Roles of Wnt pathway genes wls, wnt9a, wnt5b, frzb and gpc4 in regulating convergent-extension during zebrafish palate morphogenesis.

The Wnt signaling pathway is crucial for tissue morphogenesis, participating in cellular behavior changes, notably during the process of convergent-extension. Interactions between Wnt-secreting and receiving cells during convergent-extension remain elusive. We investigated the role and genetic interactions of Wnt ligands and their trafficking factors Wls, Gpc4 and Frzb in the context of palate morphogenesis in zebrafish. We describe that the chaperon Wls and its ligands Wnt9a and Wnt5b are expressed in the ectoderm, whereas juxtaposed chondrocytes express Frzb and Gpc4. Using wls, gpc4, frzb, wnt9a and wnt5b mutants, we genetically dissected the Wnt signals operating between secreting ectoderm and receiving chondrocytes. Our analysis delineates that non-canonical Wnt signaling is required for cell intercalation, and that wnt5b and wnt9a are required for palate extension in the anteroposterior and transverse axes, respectively.

Occult Histopathology and Its Predictors in Contralateral and Bilateral Prophylactic Mastectomies.

BACKGROUND: The last decade has seen an increasing prevalence of prophylactic mastectomies with decreasing age of patients treated for breast cancer. Data are limited on the prevalence of histopathologic abnormalities in this population. This study aimed to measure the prevalence of histopathologic findings in contralateral prophylactic mastectomy (CPM) and bilateral prophylactic mastectomy (BPM) patients and identify predictors of findings. METHODS: Our institution's prophylactic mastectomies from 2004 to 2011 were reviewed. Breast specimens with prior malignancies were excluded. Patient factors and pathology reports were collected. Independent predictive factors were identified with univariate and multivariate logistic analysis. RESULTS: A total of 524 specimens in 454 patients were identified. Malignancy was found in 7.0% of CPM and 5.7% of BPM specimens. In CPM patients, ipsilateral lobular carcinoma-in situ [odds ratio (OR) 4.0] and mammogram risk group (OR 2.0) were predictive of malignancy. Age group (OR 1.5), ipsilateral lobular carcinoma-in situ (OR 2.3), and prior bilateral salpingo-oophorectomy (OR 0.3) were predictive of moderate- to high-risk histopathology. Only increasing age group was predictive of increased moderate- to high-risk histopathology in BPM patients (OR 2.3). There were no independent predictors of malignancy in BPM. BRCA status was not predictive in either CPM or BPM. CONCLUSIONS: Patients with lobular carcinoma-in situ in the index breast or high-risk mammograms have a higher prevalence of malignancies. Although BRCA patients may benefit from prophylactic mastectomy, the genetic diagnosis does not increase the prevalence of detecting occult pathology. BPM patients can be counseled about relative risk, where occult pathology increases with age.

X-ray micro-diffraction studies on biological samples at the BioCAT Beamline 18-ID at the Advanced Photon Source.

The small source sizes of third-generation synchrotron sources are ideal for the production of microbeams for diffraction studies of crystalline and non-crystalline materials. While several such facilities have been available around the world for some time now, few have been optimized for the handling of delicate soft-tissue specimens under cryogenic conditions. Here the development of a new X-ray micro-diffraction instrument at the Biophysics Collaborative Access Team beamline 18-ID at the Advanced Photon Source, and its use with newly developed cryo-diffraction techniques for soft-tissue studies, are described. The combination of the small beam sizes delivered by this instrument, the high delivered flux and successful cryo-freezing of rat-tail tendon has enabled us to record data to better than 4 Šresolution. The ability to quickly raster scan samples in the beam allows selection of ordered regions in fibrous samples for markedly improved data quality. Examples of results of experiments obtainable using this instrument are presented.

Perception of pacifier use among caregivers of infants 0-1 years of age.

