•Deletion of Pofut2 (Protein O-fucosyltransferase 2) in mouse developing limb mesenchyme blocks O-fucosylation of Thrombospondin type-1 repeats (TSR) and significantly shortens limb bones.•Loss of ...TSR O-fucosylation alters collagen and fibrillins distribution during primary endochondral ossification.•Altered ECM remodeling enhances TGF-β and reduces BMP signaling in developing chondrocytes leading to a reduced hypertrophic zone.•Changes in the ECM composition and signaling likely result from simultaneous reduction in the function and/or level of multiple POFUT2 substrates including members of the ADAMTS family.
Many extracellular matrix (ECM) associated proteins that influence ECM properties have Thrombospondin type 1 repeats (TSRs) which are modified with O-linked fucose. The O-fucose is added in the endoplasmic reticulum to folded TSRs by the enzyme Protein O-fucosyltransferase-2 (POFUT2) and is proposed to promote efficient trafficking of substrates. The importance of this modification for function of TSR-proteins is underscored by the early embryonic lethality of mouse embryos lacking Pofut2. To overcome early lethality and investigate the impact of the Pofut2 knockout on the secretion of POFUT2 substrates and on extracellular matrix properties in vivo, we deleted Pofut2 in the developing limb mesenchyme using Prrx1-Cre recombinase. Loss of Pofut2 in the limb mesenchyme caused significant shortening of the limbs, long bones and tendons and stiff joint resembling the musculoskeletal dysplasias in human and in mice with mutations in ADAMTS or ADAMTSL proteins. Limb shortening was evident at embryonic day 14.5 where loss of O-fucosylation led to an accumulation of fibrillin 2 (FBN2), decreased BMP and IHH signaling, and increased TGF-β signaling. Consistent with these changes we saw a decrease in the size of the hypertrophic zone with lower levels of Collagen-X. Unexpectedly, we observed minimal effects of the Pofut2 knockout on secretion of two POFUT2 substrates, CCN2 or ADAMTS17, in the developing bone. In contrast, CCN2 and two other POFUT2 substrates important for bone development, ADAMTS6 and 10, showed a decrease in secretion from POFUT2-null HEK293T cells in vitro. These combined results suggest that the impact of the Pofut2 mutation is cell-type specific. In addition, these observations raise the possibility that the O-fucose modification on TSRs extends beyond promoting efficient trafficking of POFUT2 substrates and has the potential to influence their function in the extracellular environment.
Abstract
Peters plus syndrome (MIM #261540 PTRPLS), characterized by defects in eye development, prominent forehead, hypertelorism, short stature and brachydactyly, is caused by mutations in the ...β3-glucosyltransferase (B3GLCT) gene. Protein O-fucosyltransferase 2 (POFUT2) and B3GLCT work sequentially to add an O-linked glucose β1-3fucose disaccharide to properly folded thrombospondin type 1 repeats (TSRs). Forty-nine proteins are predicted to be modified by POFUT2, and nearly half are members of the ADAMTS superfamily. Previous studies suggested that O-linked fucose is essential for folding and secretion of POFUT2-modified proteins and that B3GLCT-mediated extension to the disaccharide is essential for only a subset of targets. To test this hypothesis and gain insight into the origin of PTRPLS developmental defects, we developed and characterized two mouse B3glct knockout alleles. Using these models, we tested the role of B3GLCT in enabling function of ADAMTS9 and ADAMTS20, two highly conserved targets whose functions are well characterized in mouse development. The mouse B3glct mutants developed craniofacial and skeletal abnormalities comparable to PTRPLS. In addition, we observed highly penetrant hydrocephalus, white spotting and soft tissue syndactyly. We provide strong genetic and biochemical evidence that hydrocephalus and white spotting in B3glct mutants resulted from loss of ADAMTS20, eye abnormalities from partial reduction of ADAMTS9 and cleft palate from loss of ADAMTS20 and partially reduced ADAMTS9 function. Combined, these results provide compelling evidence that ADAMTS9 and ADAMTS20 were differentially sensitive to B3GLCT inactivation and suggest that the developmental defects in PTRPLS result from disruption of a subset of highly sensitive POFUT2/B3GLCT targets such as ADAMTS20.
