likely by competing for shared modification sites on tubulin. We propose that tubulin glycylation regulates the assembly and dynamics of axonemal microtubules and acts either directly or indirectly by inhibiting tubulin glutamylation.

Identification of phagocytic cells, NK-like cytotoxic cell activity and the production of cellular exudates in the coelomic cavity of adult zebrafish.

Coelomic cavity (CC) cells of mature zebrafish harvested by lavage with media or trypsin-EDTA contained 0.80-1.20 x 10(5) and 2.0-3.5 x 10(5) cells, respectively. Media lavage was composed of granulocytes (60-80%), lymphocytes (10-20%), and NCC (4-10%). Granulocytes had large electron dense cytoplasmic paracrystalline granules and a segmented nucleus; they expressed plastin-1, myeloid specific peroxidase and MCSF mRNA; and they were NCAMP-1(+). Lymphocytes had B- and T-cell specific mRNA and were NCAMP-1(-) and NCCRP-1(-). NCC were 3 microm, NCAMP-1(+) and NCCRP-1(+) and did not express B- and T-cell specific mRNA. Additionally, trypsin lavage contained monocytes (marginated chromatin, low nuclear:cytoplasm ratio, sparse cytosolic granules) and macrophages (non-segmented nuclei, no margination of chromatin, abundant electron dense granules). E. coli injected into the CC were phagocytosed in a dose and time dependent fashion by granulocytes, monocytes and macrophages. NCC lysed mammalian target cells and NCAMP-1 expressing hybridoma cells in redirected lysis assays.

O-GlcNAc modifications regulate cell survival and epiboly during zebrafish development.

BACKGROUND: The post-translational addition of the monosaccharide O-linked beta-N-acetylglucosamine (O-GlcNAc) regulates the activity of a wide variety of nuclear and cytoplasmic proteins. The enzymes O-GlcNAc Transferase (Ogt) and O-GlcNAcase (Oga) catalyze, respectively, the attachment and removal of O-GlcNAc to target proteins. In adult mice, Ogt and Oga attenuate the response to insulin by modifying several components of the signal transduction pathway. Complete loss of ogt function, however, is lethal to mouse embryonic stem cells, suggesting that the enzyme has additional, unstudied roles in development. We have utilized zebrafish as a model to determine role of O-GlcNAc modifications in development. Zebrafish has two ogt genes, encoding six different enzymatic isoforms that are expressed maternally and zygotically. RESULTS: We manipulated O-GlcNAc levels in zebrafish embryos by overexpressing zebrafish ogt, human oga or by injecting morpholinos against ogt transcripts. Each of these treatments results in embryos with shortened body axes and reduced brains at 24 hpf. The embryos had 23% fewer cells than controls, and displayed increased rates of cell death as early as the mid-gastrula stages. An extensive marker analysis indicates that derivatives of three germ layers are reduced to variable extents, and the embryos are severely disorganized after gastrulation. Overexpression of Ogt and Oga delayed epiboly and caused a severe disorganization of the microtubule and actin based cytoskeleton in the extra-embryonic yolk syncytial layer (YSL). The cytoskeletal defects resemble those previously reported for embryos lacking function of the Pou5f1/Oct4 transcription factor spiel ohne grenzen. Consistent with this, Pou5f1/Oct4 is modified by O-GlcNAc in human embryonic stem cells. CONCLUSION: We conclude that O-GlcNAc modifications control the activity of proteins that regulate apoptosis and epiboly movements, but do not seem to regulate germ layer specification. O-GlcNAc modifies the transcription factor Spiel ohne grenzen/Pou5f1 and may regulate its activity.

The evolutionary origin of nodal-related genes in teleosts.

