Optically activated MEK1/2 inhibitors (Opti-MEKi) as potential antimelanoma agents.

Mitogen-activated protein kinase kinases 1/2 (MEK1/2) play critical roles in the canonical RAS/RAF/MEK/ERK pathway. Highly selective and potent non-ATP-competitive allosteric MEK1/2 inhibitors have been developed, and three of them were clinically approved for the treatment of BRAFV600 -mutant melanoma. However, the accompanying side effects of the systemically administered MEK1/2 drugs largely constrain their tolerable doses and efficacy. In this study, a series of mirdametinib-based optically activatable MEK1/2 inhibitors (opti-MEKi) were designed and synthesized. A structural-based design led to the discovery of photocaged compounds with dramatically diminished efficacy in vitro, whose activities can be spatiotemporally induced by short durations of irradiation of ultraviolet (365 nm) light. We demonstrated the robust photoactivation of MEK1/2 inhibition and antimelanoma activity in cultured human cells, as well as in a xenograft zebrafish model. Taken together, the modular approach presented herein provides a method for the optical control of MEK1/2 inhibitor activity, and these data support the further development of optically activatable agents for light-mediated antimelanoma phototherapy.

Reduced Compensatory ß-Cell Proliferation in Nfatc3-Deficient Mice Fed on High-Fat Diet.

BACKGROUND: High-fat-diet induces pancreatic ß-cell compensatory proliferation, and impairments in pancreatic ß-cell proliferation and function can lead to defects in insulin secretion and diabetes. NFATc3 is important for HFD-induced adipose tissue inflammation. But it is unknown whether NFATc3 is required for ß cell compensatory growth in mice fed with HFD. METHODS: NFATc3 mRNA and protein expression levels were quantified by RT-qPCR and Western blotting, respectively, in pancreatic islets of WT mice fed on HFD for 12-20 weeks. Adenoviral-mediated overexpression of NFATc3 were conducted in Min6 cells and cultured primary mouse islets. NFATc3-/- mice and WT control mice were fed with HFD and metabolic and functional parameters were measured. RESULTS: We observed that the NFATc3 expression level was reduced in the islets of high-fat-diet (HFD)-fed mice. Adenovirus-mediated overexpression of NFATc3 enhanced glucose-stimulated insulin secretion and ß-cell gene expression in cultured primary mouse islets. Nfatc3-/- mice initially developed similar glucose tolerance at 2-4 weeks after HFD feeding than HFD-fed WT mice, but Nfatc3-/- mice developed improved glucose tolerance and insulin sensitivity after 8 weeks of HFD feeding compared to Nfatc3+/+fed with HFD. Furthermore, Nfatc3-/- mice on HFD exhibited decreased ß-cell mass and reduced expression of genes important for ß-cell proliferation and function compared to Nfatc3+/+mice on HFD. CONCLUSIONS: The findings suggested that NFATc3 plays a role in maintaining the pancreatic ß-cell compensatory growth and gene expression in response to obesity.

SPARC promotes insulin secretion through down-regulation of RGS4 protein in pancreatic ß cells.

SPARC-deficient mice have been shown to exhibit impaired glucose tolerance and insulin secretion, but the underlying mechanism remains unknown. Here, we showed that SPARC enhanced the promoting effect of Muscarinic receptor agonist oxotremorine-M on insulin secretion in cultured mouse islets. Overexpression of SPARC down-regulated RGS4, a negative regulator of ß-cell M3 muscarinic receptors. Conversely, knockdown of SPARC up-regulated RGS4 in Min6 cells. RGS4 was up-regulated in islets from sparc -/- mice, which correlated with decreased glucose-stimulated insulin secretion (GSIS). Furthermore, inhibition of RGS4 restored GSIS in the islets from sparc -/- mice, and knockdown of RGS4 partially decreased the promoting effect of SPARC on oxotremorine-M-stimulated insulin secretion. Phosphoinositide 3-kinase (PI3K) inhibitor LY-294002 abolished SPARC-induced down-regulation of RGS4. Taken together, our data revealed that SPARC promoted GSIS by inhibiting RGS4 in pancreatic ß cells.

A study of degradation behaviour and biocompatibility of Zn-Fe alloy prepared by electrodeposition.

