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Despite its critical role in neurodevelopment and brain function, vitamin D (vit-D) homeostasis, metabolism, and kinetics within the central nervous system remain largely undetermined. Thus, it is of critical importance to establish an accurate, highly sensitive, and reproducible method to quantitate vit-D in brain tissue. Here, we present a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method and for the first time, demonstrate detection of seven major vit-D metabolites in brain tissues of C57BL/6J wild-type mice, namely 1,25(OH)2D3, 3-epi-1,25(OH)2D3, 1,25(OH)2D2, 25(OH)D3, 25(OH)D2, 24,25(OH)2D3, and 24,25(OH)2D2. Chromatographic separation was achieved on a pentaflurophenyl column with 3 mM ammonium formate water/methanol [A] and 3 mM ammonium formate methanol/isopropanol [B] mobile phase components. Detection was by positive ion electrospray tandem mass spectrometry with the EVOQ elite triple quadrupole mass spectrometer with an Advance ultra-high-performance liquid chromatograph and online extraction system. Calibration standards of each metabolite prepared in brain matrices were used to validate the detection range, precision, accuracy, and recovery. Isotopically labelled analogues, 1,25(OH)2D3-d3, 25(OH)D3-c5, and 24,25(OH)2D3-d6, served as the internal standards for the closest molecular-related metabolite in all measurements. Standards between 1 fg/mL and 10 ng/mL were injected with a resulting linear range between 0.001 and 1 ng, with an LLOD and LLOQ of 1 pg/mL and 12.5 pg/mL, respectively. The intra-/inter-day precision and accuracy for measuring brain vit-D metabolites ranged between 0.12–11.53% and 0.28–9.11%, respectively. Recovery in acetonitrile ranged between 99.09 and 106.92% for all metabolites. Collectively, the sensitivity and efficiency of our method supersedes previously reported protocols used to measure vit-D and to our knowledge, the first protocol to reveal the abundance of 25(OH)D2, 1,25(OH)D2, and 24,25(OH)2D2, in brain tissue of any species. This technique may be important in supporting the future advancement of pre-clinical research into the function of vit-D in neurophysiological and neuropsychiatric disorders, and neurodegeneration. Graphical Abstract

Essential and nonessential element concentrations in human blood provide important information on the nutritional status of individuals and can assist in the screening or diagnosis of certain disorders and their association with other causative factors. A simple and sensitive method, suitable for use with small sample volumes, for quantification of multiple trace element concentrations in whole blood and plasma has been developed using inductively coupled plasma-mass spectrometry. Method validation was performed using standard reference materials of whole blood and serum using varying sample treatments with nitric acid, water and hydrogen peroxide. The method was applied to quantify the trace element concentrations in whole blood and plasma samples (0.1 mL) from 50 adult blood donors in Queensland. The whole blood sample (5 mL) was collected in Vacutainer tubes with K2EDTA as anticoagulant. The developed method was able to quantify, in blood and plasma samples over a wide range of concentrations, several essential elements: cobalt, copper, zinc, iron, manganese and selenium; the nutritionally probably essential elements vanadium and strontium; and nonessential elements including lead, cadmium, arsenic, caesium, barium, thallium and uranium. Significant differences (P

Distinct neural stem cells (NSCs) reside in different regions of the subventricular zone (SVZ) and generate multiple olfactory bulb (OB) interneuron subtypes in the adult brain. However, the molecular mechanisms underlying such NSC heterogeneity remain largely unknown. Here, we show that the basic helix-loop-helix transcription factor Olig2 defines a subset of NSCs in the early postnatal and adult SVZ. Olig2-expressing NSCs exist broadly but are most enriched in the ventral SVZ along the dorsoventral axis complementary to dorsally enriched Gsx2-expressing NSCs. Comparisons of Olig2-expressing NSCs from early embryonic to adult stages using single cell transcriptomics reveal stepwise developmental changes in their cell cycle and metabolic properties. Genetic studies further show that cross-repression contributes to the mutually exclusive expression of Olig2 and Gsx2 in NSCs/progenitors during embryogenesis, but that their expression is regulated independently from each other in adult NSCs. Finally, lineage-tracing and conditional inactivation studies demonstrate that Olig2 plays an important role in the specification of OB interneuron subtypes. Altogether, our study demonstrates that Olig2 defines a unique subset of adult NSCs enriched in the ventral aspect of the adult SVZ.

Traditional heating using non-renewable energy resources contributes up to 50% of current carbon emission level. Different sources of renewable energy are being exploited and developed to lower the carbon emission level for continuity of healthy living environment. It is found that thermal energy is stored in minewater flooding abandoned mines. The minewater can be extracted through newly drilled boreholes or existing mineshafts. To ensure successful and sustainable operation, mineshafts have to be structurally stable. When the mines are abandoned, the water level tends to recover. Some of the configurations of the minewater heat recovery may change the temperature of part of the shaft wall. This research aims to provide some insight on the stability of mineshafts for minewater heat recovery through numerical sensitivity analyses on: (a) water level, (b) temperature fluctuations. In the presented research work, rock masses with different properties have been analyzed. Change in temperature is found to mainly change the static Young’s Modulus of intact rock and the joint roughness. However, the joint roughness is expressed indirectly using the Geological Strength Index, which has direct relationship with joint roughness and is used in stability analysis. It is found that an increase in water level reduces the integrity of the whole shaft. The degrees of stability deterioration are different at different depths and depend on the in situ stress state. Findings of this analyses can be assist in making a decision on the selection of the appropriate configuration for minewater heat recovery.