American Academy of Pediatrics approves pacifier use for soothing and calming; it recommends delaying its use until breastfeeding is well established. Though pacifiers have protective effects against sudden infant death syndrome, prolonged use of a pacifier can lead to complications. American Academy of Family Physicians discourages the use by 6 months to 1 year of age. Pacifier use guidelines are not established primarily due to a paucity of information regarding initiation, termination, benefits, and harmful effects of pacifiers by parents. We aim to investigate pacifier use among caregivers of 0-1-year-old infants. It was a descriptive study of parents or caregivers of children 0-1 year of age who completed a questionnaire focused on pacifier use. Statistical analysis was calculated using SPSS version 23. One hundred thirty-three caregivers were interviewed. One hundred eighteen (88.7%) caregivers were mothers. Ninety-one (68.4%) of caregivers identified as Hispanic and 42 (30.1%) as African American. Caregivers reported that mean pacifier use was 16 months and 3.4 h/day. One hundred six (80%) reported the most common use of the pacifier alone was to calm the baby. For the weaning method, 37 (27.8%) stated that gradual decrease of pacifiers was useful whereas 33 (24.8%) stated that abrupt removal of pacifiers was effective. Seventy-two (54.1%) reported that their family and friends recommended pacifiers. Eleven (8.3%) caregivers reported that information about pacifiers was provided by medical and day-care providers. Pacifier use was not significantly related to the feeding method during the first 2 months of life. This study identifies impressions and common misconceptions of pacifier use which may assist in the development of comprehensive guidelines.

Modelling meningococcal meningitis in the African meningitis belt.

SUMMARYMeningococcal meningitis is a major public health problem in a large area of sub-Saharan Africa known as the meningitis belt. Disease incidence increases every dry season, before dying out with the first rains of the year. Large epidemics, which can kill tens of thousands of people, occur frequently but unpredictably every 6-14 years. It has been suggested that these patterns may be attributable to complex interactions between the bacteria, human hosts and the environment. We used deterministic compartmental models to investigate how well simple model structures with seasonal forcing were able to qualitatively capture these patterns of disease. We showed that the complex and irregular timing of epidemics could be caused by the interaction of temporary immunity conferred by carriage of the bacteria together with seasonal changes in the transmissibility of infection. This suggests that population immunity is an important factor to include in models attempting to predict meningitis epidemics.

Experimental approaches for solution X-ray scattering and fiber diffraction.

X-ray scattering and diffraction from non-crystalline systems have gained renewed interest in recent years, as focus shifts from the structural chemistry information gained by high-resolution studies to the context of structural physiology at larger length scales. Such techniques permit the study of isolated macromolecules as well as highly organized macromolecular assemblies as a whole under near-physiological conditions. Time-resolved approaches, made possible by advanced synchrotron instrumentation, add a crucial dimension to many of these investigations. This article reviews experimental approaches in non-crystalline X-ray scattering and diffraction that may be used to illuminate important scientific questions such as protein/nucleic acid folding and structure-function relationships in large macromolecular assemblies.

Reverse actin sliding triggers strong myosin binding that moves tropomyosin.

Actin/myosin interactions in vertebrate striated muscles are believed to be regulated by the "steric blocking" mechanism whereby the binding of calcium to the troponin complex allows tropomyosin (TM) to change position on actin, acting as a molecular switch that blocks or allows myosin heads to interact with actin. Movement of TM during activation is initiated by interaction of Ca(2+) with troponin, then completed by further displacement by strong binding cross-bridges. We report x-ray evidence that TM in insect flight muscle (IFM) moves in a manner consistent with the steric blocking mechanism. We find that both isometric contraction, at high [Ca(2+)], and stretch activation, at lower [Ca(2+)], develop similarly high x-ray intensities on the IFM fourth actin layer line because of TM movement, coinciding with x-ray signals of strong-binding cross-bridge attachment to helically favored "actin target zones." Vanadate (Vi), a phosphate analog that inhibits active cross-bridge cycling, abolishes all active force in IFM, allowing high [Ca(2+)] to elicit initial TM movement without cross-bridge attachment or other changes from relaxed structure. However, when stretched in high [Ca(2+)], Vi-"paralyzed" fibers produce force substantially above passive response at pCa approximately 9, concurrent with full conversion from resting to active x-ray pattern, including x-ray signals of cross-bridge strong-binding and TM movement. This argues that myosin heads can be recruited as strong-binding "brakes" by backward-sliding, calcium-activated thin filaments, and are as effective in moving TM as actively force-producing cross-bridges. Such recruitment of myosin as brakes may be the major mechanism resisting extension during lengthening contractions.