Zinc is a biodegradable metal, which exhibits more moderate biodegradability than magnesium and iron, so that it has great application potential in the field of biomedical materials. Alloying of zinc and iron may lead to producing a new type of implant material Zn-Fe alloy, which might be able to meet the requirements for a moderate degradation rate. However, due to the huge difference in the melting point between zinc and iron, the preparation of Zn-Fe alloy is quite challenging and hence rarely reported. In this study, we show that Zn-Fe alloys can be successfully prepared by electrodeposition technology. The microstructures, composition, degradation properties and biocompatibility of the Zn-Fe alloys were systematically studied. The results showed that the content of iron in the alloys ranged from 0 to 8 wt%, depending on the concentration of Fe ions and the current density. In the alloys, the major's phases were η, δ and Г1, and they were mainly affected by the ion concentration in the electrolyte. In the in vitro immersion tests, the Zn-Fe alloy ZF2-1 showed the highest immersion corrosion rate, while ZF3-1 showed the highest electrochemical corrosion rate. Moreover, we found that the corrosion rates of the alloys were significantly higher than that of the pure Fe. In the in vivo experiments, we confirmed that the Zn-Fe alloy possessed good biocompatibility. These results demonstrate that the electrodeposition technology is a good method to prepare Zn-Fe alloys, and the Zn-Fe alloys prepared by this method are potentially promising materials for biomedical applications.

NFATc3 deficiency reduces the classical activation of adipose tissue macrophages

Nuclear factors of activated T cells (NFAT) c3 have a prominent role in the regulation of proinflammatory factors in immune cells. The classically activated M1 macrophages are key players in the initiation and maintenance of adipose tissue (AT) inflammation. The role of NFATc3 in obesity and AT inflammation is unknown. We set out to determine how deficiency of NFATc3 effected macrophage polarization, inflammation and insulin resistance in visceral AT of high-fat diet (HFD)-fed mice. Nfatc3−/− and WT mice were fed a HFD for 8­17 weeks. Epididymal white AT (eWAT) F4/80(+) cells were characterized by fluorescence-activated cell sorting and quantitative RT-PCR. Results showed that Nfatc3−/− mice developed HFD-induced obesity similar to WT mice, but insulin sensitivity and glucose tolerance were improved, and liver fat accumulation was reduced in Nfatc3−/− mice compared to WT control mice. Moreover, M1 macrophage content and proinflammatory factors were reduced, whereas the alternatively activated M2 macrophage content was increased in eWAT of HFD-fed Nfatc3−/− mice compared to that of WT mice. In addition, eWAT insulin signaling was improved in HFD-fed Nfatc3−/− mice. Importantly, after bone-marrow-derived macrophages had been isolated from Nfatc3−/− mice and cultured in vitro, treatment of these cells with interferon-γ and lipopolysaccharide resulted in reduction of M1 inflammatory markers, suggesting that NFATc3 promoted M1 polarization by a cell-autonomous mechanism. The results demonstrated that NFATc3 played an important role in M1 macrophage polarization, AT inflammation and insulin resistance in response to obesity through transcriptional activation of proinflammatory genes.

Design and synthesis of (aza)indolyl maleimide-based covalent inhibitors of glycogen synthase kinase 3ß.

As an important kinase in multiple signal transduction pathways, GSK-3ß has been an attractive target for chemical probe discovery and drug development. Compared to numerous reversible inhibitors that have been developed, covalent inhibitors of GSK-3ß are noticeably lacking. Here, we report the discovery of a series of covalent GSK-3ß inhibitors by optimizing both non-covalent interactions and reactive groups. Among these covalent inhibitors, compound 38b with a mild α-fluoromethyl amide reactive group emerges as a selective and covalent inhibitor against GSK-3ß, effectively inhibits the phosphorylation of glycogen synthase and tau protein, and increases ß-catenin's levels in living cells. In addition, compound 38b is highly permeable and not a substrate of P-glycoprotein.

Development of a Selective Labeling Probe for Bruton's Tyrosine Kinase Quantification in Live Cells.

As a key regulator of the B-cell receptor signaling pathway, Bruton's tyrosine kinase (Btk) has emerged as an important therapeutic target for various malignancies and autoimmune disorders. However, data on the expression profiles of Btk are lacking. Here, we report the discovery of a new, selective Btk probe and of a sandwich-type ELISA quantification method to detect endogenous Btk in live cells. We achieved selective labeling of Btk in vivo and quantified Btk levels in seven types of human lymphoma cell lines. This quantification method provides a powerful tool to study Btk in live cells that may also be useful in clinical settings.

