On the stability of the disordered molecular alloy phase of ammonia hemihydrate.

The disordered-molecular-alloy phase (DMA) of ammonia hydrates [J. S. Loveday and R. J. Nelmes, Phys. Rev. Lett. 83, 4329 (1999)] is unique in that it has substitutional disorder of ammonia and water over the molecular sites of a body centred cubic lattice. Whilst this structure has been observed in ammonia di- and mono-hydrate compositions, it has not been conclusively observed in the ammonia hemihydrate system. This work presents investigations of the structural behaviour of ammonia hemihydrate as a function of P and T. The indications of earlier studies [Ma et al. RSC Adv. 2, 4290 (2012)] that the DMA structure could be produced by compression of ammonia hemihydrate above 20 GPa at ambient temperature are confirmed. In addition, the DMA structure was found to form reversibly both from the melt, and on warming of ammonia hemihydrate phase-II, in the pressure range between 4 and 8 GPa. The route used to make the DMA structure from ammonia mono- and di-hydrates--compression at 170 K to 6 GPa followed by warming to ambient temperature--was found not to produce the DMA structure for ammonia hemihydrate. These results provide the first strong evidence that DMA is a thermodynamically stable form. A high-pressure phase diagram for ammonia hemihydrate is proposed which has importance for planetary modelling.

Coming together is a beginning: the making of an intervertebral disc.

The intervertebral disc (IVD) is a complex fibrocartilaginous structure located between the vertebral bodies that allows for movement and acts as a shock absorber in our spine for daily activities. It is composed of three components: the nucleus pulposus (NP), annulus fibrosus, and cartilaginous endplate. The characteristics of these cells are different, as they produce specific extracellular matrix (ECM) for tissue function and the niche in supporting the differentiation status of the cells in the IVD. Furthermore, cell heterogeneities exist in each compartment. The cells and the supporting ECM change as we age, leading to degenerative outcomes that often lead to pathological symptoms such as back pain and sciatica. There are speculations as to the potential of cell therapy or the use of tissue engineering as treatments. However, the nature of the cells present in the IVD that support tissue function is not clear. This review looks at the origin of cells in the making of an IVD, from the earliest stages of embryogenesis in the formation of the notochord, and its role as a signaling center, guiding the formation of spine, and in its journey to become the NP at the center of the IVD. While our current understanding of the molecular signatures of IVD cells is still limited, the field is moving fast and the potential is enormous as we begin to understand the progenitor and differentiated cells present, their molecular signatures, and signals that we could harness in directing the appropriate in vitro and in vivo cellular responses in our quest to regain or maintain a healthy IVD as we age.

Hypertrophic chondrocytes at the junction of musculoskeletal structures.

Hypertrophic chondrocytes are found at unique locations at the junction of skeletal tissues, cartilage growth plate, articular cartilage, enthesis and intervertebral discs. Their role in the skeleton is best understood in the process of endochondral ossification in development and bone fracture healing. Chondrocyte hypertrophy occurs in degenerative conditions such as osteoarthritis. Thus, the role of hypertrophic chondrocytes in skeletal biology and pathology is context dependent. This review will focus on hypertrophic chondrocytes in endochondral ossification, in which they exist in a transient state, but acting as a central regulator of differentiation, mineralization, vascularization and conversion to bone. The amazing journey of a chondrocyte from being entrapped in the extracellular matrix environment to becoming proliferative then hypertrophic will be discussed. Recent studies on the dynamic changes and plasticity of hypertrophic chondrocytes have provided new insights into how we view these cells, not as terminally differentiated but as cells that can dedifferentiate to more progenitor-like cells in a transition to osteoblasts and adipocytes, as well as a source of skeletal stem and progenitor cells residing in the bone marrow. This will provide a foundation for studies of hypertrophic chondrocytes at other skeletal sites in development, tissue maintenance, pathology and therapy.

Pressure-induced dehydration and the structure of ammonia hemihydrate-II.

The structure of the crystalline ammonia-bearing phase formed when ammonia monohydrate liquid is compressed to 3.5(1) GPa at ambient temperature has been solved from a combination of synchrotron x-ray single-crystal and neutron powder-diffraction studies. The solution reveals that rather than having the ammonia monohydrate (AMH) composition as had been previously thought, the structure has an ammonia hemihydrate composition. The structure is monoclinic with spacegroup P2(1)/c and lattice parameters a = 3.3584(5) Å, b = 9.215(1) Å, c = 8.933(1) Å and ß = 94.331(8)° at 3.5(1) GPa. The atomic arrangement has a crowned hexagonal arrangement and is a layered structure with long N-D···N hydrogen bonds linking the layers. The existence of pressure-induced dehydration of AMH may have important consequences for the behaviour and differentiation of icy planets and satellites.

