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Caspase activated DNase (CAD) induced DNA breaks promote cell differentiation and therapy-induced cancer cell resistance. CAD targeting activity is assumed to be unique to each condition, as differentiation and cancer genesis are divergent cell fates. Here, we made the surprising discovery that a subset of CAD-bound targets in differentiating muscle cells are the same genes involved in the genesis of cancer-causing translocations. In muscle cells, a prominent CAD-bound gene pair is Pax7 and Foxo1a, the mismatched reciprocal loci that give rise to alveolar rhabdomyosarcoma. We show that CAD-targeted breaks in the Pax7 gene are physiologic to reduce Pax7 expression, a prerequisite for muscle cell differentiation. A cohort of these CAD gene targets are also conserved in early differentiating T cells and include genes that spur leukemia/lymphoma translocations. Our results suggest the CAD targeting of translocation prone oncogenic genes is non-pathologic biology and aligns with initiation of cell fate transitions.

Background: Glucagon-like peptide-2 (GLP-2) enhances intestinal repair and attenuates inflammation in preclinical inflammatory bowel disease (IBD) models, making GLP-2 analogues attractive candidates for IBD therapy. Glepaglutide is a long-acting GLP-2 receptor agonist in clinical development for treatment of short bowel syndrome. Here, we investigated if glepaglutide is therapeutically beneficial in rats with small intestinal inflammation., Methods: Small intestinal inflammation was induced with indomethacin in naive Wistar rats, followed by glepaglutide administration at different disease stages. Glepaglutide was administered in co-treatment and post-treatment regimens. Small intestinal length and concentrations of inflammatory markers α-1-acid glycoprotein and myeloperoxidase were used to assess anti-inflammatory effects. Small intestinal mass was evaluated to determine intestinotrophic effects., Results: Glepaglutide co- and post-treatment significantly reduced severity of small intestinal inflammation, evidenced by reversed small intestinal shortening and decreased α-1-acid glycoprotein and/or myeloperoxidase concentration(s). Co- and post-treatment with glepaglutide also significantly increased small intestinal mass, indicating intestinal regenerative effects. Similar effects were observed in naive rats after glepaglutide treatment., Conclusion: Glepaglutide has anti-inflammatory and intestinotrophic effects without the need for pre-treatment in a rat model of small intestinal inflammation. Thus, glepaglutide is of potential clinical interest for patients with IBD., (© 2023. The Author(s).)

Genotoxic therapy such as radiation serves as a frontline cancer treatment, yet acquired resistance that leads to tumor reoccurrence is frequent. We found that cancer cells maintain viability during irradiation by reversibly increasing genome-wide DNA breaks, thereby limiting premature mitotic progression. We identify caspase-activated DNase (CAD) as the nuclease inflicting these de novo DNA lesions at defined loci, which are in proximity to chromatin-modifying CCCTC-binding factor (CTCF) sites. CAD nuclease activity is governed through phosphorylation by DNA damage response kinases, independent of caspase activity. In turn, loss of CAD activity impairs cell fate decisions, rendering cancer cells vulnerable to radiation-induced DNA double-strand breaks. Our observations highlight a cancer-selective survival adaptation, whereby tumor cells deploy regulated DNA breaks to delimit the detrimental effects of therapy-evoked DNA damage.

DNA-damaging cancer therapies induce interferon expression and stimulate the immune system, promoting therapy responses. The immune-activating STING (Stimulator of Interferon Genes) pathway is induced when DNA or double-stranded RNA (dsRNA) is detected in the cell cytoplasm, which can be caused by viral infection or by DNA damage following chemo- or radiotherapy. Here, we investigated the responses of cutaneous T-cell lymphoma (CTCL) cells to the clinically applied DNA crosslinking photochemotherapy (combination of 8-methoxypsoralen and UVA light; 8-MOP + UVA). We showed that this treatment evokes interferon expression and that the type III interferon IFNL1 is the major cytokine induced. IFNL1 upregulation is dependent on STING and on the cytoplasmic DNA sensor cyclic GMP-AMP synthase (cGAS). Furthermore, 8-MOP + UVA treatment induced the expression of genes in pathways involved in response to the tumor necrosis factor, innate immune system and acute inflammatory response. Notably, a subset of these genes was under control of the STING-IFNL1 pathway. In conclusion, our data connected DNA damage with immune system activation via the STING pathway and contributed to a better understanding of the effectiveness of photochemotherapy.

Long-chain n-3 polyunsaturated fatty acids (n-3 LC-PUFAs) are collectively recognized triglyceride-lowering agents, and their preventive action is likely mediated by changes in gene expression. However, as most studies employ fish oil, which contains a mixture of n-3 LC-PUFAs, the docosahexaenoic acid (DHA)-specific transcriptional effects on lipid metabolism are still unclear. The aim of the present study was to further elucidate the DHA-induced transcriptional effects on lipid metabolism in the liver, and to investigate the effects of co-administration with other bioactive compounds having effects on lipid metabolism. To this purpose, HepG2 cells were treated for 6 or 24 h with DHA, the short-chain fatty acid propionate (PRO), and protocatechuic acid (PCA), the main human metabolite of cyanidin-glucosides. Following supplementation, we mapped the global transcriptional changes. PRO and PCA alone had a very slight effect on the transcriptome; on the contrary, supplementation of DHA highly repressed the steroid and fatty acid biosynthesis pathways, this transcriptional modulation being not affected by co-supplementation. Our results confirm that DHA effect on lipid metabolism are mediated at least in part by modulation of the expression of specific genes. PRO and PCA could contribute to counteracting dyslipidemia through other mechanisms.