A novel layer-structured scaffold with large pore sizes suitable for 3D cell culture prepared by near-field electrospinning.

Electrospinning is a powerful method for preparing porous materials that can be applied as biomedical materials for implantation or tissue engineering or as scaffolds for 3D cell culture experiments. However, this technique is limited in practical applications because the pore size of 3D scaffolds directly prepared by conventional electrospinning is usually less than several tens of micrometres, which may not be suitable for 3D cell culture and tissue growth. To allow for satisfactory 3D cell culture and tissue engineering, the pore size of the scaffold should be controllable according to the requirement of the specific cells to be cultured. Here, we show that layer-structured scaffolds with pore sizes larger than 100µm can be obtained by stacking meshes prepared by direct-writing using the near-field electrospinning (NFES) technique. In the study, we prepared composite scaffolds made of polycaprolactone (PCL) and hydroxyapatite (HAp) via the above-mentioned method and tested the effectiveness of the novel scaffold in cell culture using mouse pre-osteoblast cells (MC3T3-E1). The pore size and the degradability of the PCL/HAp scaffolds were characterized. The results showed that the average pore size of the scaffolds was 167µm, which was controllable based on the required application; the degradation rate was controllable depending on the ratio of PCL to HAp. The biocompatibility of the scaffolds in vitro was studied, and it was found that the scaffolds showed no toxicity and that the cells could effectively attach, proliferate, and differentiate in the 3D skeleton of the scaffolds. Our studies showed that a simple modification of the preparation procedure can lead to a new way to fabricate novel layer-structured 3D scaffolds with controllable structures and pore sizes suitable for practical applications in implantation, tissue engineering and 3D cell culture.

Silk fibroin/chitosan thin film promotes osteogenic and adipogenic differentiation of rat bone marrow-derived mesenchymal stem cells.

As a biodegradable polymer thin film, silk fibroin/chitosan composite film overcomes the defects of pure silk fibroin and chitosan films, respectively, and shows remarkable biocompatibility, appropriate hydrophilicity and mechanical properties. Silk fibroin/chitosan thin film can be used not only as metal implant coating for bone injury repair, but also as tissue engineering scaffold for skin, cornea, adipose, and other soft tissue injury repair. However, the biocompatibility of silk fibroin/chitosan thin film for mesenchymal stem cells, a kind of important seed cell of tissue engineering and regenerative medicine, is rarely reported. In this study, silk fibroin/chitosan film was prepared by solvent casting method, and the rat bone marrow-derived mesenchymal stem cells were cultured on the silk fibroin/chitosan thin film. Osteogenic and adipogenic differentiation of rat bone marrow-derived mesenchymal stem cells were induced, respectively. The proliferation ability, osteogenic and adipogenic differentiation abilities of rat bone marrow-derived mesenchymal stem cells were systematically compared between silk fibroin/chitosan thin film and polystyrene tissue culture plates. The results showed that silk fibroin/chitosan thin film not only provided a comparable environment for the growth and proliferation of rat bone marrow-derived mesenchymal stem cells but also promoted their osteogenic and adipogenic differentiation. This work provided information of rat bone marrow-derived mesenchymal stem cells behavior on silk fibroin/chitosan thin film and extended the application of silk fibroin/chitosan thin film. Based on the results, we suggested that the silk fibroin/chitosan thin film could be a promising material for tissue engineering of bone, cartilage, adipose, and skin.

From 2D to 3D: The morphology, proliferation and differentiation of MC3T3-E1 on silk fibroin/chitosan matrices.

It has been widely accepted that cell culture in two-dimensional (2D) conditions may not be able to represent growth in three-dimensional (3D) conditions. Systematic comparisons between 2D and 3D cell cultures are needed to appropriately use the existing 2D results. In this work, we conducted a comparative study between 2D and 3D cell cultures of MC3T3-E1 using the same type of material (a mixture of silk fibroin (SF) and chitosan (CS)). Our results showed 3D SF/CS scaffold exhibited different effects on cell culture compared with the 2D cases. 1) The cells grown in 3D scaffold showed multiple morphologies. 2) The proliferation of cells in 3D scaffold was long-term and sustainable. 3) Cell differentiation occurred throughout the entire 3D scaffold. The results showed that cell culture in 3D SF/CS scaffold exhibited different features than 2D cases and 3D SF/CS scaffold could be a promising material for 3D cell culture.

