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Zebrafish (Danio rerio) are aquatic vertebrates with significant homology to their terrestrial counterparts. While zebrafish have a centuries-long track record in developmental and regenerative biology, their utility has grown exponentially with the onset of modern genetics. This is exemplified in studies focused on skeletal development and repair. Herein, the numerous contributions of zebrafish to our understanding of the basic science of cartilage, bone, tendon/ligament, and other skeletal tissues are described, with a particular focus on applications to development and regeneration. We summarize the genetic strengths that have made the zebrafish a powerful model to understand skeletal biology. We also highlight the large body of existing tools and techniques available to understand skeletal development and repair in the zebrafish and introduce emerging methods that will aid in novel discoveries in skeletal biology. Finally, we review the unique contributions of zebrafish to our understanding of regeneration and highlight diverse routes of repair in different contexts of injury. We conclude that zebrafish will continue to fill a niche of increasing breadth and depth in the study of basic cellular mechanisms of skeletal biology.

The over-enrichment of nitrogen (N) in the environment has contributed to severe and recurring harmful cyanobacterial blooms, especially by the non-N(2)-fixing Microcystis spp. N chemical speciation influences cyanobacterial growth, persistence and the production of the hepatotoxin microcystin, but the physiological mechanisms to explain these observations remain unresolved. Stable-labelled isotopes and metabolomics were employed to address the influence of nitrate, ammonium, and urea on cellular physiology and production of microcystins in Microcystis aeruginosa NIES-843. Global metabolic changes were driven by both N speciation and diel cycling. Tracing (15)N-labelled nitrate, ammonium, and urea through the metabolome revealed N uptake, regardless of species, was linked to C assimilation. The production of amino acids, like arginine, and other N-rich compounds corresponded with greater turnover of microcystins in cells grown on urea compared to nitrate and ammonium. However, (15)N was incorporated into microcystins from all N sources. The differences in N flux were attributed to the energetic efficiency of growth on each N source. While N in general plays an important role in sustaining biomass, these data show that N-speciation induces physiological changes that culminate in differences in global metabolism, cellular microcystin quotas and congener composition.

We report continuous measurements of quasiparticles trapping and clearing from Andreev Bound States in aluminum nanobridge Josephson junctions integrated into a superconducting-qubit-like device. We find that trapping is well modeled by independent spontaneous emission events. Above 80 mK the clearing process is well described by absorption of thermal phonons, but other temperature-independent mechanisms dominate at low temperature. We find complex structure in the dependence of the low-temperature clearing rate on the Andreev Bound State energy. Our results shed light on quasiparticle behavior in qubit-like circuits.

A major feature of Saethre-Chotzen syndrome is coronal craniosynostosis, the fusion of the frontal and parietal bones at the coronal suture. It is caused by heterozygous loss-of-function mutations in either of the bHLH transcription factors TWIST1 and TCF12. Although compound heterozygous Tcf12; Twist1 mice display severe coronal synostosis, the individual role of Tcf12 had remained unexplored. Here, we show that Tcf12 controls several key processes in calvarial development, including the rate of frontal and parietal bone growth, and the boundary between sutural and osteogenic cells. Genetic analysis supports an embryonic requirement for Tcf12 in suture formation, as combined deletion of Tcf12 in embryonic neural crest and mesoderm, but not in postnatal suture mesenchyme, disrupts the coronal suture. We also detected asymmetric distribution of mesenchymal cells on opposing sides of the wild-type frontal and parietal bones, which prefigures later bone overlap at the sutures. In Tcf12 mutants, reduced asymmetry is associated with bones meeting end-on-end, possibly contributing to synostosis. Our results support embryonic requirements of Tcf12 in proper formation of the overlapping coronal suture.

Aim The primary aim was to review current venous thromboembolism (VTE) prophylaxis prescribing against national and European guidelines at two hospitals in northwest England. A secondary aim was to standardise VTE prescribing practices. Method 3 standards were identified (NICE, BAUS and EUA) for VTE prophylaxis in nephrectomy. All simple and radical nephrectomies and nephroureterectomies were included. Open and laparoscopic cases were included. Data was collected from Royal Liverpool University Hospital (RLUH) and Arrowe Park Hospital (APH). Cases from surgical diaries between January 2019 to January 2020 were identified and compared to the 3 standards. 49 cases were identified at RLUH and 83 at APH Results At APH, 77/83 (92.7%) cases received inpatient LMWH. The remaining 6 were already on a DOAC. 98.7% received inpatient mechanical VTE prophylaxis. 85.5% of patients received extended VTE prophylaxis with no documented indication, and only 20% of open nephrectomies received 28 days LMWH. At RLUH 44/49 cases (89.7%) received inpatient LMWH. All 5 patients who did not had a documented reason why. 100% of inpatients at received inpatient mechanical VTE prophylaxis. 4 patients underwent open nephrectomy, however none of these received 28-day extended LMWH prophylaxis. Conclusions Comparing guidelines with local data reveals that prescribing practice for both in- and outpatient LMWH is variable and often is based on personal preferences. The above results have been presented locally at each institution and practice standardised with re-audit ongoing.