Fast-scanning high-flux microprobe for biological X-ray fluorescence microscopy and microXAS.

There is a growing interest in the biomedical community in obtaining information concerning the distribution and local chemical environment of metals in tissues and cells. Recently, biological X-ray fluorescence microscopy (XFM) has emerged as the tool of choice to address these questions. A fast-scanning high-flux X-ray microprobe, built around a recently commissioned pair of 200 mm-long Rh-coated silicon Kirkpatrick-Baez mirrors, has been constructed at BioCAT beamline 18ID at the Advanced Photon Source. The new optical system delivers a flux of 1.3 x 10(12) photons s(-1) into a minimum focal spot size of approximately 3-5 microm FWHM. A set of Si drift detectors and bent Laue crystal analyzers may be used in combination with standard ionization chambers for X-ray fluorescence measurements. BioCAT's scanning software allows fast continuous scans to be performed while acquiring and storing full multichannel analyzer spectra per pixel on-the-fly with minimal overhead time (<20 ms per pixel). Together, the high-flux X-ray microbeam and the rapid-scanning capabilities of the BioCAT beamline allow the collection of XFM and micro X-ray absorption spectroscopy (microXAS) measurements from as many as 48 tissue sections per day. This paper reports the commissioning results of the new instrument with representative XFM and microXAS results from tissue samples.

Tissue-Specific In Vivo Biotin Chromatin Immunoprecipitation with Sequencing in Zebrafish and Chicken.

Chromatin immunoprecipitation with sequencing (ChIP-seq) has been instrumental in understanding transcription factor (TF) binding during gene regulation. ChIP-seq requires specific antibodies against desired TFs, which are not available for numerous species. Here, we describe a tissue-specific biotin ChIP-seq protocol for zebrafish and chicken embryos which utilizes AVI tagging of TFs, permitting their biotinylation by a co-expressed nuclear biotin ligase. Subsequently, biotinylated factors can be precipitated with streptavidin beads, enabling the user to construct TF genome-wide binding landscapes like conventional ChIP-seq methods. For complete details on the use and execution of this protocol, please see Lukoseviciute et al. (2018) and Ling and Sauka-Spengler (2019).

Early chromatin shaping predetermines multipotent vagal neural crest into neural, neuronal and mesenchymal lineages.

The enteric nervous system (ENS) predominantly originates from vagal neural crest (VNC) cells that emerge from the caudal hindbrain, invade the foregut and populate the gastrointestinal tract. However, the gene regulatory network (GRN) orchestrating the early specification of VNC remains unknown. Using an EdnrB enhancer, we generated a comprehensive temporal map of the chromatin and transcriptional landscape of VNC in the avian model, revealing three VNC cell clusters (neural, neurogenic and mesenchymal), each predetermined epigenetically prior to neural tube delamination. We identify and functionally validate regulatory cores (Sox10/Tfap2B/SoxB/Hbox) mediating each programme and elucidate their combinatorial activities with other spatiotemporally specific transcription factors (bHLH/NR). Our global deconstruction of the VNC-GRN in vivo sheds light on critical early regulatory mechanisms that may influence the divergent neural phenotypes in enteric neuropathies.

Reconstruction of the Global Neural Crest Gene Regulatory Network In Vivo.

Precise control of developmental processes is encoded in the genome in the form of gene regulatory networks (GRNs). Such multi-factorial systems are difficult to decode in vertebrates owing to their complex gene hierarchies and dynamic molecular interactions. Here we present a genome-wide in vivo reconstruction of the GRN underlying development of the multipotent neural crest (NC) embryonic cell population. By coupling NC-specific epigenomic and transcriptional profiling at population and single-cell levels with genome/epigenome engineering in vivo, we identify multiple regulatory layers governing NC ontogeny, including NC-specific enhancers and super-enhancers, novel trans-factors, and cis-signatures allowing reverse engineering of the NC-GRN at unprecedented resolution. Furthermore, identification and dissection of divergent upstream combinatorial regulatory codes has afforded new insights into opposing gene circuits that define canonical and neural NC fates early during NC ontogeny. Our integrated approach, allowing dissection of cell-type-specific regulatory circuits in vivo, has broad implications for GRN discovery and investigation.

