Targeted transcriptional activation of silent oct4 pluripotency gene by combining designer TALEs and inhibition of epigenetic modifiers

Specific control of gene activity is a valuable tool to study and engineer cellular functions. Recent studies uncovered the potential of transcription activator-like effector (TALE) proteins that can be tailored to activate user-defined target genes. It remains however unclear whether and how epigenetic modifications interfere with TALE-mediated transcriptional activation. We studied the activity of five designer TALEs (dTALEs) targeting the oct4 pluripotency gene. In vitro assays showed that the five dTALEs that target distinct sites in the oct4 promoter had the expected DNA specificity and comparable affinities to their corresponding DNA targets. In contrast to their similar in vitro properties, transcriptional activation of oct4 by these distinct dTALEs varied up to 25-fold. While dTALEs efficiently upregulated transcription of the active oct4 promoter in embryonic stem cells (ESCs) they failed to activate the silenced oct4 promoter in ESC-derived neural stem cells (NSCs), indicating that as for endogenous transcription factors also dTALE activity is limited by repressive epigenetic mechanisms. We therefore targeted the activity of epigenetic modulators and found that chemical inhibition of histone deacetylases by valproic acid or DNA methyltransferases by 5-aza-2′-deoxycytidine facilitated dTALE-mediated activation of the epigenetically silenced oct4 promoter in NSCs. Notably, demethylation of the oct4 promoter occurred only if chemical inhibitors and dTALEs were applied together but not upon treatment with inhibitors or dTALEs only. These results show that dTALEs in combination with chemical manipulation of epigenetic modifiers facilitate targeted transcriptional activation of epigenetically silenced target genes

Rickets in the 21st century

Rickets may have been thought to be a \u27thing of the past\u27. In fact at the beginning of the 20th century, it has been estimated that 85% of children living in northern hemisphere urban industrialised cities had rickets. Major public health initiatives were then introduced to tackle this problem.  In \u27sunny Australia\u27 rickets has not been considered a significant health problem until the last decade or so, when a rising incidence of Vitamin D deficiency rickets occurring in southern states in particular, has been observed, however as yet there has been no systematic approach to prevention. This article outlines both the pathophysiology and clinical features of rickets, highlighting recognition of infants and children at risk

Effect of polyester and Plaster of Paris casts on determination of volumetric bone mineral density assessed by Peripheral Quantitative Computed Tomography (pQCT)

Peripheral quantitative computed tomography (pQCT) is a non-invasive, low-radiation tool for measuring volumetric bone mineral density. It has potential for use in fracture healing applications; however, the unknown attenuation effects of cast material on peripheral quantitative computed tomography have contributed to its limited use in this area. The effect of two common cast materials, polyester and Plaster of Paris was investigated by performing both in vitro and in vivo studies. The in vitro study tested the effect of increasing layers of cast material on bone density measurements performed on a hydroxyapatite phantom. Cast thickness was directly associated with a reduction in bone mineral density, with twelve layers of polyester and Plaster of Paris resulting in a 0.55 and 2.21 % decrease in bone density measurements. Precision error in situ with polyester cast material was 0.71 %, and 2.31 % with Plaster of Paris cast material. The in vivo study comprised a prospective trial with 28 healthy adult participants to evaluate the effect of the two cast materials. Trabecular bone mineral density was increased by 0.5 % in the presence of a polyester cast and decreased by 4.22 % in the presence of a Plaster of Paris cast. Cortical bone mineral density was decreased by 3.46 and 5.54 % for polyester and Plaster of Paris, respectively. This study quantified the effects of orthopaedic casts on pQCT-derived bone parameters. The results suggest applicability of commonly utilised cast materials in combination with pQCT to assess fracture healing

Reply to Comment on Di Marco, N., Kaufman, J., Rodda, C.P. Shedding Light on Vitamin D Status and Its Complexities during Pregnancy, Infancy and Childhood: An Australian Perspective. <i>Int. J. Environ. Res. Public Health</i> 2019, <i>16</i> (4), 538, doi:10.3390/ijerph16040538

We thank the author(s) for their most informative letter in response to our article [...

Comparison of the moulding ability of Plaster of Paris and polyester cast material in the healthy adult forearm

Objectives: To quantify the moulding ability of Plaster of Paris and polyester cast materials as assessed by the novel use of peripheral quantitative computed tomography. Methods: A prospective crossover study was performed in 25 healthy volunteers aged 18–65 years. Participants’ non-dominant wrist was immobilized using a synthetic polyester cast followed by a Plaster of Paris cast with three point moulding to simulate reduction of a dorsally angulated distal radius fracture. The novel use of peripheral quantitative computed tomography was used to measure the closeness of fit of each cast on an axial tomographic slice. Results and conclusions: Plaster of Paris casts were able to achieve a closer mould than polyester when measured between the bone and the cast (p = 0.002), as well as between the skin and the cast (p = 0.001). There was no difference when stratified on BMI. Using pQCT assessment, a closely moulded fit was able to be more consistently achieved when using Plaster of Paris when compared to polyester casts of the distal radius

Maternal vitamin D supplementation during pregnancy prevents vitamin D deficiency in the newborn: an open-label randomized controlled trial.