Cytochrome P450s (CYP450s) are a rapidly evolving family of enzymes, making it difficult to identify bona fide orthologs with notable lineage-specific exceptions. In ecdysozoans, a small number of the most conserved orthologs include enzymes which metabolize ecdysteroids. Ecdysone pathway components were recently shown in a decapod crustacean but with a notable absence of shade, which is important for converting ecdysone to its active form, 20-hydroxyecdysone (20HE), suggesting that another CYP450 performs a similar function in crustaceans. A CYPome temporal expression analysis throughout metamorphosis performed in this research highlights several un-annotated CYP450s displaying differential expression and provides information into expression patterns of annotated CYP450s. Using the expression patterns in the Eastern spiny lobster Sagmariasus verreauxi, followed by 3D modelling and finally activity assays in vitro, we were able to conclude that a group of CYP450s, conserved across decapod crustaceans, function as the insect shade. To emphasize the fact that these genes share the function with shade but are phylogenetically distinct, we name this enzyme system Shed.

Context Osteogenesis imperfecta (OI) is associated with reduced muscle size, dynamic muscle function, and mobility. Objective To assess the effect of whole body vibration (WBV) on bone density and geometry, muscle size and function, mobility, and balance in children with OI. Design Randomized controlled pilot trial. Setting Tertiary pediatric research center. Participants Twenty-four children (5 to 16 years) with OI types 1, 4, and limited mobility [Child Health Assessment Questionnaire (CHAQ) score ≥ 0.13] recruited in sex- and pubertal stage-matched pairs. Incident fractures in two boys (WBV arm) led to exclusion of two prepubertal pairs. Intervention Five months of WBV training (3 × 3 minutes twice daily) or regular care. Main Outcome Measures Bone and muscle variables measured by dual-energy X-ray absorptiometry (spine, hip, total body) and peripheral quantitative computed tomography (tibia). Mobility assessed by 6-minute walk tests and CHAQ; dynamic muscle function by mechanography. Results All participants had reduced walking distances and muscle function (P < 0.001). Body mass index z score was associated with higher CHAQ scores (ρ + 0.552; P = 0.005) and lower walking and two-leg jumping performance (ρ − 0.405 to −0.654, P < 0.05). The WBV and control groups did not differ in the 5-month changes in bone. Total lean mass increased more in the WBV group [+1119 g (+224 to +1744)] compared with controls [+635 g (−951 to +1006)], P = 0.01, without improving mobility, muscle function, or balance. Conclusions The increase in lean mass without changes in muscle function or bone mass suggests reduced biomechanical responsiveness of the muscle-bone unit in children with OI.

Introduction Capturing the patient experience of living with a rare disease such as X-linked hypophosphataemia (XLH) is critical for a holistic understanding of the burden of a disease. The complexity of the disease coupled with the limited population makes elicitation of the patient burden methodologically challenging. This study used qualitative information direct from patient and caregiver statements to assess the burden of XLH. Methods A thematic analysis was conducted on statements received during a National Institute for Health and Care Excellence (NICE) online public open consultation from 15 June to 6 July 2018. Researchers and clinical experts generated themes and codes based on expected aspects of XLH burden. Statements were independently coded by two reviewers, adding additional codes as required, and analysed by frequency and co-reporting across age groups. Results The majority of responses were submitted from UK-based patients with some from the USA and Australia, and the statements related to children, adolescents and adults. The findings suggest that the greatest burden experienced by children is associated with conventional therapy, co-reported with dosing regimen, adherence, distress and pain. During adolescence, the burden becomes increasingly complex and multi-factorial, with an increasing psychological burden. In adults, conventional therapy co-reported with bone deformity and orthopaedic surgery, as well as pain, mobility, fatigue and dental problems, featured highly. Discussion Whilst our study was opportunistic in nature, it has highlighted the clear and distinctive evolution of the burden of XLH, transitioning from being therapy-oriented in childhood to multi-factorial in adolescence, and finally to adulthood with its high impact on need for other interventions, function and mobility. This qualitative thematic analysis enhances the understanding of the symptom and treatment burden of XLH.

The increasing use of dual-energy X-ray absorptiometry (DXA) in children has led to the need for robust reference data for interpretation of scans in daily clinical practice. Such data need to be representative of the population being studied and be "future-proofed" to software and hardware upgrades. The aim was to combine all available pediatric DXA reference data from seven UK centers to create reference curves adjusted for age, sex, ethnicity, and body size to enable clinical application, using in vivo cross-calibration and making data back and forward compatible. Seven UK sites collected data on GE Lunar or Hologic Scanners between 1996 and 2012. Males and females aged 4 to 20 years were recruited (n = 3598). The split by ethnic group was white 2887; South Asian 385; black Afro-Caribbean 286; and mixed heritage 40. Scans of the total body and lumbar spine (L1 to L4 ) were obtained. The European Spine Phantom was used to cross-calibrate the 7 centers and 11 scanners. Reference curves were produced for L1 to L4 bone mineral apparent density (BMAD) and total body less head (TBLH) and L1 to L4 areal bone mineral density (aBMD) for GE Lunar Prodigy and iDXA (sex- and ethnic-specific) and for Hologic (sex-specific). Regression equations for TBLH BMC were produced using stepwise linear regression. Scans of 100 children were randomly selected to test backward and forward compatibility of software versions, up to version 15.0 for GE Lunar and Apex 4.1 for Hologic. For the first time, sex- and ethnic-specific reference curves for lumbar spine BMAD, aBMD, and TBLH aBMD are provided for both GE Lunar and Hologic scanners. These curves will facilitate interpretation of DXA data in children using methods recommended in ISCD guidelines. The databases have been created to allow future updates and analysis when more definitive evidence for the best method of fracture prediction in children is agreed. © 2016 American Society for Bone and Mineral Research.