Bacterial membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties that allows cells to respond to environmental conditions. Carotenoids are sterol analogs that are important for regulating membrane dynamics. The membrane of Pantoea sp. YR343 is characterized by the presence of the carotenoid zeaxanthin, and a carotenoid-deficient mutant, ΔcrtB, displays defects in root colonization, reduced secretion of indole-3-acetic acid, and defects in biofilm formation. Here we demonstrate that the loss of carotenoids results in changes to the membrane lipid composition in Pantoea sp. YR343, including increased amounts of unsaturated fatty acids in the ΔcrtB mutant membranes. These mutant cells displayed less fluid membranes in comparison to wild type cells as measured by fluorescence anisotropy of whole cells. Studies with artificial systems, however, have shown that carotenoids impart membrane rigidifying properties. Thus, we examined membrane fluidity using spheroplasts and vesicles composed of lipids extracted from either wild type or mutant cells. Interestingly, with the removal of the cell wall and membrane proteins, ΔcrtB vesicles were more fluid than vesicles made from lipids extracted from wild type cells. In addition, carotenoids appeared to stabilize membrane fluidity during rapidly changing temperatures. Taken together, these results suggest that Pantoea sp. YR343 compensates for the loss of carotenoids by changing lipid composition, which together with membrane proteins, results in reduced membrane fluidity. These changes may influence the abundance or function of membrane proteins that are responsible for the physiological changes observed in the ΔcrtB mutant cells.

Novel hybrid-wick aluminum and stainless-steel/ammonia constant conductance heat pipes (CCHPs) were developed to operate in space carrying power over long distances, as well as act as a thermosypho...

Proper function of the vertebrate skeleton requires the development of distinct articulating embryonic cartilages. Irx transcription factors are arranged in co-regulated clusters that are expressed in the developing skeletons of the face and appendages. IrxB cluster genes are required for the separation of toes in mice and formation of the hyoid joint in zebrafish, yet whether Irx genes have broader roles in skeletal development remains unclear. Here, we perform a comprehensive loss-of-function analysis of all 11 Irx genes in zebrafish. We uncover conserved requirements for IrxB genes in formation of the fish and mouse scapula. In the face, we find a requirement for IrxAb genes and irx7 in formation of anterior neural crest precursors of the jaw, and for IrxBa genes in formation of endodermal pouches and gill cartilages. We also observe extensive joint loss and cartilage fusions in animals with combinatorial losses of Irx clusters, with in vivo imaging revealing that at least some of these fusions arise through inappropriate chondrogenesis. Our analysis reveals diverse roles for Irx genes in the formation and later segmentation of the facial skeleton.

A major feature of Saethre-Chotzen syndrome is coronal craniosynostosis, the fusion of the frontal and parietal bones at the coronal suture. It is caused by heterozygous loss-of-function mutations in the basic HLH transcription factorsTWIST1andTCF12. While compound heterozygousTcf12; Twist1mice display severe coronal synostosis, the individual role ofTcf12has remained unexplored. Here we show that Tcf12 controls several key processes in calvarial development, including the rate of frontal and parietal bone growth, and the boundary between sutural and osteogenic cells. Genetic analysis supports an embryonic requirement forTcf12in suture formation, as combined deletion ofTcf12in the embryonic neural crest and mesoderm, but not in the postnatal suture mesenchyme, disrupts the coronal suture. We also detect asymmetric distribution of Grem1 + mesenchymal cells on opposing sides of the wild-type frontal and parietal bones, which prefigures later bone overlap at the sutures. InTcf12mutants, reduced asymmetry correlates with lack of bone overlap. Our results indicate a largely embryonic function of Tcf12 in controlling the rate and asymmetrical growth of calvarial bones and establishment of suture boundaries, which together ensure the proper formation of the overlapping coronal suture.