Introduction: High beat-to-beat morphological variation (divergence) on the ventricular electrogram during programmed ventricular stimulation (PVS) is associated with increased risk of ventricular fibrillation (VF), with unclear mechanisms. We hypothesized that ventricular divergence is associated with epicardial wavebreaks during PVS, and that it predicts VF occurrence., Method and Results: Langendorff-perfused rabbit hearts (n = 10) underwent 30-min therapeutic hypothermia (TH, 30°C), followed by a 20-min treatment with rotigaptide (300 nM), a gap junction modifier. VF inducibility was tested using burst ventricular pacing at the shortest pacing cycle length achieving 1:1 ventricular capture. Pseudo-ECG (p-ECG) and epicardial activation maps were simultaneously recorded for divergence and wavebreaks analysis, respectively. A total of 112 optical and p-ECG recordings (62 at TH, 50 at TH treated with rotigaptide) were analyzed. Adding rotigaptide reduced ventricular divergence, from 0.13±0.10 at TH to 0.09±0.07 (p = 0.018). Similarly, rotigaptide reduced the number of epicardial wavebreaks, from 0.59±0.73 at TH to 0.30±0.49 (p = 0.036). VF inducibility decreased, from 48±31% at TH to 22±32% after rotigaptide infusion (p = 0.032). Linear regression models showed that ventricular divergence correlated with epicardial wavebreaks during TH (p<0.001)., Conclusion: Ventricular divergence correlated with, and might be predictive of epicardial wavebreaks during PVS at TH. Rotigaptide decreased both the ventricular divergence and epicardial wavebreaks, and reduced the probability of pacing-induced VF during TH., Competing Interests: Zealand Pharma A/S provided support in the form of salaries for author [BDL] and rotigaptide. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease.

In the version of this article initially published, in the graph keys in Fig. 1i, the colors indicating 'Ob' and 'Ad' were red and blue, respectively, but should have been blue and red, respectively; the shapes indicating 'MUS' and 'BM' were a triangle and a square, respectively, but should have been a square and a triangle, respectively. The errors have been corrected in the HTML and PDF versions of the article.

Mesenchymal (stromal) stem cells (MSCs) constitute populations of mesodermal multipotent cells involved in tissue regeneration and homeostasis in many different organs. Here we performed comprehensive characterization of the transcriptional and epigenomic changes associated with osteoblast and adipocyte differentiation of human MSCs. We demonstrate that adipogenesis is driven by considerable remodeling of the chromatin landscape and de novo activation of enhancers, whereas osteogenesis involves activation of preestablished enhancers. Using machine learning algorithms for in silico modeling of transcriptional regulation, we identify a large and diverse transcriptional network of pro-osteogenic and antiadipogenic transcription factors. Intriguingly, binding motifs for these factors overlap with SNPs related to bone and fat formation in humans, and knockdown of single members of this network is sufficient to modulate differentiation in both directions, thus indicating that lineage determination is a delicate balance between the activities of many different transcription factors.

Non-alcoholic steatohepatitis (NASH) signified by hepatic steatosis, inflammation, hepatocellular injury, and fibrosis is a growing cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma. Hepatic fibrosis resulting from accumulation of extracellular matrix proteins secreted by hepatic myofibroblasts plays an important role in disease progression. Activated hepatic stellate cells (HSCs) have been identified as the primary source of myofibroblasts in animal models of hepatotoxic liver injury; however, so far HSC activation and plasticity have not been thoroughly investigated in the context of NASH-related fibrogenesis. Here we have determined the time-resolved changes in the HSC transcriptome during development of Western diet- and fructose-induced NASH in mice, a NASH model recapitulating human disease. Intriguingly, HSC transcriptional dynamics are highly similar across disease models pointing to HSC activation as a point of convergence in the development of fibrotic liver disease. Bioinformatic interrogation of the promoter sequences of activated genes combined with loss-of-function experiments indicates that the transcriptional regulators ETS1 and RUNX1 act as drivers of NASH-associated HSC plasticity. Taken together, our results implicate HSC activation and transcriptional plasticity as key aspects of NASH pathophysiology.

