Stereoselective synthesis of novel 2'-(S)-CCG-IV analogues as potent NMDA receptor agonists.

We developed a versatile stereoselective route for the synthesis of new 2'-(S)-CCG-IV analogues. The route allows for late stage diversification and thereby provides access to a great variety of conformationally restricted cyclopropyl glutamate analogues. A selection of the 2'-(S)-CCG-IV analogues were evaluated using two-electrode voltage-clamp electrophysiology at recombinant GluN1/GluN2A-D receptors, demonstrating that agonists can be developed with GluN2 subunit-dependent potency and agonist efficacy. We also describe a crystal structure of the GluN2A agonist binding domain in complex with 2'-butyl-(S)-CCG-IV that determines the position of 2'-substituents in (S)-CCG-IV agonists in the glutamate binding site and provides further insight to the structural determinants of their agonist efficacy. The stereoselective synthesis described here enables versatile and straight-forward modifications to diverse analogues of interest for the development of potent subtype-specific NMDA receptor agonists and other applications.

Delivery of dimethyloxalylglycine in calcined bone calcium scaffold to improve osteogenic differentiation and bone repair.

As hypoxia plays a vital role in the angiogenic-osteogenic coupling, using proline hydroxylase inhibitors to manipulate hypoxia-inducible factors has become a strategy to improve the osteogenic properties of biomaterials. Dimethyloxallyl glycine (DMOG) is a 2-ketoglutarate analog, a small molecular compound that competes for 2-ketoglutaric acid to inhibit proline hydroxylase. In order to improve the osteogenic ability of calcined bone calcium (CBC), a new hypoxia-mimicking scaffold (DMOG/Collagen/CBC) was prepared by immersing it in the DMOG-Collagen solution, followed by freeze-drying. All coated CBC scaffolds retained the inherent natural porous architecture and showed excellent biocompatibility. A slow release of DMOG by the DMOG-loaded CBC scaffolds for up to one week was observed inin vitroexperiments. Moreover, the DMOG/Collagen/CBC composite scaffold was found to significantly stimulate bone marrow stromal cells to express osteogenic and angiogenic genesin vitro. In addition, the osteogenic properties of three kinds of scaffolds, raw CBC, Collagen/CBC, and DMOG/Collagen/CBC, were evaluated by histology using the rabbit femoral condyle defect model. Histomorphometric analyses showed that the newly formed bone (BV/TV) in the DMOG/Collagen/CBC group was significantly higher than that of the Collagen/CBC group. However, immunostaining of CD31 and Runx2 expression between these two groups showed no significant difference at this time point. Our results indicate that DMOG-coated CBC can promote osteogenic differentiation and bone healing, and show potential for clinical application in bone tissue engineering.

Bone augmentation using small molecules with biodegradable calcium sulfate particles in a vertical onlay graft model in the rabbit calvarium.

Small molecules including sodium butyrate (SB) and dimethyloxalylglycine (DMOG) can promote bone regeneration via inhibitive effects eliciting cellular responses through signaling cascades. The purpose of this study was to determine the synergistic effects of SB and DMOG loaded on calcium sulfate (CaS) on bone regeneration in the challenging vertical augmentation model in the rabbit calvarium. Four plastic cylinders screwed on the calvarium of each of 10 rabbits were randomly grafted with CaS, CaS/SB, CaS/DMOG, or CaS/DMOG/SB. All specimens were assessed by radiographic, histologic, and histomorphometric analyses. In the radiographic analysis, three different layers (new bone, degraded CaS, and pristine CaS layers) could be distinguished within the cylinder in all groups at 2 weeks. Newly formed bone grew up from basal bone, and CaS in contact with newly formed bone was degraded into small particles to form a different layer. At 8 weeks, most of the pristine CaS had been absorbed and hardly seen within the cylinder. In the histomorphometric analysis, all groups showed comparable new bone areas and heights at 2 and 8 weeks. The DMOG group showed a significant increase in new bone area at 8 weeks compared with 2 weeks, but there was no significant difference among the groups at 8 weeks. The DMOG group showed significantly lower values for the residual material area than the control group at 2 weeks. Within the limitations of this study, SB and DMOG seem to exert smaller synergistic effects on bone regeneration compared to CaS alone in vertical bone augmentation.

