mTOR signaling contributes to chondrocyte differentiation.

The mammalian Target Of Rapamycin (mTOR) is a nutrient-sensing protein kinase that regulates numerous cellular processes. Fetal rat metatarsal explants were used as a physiological model to study the effect of mTOR inhibition on chondrogenesis. Insulin significantly enhanced their growth. Rapamycin significantly diminished this response to insulin through a selective effect on the hypertrophic zone. Cell proliferation (bromodeoxyuridine incorporation) was unaffected by rapamycin. Similar observations were made when rapamycin was injected to embryonic day (E) 19 fetal rats in situ. In the ATDC5 chondrogenic cell line, rapamycin inhibited proteoglycan accumulation and collagen X expression. Rapamycin decreased content of Indian Hedgehog (Ihh), a regulator of chondrocyte differentiation. Addition of Ihh to culture medium reversed the effect of rapamycin. We conclude that modulation of mTOR signaling contributes to chondrocyte differentiation, perhaps through its ability to regulate Ihh. Our findings support the hypothesis that nutrients, acting through mTOR, directly influence chondrocyte differentiation and long bone growth.     

Errors in Patient Positioning for Bone Mineral Density Assessment by Dual X-Ray Absorptiometry: Effect of Technologist Retraining

Improper positioning is one of the factors that can lead to incorrect bone mineral density (BMD) results. This study aimed to assess the frequencies of erroneous positioning during three periods: before retraining of the technologists (BR), after retraining (AR), and at the current timepoint 8 years after retraining (C). The BMD images of the first 150 consecutive patients who underwent DXA of the lumbar spine and hip during each of the three periods were retrospectively reviewed. Patients were excluded if they had severe scoliosis, rendering proper positioning impossible. Each BMD image was assessed by an International Society of Clinical Densitometry certified clinical densitometrist who was blinded to the date of the initial examination. For the lumbar spine in the BR group, the criteria frequently not met were inclusion of both iliac crests (33.8%), straightness (30.3%), and midline positioning (20.4%); the respective frequencies were significantly reduced to 0.8%?5.6%, 2.1%?3.0%, and 0%?2.8% in the AR and C groups (p < 0.05). For the hip in the BR group, the criteria frequently not met were straightness (52.8%) and internal rotation (21.8%); the respective frequencies were significantly reduced to 0%?4.2% and 8.3%?8.4% in the AR and C groups (p < 0.05). Overall improper positioning in the BR group was 49.3% and 57.3% at the lumbar spine and the hip, respectively; the respective frequencies were reduced to 9.3% and 12.7% in the AR group, and to 2.7% and 7.3% in the C group. The least significant change values for the lumbar spine, femoral neck, and total hip also became smaller after retraining. Retraining the technologists improved patient positioning, as evidenced by the decreased frequencies of erroneous positioning and the improved least significant change values after the retraining.     

Exacerbation of the ochronosis of alkaptonuria due to renal insufficiency and improvement after renal transplantation

In alkaptonuria, homogentisate 1,2-dioxygenase deficiency causes tissue accumulation of homogentisic acid (HGA), followed by signs and symptoms of ochronosis. These include massive urinary excretion of HGA, arthritis and joint destruction, pigmentation of cartilage and connective tissue, and cardiac valve deterioration. We describe a 46-year-old man with alkaptonuria and diabetic renal failure whose plasma HGA concentration was twice that of any other alkaptonuria patient, and whose ochronosis progressed much more rapidly than that of his two alkaptonuric siblings. After renal transplantation, the plasma HGA normalized, and the daily urinary excretion of HGA decreased by 2-3g. This case illustrates the critical role of renal tubular secretion in eliminating HGA from the body, and suggests that renal transplantation in a uremic patient not only restores HGA excretion, but may also provide homogentisate 1,2-dioxygenase activity for the metabolism of HGA.     

Real-time quantitative polymerase chain reaction for enumeration of Streptococcus mutans from oral samples

This study compared SYBR Green real-time quantitative PCR (qPCR) with standard plate counting for the enumeration of Streptococcus mutans in oral samples. Oral samples (n = 710) were collected from high-caries-risk children for quantification of S. mutans by qPCR using primer pairs. The S. mutans copy number was calculated with reference to a qPCR quantification cycle (Cq) standard curve and compared with the absorbance value at 600 nm of a standard suspension of S. mutans UA159. The S. mutans copy number results were evaluated in relation to standard plate count (SPC) results obtained from each sample following culture on Petri plates containing S. mutans selective media and reported as colony-forming units (CFUs). The mean S. mutans copy number calculated from qPCR was higher than the SPC CFUs (1.3 × 10(6) and 1.5 × 10(5) CFUs, respectively). The qPCR values were usually higher in individual samples and qPCR detected the presence of S. mutans 84% (231/276) of the time that the SPC did not, compared with 33% (4/12) of the time when qPCR failed to detect S. mutans and the SPC did. The qPCR technique was found to be more sensitive for detection of S. mutans from oral samples, a method that is not dependent on the viability of the sample taken and therefore is proposed as a more reliable and efficient means of quantification of S. mutans.     

The role of insulin in chondrogenesis

The ATDC5 chondrogenic cell line is typically induced to differentiate by exposure to insulin at high concentration (10 microg/ml, approximately 1600 nM). Differentiation can also be induced by physiological concentrations of insulin-like growth factor-I (IGF-I). Unlike previous reports, we observed a stimulation of differentiation, as measured by collagen X expression and Alcian Blue staining for proteoglycan synthesis, upon exposure to insulin at concentrations (10-50 nM) consistent with signaling via the insulin receptor. Analysis of lysates from proliferating and hypertrophic ATDC5 cells demonstrated that exposure to 50 nM insulin induced tyrosine phosphorylation of insulin receptors but not IGF-I receptors or hybrid receptors. In contrast to the potent effects of IGF-I to stimulate both ATDC5 proliferation and differentiation, insulin was not as potent as IGF-I as a proliferating agent but more selectively a differentiating agent. Consistent with this result, insulin was less potent than IGF-I in inducing activation of the Erk1/Erk2 mitogenic signaling pathway. Furthermore, Erk pathway inhibition did not enhance the differentiating effects of insulin as it does in the case of IGF-I exposure. Extending our observations to fetal rat metatarsal explants, we observed significant stimulation of bone growth by 50 nM insulin. This could be accounted for by a disproportionate stimulatory effect on growth of the hypertrophic zone. The proliferative zone was not significantly affected. Based on our results in both ATDC5 cells and metatarsal explants, we conclude that the insulin functioning through insulin receptor has a dominant effect as an inducer of chondrocyte differentiation. These results support assignment of a physiological role for this hormone in linear bone growth.     

<Emphasis Type="Italic">PRKAG2</Emphasis> mutations presenting in infancy

PRKAG2 encodes the γ2 subunit of AMP-activated protein kinase (AMPK), which is an important regulator of cardiac metabolism. Mutations in PRKAG2 cause a cardiac syndrome comprising ventricular hypertrophy, pre-excitation, and progressive conduction-system disease, which is typically not diagnosed until adolescence or young adulthood. However, significant variability exists in the presentation and outcomes of patients with PRKAG2 mutations, with presentation in infancy being underrecognized. The diagnosis of PRKAG2 can be challenging in infants, and we describe our experience with three patients who were initially suspected to have Pompe disease yet ultimately diagnosed with mutations in PRKAG2. A disease-causing PRKAG2 mutation was identified in each case, with a novel missense mutation described in one patient. We highlight the potential for patients with PRKAG2 mutations to mimic Pompe disease in infancy and the need for confirmatory testing when diagnosing Pompe disease.