Purpose Care of children with spina bifida has significantly advanced in the last half century, resulting in gains in longevity and quality of life for affected children and caregivers. Bladder ...dysfunction is the norm in patients with spina bifida and may result in infection, renal scarring and chronic kidney disease. However, the optimal urological management for spina bifida related bladder dysfunction is unknown. Materials and Methods In 2012 the Centers for Disease Control and Prevention convened a working group composed of pediatric urologists, nephrologists, epidemiologists, methodologists, community advocates and Centers for Disease Control and Prevention personnel to develop a protocol to optimize urological care of children with spina bifida from the newborn period through age 5 years. Results An iterative quality improvement protocol was selected. In this model participating institutions agree to prospectively treat all newborns with spina bifida using a single consensus based protocol. During the 5-year study period outcomes will be routinely assessed and the protocol adjusted as needed to optimize patient and process outcomes. Primary study outcomes include urinary tract infections, renal scarring, renal function and bladder characteristics. The protocol specifies the timing and use of testing (eg ultrasonography, urodynamics) and interventions (eg intermittent catheterization, prophylactic antibiotics, antimuscarinic medications). Starting in 2014 the Centers for Disease Control and Prevention began funding 9 study sites to implement and evaluate the protocol. Conclusions The Centers for Disease Control and Prevention Urologic and Renal Protocol for the Newborn and Young Child with Spina Bifida began accruing patients in 2015. Assessment in the first 5 years will focus on urinary tract infections, renal function, renal scarring and clinical process improvements.
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Controllable surface morphology is requisite across a gamut of processes, industries, and applications. The surface morphology of silica-coated polystyrene microspheres was ...controllably modified to enable generation of both smooth and bumpy, or raspberry-like, surfaces. Although smooth and raspberry-like silica shells on polystyrene templates have been demonstrated extensively, the method described here used readily available materials to produce radical changes in surface morphology from a single polystyrene template coated in silica through a facile sol-gel reaction processes. Silica shells were deposited via a sol-gel process (using tetraethyl orthosilicate as the silica precursor) onto 1 to 2 μm diameter anionic polystyrene spheres, fabricated by emulsifier-free polymerization. By varying the concentration of silica precursor and ammonium hydroxide catalyst and altering the electrostatic surface interactions via addition of a cationic polymeric brush, an array of surface topologies was generated. Incremented addition of the ammonium hydroxide base catalyst and sol-gel precursors generated smooth silica shells, whereas identical one-pot reactions led to raspberry-like shells. This modification of sol-gel deposition on large polystyrene cores via means of reactant addition offers additional control over sol-gel shell morphologies.
Targeting epigenetic mechanisms has shown promise against several cancers but has so far been unsuccessful against glioblastoma (GBM). Altered histone 3 lysine 4 methylation and increased ...lysine‐specific histone demethylase 1A (LSD1) expression in GBM tumours nonetheless suggest that epigenetic mechanisms are involved in GBM. We engineered a dual‐action prodrug, which is activated by the high hydrogen peroxide levels associated with GBM cells. This quinone methide phenylaminecyclopropane prodrug releases the LSD1 inhibitor 2‐phenylcyclopropylamine with the glutathione scavenger para‐quinone methide to trigger apoptosis in GBM cells. Quinone methide phenylaminocyclopropane impaired GBM cell behaviours in two‐dimensional and three‐dimensional assays, and triggered cell apoptosis in several primary and immortal GBM cell cultures. These results support our double‐hit hypothesis of potentially targeting LSD1 and quenching glutathione, in order to impair and kill GBM cells but not healthy astrocytes. Our data suggest this strategy is effective at selectively targeting GBM and potentially other types of cancers.
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Glioblastoma (GBM) is the most common primary and malignant brain tumour in adults. We report here a prodrug that releases the LSD1 inhibitor trans‐2‐phenylcyclopropylamine (2‐PCPA) and quinone methide simultaneously through oxidation of a boronate trigger by hydrogen peroxide. This quinone methide phenylaminecyclopropane (Q‐PAC) prodrug impaired GBM cell behaviours in two‐dimensional and three‐dimensional assays, and triggered cell apoptosis in several primary and immortal GBM cell cultures, but not healthy cells.
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Endolymphatic sac tumors are rare neoplasms originating in the endolymphatic sac. Current literature is limited to case reports and small case series. The objective of this study was to ...systematically review the literature to better describe clinical presentation, treatment options, and outcomes in endolymphatic sac tumors.