The aim of this study was to investigate whether inhibition of connexin 43 gap junction-uncoupling is sufficient to prevent retinal vascular cell loss under high glucose condition and reduce cell monolayer permeability. Rat retinal endothelial cells were grown for 3, 5, and 7 days in normal (5 mM) or high glucose (30 mM) medium; in parallel, cells grown in high glucose medium were exposed for 3, 5, and 7 days to 100 nM danegaptide, which stabilizes connexin 43-mediated cell coupling. Additionally, cells grown in normal medium were treated with a connexin 43 blocker as a negative control. To determine gap junction intercellular communication, scrape load dye transfer assay was performed at the three time points. Cells were assessed for apoptosis and cell monolayer permeability by differential dye staining and in vitro permeability assays, respectively. Cells treated with danegaptide preserved gap junction intercellular communication, decreased cell death, and reduced cell monolayer permeability. Scrape load dye transfer assay indicated that cells exposed to danegaptide for 3, 5, and 7 days under high glucose condition maintained gap junction intercellular communication. Importantly, danegaptide significantly prevented high glucose-induced apoptosis at all three time points, and inhibited cell monolayer permeability by day 5. Cells exposed to a connexin 43 blocker, which decreased cell coupling, showed excess apoptosis and cell monolayer permeability. These findings suggest that prevention of high glucose-induced compromised cell-cell coupling may be a useful strategy for inhibiting apoptosis and excess vascular permeability associated with diabetic retinopathy., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chromatin immunoprecipitation (ChIP) is a powerful technique allowing for investigation of protein-DNA interactions in living cells. Here, we provide a detailed step-by-step protocol for ChIP and highlight important considerations, challenges and pitfalls often encountered in the ChIP procedure. Furthermore, we present data of key quality control (QC) steps and exemplify material performance validation on transcription factor ChIP to provide a QC guide for setting up ChIP. Finally, we provide guidelines for scaling of the ChIP procedure to ChIP sequencing (ChIP-seq) and discuss important considerations associated with this.

Organismal development and function requires multiple and accurate signal transduction pathways to ensure that proper balance between cell proliferation, differentiation, inactivation, and death is achieved. Cell death via apoptotic caspase signal transduction is extensively characterized and integral to this balance. Importantly, the view of apoptotic signal transduction has expanded over the previous decades. Subapoptotic caspase signaling has surfaced as mechanism that can promote the adoption of a range of cellular fates. An emerging mechanism of subapoptotic caspase signaling is the activation of the caspase-activated DNase (CAD) through controlled cleavage of the inhibitor of CAD (ICAD). CAD-induced DNA breaks incite a DNA damage response, frequently invoking p53 signaling, that transduces a change in cell fate. Cell differentiation and senescence are fates demonstrated to arise from CAD-induced DNA breaks. Furthermore, an apparent consequence of CAD activity is also emerging, as a potential source of oncogenic mutations. This review will discuss the mechanisms underlying CAD-induced DNA breaks and highlight how CAD activity promotes diverse cell fates., (© 2016 Federation of European Biochemical Societies.)

Glucose is an important inducer of insulin secretion, but it also stimulates long-term adaptive changes in gene expression that can either promote or antagonize the proliferative potential and function of β cells. Here, we have generated time-resolved profiles of enhancer and transcriptional activity in response to glucose in the INS-1E pancreatic β cell line. Our data outline a biphasic response with a first transcriptional wave during which metabolic genes are activated, and a second wave where cell-cycle genes are activated and β cell identity genes are repressed. The glucose-sensing transcription factor ChREBP directly activates first wave enhancers, whereas repression and activation of second wave enhancers are indirect. By integrating motif enrichment within late-regulated enhancers with expression profiles of the associated transcription factors, we have identified multiple putative regulators of the second wave. These include RORγ, the activity of which is important for glucose-induced proliferation of both INS-1E and primary rat β cells., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cofactor squelching is the term used to describe competition between transcription factors (TFs) for a limited amount of cofactors in a cell with the functional consequence that TFs in a given cell interfere with the activity of each other. Since cofactor squelching was proposed based primarily on reporter assays some 30 years ago, it has remained controversial, and the idea that it could be a physiologically relevant mechanism for transcriptional repression has not received much support. However, recent genome-wide studies have demonstrated that signal-dependent TFs are very often absent from the enhancers that are acutely repressed by those signals, which is consistent with an indirect mechanism of repression such as squelching. Here we review these recent studies in the light of the classical studies of cofactor squelching, and we discuss how TF cooperativity in so-called hotspots and super-enhancers may sensitize these to cofactor squelching., (© 2016 WILEY Periodicals, Inc.)

DNA damage activates the ATM and ATR kinases that coordinate checkpoint and DNA repair pathways. An essential step in homology-directed repair (HDR) of DNA breaks is the formation of RAD51 nucleofilaments mediated by PALB2-BRCA2; however, roles of ATM and ATR in this critical step of HDR are poorly understood. Here, we show that PALB2 is markedly phosphorylated in response to genotoxic stresses such as ionizing radiation and hydroxyurea. This response is mediated by the ATM and ATR kinases through three N-terminal S/Q-sites in PALB2, the consensus target sites for ATM and ATR Importantly, a phospho-deficient PALB2 mutant is unable to support proper RAD51 foci formation, a key PALB2 regulated repair event, whereas a phospho-mimicking PALB2 version supports RAD51 foci formation. Moreover, phospho-deficient PALB2 is less potent in HDR than wild-type PALB2. Further, this mutation reveals a separation in PALB2 function, as the PALB2-dependent checkpoint response is normal in cells expressing the phospho-deficient PALB2 mutant. Collectively, our findings highlight a critical importance of PALB2 phosphorylation as a novel regulatory step in genome maintenance after genotoxic stress., (© 2016 The Authors.)

