Insights into digit evolution from a fate map study of the forearm using Chameleon, a new transgenic chicken line.

The cellular and genetic networks that contribute to the development of the zeugopod (radius and ulna of the forearm, tibia and fibula of the leg) are not well understood, although these bones are susceptible to loss in congenital human syndromes and to the action of teratogens such as thalidomide. Using a new fate-mapping approach with the Chameleon transgenic chicken line, we show that there is a small contribution of SHH-expressing cells to the posterior ulna, posterior carpals and digit 3. We establish that although the majority of the ulna develops in response to paracrine SHH signalling in both the chicken and mouse, there are differences in the contribution of SHH-expressing cells between mouse and chicken as well as between the chicken ulna and fibula. This is evidence that, although zeugopod bones are clearly homologous according to the fossil record, the gene regulatory networks that contribute to their development and evolution are not fixed.

Homozygous SMAD6 variants in two unrelated patients with craniosynostosis and radioulnar synostosis.

BACKGROUND: SMAD6 encodes an intracellular inhibitor of the bone morphogenetic protein (BMP) signalling pathway. Until now, rare heterozygous loss-of-function variants in SMAD6 were demonstrated to increase the risk of disparate clinical disorders including cardiovascular disease, craniosynostosis and radioulnar synostosis. Only two unrelated patients harbouring biallelic SMAD6 variants presenting a complex cardiovascular phenotype and facial dysmorphism have been described. CASES: Here, we present the first two patients with craniosynostosis harbouring homozygous SMAD6 variants. The male probands, both born to healthy consanguineous parents, were diagnosed with metopic synostosis and bilateral or unilateral radioulnar synostosis. Additionally, one proband had global developmental delay. Echocardiographic evaluation did not reveal cardiac or outflow tract abnormalities. MOLECULAR ANALYSES: The novel missense (c.[584T>G];[584T>G], p.[(Val195Gly)];[(Val195Gly)]) and missense/splice-site variant (c.[817G>A];[817G>A], r.[(817g>a,817delins[a;817+2_817+228])];[(817g>a,817delins[a;817+2_817+228])], p.[(Glu273Lys,Glu273Serfs*72)];[(Glu273Lys,Glu273Serfs*72)]) both locate in the functional MH1 domain of the protein and have not been reported in gnomAD database. Functional analyses of the variants showed reduced inhibition of BMP signalling or abnormal splicing, respectively, consistent with a hypomorphic mechanism of action. CONCLUSION: Our data expand the spectrum of variants and phenotypic spectrum associated with homozygous variants of SMAD6 to include craniosynostosis.

Lewy body radius growth: The hypothesis of the cube root of time dependency.

This paper presents a model for the growth of Lewy bodies (LBs), which are pathological hallmarks of Parkinson's disease (PD). The model simulates the growth of classical LBs, consisting of a core and a halo. The core is assumed to comprise lipid membrane fragments and damaged organelles, while the halo consists of radiating alpha-synuclein (α-syn) fibrils. The Finke-Watzky model is employed to simulate the aggregation of lipid fragments and α-syn monomers. Analytical and numerical exploration of the governing equations yielded approximate solutions applicable for larger times. The application of these approximate solutions to simulate LB radius growth led to the discovery of the cube root hypothesis, which posits that the LB radius is proportional to the cube root of its growth time. Sensitivity analysis revealed that the LB radius is unaffected by the kinetic rates of nucleation and autocatalytic growth, with growth primarily regulated by the production rates of lipid membrane fragments and α-syn monomers. The model indicates that the formation of large LBs associated with PD is dependent on the malfunction of the machinery responsible for the degradation of lipid membrane fragments, α-syn monomers, and their aggregates.

Assessment of models for calculating the hydrodynamic radius of intrinsically disordered proteins.