PubMed, Embase, and Cochrane Library.
A systematic review was performed following Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines describing human endolymphatic sac tumors. Risk of bias was assessed using a validated critical appraisal checklist for case series. Studies without inclusion of individual patient characteristics, corresponding treatments, and outcomes were excluded. Heterogeneity of data precluded meta-analysis.
A total of 82 studies met inclusion criteria, and 253 discrete tumors were analyzed. A total of 23.4% of patients had von Hippel-Lindau disease. Von Hippel-Lindau-associated tumors affected females to males in a 2.4:1 ratio. Patients with von Hippel-Lindau disease displayed earlier average age at diagnosis compared to the sporadic cohort. Surgery was the primary treatment modality and was performed in 88.9% of cases. Adjuvant radiation therapy was employed in 18.7% of cases; 16.2% cases recurred, and 10.6% had progression of residual disease after treatment. Mean time to recurrence or progression was 53.1 ± 52.4 months with a range of 3 to 240 months.
Endolymphatic sac tumors require a high degree of suspicion for early diagnosis. Complete resection is the standard of care. No strong evidence supports routine use of adjuvant radiation therapy. Given the high rate of recurrence and wide-ranging time to recurrence, long-term follow-up is necessary.
In 2008, Browder and colleagues published a meta-analysis on mathematics instruction for learners with significant cognitive disabilities and found that most skills taught to these students were only ...from two of the five strands recommended by the National Council for Teachers of Mathematics (i.e., Number and Operations, and Measurement). A review of the literature since Browder et al. yielded an additional 29 studies. When results from both reviews were compared, a greater percentage of studies taught skills from three strands (i.e., Number and Operations, Geometry, and Algebra), whereas the percentage teaching skills from the Measurement strand decreased and the percentage teaching skills from the Data Analysis and Probability strand was unchanged. In addition, a systematic evaluation of the studies' instructional components found evidence to support the use of systematic instruction, in vivo instruction, system of least prompts strategy, constant time delay strategy, and task-analytic instruction as evidence-based practices for teaching mathematics to learners with significant cognitive disabilities. Implications for practice include the use of systematic instruction and in vivo procedures, the need for practitioners to have a deep understanding of mathematics, and the importance of relevancy when teaching a variety of mathematics skills.
Background
Finger prick blood glucose (BG) monitoring remains a mainstay of management in people with type 2 diabetes (T2DM) who take sulphonylurea (SU) drugs or insulin.
We recently examined patient ...experience of BG monitoring in people with type 1 diabetes (T1DM). There has not been any recent comprehensive assessment of the performance of BG monitoring strips or the patient experience of BG strips in people with T2DM in the UK.
Methods
An online self‐reported questionnaire containing 44 questions, prepared following consultation with clinicians and patients, was circulated to people with T2DM. 186 responders provided completed responses (25.5% return rate). Fixed responses were coded numerically (eg not confident = 0 fairly confident = 1).
Results
Of responders, 84% were treated with insulin in addition to other agents. 75% reported having had an HbA1c check in the previous 6 months.
For those with reported HbA1c ≥ 65 mmol/mol, a majority of people (70%) were concerned or really concerned about the shorter term consequences of running a high HbA1c This contrasted with those who did not know their recent HbA1c, of whom only 33% were concerned/really concerned and those with HbA1c <65 mmol/mol of whom 35% were concerned.
Regarding BG monitoring/insulin adjustment, only 25% of responders reported having sufficient information with 13% believing that the accuracy and precision of their BG metre was being independently checked. Only 9% recalled discussing BG metre accuracy when their latest metre was provided and only 7% were aware of the International Standardisation Organisation (ISO) standards for BG metres. 77% did not recall discussing BG metre performance with a healthcare professional.
Conclusion
The group surveyed comprised engaged people with T2DM but even within this group there was significant variation in (a) awareness of shorter term risks, (b) confidence in their ability to implement appropriate insulin dosage (c) awareness of the limitations of BG monitoring technology. There is clearly an area where changes in education/support would benefit many.
We conducted an online survey of experience of BG monitoring in people with T2DM. Perceptions of shorter vs longer term consequences of hyperglycaemia varied widely. Only 9% recalled discussing BG metre accuracy when their latest metre was provided and 77% did not recall discussing BG metre performance with a healthcare professional ‐ this is clearly an area where changes in education and support would benefit many.
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