This paper uses recently-released material from the ‘migrated archives’ to provide an original counterinsurgency analysis of the TNKU revolt in Brunei and Sarawak from December 1962 to May 1963. It argues that, despite a failure to act upon intelligence predicting the outbreak of insurgency, Britain developed a highly effective counterinsurgency organisation. These records also indicate that decision-makers drew inspiration from the Malayan Emergency to inform success in Brunei. Although Malaya has been challenged as a counterinsurgency paradigm, the Brunei operations show the utility of striking a balance between inappropriately copying from past campaigns and developing best practices applicable to the unique environment of Borneo. In turn, the evolution of effective operational practices in Brunei informed their successful application to the subsequent Indonesian Confrontation.

Various food constituents have been proposed as disease-modifying agents for Alzheimer's disease (AD), due to epidemiological evidence of their beneficial effects, and for their ability to ameliorate factors linked to AD pathogenesis, namely by: chelating iron, copper and zinc; scavenging reactive oxygen species; and suppressing the fibrillation of amyloid-beta peptide (Aβ). In this study, nine different food constituents (l-ascorbic acid, caffeic acid, caffeine, curcumin, (-)-epigallocatechin gallate (EGCG), gallic acid, propyl gallate, resveratrol, and α-tocopherol) were investigated for their effects on the above factors, using metal chelation assays, antioxidant assays, and assays of Aβ42 fibrillation. An assay method was developed using 5-Br-PAPS to examine the complexation of Zn(II) and Cu(II). EGCG, gallic acid, and curcumin were identified as a multifunctional compounds, however their poor brain uptake might limit their therapeutic effects. The antioxidants l-ascorbic acid and α-tocopherol, with better brain uptake, deserve further investigation for specifically addressing oxidative stress within the AD brain.

This article challenges traditional accounts of the 1946 Cold War Crisis in Iran by moving beyond Soviet–American confrontation to focus on British policy. In contrast to the United States, Britain was a major stake-holding power in Iran due to the valuable holdings of the Anglo–Iranian Oil Company (AIOC). By comparing the reactions of the AIOC and Foreign Office, continuity between the events of the 1946 Crisis and later developments in the Mosaddegh premiership becomes apparent. Soviet interference in Azerbaijan prompted great concern from representatives in Iran, but the central Foreign Office pursued a more cautious policy. Only concerns relating to the growth of domestic Iranian communism in the form of the Tudeh Party and the threat this entailed to the British concession prompted the Foreign Office and AIOC to take measures rendering them partially complicit in the internationalization of Iranian politics, setting an important precedent for future action. This article evaluates the policy-m...

Background Bile acids (BAs) are known mediators of glucose metabolism that are altered in type 2 diabetes mellitus (T2DM) and gestational diabetes mellitus (GDM). We hypothesised that post-prandial BA fractions are changed in women with Insulin resistance (IR) after recovery from GDM using homeostatic model assessment (HOMA-IR). Methods 45 women median age 44(31–47) with previous GDM, including 20 with HOMA-IR >2.8 and 25 age-matched controls with HOMA-IR ≤ 2.8 were studied. After an overnight fast, all underwent an oral glucose tolerance test . Blood samples were collected at baseline and every 30 min for 120 min and analysed for glucose on automated platform and for total BAs, their conjugates and fractions using liquid-chromatography tandem mass-spectrometry. Baseline samples were analysed for insulin on automated platform. Delta (Δ) change (difference between baseline and maximal post-prandial response) were calculated. Data is presented as median (IQR). Results Fasting primary and unconjugated BAs were higher in women with HOMA-IR >2.8 vs. those with HOMA-IR ≤ 2.8 [0.24 (0.16–0.33) vs 0.06(0.04–0.22) μmol/L and 0.91(0.56–1.84) μmol/L vs. 0.69(0.32–0.89) μmol/L respectively. ∆ taurine-conjugated BAs was higher in women with HOMA-IR ≤ 2.8 than those with HOMA-IR > 2.8 [0.33(0.20–0.54) vs 0.23(0.13–0.34) μmol/L]. Fasting glucose and non-12α-hydroxylated BAs were negatively correlated in women with HOMA-IR >2.8 (all p Conclusions Following GDM, individuals with HOMA-IR >2.8 have altered conjugated and non-12α-hydroxylated fractions of BAs. It remains to be elucidated if the altered BA metabolism is a contributing factor to the pathogenesis or a consequence of GDM.

Genetic forms of vitamin D-dependent rickets (VDDRs) are due to mutations impairing activation of vitamin D or decreasing vitamin D receptor responsiveness. Here we describe two unrelated patients with early-onset rickets, reduced serum levels of the vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and deficient responsiveness to parent and activated forms of vitamin D. Neither patient had a mutation in any genes known to cause VDDR; however, using whole exome sequencing analysis, we identified a recurrent de novo missense mutation, c.902T>C (p.I301T), in CYP3A4 in both subjects that alters the conformation of substrate recognition site 4 (SRS-4). In vitro, the mutant CYP3A4 oxidized 1,25-dihydroxyvitamin D with 10-fold greater activity than WT CYP3A4 and 2-fold greater activity than CYP24A1, the principal inactivator of vitamin D metabolites. As CYP3A4 mutations have not previously been linked to rickets, these findings provide insight into vitamin D metabolism and demonstrate that accelerated inactivation of vitamin D metabolites represents a mechanism for vitamin D deficiency.