Diffusion measurements by pulsed-field gradient NMR and fluorescence correlation spectroscopy can be used to probe the hydrodynamic radius of proteins, which contains information about the overall dimension of a protein in solution. The comparison of this value with structural models of intrinsically disordered proteins is nonetheless impaired by the uncertainty of the accuracy of the methods for computing the hydrodynamic radius from atomic coordinates. To tackle this issue, we here build conformational ensembles of 11 intrinsically disordered proteins that we ensure are in agreement with measurements of compaction by small-angle x-ray scattering. We then use these ensembles to identify the forward model that more closely fits the radii derived from pulsed-field gradient NMR diffusion experiments. Of the models we examined, we find that the Kirkwood-Riseman equation provides the best description of the hydrodynamic radius probed by pulsed-field gradient NMR experiments. While some minor discrepancies remain, our results enable better use of measurements of the hydrodynamic radius in integrative modeling and for force field benchmarking and parameterization.

Diversity of hydrodynamic radii of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) form an important class of biomolecules regulating biological processes in higher organisms. The lack of a fixed spatial structure facilitates them to perform their regulatory functions and allows the efficiency of biochemical reactions to be controlled by temperature and the cellular environment. From the biophysical point of view, IDPs are biopolymers with a broad configuration state space and their actual conformation depends on non-covalent interactions of its amino acid side chain groups at given temperature and chemical conditions. Thus, the hydrodynamic radius (Rh) of an IDP of a given polymer length (N) is a sequence- and environment-dependent variable. We have reviewed the literature values of hydrodynamic radii of IDPs determined experimentally by SEC, AUC, PFG NMR, DLS, and FCS, and complement them with our FCS results obtained for a series of protein fragments involved in the regulation of human gene expression. The data collected herein show that the values of hydrodynamic radii of IDPs can span the full space between the folded globular and denatured proteins in the Rh(N) diagram.

Nanoscale perfluorocarbon expediates bone fracture healing through selectively activating osteoblastic differentiation and functions.

BACKGROUND AND RATIONALE: Fracture incidence increases with ageing and other contingencies. However, the strategy of accelerating fracture repair in clinical therapeutics remain a huge challenge due to its complexity and a long-lasting period. The emergence of nano-based drug delivery systems provides a highly efficient, targeted and controllable drug release at the diseased site. Thus far, fairly limited studies have been carried out using nanomedicines for the bone repair applications. Perfluorocarbon (PFC), FDA-approved clinical drug, is received increasing attention in nanomedicine due to its favorable chemical and biologic inertness, great biocompatibility, high oxygen affinity and serum-resistant capability. In the premise, the purpose of the current study is to prepare nano-sized PFC materials and to evaluate their advisable effects on promoting bone fracture repair. RESULTS: Our data unveiled that nano-PFC significantly enhanced the fracture repair in the rabbit model with radial fractures, as evidenced by increased soft callus formation, collagen synthesis and accumulation of beneficial cytokines (e.g., vascular endothelial growth factor (VEGF), matrix metalloprotein 9 (MMP-9) and osteocalcin). Mechanistic studies unraveled that nano-PFC functioned to target osteoblasts by stimulating their differentiation and activities in bone formation, leading to accelerated bone remodeling in the fractured zones. Otherwise, osteoclasts were not affected upon nano-PFC treatment, ruling out the potential target of nano-PFC on osteoclasts and their progenitors. CONCLUSIONS: These results suggest that nano-PFC provides a potential perspective for selectively targeting osteoblast cell and facilitating callus generation. This study opens up a new avenue for nano-PFC as a promising agent in therapeutics to shorten healing time in treating bone fracture.

The stability of Magoh and Y14 depends on their heterodimer formation and nuclear localization.

Reduced expression of the Y14 gene is a cause of Thrombocytopenia-absent radius (TAR) syndrome. This gene contains a conserved RNA recognition motif (RRM) in the central region and nuclear localization/export sequences (NLS/NES) in the N-terminal. Y14 and Magoh proteins form tight heterodimers and are the core of exon junction complexes (EJCs), which mediate various processes of mRNA metabolism after transcription. In this report, we found that protein expression levels of exogenously expressed Magoh L136R and Y14 L118R (leucine-to-arginine substitution at amino acid residue 136 and 118 respectively, that results in the formation of the complex being lost) are lower than their wild-types. This reduction is likely caused by protein levels, as no difference in mRNA levels was detected. Meanwhile, a cycloheximide chase assay determined that the degradation rates of Magoh L136R and Y14 L118R were faster than their wild-types. Both Y14 L118R and Magoh L136R lost the ability to form heterodimers with corresponding wild-type proteins. However, Y14 L118R is able to still localize in the nucleus which causes the stability of Y14 L118R to be higher than Magoh L136R. These results reveal that the stability of Magoh and Y14 is not only dependent on the heterodimer structure, but also dependent on nuclear localization.