Because of an extra whole-genome duplication, zebrafish and other teleosts have two copies of genes that are present in a single copy in tetrapod genomes. Some zebrafish genes, however, are present in triplicate. For example, the nodal-related genes encode secreted proteins of the transforming growth factor beta superfamily that are required in all vertebrates to induce the mesoderm and endoderm, pattern all three germ layers, and establish the left-right axis. Zebrafish have three nodal-related genes, called ndr1/squint, ndr2/cyclops, and ndr3/southpaw. As part of an analysis of enhancer elements controlling zebrafish nodal-related gene expression, we analyzed the nodal loci in the sequenced genomes of five teleost species and four tetrapod species. Each teleost genome contains three nodal-related genes, indicating that squint, cyclops, and southpaw orthologues were present early in the teleost lineage. The genes flanking the nodal-related genes are also conserved, demonstrating a high degree of conserved synteny. Although we found little homology outside of the coding sequences in this region, pufferfish enhancer sequences work in zebrafish embryos to drive reporter gene expression in the squint expression pattern. This indicates a high degree of functional conservation of enhancer elements within the teleosts. We conclude that the ancestral squint and cyclops genes arose during the teleost-specific whole-genome duplication event and that southpaw emerged from a subsequent duplication event involving ancestral squint.

Nodal signals mediate interactions between the extra-embryonic and embryonic tissues in zebrafish.

In many vertebrates, extra-embryonic tissues are important signaling centers that induce and pattern the germ layers. In teleosts, the mechanism by which the extra-embryonic yolk syncytial layer (YSL) patterns the embryo is not understood. Although the Nodal-related protein Squint is expressed in the YSL, its role in this tissue is not known. We generated a series of stable transgenic lines with GFP under the control of squint genomic sequences. In all species, nodal-related genes induce their own expression through a positive feedback loop. We show that two tissue specific enhancers in the zebrafish squint gene mediate the response to Nodal signals. Expression in the blastomeres depends upon a conserved Nodal response element (NRE) in the squint first intron, while expression in the extra-embryonic enveloping layer (EVL) is mediated by an element upstream of the transcription start site. Targeted depletion experiments demonstrate that the zebrafish Nodal-related proteins Squint and Cyclops are required in the YSL for endoderm and head mesoderm formation. Thus, Nodal signals mediate interactions between embryonic and extra-embryonic tissues in zebrafish that maintain nodal-related gene expression in the margin. Our results demonstrate a high degree of functional conservation between the extra-embryonic tissues of mouse and zebrafish.

The role of maternal Activin-like signals in zebrafish embryos.

Maternal Activin-like proteins, a subgroup of the TGF-beta superfamily, play a key role in establishing the body axes in many vertebrates, but their role in teleosts is unclear. At least two maternal Activin-like proteins are expressed in zebrafish, including the Vg1 orthologue, zDVR-1, and the nodal-related gene, Squint. Our analysis of embryos lacking both maternal and zygotic squint function revealed that maternal squint is required in some genetic backgrounds for the formation of dorsal and anterior tissues. Conditional inactivation of the ALK4, 5 and 7 receptors by SB-505124 treatment during the cleavage stages ruled out a role for maternal Squint, zDVR-1, or other Activin-like ligands before the mid-blastula transition, when the dorsal axis is established. Furthermore, we show that maternal Squint and zDVR-1 are not required during the cleavage stages to induce zygotic nodal-related gene expression. nodal-related gene expression decreases when receptor inhibition continues past the mid-blastula transition, resulting in a progressive loss of mesoderm and endoderm. We conclude that maternally expressed Activin-like signals do not act before the mid-blastula transition in zebrafish, but do have a variably penetrant role in the later stages of axis formation. This contrasts with the early role for these signals during Xenopus development.

Mycobacterium haemophilum infections of zebrafish (Danio rerio) in research facilities.

In May 2005, a disease outbreak was investigated at a zebrafish (Danio rerio) research facility experiencing severe losses. Mycobacterium haemophilum was isolated from these fish and the disease was subsequently recreated in experimentally infected zebrafish. Fish exhibited signs characteristic of mycobacteriosis, including granuloma formation and severe, diffuse, chronic inflammation. Bacteria were observed in multiple tissues, including the central nervous system. Biofilm samples from the outbreak facility were PCR positive for M. haemophilum, suggesting biofilms might act as a reservoir for infection. Zebrafish appear to be particularly vulnerable to M. haemophilum, and measures such as quarantine and treatment of incoming water should be implemented to minimize the likelihood of introduction of this bacterium to zebrafish research facilities. Zebrafish are already a well-established laboratory animal model for genetics, toxicology and disease, their susceptibility to M. haemophilum may make them useful for the study of this bacterium in the future.