PRIMUS: Comprehensive proteomics of mouse intervertebral discs that inform novel biology and relevance to human disease modelling.

Mice are commonly used to study intervertebral disc (IVD) biology and related diseases such as IVD degeneration. Discs from both the lumbar and tail regions are used. However, little is known about compartmental characteristics in the different regions, nor their relevance to the human setting, where a functional IVD unit depends on a homeostatic proteome. Here, we address these major gaps through comprehensive proteomic profiling and in-depth analyses of 8-week-old healthy murine discs, followed by comparisons with human. Leveraging on a dataset of over 2,700 proteins from 31 proteomic profiles, we identified key molecular and cellular differences between disc compartments and spine levels, but not gender. The nucleus pulposus (NP) and annulus fibrosus (AF) compartments differ the most, both in matrisome and cellularity contents. Differences in the matrisome are consistent with the fibrous nature required for tensile strength in the AF and hydration property in the NP. Novel findings for the NP cells included an enrichment in cell junction proteins for cell-cell communication (Cdh2, Dsp and Gja1) and osmoregulation (Slc12a2 and Wnk1). In NP cells, we detected heterogeneity of vacuolar organelles; where about half have potential lysosomal function (Vamp3, Copb2, Lamp1/2, Lamtor1), some contain lipid droplets and others with undefined contents. The AF is enriched in proteins for the oxidative stress responses (Sod3 and Clu). Interestingly, mitochondrial proteins are elevated in the lumbar than tail IVDs that may reflect differences in metabolic requirement. Relative to the human, cellular and structural information are conserved for the AF. Even though the NP is more divergent between mouse and human, there are similarities at the level of cell biology. Further, common cross-species markers were identified for both NP (KRT8/19, CD109) and AF (COL12A1). Overall, mouse is a relevant model to study IVD biology, and an understanding of the limitation will facilitate research planning and data interpretation, maximizing the translation of research findings to human IVDs.

Development of a standardized histopathology scoring system using machine learning algorithms for intervertebral disc degeneration in the mouse model-An ORS spine section initiative.

Mice have been increasingly used as preclinical model to elucidate mechanisms and test therapeutics for treating intervertebral disc degeneration (IDD). Several intervertebral disc (IVD) histological scoring systems have been proposed, but none exists that reliably quantitate mouse disc pathologies. Here, we report a new robust quantitative mouse IVD histopathological scoring system developed by building consensus from the spine community analyses of previous scoring systems and features noted on different mouse models of IDD. The new scoring system analyzes 14 key histopathological features from nucleus pulposus (NP), annulus fibrosus (AF), endplate (EP), and AF/NP/EP interface regions. Each feature is categorized and scored; hence, the weight for quantifying the disc histopathology is equally distributed and not driven by only a few features. We tested the new histopathological scoring criteria using images of lumbar and coccygeal discs from different IDD models of both sexes, including genetic, needle-punctured, static compressive models, and natural aging mice spanning neonatal to old age stages. Moreover, disc sections from common histological preparation techniques and stains including H&E, SafraninO/Fast green, and FAST were analyzed to enable better cross-study comparisons. Fleiss's multi-rater agreement test shows significant agreement by both experienced and novice multiple raters for all 14 features on several mouse models and sections prepared using various histological techniques. The sensitivity and specificity of the new scoring system was validated using artificial intelligence and supervised and unsupervised machine learning algorithms, including artificial neural networks, k-means clustering, and principal component analysis. Finally, we applied the new scoring system on established disc degeneration models and demonstrated high sensitivity and specificity of histopathological scoring changes. Overall, the new histopathological scoring system offers the ability to quantify histological changes in mouse models of disc degeneration and regeneration with high sensitivity and specificity.

Surviving endoplasmic reticulum stress is coupled to altered chondrocyte differentiation and function.

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded alpha1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia.

Matrix remodeling during intervertebral disc growth and degeneration detected by multichromatic FAST staining.

