Muscle insulin resistance is a primary metabolic defect underlying the etiology of type 2 diabetes. Consumption of a high-fat/high-calorie diet (HFD) is associated with the development of insulin resistance and type 2 diabetes, while calorie restriction (CR) (60% of ad libitum [AL] food intake) robustly reverses muscle insulin resistance. Remarkably, the signaling mechanisms linking nutrient status to muscle insulin sensitivity are incompletely defined. Considering that the prevalence of diabetes continues to grow at an alarming rate, addressing this fundamental gap in knowledge has the potential to significantly impact human health. Thus, the objective of this dissertation was to define the mechanisms underlying muscle insulin action in the context of nutrient availability. Sirtuin 1 (SIRT1), a NAD+-dependent protein deacetylase, has been proposed to link perturbations in nutrient availability to muscle insulin action. In line with this, SIRT1 activity in skeletal muscle is increased by CR and decreased by HFD. Furthermore, mice with transgenic overexpression of SIRT1 exhibit a metabolic profile resembling CR, and small molecule activators of SIRT1 enhance insulin sensitivity in rodent models of insulin resistance. Nevertheless, little is known regarding the regulatory role of SIRT1 in muscle insulin action, particularly in vivo. We recently identified the SIRT1 target, signal transducer and activator of transcription 3 (STAT3), as an important regulator of muscle insulin sensitivity in response to CR. Furthermore, STAT3 is activated in insulin resistant states and inhibition of STAT3 in liver restores hepatic insulin sensitivity. Thus, we hypothesized that SIRT1 and STAT3 regulate muscle insulin sensitivity in response to nutrient availability. To address this hypothesis, we investigated muscle insulin signaling and sensitivity in response to HFD and CR using novel mouse models in which we modulated SIRT1 and STAT3 activity in skeletal muscle. Our findings demonstrate that muscle-specific overexpression of SIRT1 does not enhance muscle insulin action under AL or CR conditions or prevent HFD-induced obesity or insulin resistance. Finally, we showed that knockout of STAT3 in skeletal muscle does not enhance muscle insulin sensitivity on a control diet or HFD. Taken together, the studies from this dissertation broaden our understanding of the roles of SIRT1 and STAT3 in muscle insulin action in response to nutrient availability

The pediatric lower extremity has well known growth patterns. When deformities or growth disturbances occur, there are several methods to measure and predict the resulting discrepancy, including the Green-Anderson, Moseley, and Multiplier methods. Many techniques exist to correct leg length discrepancy and deformity such and temporary epiphysiodesis, permanent epiphysiodesis, external fixators, and internal lengthening devices. All of these methods have numerous complications and limitations; however, with careful planning and patient selection, length and alignment can be improved with high patient satisfaction.

Background

The Ponseti method is the standard of care for managing idiopathic congenital talipes equinovarus (clubfoot) in the outpatient setting, but there are no clinical guidelines for inpatient treatment. Children in the neonatal intensive care unit (NICU) with clubfoot often delay treatment initiation due to medical reasons.

Methods

We systematically reviewed literature related to the treatment of clubfoot in the NICU, non-idiopathic clubfoot, and older infants, as well as barriers to care.

Results

In a mixed NICU population of syndromic and idiopathic clubfoot, the Ponseti method has good functional outcomes with minimal interference with medical management. The Ponseti method has good functional outcomes with reduced need for extensive surgical procedures in non-idiopathic clubfoot and idiopathic clubfoot with delayed presentation (under one year of age).

Conclusions

It is possible to begin Ponseti treatment in the NICU without compromising medical management. It is not clear if this confers an advantage over waiting for outpatient casting.

Rationale

The sciatic nerve runs a predictable course combining L4-S3 nerve roots through the true pelvis and under the greater sciatic notch. There are reports of bony protuberances from the sacrum and ilium in cases of spinal dysraphism; however advanced imaging, treatment, or outcomes are not described. There are no cases with associated fibular hemimelia in the current literature.