The determination of both the water-soluble and lipid-soluble vitamin D compounds in human biological fluids is necessary to illuminate potentially significant biochemical mechanisms. The lack of analytical methods to quantify the water-soluble forms precludes studies on their role and biological functions; currently available liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods are able to determine only a single sulfated form of Vitamin D. We describe here a highly sensitive and specific LC-MS/MS method for the quantification of four sulfated forms of vitamin D: vitamins D2- and D3-sulfate (D2-S and D3-S) and 25-hydroxyvitamin D2- and D3-sulfate (25(OH)D2-S and 25(OH)D3-S). A comparative evaluation showed that the ionization efficiencies of underivatized forms in negative ion mode electrospray ionisation (ESI) are superior to those of the derivatized (using 4-phenyl-l,2,4-triazoline-3,5-dione (PTAD)) forms in positive ion mode ESI. Separation was optimised to minimise co-elution with endogenous matrix compounds, thereby reducing ion suppression/enhancement effects. Isotopically labelled analogues of each compound were used as internal standards to correct for ion suppression/enhancement effects. The method was validated and then applied for the analysis of breastmilk and human serum. The detection limits, repeatability standard deviations, and recoveries ranged from 0.20 to 0.28fmol, 2.8 to 10.2%, and 81.1 to 102%, respectively.

Intravenous pamidronate has been used in the treatment of osteogenesis imperfecta (OI) in children for over 20 years. The more potent zoledronate is an attractive alternative as it is administered less frequently. This study compares the clinical efficacy of intravenous pamidronate (1.5 mg/kg/day over 2 days, every 3 months) versus zoledronate (0.05 mg/kg/dose every 6 months) in 40 children (20 per group) with mild to moderate OI and the treatment costs of the two drugs in a tertiary centre for children with osteoporosis. Lumbar spine bone mineral density and fracture rate did not differ between drug groups following 1 and 2 years of treatment, respectively. Total cost per treatment course per patient was £1157 for pamidronate and £498 for zoledronate. Therefore, zoledronate is a considerably cheaper alternative to pamidronate with comparable efficacy, resulting in substantial annual savings for healthcare providers and a more convenient option for patients due to fewer hospital visits.

Among the high bone mass disorders, the osteopetroses reflect osteoclast failure that prevents skeletal resorption and turnover, leading to reduced bone growth and modeling and characteristic histopathological and radiographic findings. We report an 11-year-old boy with a new syndrome that radiographically mimics osteopetrosis (OPT), but features rapid skeletal turnover. He presented at age 21 months with a parasellar, osteoclast-rich giant cell granuloma. Radiographs showed a dense skull, generalized osteosclerosis and cortical thickening, medullary cavity narrowing, and diminished modeling of tubular bones. His serum alkaline phosphatase was >5000 IU/L (normal

Context: Acid labile subunit (ALS) deficiency, caused by IGFALS mutations, is a subtype of primary IGF-I deficiency (PIGFD) and has been associated with insulin resistance (IR) and osteopenia. Whether patients respond to recombinant human IGF-I (rhIGF-I) is unknown. Objective and Design: This study determined the 14-hour pharmacokinetic response of free and total IGF-I and IGF binding protein 3 (IGFBP-3) to a single sc dose of rhIGF-I (120 μg/kg) in four ALS-deficient patients, compared with severe PIGFD, moderate PIGFD, and controls. Intravenous glucose tolerance tests, fasting blood levels, dual-energy X-ray absorptiometry, peripheral quantitative computed tomography, and metacarpal radiogrammetry were performed in the four patients and 12 heterozygous family members. Results: IGF-I and IGFBP-3 increased above baseline (P < .05) for 2.5 hours, returning to baseline 7 hours after rhIGF-I injection. Mean (SD) IGF-I Z-score increased by 2.49 (0.90), whereas IGFBP-3 Z-score increased by 0.57 (0.10) only. IGF-I elimination rates in ALS deficiency were similar, but the IGF-I increment was lower than those for severe PIGFD. Significant gene dosage effects were found for all IGF-I peptides, height, forearm muscle size, and metacarpal width. Bone analysis showed that ALS deficiency creates a phenotype of slender bones with normal size-corrected density. Abnormal glucose handling and IR was found in three of four patients and 6 of 12 carriers. Conclusions: These gene dosage effects demonstrate that one functional IGFALS allele is insufficient to maintain normal ALS levels, endocrine IGF-I action, full growth potential, muscle size, and periosteal expansion. Similar gene dosage effects may exist for parameters of IR. Despite similar IGF-I elimination compared with severe PIGFD, ALS-deficient patients cannot mount a similar response. Alternative ways of rhIGF-I administration should be sought.

The occurrence of vitamin D deficiency has become an issue of serious concern in the worldwide population. As a result numerous analytical methods have been developed, for a variety of matrices, during the last few years to measure vitamin D analogs and metabolites. This review employs a comprehensive search of all vitamin D methods developed during the last 5 years for all applications, using ISI Web of Science®, Scifinder®, Science Direct, Scopus and PubMed. Particular emphasis is given to sample-preparation methods and the different forms of vitamin D measured across different fields of applications such as biological fluids, food and pharmaceutical preparations. This review compares and critically evaluates a wide range of approaches and methods, and hence it will enable readers to access developments across a number of applications and to select or develop the optimal analytical method for vitamin D for their particular application.