Background: Previous studies suggest that intake of specific bioactive compounds may have beneficial clinical effects on adipose tissue partly due to their anti-inflammatory and insulin-sensitizing properties. With the overall aim to contribute to better understanding of the mechanisms of selected bioactive nutrients on fat metabolism, we investigated their role on human white adipocyte function., Methods: The influence of the omega-3-fatty acid docosahexaenoic acid (DHA), the anthocyanin (AC) cyanidin-3-glucoside and its metabolite protocatechuic acid, and the beta-glucan metabolite propionic acid (PI) on adipokine secretion, fatty acid metabolism (lipolysis/lipogenesis) and adipocyte differentiation (lipid accumulation) was studied in human fat cells differentiated in vitro. To investigate possible synergistic, additive or antagonistic effects, DHA was also combined with AC or PI., Results: Each compound, alone or together with DHA, suppressed basal adipocyte lipolysis compared to control treated cells. DHA alone attenuated the secretion of pro-inflammatory adipokines such as chemerin, interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1/CCL2), whereas AC suppressed only the latter two. Treatment with PI decreased IL-6, tumour necrosis factor alpha (TNFα) and adiponectin secretion. A combination of DHA and AC decreased TNFα secretion and increased insulin-stimulated lipogenesis. No effect was found on adipocyte differentiation. At the selected concentrations, none of the compounds was found to be cytotoxic., Conclusion: The studied bioactive food compounds or their metabolites have beneficial effects in human primary fat cells measured as decreased basal lipolytic activity and secretion of inflammatory markers. A minor effect was also observed on insulin-stimulated glucose uptake albeit only with the combination of DHA and AC. Taken together, our results may link the reported health benefits of the selected bioactives on metabolic disorders such as insulin resistance, hypertension and dyslipidemia to effects on white adipocytes.

Transient DNA strand break formation has been identified as an effective means to enhance gene expression in living cells. In the muscle lineage, cell differentiation is contingent upon the induction of caspase-mediated DNA strand breaks, which act to establish the terminal gene expression program. This coordinated DNA nicking is rapidly resolved, suggesting that myoblasts may deploy DNA repair machinery to stabilize the genome and entrench the differentiated phenotype. Here, we identify the base excision repair pathway component XRCC1 as an indispensable mediator of muscle differentiation. Caspase-triggered XRCC1 repair foci form rapidly within differentiating myonuclei, and then dissipate as the maturation program proceeds. Skeletal myoblast deletion of Xrcc1 does not have an impact on cell growth, yet leads to perinatal lethality, with sustained DNA damage and impaired myofiber development. Together, these results demonstrate that XRCC1 manages a temporally responsive DNA repair process to advance the muscle differentiation program.

Background: Therapeutic hypothermia (TH) may increase the susceptibility to ventricular arrhythmias by decreasing ventricular conduction velocity (CV) and facilitating arrhythmogenic spatially discordant alternans (SDA)., Objective: The purpose of this study was to test the hypothesis that rotigaptide, a gap junction enhancer, can increase ventricular CV, delay the onset of SDA, and decrease the susceptibility to pacing-induced ventricular fibrillation (PIVF) during TH., Methods: Langendorff-perfused isolated rabbit hearts were subjected to 30-minute moderate hypothermia (33°C) followed by 20-minute treatment with rotigaptide (300 nM, n = 8) or vehicle (n = 5). The same protocol was also performed at severe hypothermia (30°C; n = 8 for rotigaptide, n = 5 for vehicle). Using an optical mapping system, epicardial CV and SDA threshold were evaluated by S1 pacing. Ventricular fibrillation inducibility was evaluated by burst pacing for 30 seconds at the shortest pacing cycle length (PCL) that achieved 1:1 ventricular capture., Results: Rotigaptide increased ventricular CV during 33°C (PCL 300 ms, from 76 ± 6 cm/s to 84 ± 7 cm/s, P = .039) and 30°C (PCL 300 ms, from 62 ± 6 cm/s to 68 ± 4 cm/s, P = .008). Rotigaptide decreased action potential duration dispersion at 33°C (P = .01) and 30°C (P = .035). During 30°C, SDA thresholds (P = .042) and incidence of premature ventricular complexes (P = .025) were decreased by rotigaptide. PIVF inducibility was decreased by rotigaptide at 33°C (P = .039) and 30°C (P = .042). Rotigaptide did not change connexin43 expressions and distributions during hypothermia., Conclusion: Rotigaptide protects the hearts against ventricular arrhythmias by increasing ventricular CV, delaying the onset of SDA, and reducing repolarization heterogeneity during TH. Enhancing cell-to-cell coupling by rotigaptide might be a novel approach to prevent ventricular arrhythmias during TH., (Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

The proinflammatory cytokine tumor necrosis factor (TNF) plays a central role in low-grade adipose tissue inflammation and development of insulin resistance during obesity. In this context, nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) is directly involved and required for the acute activation of the inflammatory gene program. Here, we show that the major transactivating subunit of NFκB, v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), is also required for acute TNF-induced suppression of adipocyte genes. Notably, this repression does not involve RELA binding to the associated enhancers but rather loss of cofactors and enhancer RNA (eRNA) selectively from high-occupancy sites within super-enhancers. Based on these data, we have developed models that, with high accuracy, predict which enhancers and genes are repressed by TNF in adipocytes. We show that these models are applicable to other cell types where TNF represses genes associated with super-enhancers in a highly cell-type-specific manner. Our results propose a novel paradigm for NFκB-mediated repression, whereby NFκB selectively redistributes cofactors from high-occupancy enhancers, thereby specifically repressing super-enhancer-associated cell identity genes., (© 2015 Schmidt et al.; Published by Cold Spring Harbor Laboratory Press.)

