Structure-based identification of CaMKIIα-interacting MUPP1 PDZ domains and rational design of peptide ligands to target such interaction in human fertilization.

The recognition and association between Ca(2+)/calmodulin-activated protein kinase II-α (CaMKIIα) and multi-PDZ domain protein 1 (MUPP1) plays an important role in sperm acrosome reaction and human fertilization, which is mediated by the binding of CaMKIIα's C-terminal tail to one or more PDZ domains of the scaffolding protein MUPP1. In this study, we attempt to identify the CaMKIIα-interacting MUPP1 PDZ domains and to design peptide ligands that can potently target and then competitively disrupt such interaction. Here, a synthetic biology approach was proposed to systematically characterize the structural basis, energetic property, dynamic behavior and biological implication underlying the intermolecular interactions between the C-terminal peptide of CaMKIIα and all the 13 PDZ domains of MUPP1. These domains can be grouped into four clusters in terms of their sequence, structure and physiochemical profile; different clusters appear to recognize different classes of PDZ-binding motifs. The cluster 3 includes two members, i.e. MUPP1 PDZ 5 and 11 domains, which were suggested to bind class II motif Φ-X-Φ(-COOH) of the C-terminal peptide SGAPSV(-COOH) of CaMKIIα. Subsequently, the two domains were experimentally measured as the moderate- and high-affinity binders of the peptide by using fluorescence titration (dissociation constants K d = 25.2 ± 4.6 and 0.47 ± 0.08 µM for peptide binding to PDZ 5 and 11, respectively), which was in line with theoretical prediction (binding free energies ΔG total = -7.6 and -9.2 kcal/mol for peptide binding to PDZ 5 and 11, respectively). A systematic mutation of SGAPSV(-COOH) residues suggested few favorable amino acids at different residue positions of the peptide, which were then combined to generate a number of potent peptide mutants for PDZ 11 domain. Consequently, two peptides (SIAPNV(-COOH) and SIVMNV(-COOH)) were identified to have considerably improved affinity with K d increase by ~tenfold relative to wild type peptide. Thus, the two peptides are considered as promising lead entities to develop therapeutic molecular agents with high efficacy and specificity to target CaMKIIα-MUPP1 interaction. Other five designed peptides (SILPSV(-COOH), SGLPNV(-COOH), SIVMSV(-COOH), SIVPNV(-COOH) and SIAMNV(-COOH)) possessed comparable affinity with the wild type, and they may be further optimized to obtain higher potency.

Exosomal lncRNA KLF3-AS1 derived from bone marrow mesenchymal stem cells stimulates angiogenesis to promote diabetic cutaneous wound healing.

AIMS: We focused on BMSC-derived exosomal lncRNA KLF3-AS1 and its significance in diabetic cutaneous wound healing. METHODS: Potential interaction between KLF3-AS1 and miR-383, miR-383 and VEGFA were predicted using bioinformatic analysis and validated by luciferase reporter, RIP, and FISH assays. The proliferation, apoptosis, migration and tube formation of HUVECs were evaluated by CCK-8, flow cytometry, wound healing, and tube formation assays, respectively. A murine diabetic cutaneous wound model was used to investigate therapeutic effects of exosomal KLF3-AS1 in vivo. Histological alterations in skin tissues were examined using HE, Masson staining, and immunostaining of CD31. RESULTS: BMSC-derived exosomal KLF3-AS1 sufficiently promoted proliferation, migration, and tube formation, while inhibited apoptosis of HUVECs challenged by high glucose. The protective effects of exosomal KLF3-AS1 were achieved at least partially by down-regulating miR-383, and boosting the expression of its target, VEGFA. In vivo, exosomes from KLF3-AS1-expressing BMSCs demonstrated the best effects in promoting cutaneous wound healing in diabetic mice, which were associated with minimal weight loss, increased blood vessel formation, reduced inflammation, decreased miR-383 expression, and up-regulated VEGFA. CONCLUSIONS: Exosomal lncRNA KLF3-AS1 derived from BMSCs induces angiogenesis to promote diabetic cutaneous wound healing.

Rational design of an orthogonal noncovalent interaction system at the MUPP1 PDZ11 complex interface with CaMKIIα-derived peptides in human fertilization.