The controlled release of diflunisal and fluconazole from tablets made of novel polymers, poly(acrylic acid) (PAA) crosslinked with either β-cyclodextrin (βCD) or hydroxypropyl-βCD (HPβCD), was investigated and Carbopol 934P (Carbopol) was used as a highly crosslinked PAA for comparison. Diflunisal strongly associates with βCD-PAA and HPβCD-PAA polymers (Ka of 486 and 6,055 M(-1) respectively); thus, it was physically mixed into the conjugates and also precomplexed to identify whether decomplexation has any influence on release kinetics. Fluconazole has poor complexing ability (Ka of 34 M(-1) with HPβCD-PAA); thus, it was only tested as a physical mixture. Swelling and adhesion studies were conducted on all tablet combinations and adhesivity of the CD-PAA polymer tablets was maintained. Diflunisal release was much slower from HPβCD-PAA tablets than from βCD-PAA, suggesting that a higher degree of complexation retards release. The precomplexed diflunisal release was also slower than the physically mixed diflunisal of the corresponding conjugate. The release closely followed zero-order kinetics for HPβCD-PAA, but was more sigmoidal for βCD-PAA and especially Carbopol. Conversely, poorly associating fluconazole released in almost exactly the same way across both polymers and Carbopol, indicating that the release kinetics of poorly associating drugs are not influenced by the presence of cyclodextrins. In view of the varying profiles and release rates shown with diflunisal for the different polymers, the fluconazole data support the concept that adequate complexation can indeed modulate the release kinetics of drugs.

Chronic inflammation is linked with the generation and progression of various diseases such as cancer, diabetes and atherosclerosis, and anti-inflammatory drugs therefore have the potential to assist in the treatment of these conditions. Carica papaya is a tropical plant that is traditionally used in the treatment of various ailments including inflammatory conditions. A literature search was conducted by using the keywords "papaya", "anti-inflammatory and inflammation" and "immunomodulation and immune" along with cross-referencing. Both in vitro and in vivo investigation studies were included. This is a review of all studies published since 2000 on the anti-inflammatory activity of papaya extracts and their effects on various immune-inflammatory mediators. Studies on the anti-inflammatory activities of recognized phytochemicals present in papaya are also included. Although in vitro and in vivo studies have shown that papaya extracts and papaya-associated phytochemicals possess anti-inflammatory and immunomodulatory properties, clinical studies are lacking.

Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50–70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes., Author Summary Osteogenesis imperfecta (OI) is a heritable disorder of connective tissues characterized by fracture susceptibility and growth deficiency. Most OI cases are caused by autosomal dominant mutations in the genes encoding type I collagen, COL1A1 and COL1A2. Delineation of novel gene defects causing dominant and recessive forms of OI has led to the understanding that the bone pathology results not only from abnormalities in type I collagen quantity and primary structure, but also from defects in post-translational modification, folding, intracellular transport and extracellular matrix incorporation. Recently, mutations in TMEM38B, which encodes the integral ER membrane K+ channel TRIC-B, have been identified as causative for the OI phenotype. However, the mechanism by which absence of TRIC-B causes OI has not been reported. Using cell lines established from three independent probands, we have demonstrated that absence of TRIC-B leads to abnormal ER Ca2+ flux and store-operated calcium entry (SOCE), although ER steady state Ca2+ is normal. Disruption of intracellular calcium dynamics alters the expression and activity of multiple collagen interacting chaperones and modifying enzymes within the ER. Thus TRIC-B deficiency causes OI by dysregulation of collagen synthesis, through the impairment of calcium-dependent gene expression and protein-protein interactions within the ER.

Since the 1990s, Knowledge Management Systems (KMS) have been largely unsuccessful in the collection of tacit knowledge. This includes direct input by the holder of the tacit knowledge into a KMS or through an intermediary such as the collection of tacit knowledge through interviews and videos then posting the data to a KMS. The reasons span the organizational, technological, and individual. In this paper, we concentrate on the tacit knowledge reasons that spans the technological and individual. We believe that tacit knowledge can be effectively and consistently collected from the contributors themselves and placed into a KMS by the contributors using a storytelling-based approach. This study extends past research that collected stories using interviews and videos. The methodology utilizes a storytelling-based KMS, developed in a relational database management system (RDBMS), for Domain Experts (DEs) to contribute their tacit knowledge. It is hypothesized that having contributors use stories to enter their tacit knowledge themselves into a KMS will overcome their reluctance thus making the overall process successful. Once tacit knowledge is entered into a KMS, it becomes explicit knowledge available to anyone who requires it.