Patient concerns

This is a 4-year-old girl with tethered cord, acetabular dysplasia with hip subluxation, congenital short femur, anterior cruciate ligament (ACL) deficiency, and fibular hemimelia with her sciatic nerve coursing through the ilium.

Diagnosis

Aberrant course of the sciatic nerve through the ilium in the setting of spinal dysraphism.

Outcomes

The hip subluxation was treated with a femoral varus derotation osteotomy and Salter osteotomy with transposition of the sciatic nerve into the greater sciatic notch resulting in a stable hip with no sciatic nerve symptoms at last follow-up.

Lessons

The combination of spinal dysraphism with acetabular dysplasia should be a warning for anomalous sciatic nerveanatomy, possibly through the ilium. Preoperative imaging (MRI, CT scan) may be obtained and carefully reviewed for the course of the sciatic nerve prior to pelvic or femoral osteotomy. Decompressing the sciatic nerve from the aberrant foramen may be considered as part of the procedure.

Objective

Increased signal transducer and activator of transcription 3 (STAT3) signaling has been implicated in the development of skeletal muscle insulin resistance, though its contribution, in vivo, remains to be fully defined. Therefore, the aim of this study was to determine whether knockout of skeletal muscle STAT3 would prevent high-fat diet (HFD)-induced insulin resistance.

Methods

We used Cre-LoxP methodology to generate mice with muscle-specific knockout (KO) of STAT3 (mKO). Beginning at 10 weeks of age, mKO mice and their wildtype/floxed (WT) littermates either continued consuming a low fat, control diet (CON; 10% of calories from fat) or were switched to a HFD (60% of calories from fat) for 20 days. We measured body composition, energy expenditure, oral glucose tolerance and in vivo insulin action using hyperinsulinemic-euglycemic clamps. We also measured insulin sensitivity in isolated soleus and extensor digitorum longus muscles using the 2-deoxy-glucose (2DOG) uptake technique.

Results

STAT3 protein expression was reduced ∼75-100% in muscle from mKO vs. WT mice. Fat mass and body fat percentage did not differ between WT and mKO mice on CON and were increased equally by HFD. There were also no genotype differences in energy expenditure or whole-body fat oxidation. As determined, in vivo (hyperinsulinemic-euglycemic clamps) and ex vivo (2DOG uptake), skeletal muscle insulin sensitivity did not differ between CON-fed mice, and was impaired similarly by HFD.

Conclusions

These results demonstrate that STAT3 activation does not underlie the development of HFD-induced skeletal muscle insulin resistance.

Smad4 is an intracellular effector of the TGFβ family that has been implicated in Myhre syndrome, a skeletal dysplasia characterized by short stature, brachydactyly and stiff joints. The TGFβ pathway also plays a critical role in the development, organization and proliferation of the growth plate, although the exact mechanisms remain unclear. Skeletal phenotypes in Myhre syndrome overlap with processes regulated by the TGFβ pathway, including organization and proliferation of the growth plate and polarity of the chondrocyte. We used in vitro and in vivo models of Smad4 deficiency in chondrocytes to test the hypothesis that deregulated TGFβ signaling leads to aberrant extracellular matrix production and loss of chondrocyte polarity. Specifically, we evaluated growth plate chondrocyte polarity in tibiae of Col2-Cre^+/-;Smad4^fl/fl mice and in chondrocyte pellet cultures. In vitro and in vivo, Smad4 deficiency decreased aggrecan expression and increased MMP13 expression. Smad4 deficiency disrupted the balance of cartilage matrix synthesis and degradation, even though the sequential expression of growth plate chondrocyte markers was intact. Chondrocytes in Smad4-deficient growth plates also showed evidence of polarity defects, with impaired proliferation and ability to undergo the characteristic changes in shape, size and orientation as they differentiated from resting to hypertrophic chondrocytes. Therefore, we show that Smad4 controls chondrocyte proliferation, orientation, and hypertrophy and is important in regulating the extracellular matrix composition of the growth plate.

