Regulation of GSK3 cellular location by FRAT modulates mTORC1-dependent cell growth and sensitivity to rapamycin.

The mTORC1 pathway regulates cell growth and proliferation by properly coupling critical processes such as gene expression, protein translation, and metabolism to the availability of growth factors and hormones, nutrients, cellular energetics, oxygen status, and cell stress. Although multiple cytoplasmic substrates of mTORC1 have been identified, how mTORC1 signals within the nucleus remains incompletely understood. Here, we report a mechanism by which mTORC1 modulates the phosphorylation of multiple nuclear events. We observed a significant nuclear enrichment of GSK3 when mTORC1 was suppressed, which promotes phosphorylation of several proteins such as GTF2F1 and FOXK1. Importantly, nuclear localization of GSK3 is sufficient to suppress cell proliferation. Additionally, expression of a nuclear exporter of GSK3, FRAT, restricts the nuclear localization of GSK3, represses nuclear protein phosphorylation, and prevents rapamycin-induced cytostasis. Finally, we observe a correlation between rapamycin resistance and FRAT expression in multiple-cancer cell lines. Resistance to Food and Drug Administration (FDA)-approved rapamycin analogs (rapalogs) is observed in many tumor settings, but the underling mechanisms remain incompletely understood. Given that FRAT expression levels are frequently elevated in various cancers, our observations provide a potential biomarker and strategy for overcoming rapamycin resistance.

Malonyl-CoA is a conserved endogenous ATP-competitive mTORC1 inhibitor.

Cell growth is regulated by the mammalian/mechanistic target of rapamycin complex 1 (mTORC1), which functions both as a nutrient sensor and a master controller of virtually all biosynthetic pathways. This ensures that cells are metabolically active only when conditions are optimal for growth. Notably, although mTORC1 is known to regulate fatty acid biosynthesis, how and whether the cellular lipid biosynthetic capacity signals back to fine-tune mTORC1 activity remains poorly understood. Here we show that mTORC1 senses the capacity of a cell to synthesise fatty acids by detecting the levels of malonyl-CoA, an intermediate of this biosynthetic pathway. We find that, in both yeast and mammalian cells, this regulation is direct, with malonyl-CoA binding to the mTOR catalytic pocket and acting as a specific ATP-competitive inhibitor. When fatty acid synthase (FASN) is downregulated/inhibited, elevated malonyl-CoA levels are channelled to proximal mTOR molecules that form direct protein-protein interactions with acetyl-CoA carboxylase 1 (ACC1) and FASN. Our findings represent a conserved and unique homeostatic mechanism whereby impaired fatty acid biogenesis leads to reduced mTORC1 activity to coordinately link this metabolic pathway to the overall cellular biosynthetic output. Moreover, they reveal the existence of a physiological metabolite that directly inhibits the activity of a signalling kinase in mammalian cells by competing with ATP for binding.

Biomechanical testing of a shape memory alloy suture in a meniscal suture model.

PURPOSE: The purpose of this study was to evaluate the biomechanical results of meniscal repair in vitro by a nitinol suture and compare them with Ethibond (Ethicon, Somerville, NJ) and nylon. METHODS: The 6 testing groups consisted of nitinol, Ethibond, and nylon sutures (No. 2-0 and No. 3-0). Sixty bovine menisci with a vertical longitudinal tear were repaired with 1 horizontal mattress suture and were fully immersed into a water bath, adjusted to a tensile testing machine. All specimens were subjected to tensile testing, and force/displacement curves were obtained. Load to 5-mm gap, load to failure, tensile strength, stiffness, and mode of failure for each suture group were recorded. Statistical analysis included analysis of variance with Bonferroni correction for the post hoc multiple comparisons. RESULTS: Nylon sutures achieved the lowest scores in all measurements. Nitinol achieved better scores, but not significantly better scores, than Ethibond in load to 5-mm gap and stiffness. The No. 2-0 and No. 3-0 nitinol suture repair showed the highest mean tensile strength and load to failure, with significant differences, being 36% and 45% stronger, respectively, than Ethibond. Modes of failure included pulling through the inner segment of meniscus and rupture of the suture at the knot. CONCLUSIONS: This study shows the superior load-to-failure and tensile strength characteristics of nitinol. However, in terms of stiffness and gap resistance force, the results were equivalent to those of Ethibond. Nitinol is an interesting and promising suture. CLINICAL RELEVANCE: Nitinol can be elongated and become soft and flexible for proper suturing at low temperature. At body temperature, it can contract to its original length, providing stronger knots. This may result in more efficient primary stability of meniscal repair, minimizing the chances of loosening during healing and allowing earlier rehabilitation.