Time-dependent patterning of the mesoderm and endoderm by Nodal signals in zebrafish.

BACKGROUND: The vertebrate body plan is generated during gastrulation with the formation of the three germ layers. Members of the Nodal-related subclass of the TGF-beta superfamily induce and pattern the mesoderm and endoderm in all vertebrates. In zebrafish, two nodal-related genes, called squint and cyclops, are required in a dosage-dependent manner for the formation of all derivatives of the mesoderm and endoderm. These genes are expressed dynamically during the blastula stages and may have different roles at different times. This question has been difficult to address because conditions that alter the timing of nodal-related gene expression also change Nodal levels. We utilized a pharmacological approach to conditionally inactivate the ALK 4, 5 and 7 receptors during the blastula stages without disturbing earlier signaling activity. This permitted us to directly examine when Nodal signals specify cell types independently of dosage effects. RESULTS: We show that two drugs, SB-431542 and SB-505124, completely block the response to Nodal signals when added to embryos after the mid-blastula transition. By blocking Nodal receptor activity at later stages, we demonstrate that Nodal signaling is required from the mid-to-late blastula period to specify sequentially, the somites, notochord, blood, Kupffer's vesicle, hatching gland, heart, and endoderm. Blocking Nodal signaling at late times prevents specification of cell types derived from the embryo margin, but not those from more animal regions. This suggests a linkage between cell fate and length of exposure to Nodal signals. Confirming this, cells exposed to a uniform Nodal dose adopt progressively more marginal fates with increasing lengths of exposure. Finally, cell fate specification is delayed in squint mutants and accelerated when Nodal levels are elevated. CONCLUSION: We conclude that (1) Nodal signals are most active during the mid-to-late blastula stages, when nodal-related gene expression and the movement of responding cells are at their most dynamic; (2) Nodal signals specify cell fates along the animal-vegetal axis in a time-dependent manner; (3) cells respond to the total cumulative dose of Nodal signals to which they are exposed, as a function of distance from the source and duration of exposure.

The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm.

Nodal signals, a subclass of the TGFbeta superfamily of secreted factors, induce formation of mesoderm and endoderm in vertebrate embryos. We have examined the possible dorsoventral and animal-vegetal patterning roles for Nodal signals by using mutations in two zebrafish nodal-related genes, squint and cyclops, to manipulate genetically the levels and timing of Nodal activity. squint mutants lack dorsal mesendodermal gene expression at the late blastula stage, and fate mapping and gene expression studies in sqt(-/-); cyc(+/+) and sqt(-/-); cyc(+/-) mutants show that some dorsal marginal cells inappropriately form hindbrain and spinal cord instead of dorsal mesendodermal derivatives. The effects on ventrolateral mesendoderm are less severe, although the endoderm is reduced and muscle precursors are located nearer to the margin than in wild type. Our results support a role for Nodal signals in patterning the mesendoderm along the animal-vegetal axis and indicate that dorsal and ventrolateral mesoderm require different levels of squint and cyclops function. Dorsal marginal cells were not transformed toward more lateral fates in either sqt(-/-); cyc(+/-) or sqt(-/-); cyc(+/+) embryos, arguing against a role for the graded action of Nodal signals in dorsoventral patterning of the mesendoderm. Differential regulation of the cyclops gene in these cells contributes to the different requirements for nodal-related gene function in these cells. Dorsal expression of cyclops requires Nodal-dependent autoregulation, whereas other factors induce cyclops expression in ventrolateral cells. In addition, the differential timing of dorsal mesendoderm induction in squint and cyclops mutants suggests that dorsal marginal cells can respond to Nodal signals at stages ranging from the mid-blastula through the mid-gastrula.