The Effect of Chirality on the Application of 5-[18F]Fluoro-Aminosuberic Acid ([18F]FASu) for Oxidative Stress Imaging.

PURPOSE: The cystine transporter, system xC-, plays a crucial role in sustaining redox homeostasis and is reported to be overexpressed in several cancer subtypes. 5-[18F]Fluoroaminosuberic acid ([18F]FASu) is a novel positron emission tomography (PET) tracer, which exhibits specific uptake via system xC-. [18F]FASu synthesis by the commonly used Kryptofix 2.2.2/K2CO3-facilitated fluorination method results in four diastereomers, as a result of 2 chiral centers at positions 2- and 5- of the tracer. We recently reported the synthesis of the optically pure 2S-[18F]FASu from chiral precursors. Our preliminary results indicated preferential uptake of the 2S-isomer by tumor cells compared to 2R-[18F]FASu. Few studies have investigated the biodistribution of chiral 18F-labeled amino acids. The aim of this study was to evaluate the imaging utility and biodistribution of the 5-position diastereomers as well as the racemic (2S,5R/S-) mixture in three different tumor models. PROCEDURES: In vitro tracer uptake experiments and Western blotting were performed in breast cancer (MDA-MB-231), glioblastoma (U-87), and prostate (PC-3) cancer cell lines. PET imaging and biodistribution studies were conducted in xenograft-bearing immunocompromised Rag2M female mice. RESULTS: All three tracer conformations allowed for the visualization of tumor xenografts at 1 h (for U-87 and PC-3 tumors) or 2 h (in the case of MDA-MB-231 xenografts) post-injection, with the racemate (2S,5R/S-) displaying similar image contrast as compared to the 5- position diastereomers and the 2S,5S-[18F]FASu conformation exhibiting relatively higher contrast for imaging U-87 and PC-3 xenografts. Tumor uptake of the isomers was blocked by an excess of the non-radioactive standard, aminosuberic acid (ASu), confirming target specificity. All three isomers were excreted via the renal pathway. Biodistribution analyses showed that PC-3 tumors had the highest tracer uptake, and the accumulation (%ID/g) of the 2S,5R/S-, 2S,5S-, and 2S,5R- isomers was 9.19 ± 1.14, 8.00 ± 1.41, and 7.16 ± 2.13 at 1 h post-injection, respectively. This gave corresponding tumor-to-muscle ratios of 33.68 ± 9.52, 31.42 ± 4.54, and 25.33 ± 4.97, respectively. CONCLUSION: Our data suggest that pure 2S-[18F]FASu can be used to noninvasively image system xC- in a variety of cancers, either as the racemic mixture (2S,5R/S-) or optically pure form. Furthermore, this work shows potential utility of [18F]FASu for detection of glioblastoma and prostate cancer.

Manipulating selectivity of covalently-bonded hyperbranched anion exchangers toward organic acids. Part II: Effect of mono- and dicarboxylic amino acids in the internal part of the functional layer.

Four covalently-bonded hyperbranched anion exchangers based on poly(styrene-divinylbenzene) (PS-DVB) substrate with different structure of the functional layer were prepared using mono- and dianionic amino acids such as glycine, ß-alanine, aspartic acid, and glutamic acid in the internal part of the functional layer. Selectivity of all anion exchangers toward weakly retained organic acids was investigated at different temperatures in order to evaluate the effect of the number of carboxylic groups in the functional layer and its hydrophilicity on the separation. It was found that dianionic amino acids used in the first modification cycle of hyperbranching provide the best resolution for mono- and divalent organic acids, which makes the number of carboxylic groups in the structure of amino acid a key factor in the separation of such analytes with covalently-bonded hyperbranched anion exchangers, while the role of amino acid hydrophilicity is not that significant. Stationary phases prepared using aspartic and glutamic acids provided baseline resolution for quinic, glycolic, acetic, lactic, formic, and galacturonic acids, which are not resolved to baseline with modern commercially available anion exchangers; the increase of temperature was found to be favorable for improving the resolution even further.

Nano polydopamine crosslinked thiol-functionalized hyaluronic acid hydrogel for angiogenic drug delivery.