The recognition and association between the Ca2+/calmodulin-activated protein kinase II-α (CaMKIIα) and the multi-PDZ domain protein 1 (MUPP1) plays an important role in the sperm acrosome reaction and human fertilization. Previously, we have demonstrated that the MUPP1 PDZ11 domain is the primary binding partner of the CaMKIIα C-terminal tail, which can be targeted by a rationally designed sia peptide with nanomolar affinity. Here, we further introduced an orthogonal noncovalent interaction (ONI) system between a native hydrogen bond and a designed halogen bond across the complex interface of the PDZ11 domain with the sia [Asn-1Phe] peptide mutant, where the halogen bond was formed by substituting the o-hydrogen atom of the benzene ring of the peptide Phe-1 residue with a halogen atom (F, Cl, Br or I). Molecular dynamics simulations and high-level theoretical calculations suggested that bromine (Br) is a good compromise between the halogen-bonding strength and steric hindrance effect due to introduction of a bulkier halogen atom into the tightly packed complex interface. Fluorescence spectroscopy assays revealed that the resulting o-Br-substituted peptide (Kd = 18 nM) exhibited an ∼7.6-fold affinity increase relative to its native counterpart (Kd = 137 nM). In contrast, the p-Br-substituted peptide, a negative control that is unable to establish the ONI according to structure-based analysis, has decreased affinity (Kd = 210 nM) upon halogenation.

A comparison of the Cook-Swartz Doppler with conventional clinical methods for free flap monitoring: A systematic review and a meta-analysis.

INTRODUCTION: Currently there is no consensus on what is the optimal method for monitoring free flaps. Our meta-analysis compared the free flap success and salvage rates of Cook-Swartz Implantable Doppler monitoring with clinical monitoring to gain insight into the relative benefit of these systems. METHODS: Medline, Cochrane, EMBASE, and Google Scholar databases were searched until January 16, 2016. Search terms included free flap surgery, free flap microsurgery and implantable Doppler. Studies were included if they involved the comparison of Cook-Swartz Doppler and clinical assessment for monitoring free flap function. Studies using free flap monitoring as an outcome measure for drug treatment were also excluded. Sensitivity analysis using the leave-one-out approach was used to assay the reliability of the findings. RESULTS: Initial search identified 14 studies, of which five studies were included in the meta-analysis. Cook-Swartz Doppler had significantly better rate of free flap success and salvage than clinical monitoring methods (P values ≤ 0.006). Data did not markedly changed when each study was removed in turn, showing reliability of the findings. DISCUSSION: The Cook-Swartz Doppler as a monitoring method may result in a higher rate of free flap success and salvaging but also a greater frequency of false positives than conventional methods. Our analysis is limited by designs of included studies and by heterogeneity of clinical monitoring techniques. CONCLUSIONS: More studies are needed to evaluate if Cook-Swartz Doppler can be used alone, or to be better used as an adjunctive technique to complement the clinical method of monitoring.

Comparison of pemetrexed and docetaxel as salvage chemotherapy for the treatment for nonsmall-cell lung cancer after the failure of epidermal growth factor receptor-tyrosine kinase inhibitors.

OBJECTIVE: To compare the therapeutic effects and adverse reactions of pemetrexed and docetaxel as salvage chemotherapy in patients with nonsmall-cell lung cancer (NSCLC) after the failure of epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI). METHODS: In this randomized Phase 2 trial, patients with NSCLC who had previously failed EGFR-TKI therapy were randomized to receive intravenous pemetrexed (500 mg/m(2) for 21 days [one cycle]) or docetaxel (75 mg/m(2) for 21 days [one cycle]). Therapeutic effects were evaluated according to Response Evaluation Criteria in Solid Tumours standards and adverse effects were evaluated according to the US National Cancer Institute Common Terminology Criteria for Adverse Events. RESULTS: There was no statistically significant difference in disease control rate, response rate, median survival and 1-year survival between treatment groups. Rates of nausea, myelosuppression, renal damage and hair loss were significantly higher in the docetaxel group than the pemetrexed group. CONCLUSION: Pemetrexed is effective and well tolerated as salvage chemotherapy in patients with NSCLC after EGFR-TKI failure and may be a suitable therapeutic option in these patients.

Comparison of short-term efficacy and safety of TIROX and DCF regimens for advanced gastric cancer.

OBJECTIVE: To compare the short-term efficacy and safety profile of the S-1 + irinotecan + oxaliplatin (TIROX) and docetaxel + cisplatin + flurouracil (DCF) anticancer regimens in patients with advanced gastric cancer. METHODS: Patients with recurrent or metastatic gastric cancer diagnosed by pathology were randomly divided into two groups to receive six cycles of either the TIROX regimen (21-day cycle) or the DCF regimen (21-day cycle). After six chemotherapy cycles, the short-term efficacy was evaluated according to the Response Evaluation Criteria in Solid Tumors guidelines and adverse reactions were recorded according to National Cancer Institute Common Toxicity Criteria 2.0 standards. RESULTS: A total of 60 patients were enrolled in the study. The response rate (complete response + partial response) was significantly higher in the TIROX group (18/30 patients; 60.0%) compared with the DCF group (10/30 patients; 33.3%). The rates of grade III-IV leucopenia and neurotoxicity were significantly higher in the TIROX group than the DCF group. CONCLUSION: The TIROX regimen was effective for the treatment of advanced gastric cancer, but it was associated with leucopenia and neurotoxicity.