Early hyperglycemia is associated with poor gross motor outcome in asphyxiated term newborns.

BACKGROUND: Hyperglycemia after ischemic stroke in adults and after near-drowning in children is associated with a poor neurological outcome. Anaerobic metabolism of glucose leads to buildup of lactic acid, free radical production, mitochondrial failure, and ultimately an increase in neurological injury. In asphyxiated infants, high lactate peaks are seen in the basal ganglia with magnetic resonance spectroscopy. Because motor disability in asphyxiated full-term newborns often relates to injury in the basal ganglia, we hypothesized that hyperglycemia and associated buildup of lactic acid may lead to worse gross motor outcome. METHODS: Glucose, blood gas values, and demographic data were abstracted from the medical records of 41 term infants with asphyxia and without confounding diagnoses. Their Gross Motor Function Classification System scores were determined from the medical record or by structured telephone interviews. RESULTS: The outcomes of 14 infants were considered poor on the basis of death within the first 6 months or moderate-to-severe cerebral palsy (Gross Motor Function Classification System score 1-5). The other 27 infants had no gross motor disability (Gross Motor Function Classification System score 0). The highest recorded blood glucose correlated with poor outcome (P = 0.046 by logistic regression). Infants with hyperglycemia (blood glucose > 150 mg/dL) were more likely to have poor outcome (P = 0.017; odds ratio: 5.9; 95% confidence interval: 1.4-24.7). CONCLUSIONS: High blood glucose in the first 12 hours is associated with poor gross motor outcome in this cohort of asphyxiated term infants. Clinicians should avoid hyperglycemia in managing term infants with asphyxia.

Mutation in osteoactivin decreases bone formation in vivo and osteoblast differentiation in vitro.

We have previously identified osteoactivin (OA), encoded by Gpnmb, as an osteogenic factor that stimulates osteoblast differentiation in vitro. To elucidate the importance of OA in osteogenesis, we characterized the skeletal phenotype of a mouse model, DBA/2J (D2J) with a loss-of-function mutation in Gpnmb. Microtomography of D2J mice showed decreased trabecular mass, compared to that in wild-type mice [DBA/2J-Gpnmb(+)/SjJ (D2J/Gpnmb(+))]. Serum analysis showed decreases in OA and the bone-formation markers alkaline phosphatase and osteocalcin in D2J mice. Although D2J mice showed decreased osteoid and mineralization surfaces, their osteoblasts were increased in number, compared to D2J/Gpnmb(+) mice. We then examined the ability of D2J osteoblasts to differentiate in culture, where their differentiation and function were decreased, as evidenced by low alkaline phosphatase activity and matrix mineralization. Quantitative RT-PCR analyses confirmed the decreased expression of differentiation markers in D2J osteoblasts. In vitro, D2J osteoblasts proliferated and survived significantly less, compared to D2J/Gpnmb(+) osteoblasts. Next, we investigated whether mutant OA protein induces endoplasmic reticulum stress in D2J osteoblasts. Neither endoplasmic reticulum stress markers nor endoplasmic reticulum ultrastructure were altered in D2J osteoblasts. Finally, we assessed underlying mechanisms that might alter proliferation of D2J osteoblasts. Interestingly, TGF-ß receptors and Smad-2/3 phosphorylation were up-regulated in D2J osteoblasts, suggesting that OA contributes to TGF-ß signaling. These data confirm the anabolic role of OA in postnatal bone formation.

Osteoactivin induces transdifferentiation of C2C12 myoblasts into osteoblasts.

Osteoactivin (OA) is a novel osteogenic factor important for osteoblast differentiation and function. Previous studies showed that OA stimulates matrix mineralization and transcription of osteoblast specific genes required for differentiation. OA plays a role in wound healing and its expression was shown to increase in post fracture calluses. OA expression was reported in muscle as OA is upregulated in cases of denervation and unloading stress. The regulatory mechanisms of OA in muscle and bone have not yet been determined. In this study, we examined whether OA plays a role in transdifferentiation of C2C12 myoblast into osteoblasts. Infected C2C12 with a retroviral vector overexpressing OA under the CMV promoter were able to transdifferentiate from myoblasts into osteoblasts. Immunofluorescence analysis showed that skeletal muscle marker MF-20 was severely downregulated in cells overexpressing OA and contained significantly less myotubes compared to uninfected control. C2C12 myoblasts overexpressing OA showed an increase in expression of bone specific markers such as alkaline phosphatase and alizarin red staining, and also showed an increase in Runx2 protein expression. We also detected increased levels of phosphorylated focal adhesion kinase (FAK) in C2C12 myoblasts overexpressing OA compared to control. Taken together, our results suggest that OA is able to induce transdifferentiation of myoblasts into osteoblasts through increasing levels of phosphorylated FAK.

Measurement of bacterial volume by transmission-through-dye imaging.

Transmission-through-dye (TTD) microscopy makes possible direct measurement of bacterial volume, irrespective of cell shape. The technique can be realized on any brightfield microscope and is applicable to bacteria of all shapes. TTD imaging requires that intact bacteria be immobilized on a flat transparent surface, such as a glass coverslip.