Langerhans cell histiocytosis and multiple reticulohistiocytomas in a patient with TAR syndrome: An association not previously described.

We describe a patient with thrombocytopenia-absent radius (TAR) syndrome, multisystemic Langerhans cell histiocytosis and multiple reticulohistiocytomas. A mutational study by massive sequencing identified the Val600Glu (V600E) BRAF mutation in the Langerhans cell histiocytosis lesions, but no molecular alterations were found in the reticulohistiocytoma lesions. The concomitant presence in the same patient of more than one type of histiocytosis from two different groups recognized in the most recent Histiocyte Society classification is an extremely rare event. Our case is the first reported case of multisystemic Langerhans cell histiocytosis and multiple reticulohistiocytomas in a patient with TAR syndrome.

Mutations in MECOM, Encoding Oncoprotein EVI1, Cause Radioulnar Synostosis with Amegakaryocytic Thrombocytopenia.

Radioulnar synostosis with amegakaryocytic thrombocytopenia (RUSAT) is an inherited bone marrow failure syndrome, characterized by thrombocytopenia and congenital fusion of the radius and ulna. A heterozygous HOXA11 mutation has been identified in two unrelated families as a cause of RUSAT. However, HOXA11 mutations are absent in a number of individuals with RUSAT, which suggests that other genetic loci contribute to RUSAT. In the current study, we performed whole exome sequencing in an individual with RUSAT and her healthy parents and identified a de novo missense mutation in MECOM, encoding EVI1, in the individual with RUSAT. Subsequent analysis of MECOM in two other individuals with RUSAT revealed two additional missense mutations. These three mutations were clustered within the 8(th) zinc finger motif of the C-terminal zinc finger domain of EVI1. Chromatin immunoprecipitation and qPCR assays of the regions harboring the ETS-like motif that is known as an EVI1 binding site showed a reduction in immunoprecipitated DNA for two EVI1 mutants compared with wild-type EVI1. Furthermore, reporter assays showed that MECOM mutations led to alterations in both AP-1- and TGF-ß-mediated transcriptional responses. These functional assays suggest that transcriptional dysregulation by mutant EVI1 could be associated with the development of RUSAT. We report missense mutations in MECOM resulting in a Mendelian disorder that provide compelling evidence for the critical role of EVI1 in normal hematopoiesis and in the development of forelimbs and fingers in humans.

Mesenchymal stem cells seeded onto tissue-engineered osteoinductive scaffolds enhance the healing process of critical-sized radial bone defects in rat.

Long bone defects comprise one of the most prevalent clinical problems worldwide and the current bone grafting materials have major limitations to repair them. Although tremendous efforts have been made to repair critical-sized long bone defects in animal models, designing an optimal bone tissue-engineered substitute remains one of the main challenges. Hence, this study aims to closely mimic a natural bone healing process by a tissue-engineered construct including osteoinductive materials pre-seeded with bone marrow-derived mesenchymal stem cells (BMSCs). Bioactive glass (BG) was incorporated into the gelatin/nano-hydroxyapatite (G/nHAp) scaffold (conventional one) to improve the bone regeneration process via its osteoinductivity and angiogenic activity. The fabricated G/nHAp and gelatin/nano-hydroxyapatite/bioactive glass (G/nHAp/BG) scaffolds were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM) and analyzed for porosity and degradation rate. The osteogenic capability of fabricated scaffolds with or without BMSCs was then evaluated in vitro and in vivo. Critical-sized radial bone defects in rats were randomly filled with cell-free and BMSC-seeded scaffolds, autograft and a group left empty without any treatment. In vitro analysis showed that the G/nHAp/BG scaffold significantly increased the expression level of osteogenic and angiogenic markers in comparison to the G/nHAp-treated and control groups (P < 0.05). Moreover, the defects treated with the BMSC-seeded scaffolds showed superior bone formation and structural properties compared to the cell-free scaffolds 4 and 12 weeks post surgery. The radiological and histomorphological properties of defects treated by BMSC-seeded scaffolds, especially the BMSC-seeded G/nHAp/BG scaffold, were comparable to those of the autograft group. It is concluded that the combination of osteoconductive materials (i.e., nHAp) with the bioactive ones such as bioactive glass can effectively accelerate the bone regeneration process. In addition, our results demonstrated that the BMSCs have the potential to drastically increase the bone regeneration ability of osteoinductive scaffolds.