Various imaging techniques have been used to assess degeneration of the intervertebral disc, including many histological methods, but cartilage-oriented histological stains do not clearly show the comparatively complex structures of the disc. In addition, there is no integrated method to assess efficiently both the compartmental organization and matrix composition in disc samples. In this study, a novel histological method, termed FAST staining, has been developed to investigate disc growth and degeneration by sequential staining with fast green, Alcian blue, Safranin-O, and tartrazine to generate multichromatic histological profiles (FAST profiles). This identifies the major compartments of the vertebra-disc region, including the cartilaginous endplate and multiple zones of the annulus fibrosus, by specific FAST profile patterns. A disc degeneration model in rabbit established using a previously described puncture method showed gradual but profound alteration of the FAST profile during disc degeneration, supporting continual alteration of glycosaminoglycan. Changes of the FAST profile pattern in the nucleus pulposus and annulus fibrosus of the postnatal mouse spine suggested matrix remodeling activity during the growth of intervertebral discs. In summary, we developed an effective staining method capable of defining intervertebral disc compartments in detail and showing matrix remodeling events within the disc. The FAST staining method may be used to develop a histopathological grading system to evaluate disc degeneration or malformation.

The hedgehog regulated oncogenes Gli1 and Gli2 block myoblast differentiation by inhibiting MyoD-mediated transcriptional activation.

The mechanism by which activation of the Hedgehog (Hh) pathway modulates differentiation and promotes oncogenesis in specific tissues is poorly understood. We therefore, analysed rhabdomyosarcomas from mice that were haploinsufficient for the Hh-binding protein, Hip1, or for the Hh receptor, Patched 1 (Ptch1). Transfection of the Hh-regulated transcription factor Gli1, which is expressed in a subset of mouse and human rhabdomyosarcomas, suppressed differentiation of myogenic rhabdomyosarcoma lines generated from Hip1+/- and Ptch1+/- mice. The closely related factor, Gli2, had similar effects. Gli1 and Gli2 inhibited myogenesis by repressing the capacity of MyoD to activate transcription. Deletion analysis of Gli1 indicated that multiple domains of Gli1 are required for efficient inhibition of MyoD. Gli1 reduced the ability of MyoD to heterodimerize with E12 and bind DNA, providing one mechanism whereby the Gli proteins modulate the activity of MyoD. This novel activity of Gli proteins provides new insights into how Hh signaling modulates terminal differentiation through inhibition of tissue-specific factors such as MyoD. This mechanism may contribute to the broad role of Hh signaling and the Gli proteins in differentiation decisions and cancer formation.

High-pressure/high-temperature phase diagram of zinc.

The phase diagram of zinc (Zn) has been explored up to 140 GPa and 6000 K, by combining optical observations, x-ray diffraction, and ab initio calculations. In the pressure range covered by this study, Zn is found to retain a hexagonal close-packed (hcp) crystal symmetry up to the melting temperature. The known decrease of the axial ratio (c/a) of the hcp phase of Zn under compression is observed in x-ray diffraction experiments from 300 K up to the melting temperature. The pressure at which c/a reaches [Formula: see text] (≈10 GPa) is slightly affected by temperature. When this axial ratio is reached, we observed that single crystals of Zn, formed at high temperature, break into multiple poly-crystals. In addition, a noticeable change in the pressure dependence of c/a takes place at the same pressure. Both phenomena could be caused by an isomorphic second-order phase transition induced by pressure in Zn. The reported melt curve extends previous results from 24 to 135 GPa. The pressure dependence obtained for the melting temperature is accurately described up to 135 GPa by using a Simon-Glatzel equation: [Formula: see text], where P is the pressure in GPa. The determined melt curve agrees with previous low-pressure studies and with shock-wave experiments, with a melting temperature of 5060(30) K at 135 GPa. Finally, a thermal equation of state is reported, which at room-temperature agrees with the literature.

Histological and reference system for the analysis of mouse intervertebral disc.

A new scoring system based on histo-morphology of mouse intervertebral disc (IVD) was established to assess changes in different mouse models of IVD degeneration and repair. IVDs from mouse strains of different ages, transgenic mice, or models of artificially induced IVD degeneration were assessed. Morphological features consistently observed in normal, and early/later stages of degeneration were categorized into a scoring system focused on nucleus pulposus (NP) and annulus fibrosus (AF) changes. "Normal NP" exhibited a highly cellularized cell mass that decreased with natural ageing and in disc degeneration. "Normal AF" consisted of distinct concentric lamellar structures, which was disrupted in severe degeneration. NP/AF clefts indicated more severe changes. Consistent scores were obtained between experienced and new users. Altogether, our scoring system effectively differentiated IVD changes in various strains of wild-type and genetically modified mice and in induced models of IVD degeneration, and is applicable from the post-natal stage to the aged mouse. This scoring tool and reference resource addresses a pressing need in the field for studying IVD changes and cross-study comparisons in mice, and facilitates a means to normalize mouse IVD assessment between different laboratories. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:233-243, 2018.