Crosslinking of polymeric network using nanoparticles by physical or chemical method to obtain hydrogel is an emerging approach. Herein, we synthesized Polydopamine (PDA) nanoparticles via oxidative self-polymerization of dopamine in water-ethanol mixture. Thiol-functionalized hyaluronic acid was developed using cysteamine and hyaluronic acid (HA-Cys) via 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide - N-hydroxysuccinimide (EDC-NHS) crosslinking chemistry. Developed HA-Cys conjugate was cross-linked using PDA nanoparticles via Michael-type addition reaction. Synthesized nanoparticles were monodisperse with size of 124 ± 8 nm and had spherical morphology. FTIR characterization confirmed successful synthesis of HA-Cys conjugate and subsequent crosslinking with PDA nanoparticles. Rheological characterization revealed that hydrogels were injectable in nature with good mechanical stability. Dimethyloxalylglycine (DMOG) loaded PDA nanoparticle showed sustained drug release for period of 7 days from composite hydrogel. Hydrogel microenvironment facilitated enhanced endothelial cell migration, proliferation and attachment. Furthermore, in response to release of DMOG from developed hydrogel, cells showed enhanced capillary tube formation in vitro. Overall, these results demonstrate that PDA cross-linked thiol-functionalized hydrogel was developed in a facile manner under physiological conditions. These developed hydrogels could be potentially used in tissue engineering and drug delivery.

Electrochemical behavior of titanium and platinum in dicarboxilic amino acids solution.

Titanium and platinum samples as components of bimetallic implants for the osteoregeneration process have been modified in solutions modeling biological systems and studied by means of cyclic voltammetry, electrochemical impedance spectroscopy and scanning probe microscopy. While aspartic and glutamic acids did not adsorb significantly on platinum in the potential region investigated, the presence of the amino acids affects oxide layer growth on the titanium surface under anodic polarization. The two studied amino acids behave differently on the titanium electrode surface due to differences in adsorption modes of these substances. The adsorption of the glutamic acid depends on the polarization potential to a large extent, and most of quantitative adsorption characteristics (EIS data, the surface roughness) undergo drastic change at the polarization potential value of 750 mV (vs. Ag/AgCl) in the presence of this substance. Equivalent circuit modeling of the surface processes has been carried out, and a scheme for aspartic and glutamic acid adsorption onto the titanium surface has proposed.

Deferoxamine but Not Dimethyloxalylglycine, L-Mimosine, or Cobalt Dichloride Can Interfere with the MTT Assay.

Hypoxia mimetic agents (HMAs) have been shown to have a positive influence on cellular functions in a multitude of tissue regenerative strategies. Novel experimental approaches use biomaterials as carriers for controlled delivery of these HMAs. Here, the cytotoxic aspects of biocompatibility are of key relevance. The MTT assay is widely used to evaluate cytotoxicity and proliferation. Based on the implications from the proceeding research we hypothesized that specific HMAs such as deferoxamine at high concentrations can interfere with the MTT assay. Thus, the aim of this study was to test the repercussions of the HMAs dimethyloxalylglycine, deferoxamine, L-mimosine, and CoCl2 on the validity of the MTT assay. Murine MC3T3-E1 cells were cultured in serum-free alphaMEM and in alphaMEM supplemented with 10 % fetal bovine serum with the HMAs dimethyloxalylglycine, deferoxamine, L-mimosine, and CoCl2, respectively, at 3 mM-0.3 mM for 24 h (experimental groups). Cells without HMAs served as control (control groups). The same experiments were performed with medium and phosphate buffered saline (PBS) without cells. In all settings MTT solution was added to PBS-washed or unwashed culture plates for the last two hours of the incubation period. Then MTT solution was removed and dimethyl sulfoxide was added to dissolve the formazan crystals and absorption was measured. Our data show that the presence of deferoxamine can interfere with the MTT assay if not removed before the addition of MTT. This is particularly important when evaluating cell viability in setups where deferoxamine-loaded biomaterials are used.

MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG.