Age-dependent alterations in osteoblast and osteoclast activity in human cancellous bone.

It is assumed that the activity of osteoblasts and osteoclasts is decreased in bone tissue of aged individuals. However, detailed investigation of the molecular signature of human bone from young compared to aged individuals confirming this assumption is lacking. In this study, quantitative expression analysis of genes related to osteogenesis and osteoclastogenesis of human cancellous bone derived from the distal radius of young and aged individuals was performed. Furthermore, we additionally performed immunohistochemical stainings. The young group included 24 individuals with an average age of 23.2 years, which was compared to cancellous bone derived from 11 body donators with an average age of 81.0 years. In cancellous bone of young individuals, the osteogenesis-related genes RUNX-2, OSTERIX, OSTEOPONTIN and OSTEOCALCIN were significantly up-regulated compared to aged individuals. In addition, RANKL and NFATc1, both markers for osteoclastogenesis, were significantly induced in cancellous bone of young individuals, as well as the WNT gene family member WNT5a and the matrix metalloproteinases MMP-9. However, quantitative RT-PCR analysis of BMP-2, ALP, FGF-2, CYCLIN-D1, MMP-13, RANK, OSTEOPROTEGERIN and TGFb1 revealed no significant difference. Furthermore, Tartrate-resistant acid phosphatase (TRAP) staining was performed which indicated an increased osteoclast activity in cancellous bone of young individuals. In addition, pentachrome stainings revealed significantly less mineralized bone matrix, more osteoid and an increased bone density in young individuals. In summary, markers related to osteogenesis as well as osteoclastogenesis were significantly decreased in the aged individuals. Thus, the present data extends the knowledge about reduced bone regeneration and healing capacity observed in aged individuals.

Hierarchical Microspheres Constructed from Chitin Nanofibers Penetrated Hydroxyapatite Crystals for Bone Regeneration.

Chitin exists abundantly in crab and shrimp shells as the template of the minerals, which inspired us to mineralize it for fabricating bone grafting materials. In the present work, chitin nanofibrous microspheres were used as the matrix for in situ synthesis of hydroxyapatite (HA) crystals including microflakes, submicron-needles, and submicron-spheres, which were penetrated by long chitin nanofibers, leading to the hierarchical structure. The shape and size of the HA crystals could be controlled by changing the HA synthesis process. The tight interface adhesion between chitin and HA through the noncovanlent bonds occurred in the composite microspheres, and HAs were homogeneously dispersed and bounded to the chitin nanofibers. In our findings, the inherent biocompatibilities of the both chitin and HA contributed the bone cell adhesion and osteoconduction. Moreover, the chitin microsphere with submicron-needle and submicron-sphere HA crystals remarkably promoted in vitro cell adhesion and in vivo bone healing. It was demonstrated that rabbits with 1.5 cm radius defect were almost cured completely within three months in a growth factor- and cell-free state, as a result of the unique surface microstructure and biocompatibilities of the composite microspheres. The microsphere scaffold displayed excellent biofunctions and an appropriate biodegradability. This work opened up a new avenue to construct natural polymer-based organic-inorganic hybrid microspheres for bone regeneration.