RNA-seq is a sensitive and accurate technique to compare steady-state levels of RNA between different cellular states. However, as it does not provide an account of transcriptional activity per se, other technologies are needed to more precisely determine acute transcriptional responses. Here, we have developed an easy, sensitive and accurate novel computational method, IRNA-SEQ: , for genome-wide assessment of transcriptional activity based on analysis of intron coverage from total RNA-seq data. Comparison of the results derived from iRNA-seq analyses with parallel results derived using current methods for genome-wide determination of transcriptional activity, i.e. global run-on (GRO)-seq and RNA polymerase II (RNAPII) ChIP-seq, demonstrate that iRNA-seq provides similar results in terms of number of regulated genes and their fold change. However, unlike the current methods that are all very labor-intensive and demanding in terms of sample material and technologies, iRNA-seq is cheap and easy and requires very little sample material. In conclusion, iRNA-seq offers an attractive novel alternative to current methods for determination of changes in transcriptional activity at a genome-wide level., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Cells respond to DNA damage by activating cell cycle checkpoints to delay proliferation and facilitate DNA repair. Here, to uncover new checkpoint regulators, we perform RNA interference screening targeting genes involved in ubiquitylation processes. We show that the F-box protein cyclin F plays an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein cyclin F with suppression of the B-Myb/cyclin A pathway to ensure a DNA damage-induced checkpoint response in G2.

Introduction: Lixisenatide is a once-daily short-acting glucagon-like peptide-1 (GLP-1) receptor agonist (GLP-1RA) used in the treatment of type 2 diabetes mellitus (T2DM). It is used in combination with oral antidiabetics and/or basal insulin in patients inadequately controlled on these medications and who are undergoing diet and lifestyle modification. GLP-1RAs glucose-dependently increase insulin secretion, decrease glucagon secretion, and slow gastric emptying, thereby improving glycemic control. GLP-1RAs are associated with body weight benefits and low rates of hypoglycemia which are welcome in patients with T2DM., Areas Covered: The authors describe the identification of GLP-1RAs as suitable targets for modification with structure-inducing probe technology to improve stability and resistance to proteolytic degradation. Clinical studies have assessed lixisenatide across > 5000 patients as a monotherapy or add-on to a variety of commonly used antidiabetic medications. These studies highlighted the effects of lixisenatide on gastric emptying, explaining its particular improvements in postprandial plasma glucose (PPG) excursions and underscoring its efficacy in combination with insulin glargine. Lixisenatide was well tolerated, with nausea and vomiting being the most frequently reported adverse events., Expert Opinion: The once-daily administration of lixisenatide as well as its substantial sustained effect on gastric emptying and, hence, PPG excursions are all important features compared with the other GLP-1RAs. The combination of two injectables, such as basal insulin to lower fasting plasma glucose and a GLP-1RA that curtails PPG excursions, is clinically valuable and could differentiate lixisenatide from other GLP-1RAs, especially from those continuously acting GLP-1RAs with little effect on gastric emptying and PPG excursions.

Osteoclasts are formed by the fusion of mononuclear precursor cells of the monocyte-macrophage lineage. Among several putative mechanisms, gap-junctional intercellular communication (GJC) has been proposed to have a role in osteoclast fusion and bone resorption. We examined the role of GJC in osteoclastogenesis and in vitro bone resorption with mouse bone marrow hematopoietic stem cells and RAW 264.7 cells. Blocking of gap junctions with 18-α-glycyrrhetinic acid (18GA) led to inhibition of osteoclastogenesis and in vitro bone resorption. Similarly, the GJC inhibitor GAP27 inhibited osteoclast formation. GJC modulation with the antiarrhythmic peptides (AAPs) led to increased amounts of multinuclear RAW 264.7 osteoclasts as well as increased number of nuclei per multinuclear cell. In the culture of bone marrow hematopoietic stem cells in the presence of bone marrow stromal cells AAP reduced the number of osteoclasts, and coculture of MC3T3-E1 preosteoblasts with RAW 264.7 macrophages prevented the action of AAPs to promote osteoclastogenesis. The present data indicate that AAPs modulate the fusion of the pure culture of cells of the monocyte-macrophage lineage. However, the fusion is influenced by GJC in cells of the osteoblast lineage.