A genome-wide assessment of the ancestral neural crest gene regulatory network.

The neural crest (NC) is an embryonic cell population that contributes to key vertebrate-specific features including the craniofacial skeleton and peripheral nervous system. Here we examine the transcriptional and epigenomic profiles of NC cells in the sea lamprey, in order to gain insight into the ancestral state of the NC gene regulatory network (GRN). Transcriptome analyses identify clusters of co-regulated genes during NC specification and migration that show high conservation across vertebrates but also identify transcription factors (TFs) and cell-adhesion molecules not previously implicated in NC migration. ATAC-seq analysis uncovers an ensemble of cis-regulatory elements, including enhancers of Tfap2B, SoxE1 and Hox-α2 validated in the embryo. Cross-species deployment of lamprey elements identifies the deep conservation of lamprey SoxE1 enhancer activity, mediating homologous expression in jawed vertebrates. Our data provide insight into the core GRN elements conserved to the base of the vertebrates and expose others that are unique to lampreys.

Titin-based modulation of calcium sensitivity of active tension in mouse skinned cardiac myocytes.

We studied the effect of titin-based passive force on the length dependence of activation of cardiac myocytes to explore whether titin may play a role in the generation of systolic force. Force-pCa relations were measured at sarcomere lengths (SLs) of 2.0 and 2.3 microm. Passive tension at 2.3 microm SL was varied from approximately 1 to approximately 10 mN/mm(2) by adjusting the characteristics of the stretch imposed on the passive cell before activation. Relative to 2.0 microm SL, the force-pCa curve at 2.3 microm SL and low passive tension showed a leftward shift (pCa(50) [change in pCa at half-maximal activation]) of 0.09+/-0.02 pCa units while at 2.3 microm SL and high passive tension the shift was increased to 0.25+/-0.03 pCa units. Passive tension also increased pCa(50) at reduced interfilament lattice spacing achieved with dextran. We tested whether titin-based passive tension influences the interfilament lattice spacing by measuring the width of the myocyte and by using small-angle x-ray diffraction of mouse left ventricular wall muscle. Cell width and interfilament lattice spacing varied inversely with passive tension, in the presence and absence of dextran. The passive tension effect on length-dependent activation may therefore result from a radial titin-based force that modulates the interfilament lattice spacing.

The in situ supermolecular structure of type I collagen.

BACKGROUND: The proteins belonging to the collagen family are ubiquitous throughout the animal kingdom. The most abundant collagen, type I, readily forms fibrils that convey the principal mechanical support and structural organization in the extracellular matrix of connective tissues such as bone, skin, tendon, and vasculature. An understanding of the molecular arrangement of collagen in fibrils is essential since it relates molecular interactions to the mechanical strength of fibrous tissues and may reveal the underlying molecular pathology of numerous connective tissue diseases. RESULTS: Using synchrotron radiation, we have conducted a study of the native fibril structure at anisotropic resolution (5.4 A axial and 10 A lateral). The intensities of the tendon X-ray diffraction pattern that arise from the lateral packing (three-dimensional arrangement) of collagen molecules were measured by using a method analogous to Rietveld methods in powder crystallography and to the separation of closely spaced peaks in Laue diffraction patterns. These were then used to determine the packing structure of collagen by MIR. CONCLUSIONS: Our electron density map is the first obtained from a natural fiber using these techniques (more commonly applied to single crystal crystallography). It reveals the three-dimensional molecular packing arrangement of type I collagen and conclusively proves that the molecules are arranged on a quasihexagonal lattice. The molecular segments that contain the telopeptides (central to the function of collagen fibrils in health and disease) have been identified, revealing that they form a corrugated arrangement of crosslinked molecules that strengthen and stabilize the native fibril.

In Vitro Chromatin Assembly: Strategies and Quality Control.

Chromatin accessibility is modulated by structural transitions that provide timely access to the genetic and epigenetic information during many essential nuclear processes. These transitions are orchestrated by regulatory proteins that coordinate intricate structural modifications and signaling pathways. In vitro reconstituted chromatin samples from defined components are instrumental in defining the mechanistic details of such processes. The bottleneck to appropriate in vitro analysis is the production of high quality, and quality-controlled, chromatin substrates. In this chapter, we describe methods for in vitro chromatin reconstitution and quality control. We highlight the strengths and weaknesses of various approaches and emphasize quality control steps that ensure reconstitution of a bona fide homogenous chromatin preparation. This is essential for optimal reproducibility and reliability of ensuing experiments using chromatin substrates.