Efficacy of Fu-Yuan Capsule in the Healing of Fractures of the Lower End of the Radius in a Rabbit Model.

OBJECTIVE: The aim of the study was to explore the efficacy of Fu-Yuan Capsule in the healing of fractures of the lower end of the radius in a rabbit model. METHODS: After establishing a rabbit fracture model, all animals were randomly divided into the model group (n = 24), the Fu-Yuan Capsule group (n = 24), and the Shenyang Hongyao group (n = 24). The X-ray was applied to observe the course of fracture healing at 2, 4, 6, and 8 weeks after treatment. Haematoxylin-eosin staining and immunohistochemical staining were used to determine the histological change and the expression of bone morphogenetic protein-2 (BMP-2). Serum alkaline phosphatase (ALP), calcium, and phosphorus levels were detected before and after treatment. RESULTS: X-ray showed that the Fu-Yuan Capsule and Shenyang Hongyao groups exhibited abundant callus shadow areas than the model group in a time-dependent manner. In the model group, the fractures exhibited poor recovery with fibrous callus and obstructed bone marrow cavity. In the Fu-Yuan Capsule and Shenyang Hongyao groups, the fracture showed good recovery and restored normal structure with an effective remodeling of the lamellar bone. Immunohistochemical staining showed that the Fu-Yuan Capsule and Shenyang Hongyao groups had higher expressions of BMP-2 than the model group. Furthermore, serum ALP and calcium-phosphorus product in the Fu-Yuan Capsule and Shenyang Hongyao groups were higher than what they were in the model group. CONCLUSIONS: These results suggest that Fu-Yuan Capsule could promote the fracture healing through upregulating BMP-2 expression and increasing serum ALP and calcium-phosphorus product.

Topographical Anatomy of the Distal Ulna Attachment of the Radioulnar Ligament.

PURPOSE: The deep component of the distal radioulnar ligament provides translational stability and rotational guidance to the forearm. However, controversy exists regarding the importance of this structure as well as the nature of its attachment to the distal ulna. We aimed to evaluate the topographic anatomy of the distal ulna attachment of both the superficial and the deep components of the radioulnar ligament and to assess the relationship between its internal and its external morphometry. METHODS: Thirteen human distal ulnae attached by ulnar part of the distal radioulnar ligament were scanned using micro-computed tomography and reconstructed in 3 dimensions. In addition, the distal radioulnar ligaments were examined under polarized light microscopy to determine the histological characteristics of collagen contained within the ligaments. RESULTS: The deep limbs have broad marginal insertions at the fovea, whereas the superficial limbs have a circular and condensed insertion to the ulnar styloid. The center of the deep limb was separated from the base of the ulnar styloid by a mean of 2.0 ± 0.76 mm, and this distance was positively correlated with the width of the ulnar styloid. The mean distance between the center of the ulnar head and the center of the fovea was 2.4 ± 0.58 mm. The proportion of collagen type I was lower in the deep limb than in the superficial limb. CONCLUSIONS: This new observation of the footprint of the radioulnar ligament in the distal ulna indicates that the deep limb may serve as an internal capsular ligament of the distal radioulnar joint, whereas the superficial limb as the external ligament. CLINICAL RELEVANCE: Knowledge of the topographic anatomy of the radioulnar ligament's attachment to the distal ulna may provide a better understanding of distal radioulnar ligament-related pathologies.

Bone mineral density and bone microarchitecture after long-term suppressive levothyroxine treatment of differentiated thyroid carcinoma in young adult patients.