To prevent accumulation of mutations, cells respond to DNA lesions by blocking cell cycle progression and initiating DNA repair. Homology-directed repair of DNA breaks requires CtIP-dependent resection of the DNA ends, which is thought to play a key role in activation of ATR (ataxia telangiectasia mutated and Rad3 related) and CHK1 kinases to induce the cell cycle checkpoint. In this paper, we show that CHK1 was rapidly and robustly activated before detectable end resection. Moreover, we show that the key resection factor CtIP was dispensable for initial ATR-CHK1 activation after DNA damage by camptothecin and ionizing radiation. In contrast, we find that DNA end resection was critically required for sustained ATR-CHK1 checkpoint signaling and for maintaining both the intra-S- and G2-phase checkpoints. Consequently, resection-deficient cells entered mitosis with persistent DNA damage. In conclusion, we have uncovered a temporal program of checkpoint activation, where CtIP-dependent DNA end resection is required for sustained checkpoint signaling.

The peptides encoded by the VGF gene are gaining biomedical interest and are increasingly being scrutinized as biomarkers for human disease. An endocrine/neuromodulatory role for VGF peptides has been suggested but never demonstrated. Furthermore, no study has demonstrated so far the existence of a receptor-mediated mechanism for any VGF peptide. In the present study, we provide a comprehensive in vitro, ex vivo and in vivo identification of a novel pro-lipolytic pathway mediated by the TLQP-21 peptide. We show for the first time that VGF-immunoreactivity is present within sympathetic fibres in the WAT (white adipose tissue) but not in the adipocytes. Furthermore, we identified a saturable receptor-binding activity for the TLQP-21 peptide. The maximum binding capacity for TLQP-21 was higher in the WAT as compared with other tissues, and selectively up-regulated in the adipose tissue of obese mice. TLQP-21 increases lipolysis in murine adipocytes via a mechanism encompassing the activation of noradrenaline/β-adrenergic receptors pathways and dose-dependently decreases adipocytes diameters in two models of obesity. In conclusion, we demonstrated a novel and previously uncharacterized peripheral lipolytic pathway encompassing the VGF peptide TLQP-21. Targeting the sympathetic nerve-adipocytes interaction might prove to be a novel approach for the treatment of obesity-associated metabolic complications.

Background: Gap junctions are potential targets for pharmacologic intervention. We previously developed a series of peptide sequences that prevent closure of connexin43 (Cx43) channels, bind to cardiac Cx43, and prevent acidification-induced uncoupling of cardiac gap junctions., Objective: The purpose of this study was to identify and validate the minimum core active structure in peptides containing an RR-N/Q-Y motif. Based on that information, we sought to generate a peptidomimetic molecule that acts on the chemical regulation of Cx43 channels., Methods: Experiments were based on a combination of biochemical, spectroscopic, and electrophysiologic techniques as well as molecular modeling of active pharmacophores with Cx43 activity., Results: Molecular modeling analysis indicated that the functional elements of the side chains in the motif RRXY form a triangular structure. Experimental data revealed that compounds containing such a structure bind to Cx43 and prevent Cx43 chemical gating. These results provided us with the first platform for drug design targeted to the carboxyl terminal of Cx43. Using that platform, we designed and validated a peptidomimetic compound (ZP2519; molecular weight 619 Da) that prevented octanol-induced uncoupling of Cx43 channels and pH gating of cardiac gap junctions., Conclusion: Structure-based drug design can be applied to the development of pharmacophores that act directly on Cx43. Small molecules containing these pharmacophores can serve as tools to determine the role of gap junction regulation in the control of cardiac rhythm. Future studies will determine whether these compounds can function as pharmacologic agents for the treatment of a selected subset of cardiac arrhythmias., (Copyright © 2010 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

In a series of discoveries over the preceding decade, a number of laboratories have unequivocally established that apoptotic proteins and pathways are well conserved cell fate determinants, which act independent of a cell death response. Within this context, the role for apoptotic proteins in the induction of cell differentiation has been widely documented. Despite these discoveries, little information has been forthcoming regarding a conserved mechanism by which apoptotic proteins achieve this non-death outcome. In the following discussion, we will explore the premise that the penultimate step in apoptosis, genome wide DNA damage/strand breaks act as a conserved genomic reprogramming event necessary for cell differentiation (Larsen et al. Proc Natl Acad Sci USA 2010; 107:4230-5). Moreover, we hypothesis that directed DNA damage, as mediated by known apoptotic proteins, may participate in numerous forms of regulated gene expression.

Caspase 3 is required for the differentiation of a wide variety of cell types, yet it remains unclear how this apoptotic protein could promote such a cell-fate decision. Caspase signals often result in the activation of the specific nuclease caspase-activated DNase (CAD), suggesting that cell differentiation may be dependent on a CAD-mediated modification in chromatin structure. In this study, we have investigated if caspase 3/CAD plays a role in initiating the DNA strand breaks that are known to occur during the terminal differentiation of skeletal muscle cells. Here, we show that inhibition of caspase 3 or reduction of CAD expression leads to a dramatic loss of strand-break formation and a block in the myogenic program. Caspase-dependent induction of differentiation results in CAD targeting of the p21 promoter, and loss of caspase 3 or CAD leads to a significant down-regulation in p21 expression. These results show that caspase 3/CAD promotes cell differentiation by directly modifying the DNA/nuclear microenvironment, which enhances the expression of critical regulatory genes.