α-Ketoglutarate (αKG) is a key node in many important metabolic pathways. The αKG analog N-oxalylglycine (NOG) and its cell-permeable prodrug dimethyloxalylglycine (DMOG) are extensively used to inhibit αKG-dependent dioxygenases. However, whether NOG interference with other αKG-dependent processes contributes to its mode of action remains poorly understood. Here we show that, in aqueous solutions, DMOG is rapidly hydrolyzed, yielding methyloxalylglycine (MOG). MOG elicits cytotoxicity in a manner that depends on its transport by monocarboxylate transporter 2 (MCT2) and is associated with decreased glutamine-derived tricarboxylic acid-cycle flux, suppressed mitochondrial respiration and decreased ATP production. MCT2-facilitated entry of MOG into cells leads to sufficiently high concentrations of NOG to inhibit multiple enzymes in glutamine metabolism, including glutamate dehydrogenase. These findings reveal that MCT2 dictates the mode of action of NOG by determining its intracellular concentration and have important implications for the use of (D)MOG in studying αKG-dependent signaling and metabolism.

An aligned porous electrospun fibrous membrane with controlled drug delivery - An efficient strategy to accelerate diabetic wound healing with improved angiogenesis.

A chronic wound in diabetic patients is usually characterized by poor angiogenesis and delayed wound closure. The exploration of efficient strategy to significantly improve angiogenesis in the diabetic wound bed and thereby accelerate wound healing is still a significant challenge. Herein, we reported a kind of aligned porous poly (l-lactic acid) (PlLA) electrospun fibrous membranes containing dimethyloxalylglycine (DMOG)-loaded mesoporous silica nanoparticles (DS) for diabetic wound healing. The PlLA electrospun fibers aligned in a single direction and there were ellipse-shaped nano-pores in situ generated onto the surface of fibers, while the DS were well distributed in the fibers and the DMOG as well as Si ion could be controlled released from the nanopores on the fibers. The in vitro results revealed that the aligned porous composite membranes (DS-PL) could stimulate the proliferation, migration and angiogenesis-related gene expression of human umbilical vein endothelial cells (HUVECs) compared with the pure PlLA membranes. The in vivo study further demonstrated that the prepared DS-PL membranes significantly improved neo-vascularization, re-epithelialization and collagen formation as well as inhibited inflammatory reaction in the diabetic wound bed, which eventually stimulated the healing of the diabetic wound. Collectively, these results suggest that the combination of hierarchical structures (nanopores on the aligned fibers) with the controllable released DMOG drugs as well as Si ions from the membranes, which could create a synergetic effect on the rapid stimulation of angiogenesis in the diabetic wound bed, is a potential novel therapeutic strategy for highly efficient diabetic wound healing. STATEMENT OF SIGNIFICANCE: A chronic wound in diabetic patients is usually characterized by the poor angiogenesis and the delayed wound closure. The main innovation of this study is to design a new kind of skin tissue engineered scaffold, aligned porous poly (l-lactic acid) (PlLA) electrospun membranes containing dimethyloxalylglycine (DMOG)-loaded mesoporous silica nanoparticles (DS), which could significantly improve angiogenesis in the diabetic wound bed and thereby accelerate diabetic wound healing. The results revealed that the electrospun fibers with ellipse-shaped nano-pores on the surface were aligned in a single direction, while there were DS particles distributed in the fibers and the DMOG as well as Si ions could be controllably released from the nanopores on the fibers. The in vitro studies demonstrated that the hierarchical nanostructures (nanopores on the aligned fibers) and the controllable released chemical active agents (DMOG drugs and Si ions) from the DS-PL membranes could exert a synergistic effect on inducing the endothelial cell proliferation, migration and differentiation. Above all, the scaffolds distinctly induced the angiogenesis, collagen deposition and re-epithelialization as well as inhibited inflammation reaction in the wound sites, which eventually stimulated the healing of diabetic wounds in vivo. The significance of the current study is that the combination of the hierarchical aligned porous nanofibrous structure with DMOG-loaded MSNs incorporated in electrospun fibers may suggest a high-efficiency strategy for chronic wound healing.

Effect of prolyl hydroxylase inhibitor-loaded collagen barrier membranes on osteoclastogenesis and osteoblastogenesis.