Bone mineral density (BMD) seems not to be decreased in young patients given long-term suppressive doses of levothyroxine (LT4), but information regarding the bone microstructure in these patients is lacking. The aim of this study was to determine whether supraphysiologic doses of LT4, initiated during childhood or adolescence for treatment of differentiated thyroid carcinoma (DTC), have any detrimental effects on bone microarchitecture as evaluated by high-resolution peripheral quantitative computed tomography (HR-pQCT). Seventeen patients (27.3 ± 7.1 years old) with DTC with subclinical hyperthyroidism since adolescence and 34 healthy volunteers matched for age, sex, and body mass index were studied by dual-energy X-ray absorptiometry (DXA) to determine the areal BMD at the lumbar spine, hip, and proximal third of the radius. Volumetric BMD and structural parameters of the trabecular and cortical bone were assessed by HR-pQCT of the distal radius and distal tibia. DTC patients were given suppressive doses of LT4 starting at a mean age of 12.6 years, and the mean duration of treatment was 14.2 years. In DTC patients, clinical parameters did not correlate with DXA or HR-pQCT parameters. No differences were found between the patients and controls with respect to BMD and Z scores at any site evaluated by DXA, and no differences were found in the bone microstructure parameters evaluated by HR-pQCT. This cross-sectional study suggests that long-standing suppressive therapy with LT4 during the attainment of peak bone mass may have no significant adverse effects on bone density or microarchitecture.

Bone density and microarchitecture in endogenous hypercortisolism.

OBJECTIVE: Osteoporosis is a serious and underestimated complication of endogenous hypercortisolism that results in an increased risk of fractures, even in patients with normal or slightly decreased bone mineral density (BMD). Alterations in bone microarchitecture, a very important component of bone quality, may explain bone fragility. The aim of this study was to investigate bone density and microarchitecture in a cohort of patients with endogenous Cushing's syndrome (CS). DESIGN: Cross-sectional study. PATIENTS: Thirty patients with endogenous active CS and fifty-one age-, sex- and body mass index-matched controls were included. MEASUREMENTS: Participants were studied for areal BMD (dual-energy X-ray absorptiometry) of the lumbar spine (LS), femoral neck (FN), total femur (TF) and radius (33%), and for volumetric bone density (vBMD) and structure using high-resolution peripheral quantitative computed tomography (HR-pQCT) of the distal radius and distal tibia. RESULTS: Patients with active CS exhibited lower areal BMD and Z-score values in the LS, FN and TF (P < 0·003 for all comparisons). At HR-pQCT, the patients with CS also had lower cortical area (P = 0·009 at the radius and P = 0·002 at the tibia), lower cortical thickness (P = 0·02 at the radius and P = 0·002 at the tibia), lower cortical density (P = 0·008 at the tibia) and lower total vBMD (P = 0·002 at the tibia). After the exclusion of hypogonadal individuals, the patients with CS maintained the same microarchitectural and densitometric alterations described above. CONCLUSIONS: Endogenous hypercortisolism has deleterious effects on bone, especially on cortical bone microstructure. These effects seem to be a more important determinant of bone impairment than gonadal status.

Bone microarchitecture and estimated strength in 499 adult Danish women and men: a cross-sectional, population-based high-resolution peripheral quantitative computed tomographic study on peak bone structure.

High-resolution peripheral quantitative computed tomography (HR-pQCT) allows in vivo assessment of cortical and trabecular bone mineral density (BMD), geometry, and microarchitecture at the distal radius and tibia in unprecedented detail. In this cross-sectional study, we provide normative and descriptive HR-pQCT data from a large population-based sample of Danish Caucasian women and men (n = 499) aged 20-80 years. In young adults (<35 years), women (n = 100) compared to men (n = 64) had smaller total and cortical areas, inferior metric trabecular indices, higher network inhomogeneity, lower cortical porosity, and lower finite element estimated bone strength. The changes in parameters with age were estimated from multiple regression analyses. In men, with age the greatest changes (from parameter minimum or maximum) until 80 years were found for cortical porosity (1.91 IQR), BV/TV (-1.09 IQR), and trabecular thickness (-0.87 IQR) in the radius and BV/TV (-1.55 IQR), cortical BMD (-1.25 IQR), and cortical porosity (1.25 IQR) in the tibia. In women changes were most pronounced for cortical porosity (4.76 IQR), trabecular inhomogeneity (3.84 IQR), and cortical BMD (-2.86 IQR) in the radius and cortical BMD (-5.06 IQR), cortical porosity (3.86 IQR), and cortical area (-1.64 IQR) in the tibia. These findings emphasize the age- and sex-related differences in bone morphology, with men having a structural advantage over women from early adult life translating into superior indices of bone strength. With age women are further disadvantaged compared to men by greater decrements in cortical and trabecular architecture in the radius and cortical architecture in the tibia.

