In vitro fluorescence assay to measure GDP/GTP exchange of guanine nucleotide exchange factors of Rho family GTPases.

Guanine nucleotide exchange factors (GEFs) are enzymes that promote the activation of GTPases through GTP loading. Whole exome sequencing has identified rare variants in GEFs that are associated with disease, demonstrating that GEFs play critical roles in human development. However, the consequences of these rare variants can only be understood through measuring their effects on cellular activity. Here, we provide a detailed, user-friendly protocol for purification and fluorescence-based analysis of the two GEF domains within the protein, Trio. This analysis offers a straight-forward, quantitative tool to test the activity of GEF domains on their respective GTPases, as well as utilize high-throughput screening to identify regulators and inhibitors. This protocol can be adapted for characterization of other Rho family GEFs. Such analyses are crucial for the complete understanding of the roles of GEF genetic variants in human development and disease.

The dynamic clustering of insulin receptor underlies its signaling and is disrupted in insulin resistance.

Insulin receptor (IR) signaling is central to normal metabolic control and is dysregulated in metabolic diseases such as type 2 diabetes. We report here that IR is incorporated into dynamic clusters at the plasma membrane, in the cytoplasm and in the nucleus of human hepatocytes and adipocytes. Insulin stimulation promotes further incorporation of IR into these dynamic clusters in insulin-sensitive cells but not in insulin-resistant cells, where both IR accumulation and dynamic behavior are reduced. Treatment of insulin-resistant cells with metformin, a first-line drug used to treat type 2 diabetes, can rescue IR accumulation and the dynamic behavior of these clusters. This rescue is associated with metformin's role in reducing reactive oxygen species that interfere with normal dynamics. These results indicate that changes in the physico-mechanical features of IR clusters contribute to insulin resistance and have implications for improved therapeutic approaches.

Tubular approach to minimally invasive microdiscectomy for pediatric lumbar disc herniation.

OBJECTIVE Lumbar disc herniation (LDH) in the pediatric population is rare and exhibits unique characteristics compared with adult LDH. There are limited data regarding the safety and efficacy of minimally invasive surgery (MIS) using tubular retractors in pediatric patients with LDH. Here, the outcomes of MIS tubular microdiscectomy for the treatment of pediatric LDH are evaluated. METHODS Twelve consecutive pediatric patients with LDH were treated with MIS tubular microdiscectomy at the authors' institution between July 2011 and October 2015. Data were gathered from retrospective chart review and from mail or electronic questionnaires. The Macnab criteria and the Oswestry Disability Index (ODI) were used for outcome measurements. RESULTS The mean age at surgery was 17 ± 1.6 years (range 13-19 years). Seven patients were female (58%). Prior to surgical intervention, 100% of patients underwent conservative treatment, and 50% had epidural steroid injections. Preoperative low-back and leg pain, positive straight leg raise, and myotomal leg weakness were noted in 100%, 83%, and 67% of patients, respectively. The median duration of symptoms prior to surgery was 9 months (range 1-36 months). The LDH level was L5-S1 in 75% of patients and L4-5 in 25%. The mean ± SD operative time was 90 ± 21 minutes, the estimated blood loss was ≤ 25 ml in 92% of patients (maximum 50 ml), and no intraoperative or postoperative complications were noted at 30 days. The median hospital length of stay was 1 day (range 0-3 days). The median follow-up duration was 2.2 years (range 0-5.8 years). One patient experienced reherniation at 18 months after the initial operation and required a second same-level MIS tubular microdiscectomy to achieve resolution of symptoms. Of the 11 patients seen for follow-up, 10 patients (91%) reported excellent or good satisfaction according to the Macnab criteria at the last follow-up. Only 1 patient reported a fair level of satisfaction by using the same criteria. Seven patients completed an ODI evaluation at the last follow-up. For these 7 patients, the mean ODI low-back pain score was 19.7% (SEM 2.8%). CONCLUSIONS To the authors' knowledge, this is the longest outcomes study and the largest series of pediatric patients with LDH who were treated with MIS microdiscectomy using tubular retractors. These data suggest that MIS tubular microdiscectomy is safe and efficacious for pediatric LDH. Larger prospective cohort studies with longer follow-up are needed to better evaluate the long-term efficacy of MIS tubular microdiscectomy versus other open and MIS techniques for the treatment of pediatric LDH.