Prolyl hydroxylase inhibitors induce a proangiogenic response and are therefore proposed to optimize regenerative approaches in periodontics and oral surgery. Here the effect of the prolyl hydroxylase inhibitors dimethyloxalylglycine and deferoxamine, released from collagen barrier membranes, on osteoclastogenesis and osteoblastogenesis was evaluated. Collagen barrier membranes were loaded with dimethyloxalylglycine and deferoxamine. Release studies were performed and supernatants were taken after 1, 3, 6, 24, and 48 h. The effect of these supernatants on osteoblast- and osteoclast-precursor cells was evaluated. Furthermore, dose response studies for dimethyloxalylglycine and deferoxamine were performed. Osteoclastogenesis was evaluated with RAW 264.7 cells based on the number of multinuclear tartrate-resistant acid phosphatase positive cells. Osteoblastogenesis was evaluated with MC3T3-E1 cells based on alkaline phosphatase. Metabolic activity and cell proliferation were assessed based on MTT and BrdU assays. Vascular endothelial growth factor production was evaluated using an immunoassay. We found that supernatants taken in the first hour from collagen barrier membranes loaded with dimethyloxalylglycine or deferoxamine reduced osteoclastogenesis. Osteoblastogenesis was not reduced significantly. Cell proliferation and metabolic activity of RAW 264.7 and MC3T3-E1 cells were inhibited by supernatants of collagen barrier membranes loaded with deferoxamine but not dimethyloxalylglycine. In RAW 264.7 cell culture, vascular endothelial growth factor production was increased only by supernatants of collagen barrier membranes loaded with dimethyloxalylglycine, but not deferoxamine. In MC3T3-E1 cell culture, supernatants of collagen barrier membranes loaded with dimethyloxalylglycine and deferoxamine both increased vascular endothelial growth factor production. Direct measurements showed that the majority of dimethyloxalylglycine and deferoxamine is released in the first hours. Dose-response studies supported the divergent effects of deferoxamine and dimethyloxalylglycine in RAW 264.7 and MC3T3-E1 cultures. Our findings show diverse effects of dimethyloxalylglycine- and deferoxamine-loaded collagen barrier membranes during osteoclastogenesis and osteoblastogenesis. Preclinical studies will reveal if the increase in vascular endothelial growth factor together with the inhibitory effect on osteoclasts can stimulate oral tissue regeneration.

Substrate-Trapped Interactors of PHD3 and FIH Cluster in Distinct Signaling Pathways.

Amino acid hydroxylation is a post-translational modification that regulates intra- and inter-molecular protein-protein interactions. The modifications are regulated by a family of 2-oxoglutarate- (2OG) dependent enzymes and, although the biochemistry is well understood, until now only a few substrates have been described for these enzymes. Using quantitative interaction proteomics, we screened for substrates of the proline hydroxylase PHD3 and the asparagine hydroxylase FIH, which regulate the HIF-mediated hypoxic response. We were able to identify hundreds of potential substrates. Enrichment analysis revealed that the potential substrates of both hydroxylases cluster in the same pathways but frequently modify different nodes of signaling networks. We confirm that two proteins identified in our screen, MAPK6 (Erk3) and RIPK4, are indeed hydroxylated in a FIH- or PHD3-dependent mechanism. We further determined that FIH-dependent hydroxylation regulates RIPK4-dependent Wnt signaling, and that PHD3-dependent hydroxylation of MAPK6 protects the protein from proteasomal degradation.

The stabilization of hypoxia inducible factor modulates differentiation status and inhibits the proliferation of mouse embryonic stem cells.