Repair of achilles tendon rupture under endoscopic control.

PURPOSE: To evaluate the functional outcomes and complications after endoscopically assisted percutaneous repair of Achilles tendon rupture. METHODS: An arthroscopically assisted percutaneous repair was performed in 20 patients between the ages of 28 and 47 years. Two patients were suffering from a long-standing rupture. Follow-up was 2.5 years. Evaluation entailed Merkel's scale for pain, functional load (weight) bearing capacity, and the heel raise test. For statistical analysis, a random effects Poisson regression model was used. RESULTS: All patients achieved good to excellent outcomes. The median score on Merkel's scale was 600. All patients were able to stand on the tiptoe of the operated leg and none had limitations placed on their daily activities. A 12% decrease in maximum torque and 16.5% decrease in work performance of the injured side were noted. There was no statistical significant difference in the heel raise test between the operated and non-operated leg. No wound problems, re-ruptures, or infections were reported. Two patients (10%) had sural neuralgia; in 1 case, it subsided without further treatment. CONCLUSIONS: Endoscopy in percutaneous Achilles tendon repair is useful in determining the initial gap and providing adequate apposition of the tendon ends. It is a safe technique with good outcomes and minimal complications. Despite its promising results, potential problems include sural neuralgia and some decrease in strength.

Integrative analysis of gut microbiota composition, host colonic gene expression and intraluminal metabolites in aging C57BL/6J mice.

The aging process is associated with diminished colonic health. In this study, we applied an integrative approach to reveal potential interactions between determinants of colonic health in aging C57BL/6J mice. Analysis of gut microbiota composition revealed an enrichment of various potential pathobionts, including Desulfovibrio spp., and a decline of the health-promoting Akkermansia spp. and Lactobacillus spp. during aging. Intraluminal concentrations of various metabolites varied between ages and we found evidence for an increased gut permeability at higher age. Colonic gene expression analysis suggested that during the early phase of aging (between 6 and 12 months), expression of genes involved in epithelial-to-mesenchymal transition and (re)organization of the extracellular matrix were increased. Differential expression of these genes was strongly correlated with Bifidobacterium spp. During the later phase of aging (between 12 and 28 months), gene expression profiles pointed towards a diminished antimicrobial defense and were correlated with an uncultured Gastranaerophilales spp. This study demonstrates that aging is associated with pronounced changes in gut microbiota composition and colonic gene expression. Furthermore, the strong correlations between specific bacterial genera and host gene expression may imply that orchestrated interactions take place in the vicinity of the colonic wall and potentially mediate colonic health during aging.

Can elbow-extension test be used as an alternative to radiographs in primary care?

OBJECTIVE: To evaluate the ability of elbow extension, with the patient in a supine position, as a diagnostic test of an insignificant injury, with the purpose of avoiding unnecessary radiographs. METHODS: Seventy patients suffering from an acute elbow injury were examined at the accident and emergency department. Inability to fully actively extend the elbow in a supine position was defined as a positive diagnostic test. Radiographs were interpreted by a consultant radiologist, blinded to all clinical examination results. Sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios along with their 95% confidence intervals were calculated for the elbow-extension test. RESULTS: Forty out of 70 patients had a positive test. Elbow fracture or dislocation was identified radiographically in 22 patients with positive test (sensitivity 92%). Two out of 30 (with negative test) had a hairline radial head fracture, which was found on radiographs (specificity 61%). CONCLUSION: Elbow extension as a diagnostic test in a primary care setting can predict severe elbow injuries and can be safely used in practices with no radiology facilities.

Rejection reaction to stabilizing bolts after ACL reconstruction: a case report.

A case of an adverse reaction to the stabilizing bolts after an ACL reconstruction is presented. A 21-year-old patient had an ACL reconstruction using the Mark II system. Six months post op, he presented to clinic having formed a pretibial swelling with serous discharge. There was mild tenderness over the femoral scar. Successive cultures of the tibial cyst effluent showed no microorganism growth and laboratory tests were normal. One year post op, symptoms and swelling persisted. X-rays showed periosteal reaction around the femoral bolt and resorption with widening of the distal tibial tunnel. An adverse reaction to the bolts was suggested as the possible cause. Surgical exploration was then performed. During the operation, free fluid evacuated from the femoral site and the tibial cyst was completely excised. All specimens were sent for culture, which were proven negative. Tibial and femoral bolts were both removed and no communication of the osseous tunnels to the joint was found. An arthroscopy was performed at the same time which showed degeneration of the ACL autograft, but an otherwise normal joint. Eighteen months later, the patient was symptom free and all inflammation indices still within the normal range.