Estrogen is used to induce puberty in peripubertal girls with hypogonadism. Although both synthetic and natural forms are available, along with different routes of administration, in the UK oral ethinyl estradiol and the low-dose oral contraceptive pill are commonly used as hormone replacement therapy for practical reasons. We present five peripubertal girls (aged 12.5–14.9 years) with hypogonadism (two with primary hypogonadism due to Turner syndrome and three with central (secondary) hypogonadism as part of multiple pituitary hormone deficiency) who for a variety of reasons have received milligram doses of estradiol (E2) in error for between 6 weeks and 6 months, instead of the expected microgram doses of ethinyl estradiol. Although there are no direct comparisons in peripubertal girls between synthetic and natural estrogens, all girls had vaginal bleeding whilst receiving the milligram doses and have ended up with reduced final heights, below the 9th centile in 1 and below the 2nd centile in 4. Whilst reduction in final height may be part of the underlying condition (especially in Turner syndrome) the two girls with height predictions performed prior to receiving the estrogen overdose have not achieved their predicted height. Estrogen is one of the few drugs which is available in both milligram and microgram formulations. Clinicians need to be alert to the possibility of patients receiving the wrong formulation and dosage in error. Learning points Girls with primary and secondary gonadal failure require assistance with pubertal induction. Although several different formulations and route of administration are available, for practical reasons, the majority of girls in the UK receive oral ethinyl estradiol. Estrogen preparations are available in both milligram and microgram formulations, with potential for receiving the wrong dose. Girls receiving milligram rather than microgram preparations all had vaginal bleeding and a short final height. Background In pediatric practice, there are several groups of female patients who fail to have spontaneous pubertal development. This includes those with primary gonadal failure such as Turner syndrome, or central gonadotropin deficiency secondary to hypothalamo-pituitary dysfunction, e.g. intracranial tumors and their treatment. The role of hormone replacement therapy (HRT) in these patients is to i) mimic physiologic puberty, ii) enhance normal growth, iii) induce and maintain normal menstruation, iv) support normal bone maturation and calcification, v) initiate and maintain normal brain cell growth and plasticity and vi) preserve the woman's psychosexual well-being (1). Whereas in adult practice in the UK the natural estrogen 17-β estradiol is more commonly prescribed as HRT, most UK pediatric endocrinologists use ethinyl estradiol to initiate puberty on the grounds of convenience (2, 3) and also unavailability of low-dose oral natural estrogen preparations. We report five cases of iatrogenic vaginal bleeding due to incorrect prescribing of estradiol in young female hypogonadal patients.

The developmental cycle of the obligate marine antibiotic producer actinobacterium Salinispora arenicola isolated from a Great Barrier Reef marine sponge was investigated in relation to mycelium and spore ultrastructure, synthesis of rifamycin antibiotic compounds, and expression of genes correlated with spore formation and with rifamycin precursor synthesis. The developmental cycle of S. arenicola M413 on solid agar medium was characterized by substrate mycelium growth, change of colony color, and spore formation; spore formation occurred quite early in colony growth but development of black colonies occurred only at late stages, correlated with a change in spore maturity in relation to cell wall layers. Rifamycins were detected throughout the growth cycle, but changed in relative quantity at particular phases in the cycle, with a marked increase after 32 days. Expression of the spore division gene ssgA and the rifK gene for 3-amino-5-hydroxybenzoate synthase responsible for rifamycin precursor synthesis was seen even at early stages of the growth cycle. ssgA expression significantly increased between days 26 and 31, but rifK expression effectively remained constant throughout the growth cycle, consistent with the early synthesis of rifamycin. Factors other than precursor synthesis may be responsible for an observed late increase in rifamycin production. A useful approach for measuring and exploring the regulation of antibiotic synthesis and gene expression in the marine natural product producer S. arenicola has been established.

A study was undertaken to investigate the enzyme-sensitive cleavage sites of β-END (1-31) in serum and in peripheral inflamed tissue. Inflammatory rat paw tissue was homogenized either with PBS (pH 7 .4) or MES (pH 5.5) buffer (1 mg tissue: 10ml buffer) and centrifuged. Rat β-END ( 1-31) was added to the resultant supenatants of the inflamed tissue extracts (to a final concentration of 0.1 mM) and to rat serum (to a final concentration of 74 μM). All samples were incubated at 37°C. Following incubation periods ranging from 0 min to 120 min, a sample (100 μL) was withdrawn and the protein precipitated by the addition of trichloroacetic acid. Each sample was centrifuged and resultant supernatant was analyzed with LC-MS (total ion current spectra) and LC-MS- MS (fragmented ion spectra). The primary enzymatic cleavage of β-END in rat serum occurs mainly at 16- 17-18-19-20 region that represents the amino acids Thr-Leu-Phe-Lys-Asn. For all primary fragments, the cleavage sites observed at pH 7.4 with inflamed tissue (except Lys-Ser at 9-10 position) were similar to those observed with rat serum. At pH 5.5, in addition to the cleavage sites detected in serum and in inflamed tissue at pH 7 .4, other sites were detected. These fragments were produced as a result of hydrolysis of β-END ( 1-31) by a variety of enzymes in the serum and inflammatory tissue. It is possible that the additional cleavage sites detected at pH 5.5 may be due to the enhanced enzymatic activity generated by the acidic environment of the inflammatory matrix.

There is plenty of recognition of the need for more innovation in the construction sector. Increasing levels of competition, rapid technological and regulatory change, the current economic climate and environmental concern all accentuate this requirement. In response, many construction firms are seeking ways to manage innovation more actively and conscientiously in order to remain competitive. However, there is little practical guidance for construction professionals on how to make innovation flourish in their teams. Those who aspire to improve the management of innovation will need to understand how innovation happens, what are the driving forces and how can they be influenced. This paper reports on an empirical investigation that was undertaken to explore some of these questions, specifically the role of organisational climate, customers, risk and complexity on the levels of innovation in teams and the various modes of innovation that prevail – with the aim of providing practitioners with clearer guidance on where efforts should be focused. The findings suggest that there are a limited number of fundamental factors that significantly influence innovation magnitude and mode. For industry professionals it is hoped that this stimulates debate and assists in establishing a much needed foundation for improved innovation management in construction.