Hypoxic conditions are suggested to affect the differentiation status of stem cells (SC), including embryonic stem cells (ESC). Hypoxia inducible factor (HIF) is one of the main intracellular molecules responsible for the cellular response to hypoxia. Hypoxia stabilizes HIF by inhibiting the activity of HIF prolyl-hydroxylases (PHD), which are responsible for targeting HIF-alpha subunits for proteosomal degradation. To address the impact of HIF stabilization on the maintenance of the stemness signature of mouse ESC (mESC), we tested the influence of the inhibition of PHDs and hypoxia (1% O2 and 5% O2) on spontaneous ESC differentiation triggered by leukemia inhibitory factor withdrawal for 24 and 48 h. The widely used panhydroxylase inhibitor dimethyloxaloylglycine (DMOG) and PHD inhibitor JNJ-42041935 (JNJ) with suggested higher specificity towards PHDs were employed. Both inhibitors and both levels of hypoxia significantly increased HIF-1alpha and HIF-2alpha protein levels and HIF transcriptional activity in spontaneously differentiating mESC. This was accompanied by significant downregulation of cell proliferation manifested by the complete inhibition of DNA synthesis and partial arrest in the S phase after 48 h. Further, HIF stabilization enhanced downregulation of the expressions of some pluripotency markers (OCT-4, NANOG, ZFP-42, TNAP) in spontaneously differentiating mESC. However, at the same time, there was also a significant decrease in the expression of some genes selected as markers of cell differentiation (e.g. SOX1, BRACH T, ELF5). In conclusion, the short term stabilization of HIF mediated by the PHD inhibitors JNJ and DMOG and hypoxia did not prevent the spontaneous loss of pluripotency markers in mESC. However, it significantly downregulated the proliferation of these cells.

The broad spectrum 2-oxoglutarate oxygenase inhibitor N-oxalylglycine is present in rhubarb and spinach leaves.

2-Oxoglutarate (2OG) and ferrous iron dependent oxygenases are involved in many biological processes in organisms ranging from humans (where some are therapeutic targets) to plants. These enzymes are of significant biomedicinal interest because of their roles in hypoxic signaling and epigenetic regulation. Synthetic N-oxalylglycine (NOG) has been identified as a broad-spectrum 2OG oxygenase inhibitor and is currently widely used in studies on the hypoxic response and chromatin modifications in animals. We report the identification of NOG as a natural product present in Rheum rhabarbarum (rhubarb) and Spinach oleracea (spinach) leaves; NOG was not observed in Escherchia coli or human embryonic kidney cells (HEK 293T). The finding presents the possibility that NOG plays a natural role in regulating gene expression by inhibiting 2OG dependent oxygenases. This has significance because tricarboxylic acid cycle (TCA) intermediate inhibition of 2OG dependent oxygenases has attracted major interest in cancer research.

Featured Article: Hypoxia-inducible factor-1α dependent nuclear entry of factor inhibiting HIF-1.

The regulation of hypoxia-inducible factor-1 (HIF-1) transcriptional activity in the nucleus is related to factor inhibiting HIF-1 (FIH-1). FIH-1 hydrolyzes asparagine at the C-terminal of HIF-1α, preventing the interaction between HIF-1α and its associated cofactors, and leading to suppressed activation of HIF-1. FIH-1 is a cytosolic protein and its entry to the nucleus has to be coordinated with HIF-1α. The present study was undertaken to examine the correlation between HIF-1α and FIH-1 in their nuclear entry. Human umbilical vein endothelial cells were treated with dimethyloxalylglycine at a final concentration of 100 µM for 4 h, resulting in an accumulation of HIF-1α and an increase of FIH-1 in the nucleus as determined by Western blot analysis. Pretreatment of the cells with copper (Cu) chelator tetraethylenepentamine at 50 µM in cultures for 24 h reduced both HIF-1α protein levels and the HIF-1α entry to the nucleus, along with decreased FIH-1 protein levels in the nucleus but no changes in the total FIH-1 protein levels in the cells. These effects were prevented by simultaneous addition of 50 µM CuSO4 with tetraethylenepentamine. Gene-silencing of HIF-1α significantly inhibited FIH-1 entry to the nucleus, but did not affect the total protein levels of FIH-1 in the cells. This work demonstrates that the nuclear entry of FIH-1 depends on HIF-1α. Cu deficiency caused a decrease of HIF-1α, leading to suppression of FIH-1 entry to the nucleus.

Synergistic effects of dimethyloxalylglycine and butyrate incorporated into α-calcium sulfate on bone regeneration.