Sutures separate the flat bones of the skull and enable coordinated growth of the brain and overlying cranium. To uncover the cellular diversity within sutures, we generated single-cell transcriptomes and performed extensive expression validation of the embryonic murine coronal suture. We identify Erg and Pthlh as markers of osteogenic progenitors in sutures, and distinct pre-osteoblast signatures between the bone fronts and periosteum. In the ectocranial layers above the suture, we observe a ligament-like population spanning the frontal and parietal bones. In the dura mater underlying the suture, we detect a chondrocyte-like signature potentially linked to cartilage formation under pathological conditions. Genes mutated in coronal synostosis are preferentially expressed in proliferative osteogenic cells, as well as meningeal layers, suggesting discrete cell types that may be altered in different syndromes. This single-cell atlas provides a resource for understanding development of the coronal suture, the suture most commonly fused in monogenic craniosynostosis.

Nonequilibrium quasiparticles are ubiquitous in superconducting electronics. These quasiparticles can trap in the internal Andreev bound states of a phase-biased Josephson junction, providing a mechanism for studying their presence and behavior. We characterize a quasiparticle trapping detector device based on a two junction aluminum nanobridge superconducting quantum interference device incorporated into a transmission line resonator. When the device is flux-biased, distinct resonant frequencies develop depending on the trapped quasiparticle number. We demonstrate continuous detection of up to 3 trapped quasiparticles, with detection of a trapped quasiparticle with a signal-to-noise ratio of 27 in 5 μs. We describe initial measurements of quasiparticle behavior and discuss the possible optimization and application of such detector devices.

Hormone replacement therapy (HRT) in one form or another has been used for over 70 years, yet there are still disagreements regarding its clinical value and its safety. Particularly in terms of its...

Metallic fuel relocation experiments were conducted in which molten uranium was dropped into the cladding of a single fuel pin where eutectic interaction between fuel and cladding led to the release of eutectic fuel mixture into a narrow sodium channel. The thermal hydraulic relocation behavior of the molten uranium was analyzed in a previous study. Here, the extent of the eutectic formation was evaluated by scanning electron microscopy and energy-dispersive spectroscopy (SEM/EDS). The SEM/EDS analysis confirmed that cladding failure was caused by eutectic penetration. The eutectic penetration initiated around the boundary between cladding and uranium fuel, progressed into the cladding, and caused the cladding failure. Near the cladding failure region, the original cladding shape was unrecognizable, and many cracks, breakthroughs, and partial losses of the cladding were observed. The main components of the cladding failure region were UFe2 and U6Fe, which are the key components in the eutectic penetration.

The objectives were to determine the optimal feeding amount of choline in a ruminally protected form to reduce the triacylglycerol (TAG) concentration in liver and to increase TAG in blood plasma of dairy cows. Pregnant, nonlactating multiparous Holstein cows (n = 77) were blocked by body condition score (3.59 ± 0.33) and assigned to treatment at 64 ± 10 d before calculated calving date. Dietary treatments were top-dressing of 0, 30, 60, 90, or 120 g/d of ruminally protected choline (RPC; Balchem Corp., New Hampton, NY) ions to supply the equivalent of 0, 6.5, 12.9, 19.4, and 25.8 g/d of choline ions. Diets were formulated to exceed nutrient requirements for maintenance and pregnancy and fed in ad libitum amounts for the first 5 d. From d 6 to 15, cows were restricted to consume approximately 31% of their net energy requirements to simulate early lactating cows in negative energy balance. Methionine intake was maintained throughout each 15-d period. Liver was biopsied at 5 and 14 d and analyzed for TAG and glycogen. Blood was sampled on d 5 and 14 and plasma analyzed for glucose, insulin, cholesterol, β-hydroxybutyrate, long-chain fatty acids, and haptoglobin. On d 14, a mixture of saturated long-chain fatty acids, ground corn, and dried molasses (50:37:13) was offered (908 g, as-is basis) 10 h after the single daily feeding. Blood samples were collected for 19 h and plasma analyzed for TAG and cholesterol to assess apparent absorption of dietary fat. Mean dry matter intake and energy balance decreased from means of 9.5 to 3.3 kg/d and from 0.6 to −9.2 Mcal of net energy for lactation/d during the ad libitum and restricted feeding periods, respectively. Plasma concentrations of the lipid-soluble choline biomolecules, namely total phosphatidylcholines, total lysophosphatidylcholines, and sphingomyelin, increased with choline supplementation. Feed restriction increased plasma concentrations of β-hydroxybutyrate and free long-chain fatty acids, whereas those of glucose, insulin, and total cholesterol decreased. During feed restriction, concentration of hepatic TAG and plasma haptoglobin decreased linearly, whereas concentration of hepatic glycogen tended to increase quadratically with increasing intake of RPC. After fat supplementation, mean plasma concentration of TAG increased by an average of 21% with intake of RPC ions, peaking at intakes of ≥6.5 g/d of RPC ion. In summary, feeding RPC ions to cows in negative energy balance had increasing lipotropic effects on the liver when consumed up to 25.8 g/d, whereas feeding only 6.5 g/d increased concentrations of hepatic glycogen and TAG in the blood.

Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules that have essential biochemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon-halogen bond-cleaving reductive dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base and assigns unsubstituted purine a biological function by demonstrating that Coα-purinyl-cobamide (purinyl-Cba) is the native prosthetic group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-auxotrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme activities generate opportunities to manipulate functionalities of microbiomes.

Understanding the relocation behavior of metallic fuel in severe accident scenarios is one of the most important factors in the safety assessment of sodium-cooled fast reactors (SFRs). In the present study, the relocation behavior of uranium metallic fuel was investigated by conducting metallic fuel relocation experiments using 19-pin bundle test sections in the Argonne’s Metallic Uranium Safety Experiment (MUSE) facility. Post-test radiographic images were taken to characterize the uranium fuel relocation within the pin bundle test section. In addition, the pressure drop in the test sections was measured after the relocation experiments to evaluate the blockage of the sodium coolant channel. The pressure drop in the pin bundle test sections was also analytically calculated by varying the porosity and the pore size of the relocated fuel. The porosity and pore size of the relocated fuel in the pin bundle test sections were estimated by comparing those calculated pressure drop values with the measured pressure drop values from the pressure drop tests. The experimental results showed that the more formation of the eutectics could lead to more fuel dispersion and less blockage due to the lower freezing point of the eutectics.

The tear film protects the terrestrial animal’s ocular surface and the lacrimal gland provides important aqueous secretions necessary for its maintenance. Despite the importance of the lacrimal gland in ocular health, molecular aspects of its development remain poorly understood. We have identified a noncoding RNA (miR-205) as an important gene for lacrimal gland development. Mice lacking miR-205 fail to properly develop lacrimal glands, establishing this noncoding RNA as a key regulator of lacrimal gland development. Specifically, more than half of knockout lacrimal glands never initiated, suggesting a critical role of miR-205 at the earliest stages of lacrimal gland development. RNA-seq analysis uncovered several up-regulated miR-205 targets that may interfere with signaling to impair lacrimal gland initiation. Supporting this data, combinatorial epistatic deletion of Fgf10, the driver of lacrimal gland initiation, and miR-205 in mice exacerbates the lacrimal gland phenotype. We develop a molecular rheostat model where miR-205 modulates signaling pathways related to Fgf10 in order to regulate glandular development. These data show that a single microRNA is a key regulator for early lacrimal gland development in mice and highlights the important role of microRNAs during organogenesis.

The surface ectoderm is the source of ectodermal appendages including hair, teeth, and many glands. The development and function of ectodermal appendages has been researched extensively, but many of the molecular mechanisms that govern the developmental programs of ectodermal appendages remain elusive. While several protein-coding genes are established as key regulators of ectodermal appendage development, the role of noncoding RNAs is an emerging area of investigation. This review highlights recent advances in studies of microRNA-mediated control of ectodermal appendage development using mouse models. We will also discuss future directions and technological advances that will drive the microRNA field forward and expand our understanding of how individual microRNAs control ectodermal appendage development.

INTRODUCTION: Lipidomics can reveal global alterations in a broad class of molecules whose functions are innately linked to physiology. Monitoring changes in the phospholipid composition of biological membranes in response to stressors can aid the development of targeted therapies. However, exact quantitation of cardiolipins is not a straightforward task due to low ionization efficiencies and poor chromatographic separation of these compounds. OBJECTIVE: The aim of this study was to develop a quantitative method for the detection of cardiolipins and other phospholipids using both a targeted and untargeted analyses with a Q-Exactive. METHODS: HILIC chromatography and high-resolution mass spectrometry with parallel reaction monitoring was used to measure changes in lipid concentration. Internal standards and fragmentation techniques allowed for the reliable quantitation of lipid species including: lysyl-phosphatidylglycerol, phosphatidylglycerol, and cardiolipin. RESULTS: The untargeted analysis was capable to detecting 6 different phospholipid classes as well as free fatty acids. The targeted analysis quantified up to 23 cardiolipins, 10 phosphatidylglycerols and 10 lysyl-phosphatidylglycerols with detection limits as low as 50 nM. Biological validation with Enterococcus faecalis demonstrates sensitivity in monitoring the incorporation of exogenously supplied free fats into membrane phospholipids. When supplemented with oleic acid, the amount of free oleic acid in the membrane was 100 times greater and the concentration of polyunsaturated cardiolipin increased to over 3.5 μM compared to controls. CONCLUSIONS: This lipidomics method is capable of targeted quantitation for challenging biologically relevant cardiolipins as well as broad, untargeted lipid profiling.