Inhibiting the integrated stress response pathway prevents aberrant chondrocyte differentiation thereby alleviating chondrodysplasia.

The integrated stress response (ISR) is activated by diverse forms of cellular stress, including endoplasmic reticulum (ER) stress, and is associated with diseases. However, the molecular mechanism(s) whereby the ISR impacts on differentiation is incompletely understood. Here, we exploited a mouse model of Metaphyseal Chondrodysplasia type Schmid (MCDS) to provide insight into the impact of the ISR on cell fate. We show the protein kinase RNA-like ER kinase (PERK) pathway that mediates preferential synthesis of ATF4 and CHOP, dominates in causing dysplasia by reverting chondrocyte differentiation via ATF4-directed transactivation of Sox9. Chondrocyte survival is enabled, cell autonomously, by CHOP and dual CHOP-ATF4 transactivation of Fgf21. Treatment of mutant mice with a chemical inhibitor of PERK signaling prevents the differentiation defects and ameliorates chondrodysplasia. By preventing aberrant differentiation, titrated inhibition of the ISR emerges as a rationale therapeutic strategy for stress-induced skeletal disorders.

Early onset of disc degeneration in SM/J mice is associated with changes in ion transport systems and fibrotic events.

Intervertebral disc degeneration (IDD) causes back pain and sciatica, affecting quality of life and resulting in high economic/social burden. The etiology of IDD is not well understood. Along with aging and environmental factors, genetic factors also influence the onset, progression and severity of IDD. Genetic studies of risk factors for IDD using human cohorts are limited by small sample size and low statistical power. Animal models amenable to genetic and functional studies of IDD provide desirable alternatives. Despite differences in size and cellular content as compared to human intervertebral discs (IVDs), the mouse is a powerful model for genetics and assessment of cellular changes relevant to human biology. Here, we provide evidence for early onset disc degeneration in SM/J relative to LG/J mice with poor and good tissue healing capacity respectively. In the first few months of life, LG/J mice maintain a relatively constant pool of notochordal-like cells in the nucleus pulposus (NP) of the IVD. In contrast, chondrogenic events are observed in SM/J mice beginning as early as one-week-old, with progressive fibrotic changes. Further, the extracellular matrix changes in the NP are consistent with IVD degeneration. Leveraging on the genomic data of two parental and two recombinant inbred lines, we assessed the genetic contribution to the NP changes and identified processes linked to the regulation of ion transport systems. Significantly, "transport" system is also in the top three gene ontology (GO) terms from a comparative proteomic analysis of the mouse NP. These findings support the potential of the SM/J, LG/J and their recombinant inbred lines for future genetic and biological analysis in mice and validation of candidate genes and biological relevance in human cohort studies. The proteomic data has been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD008784.

Topologically frustrated ionisation in a water-ammonia ice mixture.

Water and ammonia are considered major components of the interiors of the giant icy planets and their satellites, which has motivated their exploration under high P-T conditions. Exotic forms of these pure ices have been revealed at extreme (~megabar) pressures, notably symmetric, ionic, and superionic phases. Here we report on an extensive experimental and computational study of the high-pressure properties of the ammonia monohydrate compound forming from an equimolar mixture of water and ammonia. Our experiments demonstrate that relatively mild pressure conditions (7.4 GPa at 300 K) are sufficient to transform ammonia monohydrate from a prototypical hydrogen-bonded crystal into a form where the standard molecular forms of water and ammonia coexist with their ionic counterparts, hydroxide (OH-) and ammonium [Formula: see text] ions. Using ab initio atomistic simulations, we explain this surprising coexistence of neutral/charged species as resulting from a topological frustration between local homonuclear and long-ranged heteronuclear ionisation mechanisms.

A concurrent epidemic of respiratory syncytial virus and echovirus 7 infections in an intensive care nursery.

We describe concurrent outbreaks of respiratory syncytial virus (RSV) and Echovirus 7 (Echo 7) infections in a neonatal intensive care unit, including infants who had dual infections. Seventy-three infants were identified as having RSV from January through June, 1984. During the same surveillance period Echo 7 was cultured from 20 infants, and 6 infants had concurrent RSV and Echo 7 and RSV were isolated, but not concurrently. This dual outbreak of RSV and Echo 7 infections persisted for months despite infection control measures. Control procedures were complicated by: (1) cases of RSV infection at less than 72 hours of age, which had not previously been reported and which led to the reintroduction of RSV into "clean" areas; (2) the lack of a rapid diagnostic test for enterovirus infection; (3) the number of infants who were asymptomatic with each infection; and (4) the logistical problems of handling a dual pathogen outbreak in a confined setting. These problems were compounded by the many risk factors associated with nosocomial infections found in neonatal intensive care settings such as prolonged hospitalizations, endotracheal or nasogastric tubes and contact with many ancillary care personnel.