Eleven isolates of Mycobacterium species as well as an antimycobacterial Salinispora arenicola strain were cultured from the sponge Amphimedon queenslandica. The 16S rRNA, rpoB, and hsp65 genes from these Mycobacterium isolates were sequenced, and phylogenetic analysis of a concatenated alignment showed the formation of a large clade with Mycobacterium poriferae isolated previously from another sponge species. The separation of these Mycobacterium isolates into three species-level groups was evident from sequence similarity and phylogenetic analyses. In addition, an isolate that is phylogenetically related to Mycobacterium tuberculosis was recovered from the sponge Fascaplysinopsis sp. Several different mycobacteria thus appear to co-occur in the same sponge. An actinobacterium closely related to S. arenicola, a known producer of the antimycobacterial rifamycins, was coisolated from the same A. queenslandica specimen from which mycobacteria had been isolated. This Salinispora isolate was confirmed to synthesize rifamycin and displayed inhibitory effects against representatives from two of three Mycobacterium phylotype groups. Evidence for antagonism of sponge-derived Salinispora against sponge-derived Mycobacterium strains from the same sponge specimen and the production of antimycobacterial antibiotics by this Salinispora strain suggest that the synthesis of such antibiotics may have functions in competition between sponge microbial community members.

Background Mango fruits contain a broad spectrum of phenolic compounds which impart potential health benefits; their biosynthesis is catalysed by enzymes in the phenylpropanoid-flavonoid (PF) pathway. The aim of this study was to reveal the variability in genes involved in the PF pathway in three different mango varieties Mangifera indica L., a member of the family Anacardiaceae: Kensington Pride (KP), Irwin (IW) and Nam Doc Mai (NDM) and to determine associations with gene expression and mango flavonoid profiles. Results A close evolutionary relationship between mango genes and those from the woody species poplar of the Salicaceae family (Populus trichocarpa) and grape of the Vitaceae family (Vitis vinifera), was revealed through phylogenetic analysis of PF pathway genes. We discovered 145 SNPs in total within coding sequences with an average frequency of one SNP every 316 bp. Variety IW had the highest SNP frequency (one SNP every 258 bp) while KP and NDM had similar frequencies (one SNP every 369 bp and 360 bp, respectively). The position in the PF pathway appeared to influence the extent of genetic diversity of the encoded enzymes. The entry point enzymes phenylalanine lyase (PAL), cinnamate 4-mono-oxygenase (C4H) and chalcone synthase (CHS) had low levels of SNP diversity in their coding sequences, whereas anthocyanidin reductase (ANR) showed the highest SNP frequency followed by flavonoid 3’-hydroxylase (F3’H). Quantitative PCR revealed characteristic patterns of gene expression that differed between mango peel and flesh, and between varieties. Conclusions The combination of mango expressed sequence tags and availability of well-established reference PF biosynthetic genes from other plant species allowed the identification of coding sequences of genes that may lead to the formation of important flavonoid compounds in mango fruits and facilitated characterisation of single nucleotide polymorphisms between varieties. We discovered an association between the extent of sequence variation and position in the pathway for up-stream genes. The high expression of PAL, C4H and CHS genes in mango peel compared to flesh is associated with high amounts of total phenolic contents in peels, which suggest that these genes have an influence on total flavonoid levels in mango fruit peel and flesh. In addition, the particularly high expression levels of ANR in KP and NDM peels compared to IW peel and the significant accumulation of its product epicatechin gallate (ECG) in those extracts reflects the rate-limiting role of ANR on ECG biosynthesis in mango. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1784-x) contains supplementary material, which is available to authorized users.

Autosomal recessive hypophosphatemic rickets 2 (ARHR2) is a rare form of renal tubular phosphate wasting disorder. Loss of function mutations of the ecto-nucleotide pyrophosphatase/pyrophosphodiesterase 1 gene (ENPP1) causes a wide spectrum of phenotypes, ranging from lethal generalized arterial calcification of infancy to hypophosphatemic rickets with hypertension. Hearing loss was not previously thought to be one of the features of the disease entities and was merely regarded as a complication rather than a part of the disease. We report two children who presented in mid to late childhood with progressive varus deformity of their legs due to hypophosphatemic rickets caused by mutations in the ENPP1 gene. Both children had evidence of progressive hearing loss requiring the use of hearing aids. This report of two unrelated infants with compound heterozygous mutations in ENPP1 and previously published cases confirms that mild to moderate hearing loss is frequently associated with ARHR2. Early onset conductive hearing loss may further distinguish the autosomal recessive ENPP1 related type from other types of hypophosphatemia.

Involving patients and carers in service improvement projects is an essential aspect of patient-centred care, but happens only rarely. It can bring new perspectives and improve knowledge, health care engagement and health outcomes. While guidelines are available supporting public involvement in service improvement, these do not address the specific needs of patients with PD. The aim of this project is to develop an easy-to-use guide for service development project leads and patients/carers. This will facilitate the involvement of the latter in the development and implementation of PD service improvements. We used a semi-structured interview to capture good practice and barriers to service user involvement at each stage of service development. Local clinicians, patients and carers who have been involved in service improvement projects were invited to participate in telephone interviews. Key themes from initial grounded analysis of the data include: Access, including physical access to rooms and offering alternatives such as teleconferences or video calls; Representation, ensuring a wide range of patients are able to be involved; and Empowerment, enabling patient voice to be heard. The themes will describe how patients have been involved projects and how this could be done better; they will then inform further discussion by a focus group to define and develop the guide.