Lower head failure and corium-concrete interaction were predicted to occur at Fukushima Daiichi Unit 1 (1F1) by several different system-level code analyses, including MELCOR v2.1 and MAAP5. Althou...

The reactor accidents at Fukushima Daiichi have rekindled interest in late-phase severe accident behavior involving reactor pressure vessel breach and discharge of molten core melt into the containment. Two technical issues of interest in this area are core-concrete interaction and the extent to which the core debris may be quenched and rendered coolable by top flooding. The Organisation for Economic Co-operation and Development–sponsored Melt Coolability and Concrete Interaction programs at Argonne National Laboratory included the conduct of large-scale reactor material experiments and associated analysis with the objectives of resolving the ex-vessel debris coolability issue and addressing remaining uncertainties related to long-term two-dimensional molten core–concrete interactions under both wet and dry cavity conditions. These tests provided a broad database to support accident management planning as well as the development and validation of models and codes that can be used to extrapolate th...

Lower head failure and corium-concrete interaction were predicted to occur at Fukushima Daiichi Unit 1 (1F1) by several different system-level code analyses, including MELCOR v2.1 and MAAP5. Althou...

The reactor accidents at Fukushima Daiichi have rekindled interest in light water reactor (LWR) severe accident phenomenology. Postevent analyses have identified several areas that may warrant addi...

The U.S. Department of Energy (DOE) has played a major role in the U.S. response to the events at Fukushima Daiichi. During the first several weeks following the accident, U.S. assistance efforts were guided by results from a significant and diverse set of analyses. In the months that followed, a coordinated analysis activity aimed at gaining a more thorough understanding of the accident sequence was completed using laboratory-developed, system-level best-estimate accident analysis codes, while a parallel analysis was conducted by U.S. industry. A comparison of predictions for Unit 1 from these two studies indicated significant differences between MAAP and MELCOR results for key plant parameters, such as in-core hydrogen production. On that basis, a crosswalk was completed to determine the key modeling variations that led to these differences. In parallel with these activities, it became clear that there was a need to perform a technology gap evaluation on accident-tolerant components and severe a...

The majority of pregnancy loss in cattle occurs between Days 8 and 16 of gestation, coincident with the initiation of conceptus elongation and the onset of maternal recognition of pregnancy. Differences in conceptus length on the same day of gestation may be related to an inherent lack of developmental competency or may simply be a consequence of asynchrony with the maternal environment. The objective of this work was to characterize differential patterns of mRNA expression between short and long bovine conceptuses recovered on Day 15 of gestation. Embryos were produced from super-ovulated Holstein donor cows, and groups of Grade-1 and Grade-3 compact morulas were transferred into recipient heifers at Day 6.5 of their cycle. Conceptuses were recovered at Day 15 of gestation, and measured to assess overall length and area. Total RNA was extracted and analyzed on individual GeneChip Bovine Genome Arrays (Affymetrix, Santa Clara, CA). Gene expression was compared between conceptuses derived from the transfer of Grade-1 versus Grade-3 embryos; no differences were identified in the profiles of Day-15 conceptuses of these different embryo grades. When gene expression was compared between conceptuses classified as either short (mean length of 4.2 ± 0.1 mm [standard error]) or long (24.7 ± 1.9 mm) upon recovery at Day 15 of gestation, a total of 348 genes were differentially expressed. Of these, 221 genes were up-regulated and 127 were down-regulated in long compared to short conceptuses. In summary, differences in gene expression were identified between conceptuses recovered on Day 15 of gestation, based on their length. These data may be used to identify genes and cellular pathways involved in enhanced conceptus elongation that could serve as markers of successful pregnancy. Mol. Reprod. Dev. 83: 424-441, 2016. © 2016 Wiley Periodicals, Inc.