In an effort to discover potent, orally bioavailable compounds for the treatment of atrial fibrillation (AF) and ventricular tachycardia (VT), we developed a class of gap-junction modifiers typified by GAP-134 (1, R(1)=OH, R(2)=NH(2)), a compound currently under clinical evaluation. Selected compounds with the desired in-vitro profile demonstrated positive in vivo results in the mouse CaCl(2) arrhythmia model upon oral administration.

Gap junction pharmacology is a nascent field. Previous studies have identified molecules that enhance intercellular communication, and may offer potential for innovative antiarrhythmic therapy. However, their specific molecular target(s) and mechanism(s) of action remain unknown. Previously, we identified a 34-aa peptide (RXP-E) that binds the carboxyl terminal domain of Cx43 (Cx43CT) and prevents cardiac gap junction closure and action potential propagation block. These results supported the feasibility of a peptide-based pharmacology to Cx43, but the structure of the core active element in RXP-E, an essential step for pharmacological development, remained undefined. Here, we used a combination of molecular modeling, surface plasmon resonance, nuclear magnetic resonance and patch-clamp strategies to define, for the first time, a unique ensemble of pharmacophores that bind Cx43CT and prevent closure of Cx43 channels. Two particular molecules are best representatives of this family: a cyclized heptapeptide (called CyRP-71) and a linear octapeptide of sequence RRNYRRNY. These 2 small compounds offer the first structural platform for the design of Cx43-interacting gap junction openers. Moreover, the structure of these compounds offers an imprint of a region of Cx43CT that is fundamental to gap junction channel function.

Rotigaptide (3) is an antiarrhythmic peptide that improves cardiac conduction by modifying gap-junction communication. Small molecule gap-junction modifiers with improved physical properties were identified from a Zealand Pharma peptide library using pharmaceutical profiling, established SAR around 3, and a putative pharmacophore model for rotigaptide. Activity of the compounds was confirmed in a mouse cardiac conduction block model of arrhythmia. Dipeptide 9f (GAP-134) was identified as a potent, orally active gap-junction modifier for clinical development.

Previous studies suggest that dephosphorylation of connexin43 (Cx43) is related to uncoupling of gap junction communication, which plays an important role in the genesis of ischemia-induced ventricular tachycardia. We studied changes in Cx43 phosphorylation during global ischemia in the absence and presence of the antiarrhythmic peptide analogue rotigaptide (formerly known as ZP123). Phosphorylation analysis was performed on Cx43 purified from isolated perfused rat hearts using matrix-assisted laser desorption/ionization mass spectrometry and liquid chromatography electrospray ionization tandem mass spectrometry. Thirteen different serine phosphorylation sites were identified in Cx43 during non-ischemic conditions, three of which had not previously been described. Within the first 7 min of ischemia, Ser306 became fully dephosphorylated whereas Ser330 became phosphorylated. Between 15 and 30 min of ischemia, the critical time interval where gap junction uncoupling occurs, Ser297 and Ser368 also became fully dephosphorylated. During the same time period, all untreated hearts developed asystole. Treatment with rotigaptide significantly increased the time to ischemia-induced asystole and suppressed dephosphorylation of Ser297 and Ser368 at 30 min of ischemia. Our results suggest that phosphorylation of Ser297 and Ser368 may be involved in functional gating of Cx43 during ischemia and may be possible downstream targets for rotigaptide signaling.

The antiarrhythmic and cardioprotective effect of increasing gap junction intercellular communication during ischemia/reperfusion injury has not been studied. The antiarrhythmic peptide rotigaptide (previously ZP123), which maintains gap junction intercellular communication, was tested in dogs subjected to a 60-min coronary artery occlusion and 4 h of reperfusion. Rotigaptide was administered i.v. 10 min before reperfusion as a bolus + i.v. infusion at doses of 1 ng/kg bolus + 10 ng/kg/h infusion (n = 6), 10 ng/kg bolus + 100 ng/kg/h infusion (n = 5), 100 ng/kg bolus + 1000 ng/kg/h infusion (n = 8), 1000 ng/kg bolus + 10 mug/kg/h infusion (n = 6), and vehicle control (n = 5). Premature ventricular complexes (PVCs) were quantified during reperfusion. A series of four or more consecutive PVCs was defined as ventricular tachycardia (VT). The total incidence of VT was reduced significantly with the two highest doses of rotigaptide (20.3 +/- 10.9 and 4.3 +/- 4.1 events; p < 0.05) compared with controls (48.7 +/- 6.0). Total PVCs were reduced significantly from 25.1 +/- 4.2% in control animals to 11.0 +/- 4.4 and 1.7 +/- 1.3% after the two highest doses of rotigaptide. Infarct size, expressed as a percentage of the left ventricle, was reduced significantly from 13.2 +/- 1.9 in controls to 7.1 +/- 1.0 (p < 0.05) at the highest dose of rotigaptide. Ultrastructural evaluation revealed no differences in myocardial injury in the infarct area, area at risk, border zone, or normal zone in vehicle and rotigaptide-treated animals. However, rotigaptide did increase the presence of gap junctions in the area at risk (p = 0.022, Fisher's exact test). Rotigaptide had no effect on heart rate, blood pressure, heart rate-corrected QT interval, or left ventricular end-diastolic pressure. In conclusion, these results demonstrate that rotigaptide is a potent antiarrhythmic compound with cardioprotective effects and desirable safety.