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The skeletal maturity of Australian children aged 10–13 years in 2016

Skeletal maturity can be used as a biological indicator of the tempo of growth in children and adolescents. We present a description of skeletal maturity from a cohort of white Australian children and describe variation in skeletal maturity based on child age. Participants (n = 71; age 10.5–13.9 years) were recruited from the ‘Healthy, Active Preschool & Primary Years (HAPPY)’ study. Left hand-wrist radiographs were used to determine skeletal maturity using the Tanner-Whitehouse III (TW3) RUS technique. In boys, the mean skeletal maturity offset (bone age – chronological age) was −0.12 ± 0.19 years and 57.9% had delayed skeletal maturity compared to chronological age. Among those with delayed skeletal maturity, the average delay was 0.99 years (range 0.02–2.54 years). In girls, skeletal age was advanced, on average, compared to chronological age by 0.32 ± 0.20 years. Among the 39.4% of girls with delayed skeletal maturity, the average delay was 0.48 years (range: 0.01–2.28). Four children in the sample exhibited a delay in skeletal maturity greater than 2 years. In the context of secular trends towards advanced skeletal maturity observed globally, delayed skeletal maturation in this white, economically privileged cohort are surprising and warrant further exploration

A family with autosomal dominant hypocalcaemia with hypercalciuria (ADHH):Mutational analysis, phenotypic variability and treatment challenges

Autosomal dominant hypocalcaemia with hypercalciuria (ADHH) is an intriguing syndrome, in which activating mutations of the calcium sensing receptor (CaSR) have recently been recognised. We describe a kindred with seven affected individuals across three generations, including patients affected in the first decade of life. Age at diagnosis varied from birth to 50 years. Affected members had hypocalcaemia (1.53-1.85 mmol/l), hypercalciuria, low but detectable parathyroid hormone (PTH) and hypomagnesaemia. Four of seven affected individuals were symptomatic (seizures, abdominal pains and paraesthesias), unrelated to severity of hypocalcaemia. Additional complications include nephrocalcinosis (n = 3) and basal ganglia calcification, identified by CT scanning in all five individuals. Symptomatic individuals were treated with calcium and calcitriol to reduce the risk of hypocalcaemic seizures. DNA sequence analysis, identified a mutation in exon 3, codon 129 (TGC-->TAC) of the CaSR gene of seven affected family members, resulting in loss of a conserved cysteine residue, potentially disrupting CaSR receptor dimerisation. Thus, a novel mutation was identified in this family, who demonstrate variability of ADHH phenotype and also illustrate the complexities of clinical management. Optimal management of ADHH is difficult and we recommend judicious treatment to avoid an increased risk of nephrocalcinosis

Accelerometer-based osteogenic indices, moderate-to-vigorous and vigorous physical activity, and bone traits in adolescents

Objectives: We investigated the associations of accelerometry-derived osteogenic indices (OIs), moderate-to-vigorous (MVPA), and vigorous intensity physical activity (VPA) with peripheral quantitative computed tomography (pCQT) parameters in 99 adolescents aged 10–13 years. Methods: Bone parameters were assessed at the distal (4%) and shaft (66%) of the tibia using pQCT. Accelerometers were worn on the right hip for 7 consecutive days. OIs were calculated based on acceleration peak histograms either using all of the peaks (OI) or peaks with acceleration ≥5.2 g (HOI). MVPA and VPA were defined using previously published cut-points. Results: HOI was positively associated with total area (Partial correlation= 0.22, 95% CI=0.01 to 0.41), cortical area (CoA) (0.33, 95% CI=0.13 to 0.50), and stress strain index (SSI) (0.29, 95% CI=0.09 to 0.47) of tibial shaft and with total density at the distal tibia (0.23, 95% CI=0.02 to 0.42). OI was positively associated with CoA (0.31, 95% CI=0.11 to 0.49) and SSI (0.26, 95% CI=0.05 to 0.44) of tibial shaft. MVPA was positively associated with CoA (0.28, 95% CI=0.07 to 0.46) of the tibial shaft. Conclusions: OI and HOI were positively associated with pQCT parameters while MVPA and VPA demonstrated less consistent associations with them.peerReviewe