Occupancy Analysis of Water Molecules inside Channels within 25 Å Radius of the Oxygen-Evolving Center of Photosystem II in Molecular Dynamics Simulations.

At room temperature and neutral pH, the oxygen-evolving center (OEC) of photosystem II (PSII) catalyzes water oxidation. During this process, oxygen is released from the OEC, while substrate waters are delivered to the OEC and protons are passed from the OEC to the lumen through water channels known as the narrow or the O4 channel, broad or the Cl1 channel, and large or the O1 channel. Protein residues lining the surfaces of these channels play a critical role in stabilizing the hydrogen-bonding networks that assist in the process. We carried out an occupancy analysis to better understand the structural and possible substrate water dynamics in full PSII monomer molecular dynamics (MD) trajectories in both the S1 and S2 states. We find that the equilibrated positions of water molecules derived from MD-derived electron density maps largely match the experimentally observed positions in crystallography. Furthermore, the occupancy reduction in MD simulations of some water molecules inside the single-filed narrow channel also correlates well with the crystallographic data during a structural transition when the S1 state of the OEC advances to the S2 state. The overall reduced occupancies of water molecules are the source of their "vacancy-hopping" dynamic nature inside these channels, unlike water molecules inside an ice lattice where all water molecules have a fixed unit occupancy. We propose on the basis of findings in our structural and molecular dynamics analysis that the water molecule occupying a pocket formed by D1-D61, D1-S169, and O4 of the OEC could be the last steppingstone to enter into the OEC and that the broad channel may be favored for proton transfer.

RAPADILINO RECQL4 mutant protein lacks helicase and ATPase activity.

The RecQ family of helicases has been shown to play an important role in maintaining genomic stability. In humans, this family has five members and mutations in three of these helicases, BLM, WRN and RECQL4, are associated with disease. Alterations in RECQL4 are associated with three diseases, Rothmund-Thomson syndrome, Baller-Gerold syndrome, and RAPADILINO syndrome. One of the more common mutations found in RECQL4 is the RAPADILINO mutation, c.1390+2delT which is a splice-site mutation leading to an in-frame skipping of exon 7 resulting in 44 amino acids being deleted from the protein (p.Ala420-Ala463del). In order to characterize the RAPADILINO RECQL4 mutant protein, it was expressed in bacteria and purified using an established protocol. Strand annealing, helicase, and ATPase assays were conducted to characterize the protein's activities relative to WT RECQL4. Here we show that strand annealing activity in the absence of ATP is unchanged from that of WT RECQL4. However, the RAPADILINO protein variant lacks helicase and ssDNA-stimulated ATPase activity. These observations help explain the underlying molecular etiology of the disease and our findings provide insight into the genotype and phenotype association among RECQL4 syndromes.

Bone health of apprentice jockeys using peripheral quantitative computed tomography.

Our aim was to use Peripheral Quantitative Computed Tomography (pQCT) to assess the bone health of male and female apprentice jockeys and age- and sex-matched peers. 2 groups of 25 young adults (n=50) (age range 15-38 years) were comprised of male and female apprentice jockeys, and male and female controls. We used pQCT to measure the distal tibia and distal radius. After covarying for weight and limb length, apprentice jockeys displayed less tibial cortical area and lower strength strain index at 14% distal shaft, 38% mid shaft and 66% proximal sites measured distally than controls (p=0.001). No between group differences were found in cortical density, trabecular area, and trabecular density at the tibia. Compared with controls, apprentice jockeys displayed greater trabecular density at the distal radial site (p=0.001), greater strength strain index at 66% proximal site measured distally (p=0.01), and a lower strength strain index at the distal radius (p=0.006). In conclusion, only trabecular density at the distal radius and strength strain index at the proximal radius were greater in apprentice jockeys than controls. Strategies to increase bone density and bone strength in apprentice jockeys should be considered by relevant industry stakeholders and their health providers.