Silk fibroin/chitosan scaffold with tunable properties and low inflammatory response assists the differentiation of bone marrow mesenchymal stem cells.

The physical and chemical properties of the scaffold are known to play important roles in three-dimensional (3D) cell culture, which always determine the cellular fate or the results of implantation. To control these properties becomes necessary for meeting the requirements of a variety of tissue engineering applications. In this study, a series of silk fibroin/chitosan (SF/CS) scaffolds with tunable properties were prepared using freeze-drying method, and the rat bone marrow-derived mesenchymal stem cells (BM-MSCs) were seeded in these scaffolds to evaluate their availability of use in tissue engineering. The 3D structure, mechanical properties and degradation ability of SF/CS scaffold can be tuned by changing the total concentration of the precursor solution and the blending ratio between SF and CS. BM-MSCs cultured in the SF/CS scaffold exhibited excellent proliferation and multiple morphologies. The induction of osteogenic and adipogenic differentiation of BM-MSCs were successful in this scaffold when cultured in vitro. Subcutaneous implantation of the SF/CS scaffolds did not cause any inflammatory response within four weeks, which revealed good compatibility. Moreover, the implanted scaffold allowed host cells to invade, adhere, grow and form new blood vessels. With these excellent performance, SF/CS scaffold has great potential in preparing implants for tissue engineering applications.

A novel porous Fe/Fe-W alloy scaffold with a double-layer structured skeleton: Preparation, in vitro degradability and biocompatibility.

A novel porous Fe/Fe-W alloy scaffold with a double-layer structured skeleton was prepared for the first time by electrodeposition. The microstructure of the scaffold was analysed by X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS) and mercury porosimetry. Mechanical property, in vitro degradability and biocompatibility were tested by tensile test, immersion and a cytotoxicity test. The results showed that the scaffolds exhibited a cellular structure that is similar to that of cancellous bone and had a considerably large specific surface area. The skeleton of the scaffolds showed a double-layer structure that was composed of a hollow Fe skeleton wrapped in a thin layer of Fe-W alloy. The tensile strength and the apparent density are close to that of cancellous bone. It was also found that the different surface microstructures showed different effects on in vitro degradability and biocompatibility. In the immersion test, the corrosion rate decreased gradually as the immersion time increased. In the cytotoxicity test, the extraction medium of the pure Fe scaffold showed the lowest cell viability, followed by that of 1.5FeW as a close second. The extraction media of FeW, Fe1.5W and Fe2W were similar, and their cell viability was far above that of the Fe and 1.5FeW scaffolds. The structural style of the scaffolds presented in this paper is potentially useful and applicable to developing degradable scaffolds with a tailored corrosion rate.

Transcription factor Ets1 regulates expression of thioredoxin-interacting protein and inhibits insulin secretion in pancreatic ß-cells.

Long-term activation of extracellular-regulated kinase (ERK1/2) pathway has been shown to cause glucotoxicity and inhibit insulin gene expression in ß-cells. Transcription factor Ets1 is activated by ERK1/2-mediated phosphorylation at the Thr38 residue. We hypothesize that Ets1 plays an important role in mediating ERK1/2 induced glucotoxicity in ß-cells. We determined the role of Ets1 in Min6 cells and isolated mouse islets using overexpression and siRNA mediated knockdown of Ets1. The results show that Ets1 was localized in insulin-staining positive cells but not in glucagon-staining positive cells. Overexpression of Ets1 reduced glucose-stimulated insulin secretion in primary mouse islets. Overexpression of Ets1 in Min6 ß-cells and mouse islets increased expression of thioredoxin-interacting protein (TXNIP). Conversely, knockdown of Ets1 by siRNA reduced expression of TXNIP in Min6 cells. Ets1 was associated with the txnip promoter in min6 cells and transfection of 293 cells with Ets1 and p300 synergistically increased txnip promoter reporter activity. Moreover, overexpression of Ets1 inhibited Min6 cell proliferation. Our results suggest that Ets1, by promoting TXNIP expression, negatively regulates ß-cell function. Thus, over-activation of Ets1 may contribute to diet-induced ß-cell dysfunction.