The functional characterization of phosphorylation of tristetraprolin at C-terminal NOT1-binding domain.

BACKGROUND: Tristetraprolin (TTP) family proteins contain conserved tandem CCCH zinc-finger binding to AU-rich elements and C-terminal NOT1-binding domain. TTP is phosphorylated extensively in cells, and its mRNA destabilization activity is regulated by protein phosphorylation. METHODS: We generated an antibody against phospho-Serine316 located at the C-terminal NOT1-binding site and examined TTP phosphorylation in LPS-stimulated RAW264.7 cells. Knockout of TTP was created in RAW264.7 cells using CRISPR/Cas9 gene editing to explore TTP functions. RESULTS: We demonstrated that Ser316 was phosphorylated by p90 ribosomal S6 kinase 1 (RSK1) and p38-activated protein kinase (MK2) and dephosphorylated by Protein Phosphatase 2A (PP2A). A phosphorylation-mimic mutant of S316D resulted in dissociation with the CCR4-NOT deadenylase complex through weakening interaction with CNOT1. Furthermore, Ser316 and serines 52 and 178 were independently contributed to the CCR4-NOT complex recruitment in the immunoprecipitation assay using phosphor-mimic mutants. In RAW264.7 macrophages, TTP was induced, and Ser316 was phosphorylated through RSK1 and MK2 by LPS stimulation. Knockout of TTP resulted in TNFα mRNA increased due to mRNA stabilization. Overexpression of non-phosphorylated S316A TTP mutant can restore TTP activity and lead to TNFα mRNA decreased. GST pull-down and RNA pull-down analyses demonstrated that endogenous TTP with Ser316 phosphorylation decreased the interaction with CNOT1. CONCLUSIONS: Our results suggest that the TTP-mediated mRNA stability is modulated by Ser316 phosphorylation via regulating the TTP interaction with the CCR4-NOT deadenylase complex.

Resuming Oral Feeding in Patients With Oral Squamous Cell Carcinoma With Free Anterolateral Thigh Flap Reconstruction.

BACKGROUND: Quality of life and functional improvement have emerged as important goals for patients with oncologic disease. For patients with head and neck cancer, free anterolateral thigh (ALT) flaps serve as reliable reconstruction and provide functional restoration. Nevertheless, factors affecting the resumption of oral feeding are rarely described. This study aimed to evaluate and compare the functional outcomes of oral feeding for patients with different oncologic defect patterns and reconstructive ALT flap designs. METHODS: We retrospectively reviewed patients with head and neck cancer undergoing oncologic ablation and free ALT reconstruction between January 2016 and April 2018 at National Taiwan University Hospital. Patients were categorized into 2 groups as through-and-through (T&T) and non-through-and-through (non-T&T) according to the defect pattern. We further subgrouped T&T patients into lip resection/lip sparing according to lip involvement. Reconstructive ALT flaps were of 2 designs, folded (F-ALT) and chimeric (C-ALT). Outcomes of oral feeding were analyzed using descriptive statistics, and differences between groups were compared using the Student t test. RESULTS: We identified 233 patients who received oncologic ablation and free ALT flap reconstruction. There was no significant difference in functional recovery between the T&T and non-T&T groups (81.2% vs 73%, P = 0.137). However, among patients who succeeded in resuming oral feeding, lip-sparing patients had better functional recovery in terms of early oral feeding within 6 months and nasogastric tube removal compared with lip-resection patients (100% vs 83.3%, P = 0.001). Moreover, the F-ALT design resulted in a higher success rate in resuming oral feeding compared with the C-ALT design (90.5% vs 54.6%, P = 0.032). CONCLUSIONS: Patients with head and neck cancer with T&T defects were associated with higher rates of secondary flap revision and a trend of delayed oral feeding. In the long term, improved oral feeding outcome with the F-ALT design was observed compared with the C-ALT design in the specific group with T&T defect.

Orienting the superficial inferior epigastric artery (SIEA) pedicle in a stacked SIEA-deep inferior epigastric perforator free flap configuration for unilateral tertiary breast reconstruction.

Superficial inferior epigastric artery (SIEA) flaps represent a useful option in autologous breast reconstruction. However, the short-fixed pedicle can limit flap inset options. We present a challenging flap inset successfully addressed by de-epithelialization, turnover, and counterintuitive rotation. A 47-year-old woman underwent left tertiary breast reconstruction with stacked free flaps using right deep inferior epigastric perforator and left SIEA vessels. Antegrade and retrograde anastomoses to the internal mammary (IM) vessels were preferred; additionally, the thoracodorsal vessels were unavailable due to previous latissimus dorsi breast reconstruction. Optimal shaping required repositioning of the lateral ends of the flaps superiorly, which would position the ipsilateral SIEA hemi-flap pedicle lateral to and out of reach of the IM vessels. This problem was overcome by turning the SIEA flap on its long axis, allowing the pedicle to sit medially with the lateral end of the flap positioned superiorly. The de-epithelialized SIEA flap dermis was in direct contact with the chest wall, enabling its fixation. This method of flap inset provides a valuable solution for medializing the SIEA pedicle while maintaining an aesthetically satisfactory orientation. This technique could be used in ipsilateral SIEA flap breast reconstructions that do not require a skin paddle, as with stacked flaps or following nipple-sparing mastectomy.

Galectin-3 and S100A9: Novel Diabetogenic Factors Mediating Pancreatic Cancer-Associated Diabetes.

OBJECTIVE: Pancreatic cancer-associated diabetes (PCDM) is a paraneoplastic phenomenon accounting for 1% of new-onset diabetes. We aimed to identify the mediators of PCDM and evaluate their usefulness in distinguishing PCDM from type 2 diabetes. RESEARCH DESIGN AND METHODS: Secreted proteins of MIA PaCa-2 cells were identified by proteomics, and those with ≥10-fold overexpression in transcriptome analysis were assessed by bioinformatics and glucose uptake assay to identify candidate factors. Expression of factors was compared between tumors with and without PCDM by immunohistochemistry. Serum levels were measured in a training set including PC with and without PCDM, type 2 diabetes, pancreatitis, other pancreatic/peripancreatic tumors, and control subjects (n = 50 each). Cutoff values for differentiation between PCDM and type 2 diabetes from the training set were validated in a test set (n = 41 each). RESULTS: Galectin-3 and S100A9 were overexpressed in tumors with PCDM and dose-dependently suppressed insulin-stimulated glucose uptake in C2C12 myotubes. In the training set, serum galectin-3 and S100A9 levels were exclusively increased in patients with PCDM and distinguished PCDM from type 2 diabetes (area under the curve [AUC] galectin-3: 0.73 [95% CI 0.64-0.83]; S100A9: 0.79 [95% CI 0.70-0.87]). Similar results were observed in the test set (AUC galectin-3: 0.83 [95% CI 0.74-0.92]; S100A9: 0.77 [95% CI 0.67-0.87]), with sensitivity and specificity 72.1% and 86.1%, respectively, for galectin-3 and 69.8% and 58.1% for S100A9 in differentiating between PCDM and type 2 diabetes. CONCLUSIONS: Galectin-3 and S100A9 are overexpressed in PCDM tumors and mediate insulin resistance. Galectin-3 and S100A9 distinguish PCDM from type 2 diabetes in subjects with new-onset diabetes.