Osteogenesis is closely related to angiogenesis, and the combined delivery of angiogenic and osteogenic factors has been suggested to enhance bone regeneration. Small molecules have been explored as alternatives to growth factors for tissue regeneration applications. In this study, we examined the effects of the combined application of angiogenic and osteogenic small molecules on bone regeneration using a prolyl hydroxylase, dimethyloxalylglycine (DMOG), and a histone deacetylase inhibitor, butyrate. In a critical size bone defect model in rats, DMOG and butyrate, which were incorporated into α calcium sulfate (αCS), resulted in synergistic enhancements in bone and blood vessel formation, eventually leading to bone healing, as confirmed by micro-CT and histological analyses. In MC4 pre-osteoblast cultures, DMOG and butyrate enhanced the pro-angiogenic responses and osteoblast differentiation, respectively, which were evaluated based on the levels of hypoxia inducible factor (HIF)-1α protein and the expression of pro-angiogenic molecules (VEGF, home oxidase-1, glucose transporter-1) and by alkaline phosphatase (ALP) activity and the expression of osteoblast phenotype marker molecules (ALP, α1(I)col, osteocalcin, and bone sialoprotein). DMOG combined with butyrate synergistically improved osteoblast differentiation and pro-angiogenic responses, the levels of which were drastically increased in the cultures on αCS disks. Furthermore, it was demonstrated that αCS increased the level of HIF-1α and as a consequence VEGF expression, and supported osteoblast differentiation through the release of calcium ions from the αCS. Altogether, the results of this study provide evidence that a combination treatment with the small molecules DMOG and butyrate can expedite the process of bone regeneration and that αCS can be an efficient delivery vehicle for the small molecules for bone regeneration.

Nonlinear signalling networks and cell-to-cell variability transform external signals into broadly distributed or bimodal responses.

We show theoretically and experimentally a mechanism behind the emergence of wide or bimodal protein distributions in biochemical networks with nonlinear input-output characteristics (the dose-response curve) and variability in protein abundance. Large cell-to-cell variation in the nonlinear dose-response characteristics can be beneficial to facilitate two distinct groups of response levels as opposed to a graded response. Under the circumstances that we quantify mathematically, the two distinct responses can coexist within a cellular population, leading to the emergence of a bimodal protein distribution. Using flow cytometry, we demonstrate the appearance of wide distributions in the hypoxia-inducible factor-mediated response network in HCT116 cells. With help of our theoretical framework, we perform a novel calculation of the magnitude of cell-to-cell heterogeneity in the dose-response obtained experimentally.

Synthesis and anti-TMV activity of novel ß-amino acid ester derivatives containing quinazoline and benzothiazole moieties.

Here, a series of ß-amino acid ester derivatives containing quinazoline and benzothiazoles was synthesized and evaluated for anti-tobacco mosaic virus (TMV) activity. The compounds 3n, 3o, 3p and 3q showed good antiviral activity against TMV at a concentration of 500 µg/mL, with curative rates of 55.55%, 52.32%, 52.77% and 50.91%, respectively, and protection rates of 52.33%, 55.96%, 54.21% and 50.98%, respectively. These values were close to those of the commercially available antiviral agent ningnanmycin (which has curative and protection rates of 55.27% and 52.16%, respectively). To our knowledge, this is the first report of the anti-TMV activity of ß-amino acid ester derivatives containing quinazoline and benzothiazoles moieties; the results indicate that these novel compounds can potentially be used as protective agents against TMV diseases.

PHD inhibition mitigates and protects against radiation-induced gastrointestinal toxicity via HIF2.

Radiation-induced gastrointestinal (GI) toxicity can be a major source of morbidity and mortality after radiation exposure. There is an unmet need for effective preventative or mitigative treatments against the potentially fatal diarrhea and water loss induced by radiation damage to the GI tract. We report that prolyl hydroxylase inhibition by genetic knockout or pharmacologic inhibition of all PHD (prolyl hydroxylase domain) isoforms by the small-molecule dimethyloxallyl glycine (DMOG) increases hypoxia-inducible factor (HIF) expression, improves epithelial integrity, reduces apoptosis, and increases intestinal angiogenesis, all of which are essential for radioprotection. HIF2, but not HIF1, is both necessary and sufficient to prevent radiation-induced GI toxicity and death. Increased vascular endothelial growth factor (VEGF) expression contributes to the protective effects of HIF2, because inhibition of VEGF function reversed the radioprotection and radiomitigation afforded by DMOG. Additionally, mortality from abdominal or total body irradiation was reduced even when DMOG was given 24 hours after exposure. Thus, prolyl hydroxylase inhibition represents a treatment strategy to protect against and mitigate GI toxicity from both therapeutic radiation and potentially lethal radiation exposures.