The metabolism of supplementary vitamin C during the common cold.

Ascorbic acid concentrations have been measured in leukocytes and plasma following oral administration of 2000 mg vitamin C in the same subjects while they had cold symptoms and after recovery from their colds. Plasma and leukocyte concentrations rose significantly in females, but only plasma concentrations rose in males, after the loading dose during colds. In the postcold tests, only plasma concentrations rose in both sexes. There was a significant difference in plasma leukocyte regression coefficients between the cold and postcold tests in females. Ascorbic acid passes into the plasma for metabolic purposes, and its storage is less in the leukocytes, during colds. Males had worse colds than females because their catarrhal symptoms were more severe. Higher tissue concentrations of ascorbic acid tended to be associated with low total, toxic, and catarrhal symptom values. A rise in tissue ascorbic acid was associated with less severe catarrhal symptoms in females. Ascorbic acid concentrations in the plasma and tongue were significantly higher after the subjects had recovered from their cold symptoms. Increasing the loading dose of vitamin C from 500 to 2000 mg more than doubled the leukocyte concentration of ascorbic acid in females. The higher dose enabled uptake of the vitamin into the leukocytes to take place over a 4-hour period. It did not give rise to increased uptake into male leukocytes. Administration of supplementary vitamin C elevated plasma ascorbic acid. The ascorbic acid then passed into the tissues depleted of vitamin C during the cold syndrome. A single supplementary dose of 2000 mg vitamin C can replete leukocyte ascorbic acid during a 4-hour period in females, but a larger dose may be necessary in males.

The neuropsychological implications of allergenic clinical titration in the treatment of allergic diseases.

The wheal is used for assessment of the Provocation-Neutralisation Response in Clinical Titration. It is the third, slowest, and most indirect measure of the Antigen-Antibody Reaction as demonstrated in the Triple Response. The flare is produced by an axone reflex. It appears rapidly as a direct neurological response to antigen challenge. It is associated with local changes in Direct Current Potential. These are transmitted distally through the d.c. Perineural Analogue System. Neutralisation values appear in a harmonic series with increasing dilutions of antigen. Passive Allergenic Neutralisation does not require active antigen administration into the patient's tissues. Passive Neutralisation can be performed in the absence of allergenic material. It is concluded that the wheal need not be associated with the Provocation-Neutralisation technique, and that a neuropsychological basis for the Provocation-Neutralisation response should be explored.

The protective effect of auto-immune buccal urine therapy (AIBUT) against the Raynaud phenomenon.

The efficacy of Auto-Immune Buccal Urine Therapy (AIBUT) against allergic symptoms depends upon sublingual administration of the correct dose of urine as determined by bio-assay in individual patients. Succeeding effective turn-off doses occur at the troughs of a sinusoidal dose-response curve. Efficacy of the administered dose is confirmed by reduction in the severity and duration of Cold-water-induced Raynaud symptoms after administration of effective doses of unboiled urine in AIBUT. Boiled urine does not affect the Raynaud phenomenon.

Auto-immune therapy against human allergic disease: a physiological self defence factor.

Guinea-pigs sensitised to ovalbumen excrete the antigen in their urine in a therapeutic concentration which prevents anaphylactic death after injection of a challenge dose of the ovalbumen. Sublingual administration of the correct dose of urine from allergic patients also provides therapeutic control of their allergic symptoms. The effective dose is determined by bio-assay. The Neutralisation dose is recognised by disappearance of buccal sensation to the urine. Readministration of salivary, nasal, and sweat secretions from allergic patients onto the conjunctiva also controls allergic symptoms. These procedures provide effective physiological self-defence therapies against allergic challenge in humans.

Fluoride hypersensitivity in mains tap water demonstrated by skin potential changes in guinea-pigs.

Changes in skin electric potential following immune challenge provide a sensitive measure of the intensity of the allergic challenge. In guinea-pigs sensitised to Sodium Fluoride, Fluoridated Mains Tap Water, or Ovalbumen with Sodium Fluoride, and then challenged with one part in one million Sodium Fluoride, significant allergic responses were produced. Ovalbumen alone caused an allergic response only after a second challenge. Purified Water did not result in any immune response. It is concluded that Fluoridated Mains Tap Water is capable of causing an allergic response similar to that produced by Sodium Fluoride alone, and that Ovalbumen potentiates the immune response to Sodium Fluoride.