Cerebrosides alter the lyotropic and thermotropic phase transitions of DOPE:DOPC and DOPE:DOPC:sterol mixtures.

Freezing injury in rye and oat is a consequence of the formation of the inverted hexagonal (H(II)) phase in regions where the plasma membrane is brought into close proximity with cytoplasmic membranes during freeze-induced dehydration. Susceptibility to plasma membrane destabilization and H(II) phase formation during freezing is associated with alterations in plasma membrane lipid composition. This paper examines the influence of lipid composition and hydration on the propensity of lipid mixtures of DOPE:DOPC and DOPE:DOPC:sterols with added cerebrosides (CER) to form the H(II) phase during dehydration. The addition of CER to DOPE:DOPC:beta-sitosterol mixtures decreased the water content of the dispersions in a manner suggesting that most or all of the water in the dehydrated mixtures was associated with the phospholipids. The addition of CER significantly decreased the osmotic pressure at which the L(alpha) --> H(II) phase transition occurred from an osmotic pressure of 76.1 MPa for DOPE:DOPC (50:50) to 20 MPa in DOPE:DOPC:beta-sitosterol:CER (22.5:22.5:50:5) and 8 MPa in DOPE:DOPC:beta-sitosterol:CER (15:15:50:20). Experiments examining the effects of CER on the thermally-induced formation of the H(II) phase in fully hydrated mixtures and examining the influence of CER on the formation of the H(II) phase in DOPE:DOPC mixtures lacking beta-sitosterol suggested that CER facilitated the L(alpha) --> H(II) phase transition by effecting a decrease in bilayer hydration and by increased lateral packing pressures within the acyl domain of the bilayer. Taken in sum, these data indicate that the differential propensity of the rye and oat plasma membranes to undergo freeze-induced formation of the L(alpha) --> H(II) phase cannot be attributed to one lipid species. Rather, the propensity towards freeze-induced membrane destabilization is a consequence of the summation of physical characteristics of the membrane lipid components that included bilayer hydration, packing pressures within the hydrophobic domain of the membrane, the propensity of the lipid components to demix, and the relative proportions of the various lipid components.

Effects of plant sterols on the hydration and phase behavior of DOPE/DOPC mixtures.

Freeze-induced injury of protoplasts of non-acclimated rye and oat is associated with the formation of the inverted hexagonal (HII) phase in regions where the plasma membrane and various endomembranes are brought into close apposition as a result of freeze-induced dehydration. The influence of lipid composition and hydration on the propensity of mixtures of DOPE:DOPC containing either sterols or acylated steryl glucosides to form the HII phase was determined by DSC, freeze-fracture electron microscopy and X-ray diffraction. The addition of plant sterols to a mixture of DOPE/DOPC (either 1:1:1 or 1:1:2 mole ratio of DOPE/DOPC/sterols) reduced the total hydration of the mixture (expressed as wt% water) after desorption over a range of osmotic pressures of 2.8 to 286 MPa. However, most or all of the water remaining in the dehydrated lipid mixtures was associated predominantly with the phospholipids. Both sterols and acylated steryl glucosides significantly promoted both the dehydration-induced and thermally induced L alpha-->HII phase transitions in DOPE/DOPC mixtures however, acylated steryl glucosides were much more effective. In mixtures containing plant sterols, the HII phase occurred after dehydration at 20 MPa (20 degrees C), which resulted in a water content of 11.7 wt%. In contrast, mixtures containing acylated steryl glucosides were in the HII phase in excess water, i.e., they did not require dehydration to effect the L alpha-->HII phase transition. The results indicate that genotypic differences in the lipid composition of the plasma membrane of rye and oat leaves have a significant influence on the propensity for formation of the HII phase during freeze-induced dehydration.

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel's Cartilage.

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel's cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel's cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel's cartilage. Multispectral cell labeling enables scientists to demonstrate extension of the Meckel's chondrocytes. During extension phase of the Meckel's cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.