Purpose

Cerebral palsy (CP) is the most common motor disorder in childhood. Scoliosis is a common complication of CP that can reach clinically severe levels, but predictors for scoliosis in CP are not well understood. Some variables identified in the literature involve the severity of the brain injury and the presence of hip deformity. We aimed to identify associations with developing severe scoliosis in a prospective cohort of patients with cerebral palsy at higher risk for severe curve progression.

Methods

This study reviewed a prospectively collected database at a tertiary children's hospital. We evaluated a panel of potential associations with severe scoliosis-including age, sex, Gross Motor Function Classification System (GMFCS) class, history of hip surgery, epilepsy, and feeding tube presence-in a population of children with limited ambulatory ability defined as GMFCS level IV or V CP. Univariate analysis and multivariate logistic regression with stepwise selection was used for analysis.

Results

Descriptive analysis showed that female sex, higher GMFCS class, history of hip surgery, non-upright seating, pelvic obliquity, presence of epilepsy, and presence of a feeding tube were associated with an increased risk for scoliosis. Multivariate logistic regression analysis revealed that the presence of a feeding tube was associated with severe scoliosis even when controlling for GMFCS and age.

Conclusions

Feeding tube use may stratify risk for severe scoliosis progression in patients with GMFCS IV or V CP.

Purpose

There are over 500 medically related applications (apps) for mobile devices. Very few of these applications undergo testing and peer-review for accuracy. The purpose of this study is to assess the accuracy of limb deformity measurements on the Bone Ninja app compared to PACS and to determine the intra- and inter-observer variability among different orthopaedic practitioners.

Methods

Four participants (attending, senior and junior resident, and physician assistant) measured the leg length (LL), the lateral distal femoral angle (LDFA), and the medial proximal tibial angle (MPTA) of 48 limbs (24 patients), twice with both Bone Ninja and PACS. The difference between the measurements obtained with the Bone Ninja app and PACS were measured. We determined the consistency of the intra-observer intra-class correlation coefficient (ICC) for both systems.

Results

There were no statistical differences in leg length discrepancy (LLD), MPTA, or LDFA measurements between Bone Ninja and PACS (p = 0.96, 0.87, and 0.97, respectively). The intra-observer ICC for the LL, LDFA, and MPTA was similar between Bone Ninja and PACS (0.83, 0.89, and 0.96 vs. 0.96, 0.93, and 0.95, respectively). The inter-observer ICC was similar between Bone Ninja and PACS (0.95, 0.96, and 0.99 vs. 0.99, 0.98, and 0.98, respectively).

Conclusions

This study demonstrates that Bone Ninja is an accurate educational tool for measuring LLD, LDFA, and MPTA. Both systems are reliable instruments for evaluating limb length differences and angles on standing radiographs for pre-operative deformity planning and education. This is the first study to evaluate the accuracy of Bone Ninja compared to the gold standard of PACS.

A nanolipoprotein particle (NLP) is a lipid bilayer disc stabilized by two amphipathic "scaffold" apolipoproteins. It has been most notably utilized as a tool for solubilizing a variety of membrane proteins while preserving structural and functional properties. Transfer of functional proteins from NLPs into model membrane systems such as supported lipid bilayers (SLBs) would enable new opportunities, for example, two-dimensional protein crystallization and studies on protein-protein interactions. This work used fluorescence microscopy and atomic force microscopy to investigate the interaction between NLPs and SLBs. When incubated with SLBs, NLPs were found to spontaneously deliver lipid and protein cargo. The impact of membrane composition on lipid exchange was explored, revealing a positive correlation between the magnitude of lipid transfer and concentration of defects in the target SLB. Incorporation of lipids capable of binding specifically to polyhistidine tags encoded into the apolipoproteins also boosted transfer of NLP cargo. Optimal conditions for lipid and protein delivery from NLPs to SLBs are proposed based on interaction mechanisms.