Osmolytes have been proposed as treatments for neurodegenerative proteinopathies including Alzheimer's disease. However, for osmolytes to reach the clinic their efficacy must be improved. In this work, copper(I)-catalyzed azide-alkyne cycloaddition chemistry was used to synthesize glycoclusters bearing six copies of trehalose, lactose, galactose or glucose, with the aim of improving the potency of these osmolytes via multivalency. A trehalose glycocluster was found to be superior to monomeric trehalose in its ability to retard the formation of amyloid-beta peptide 40 (Aβ40) fibrils and protect neurons from Aβ40-induced cell death.

Marine sponges are a major component of benthic communities and act as a reservoir for microbial species. In terms of biomass, they are the richest source of secondary metabolite production, with the potential to influence both benthic and pelagic systems. In most cases it is the sponge-associated microbes that account for many of the secondary metabolites assigned to the host. Here we report the occurrence of cycloaspeptide A, a fungus-derived cyclic peptide, in a culturable bacterium Salinispora arenicola. We have also identified nazumamide A, a sponge-derived linear tetrapeptide currently used as a thrombin inhibitor, in Salinispora pacifica. Their structures were determined using an integrated approach consisting of: (1) HPLC-UV-Vis-QToF-MS analysis with multimode ionization (ESI and APCI) and fast polarity switching; (2) database searching and matching of monoisotopic masses, retention times, mass spectra of the precursor and product ions of the compounds of interest and the authentic reference standards thereof.

Background The most serious complication of subcutaneous fat necrosis (SCFN), a rare condition of the newborn characterized by indurated purple nodules, is hypercalcaemia. However, the mechanism for this hypercalcaemia remains unclear. Objectives To determine whether the hypercalcaemia associated with SCFN involves expression of the vitamin D-activating enzyme 25-hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase) in affected tissue. Methods Skin biopsies from two male patients with SCFN and hypercalcaemia were taken. The histological specimens were assessed using a polyclonal antibody against 1α-hydroxylase. Results Histology in both cases showed strong expression of 1α-hydroxylase protein (brown staining) within the inflammatory infiltrate associated with SCFN. This was consistent with similar experiments in other granulomatous conditions. Conclusions Hypercalcaemia in SCFN appears to be due to abundant levels of 1α-hydroxylase in immune infiltrates associated with tissue lesions. This is consistent with previous observations of extrarenal 1α-hydroxylase in skin from other granulomatous conditions such as sarcoidosis and slack skin disease.

Hypophosphatemic rickets is commonly an X-linked dominant disorder (XLH or HYP) associated with a renal tubular defect in phosphate transport and bone deformities. The XLH gene, referred to as PHEX, or formerly as PEX (phosphate regulating gene with homologies to endopeptidases on the X-chromosome), encodes a 749-amino acid protein that putatively consists of an intracellular, transmembrane, and extracellular domain. PHEX mutations have been observed in XLH patients, and we have undertaken studies to characterize such mutations in 46 unrelated XLH kindreds and 22 unrelated patients with nonfamilial XLH by single stranded conformational polymorphism and DNA sequence analysis. We identified 31 mutations (7 nonsense, 6 deletions, 2 deletional insertions, 1 duplication, 2 insertions, 4 splice site, 8 missense, and 1 within the 5′ untranslated region), of which 30 were scattered throughout the putative extracellular domain, together with 6 polymorphisms that had heterozygosity frequencies ranging from less than 1% to 43%. Single stranded conformational polymorphism was found to detect more than 60% of these mutations. Over 20% of the mutations were observed in nonfamilial XLH patients, who represented de novo occurrences of PHEX mutations. The unique point mutation (a→g) of the 5′untranslated region together with the other mutations indicates that the dominant XLH phenotype is unlikely to be explained by haplo-insufficiency or a dominant negative effect.

A cell-free extract from Escherichia coli containing an E. coli biotin synthase that was expressed to approx. 1% of soluble cell protein by cloning the E. coli bioB gene was used to investigate the biotin synthase reaction. The pH optimum was between 8 and 8.5, and the reaction velocity was dependent on the concentrations of dethiobiotin, cysteine, S-adenosylmethionine and asparagine. The catalytic-centre activity of the enzyme in vitro was estimated to be 0.95 h-1, and each molecule of enzyme turned over less than one molecule of dethiobiotin, i.e. the enzyme was not acting catalytically. HPLC analysis of reaction mixtures revealed the presence of a compound with the characteristics of an intermediate: (1) it was labelled with 14C, and therefore derived from the [14C]dethiobiotin substrate; (2) it was present only in reaction mixtures containing biotin synthase; (3) it was not derived from [14C]biotin; (4) 35S from [35S]cystine was incorporated into the intermediate during the reaction; (5) its synthesis was dependent on the presence of S-adenosylmethionine, and was decreased when free cysteine was omitted from the reaction; (6) it could be isolated from the reaction mixture by chromatography and then re-introduced into an assay as the substrate, whereupon it was converted to biotin; (7) this conversion to biotin was S-adenosylmethionine-dependent. During the reaction S-adenosylmethionine was cleaved to methionine and presumably 5ʹ-deoxyadenosine. Observation of the intermediate allowed us to perform experiments to determine the stoichiometry of S-adenosylmethionine use. We propose that two molecules of S-adenosylmethionine are used to synthesize one molecule of biotin, i.e. one from dethiobiotin to the intermediate, and a second from the intermediate to biotin.