We characterized the novel, rationally designed peptide glucagon-like peptide 1 (GLP-1) receptor agonist H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSK KKKKK-NH2 (ZP10A). Receptor binding studies demonstrated that the affinity of ZP10A for the human GLP-1 receptor was 4-fold greater than the affinity of GLP-1 (7-36) amide. ZP10A demonstrated dose-dependent improvement of glucose tolerance with an ED50 value of 0.02 nmol/kg i.p. in an oral glucose tolerance test (OGTT) in diabetic db/db mice. After 42 days of treatment, ZP10A dose-dependently (0, 1, 10, or 100 nmol/kg b.i.d.; n = 10/group), decreased glycosylated hemoglobin (HbA1C) from 8.4 +/- 0.4% (vehicle) to a minimum of 6.2 +/- 0.3% (100 nmol/kg b.i.d.; p < 0.05 versus vehicle) in db/db mice. Fasting blood glucose (FBG), glucose tolerance after an OGTT, and HbA1C levels were significantly improved in mice treated with ZP10A for 90 days compared with vehicle-treated controls. Interestingly, these effects were preserved 40 days after drug cessation in db/db mice treated with ZP10A only during the first 50 days of the study. Real-time polymerase chain reaction measurements demonstrated that the antidiabetic effect of early therapy with ZP10A was associated with an increased pancreatic insulin mRNA expression relative to vehicle-treated mice. In conclusion, long-term treatment of diabetic db/db mice with ZP10A resulted in a dose-dependent improvement of FBG, glucose tolerance, and blood glucose control. Our data suggest that ZP10A preserves beta-cell function. ZP10A is considered one of the most promising new drug candidates for preventive and therapeutic intervention in type 2 diabetes.

Antiarrhythmic peptides (AAPs) are a group of compounds with antiarrhythmic properties; however, their use has been hampered by very low plasma stability. The aim of this study was to compare the in vitro and in vivo stability of our new stable AAP analog Ac-d-Tyr-d-Pro-d-Hyp-Gly-d-Ala-Gly-NH2 (ZP123) with the previously described AAP analog AAP10. Moreover, the effect of the two compounds was examined in a murine in vivo model of ouabain-induced second degree AV-block, and the effect on dispersion of action potential duration (APD dispersion) was studied during hypokalemic-ischemia in isolated perfused rabbit hearts. The in vitro t1/2 of ZP123 in rat and human plasma was about 1,700 times longer than t1/2 of AAP10. Due to rapid elimination, it was not possible to obtain an in vivo pharmacokinetic characterization of AAP10; however, calculations suggested that the clearance of ZP123 was at least 140 times slower than for AAP10. AAP10 and ZP123 produced a dose-dependent delay in onset of ouabain-induced AV-block in mice at doses of 10-11 to 10-7 mol/kg i.v. ZP123 and 10-11 to 10-6 mol/kg i.v. AAP10. Maximal efficacy of ZP123 was reached at a 10-fold lower dose (10-8 mol/kg i.v.) than with AAP10. In the isolated rabbit hearts, ZP123 and AAP10 had no effect on dispersion during control conditions. The increased APD dispersion during hypokalemic ischemia is considered a major arrhythmic substrate and only ZP123 prevented the increase in APD dispersion. In conclusion, ZP123 is a new potent AAP analog with improved stability.

Antiarrhythmic peptides such as AAP10 (Gly-Ala-Gly-4Hyp-Pro-Tyr-CONH(2)) have antiarrhythmic properties related to their stimulatory effect on gap junctional coupling. However, most of these peptides are not stable in enzymatic environment which limits studies with these compounds in vivo. ZP123 is a new antiarrhythmic peptide constructed using a retro-all-D-amino acid design of the AAP10 template (Ac-D-Tyr-D-Pro-D-4Hyp-Gly-D-Ala-Gly-NH(2)). The aim of this study was to compare the effects of AAP10 and ZP123 on epicardial activation and repolarization patterns in isolated perfused rabbit hearts. In addition, we tested the effect of these compounds on PKC activation in cultured HeLa-Cx43 cells. Rabbit hearts were perfused according to the Langendorff technique with Tyrode solution at constant pressure (70 cm H(2)O). After 45 min equilibration, either AAP10 (n = 7) or ZP123 (n = 7) was infused intracoronarily in concentrations of 0.1, 1, 10, 100, and 1000 nM (15 min for each concentration) in the presence of 0.05% bovine serum albumine. 256 AgCl electrodes were attached to the hearts surface and connected to the inputs of a 256 channel mapping system in a unipolar circuit (4 kHz/channel, 0.04 mV vertical resolution, 1 mm spatial resolution). For each electrode the activation and repolarization timepoint were determined. We found that both peptides significantly reduced epicardial dispersion by a maximum of about 20% thereby enhancing the homogeneity of epicardial action potential duration, while the action potential duration itself was not affected. The beat-to-beat variability of the epicardial activation pattern was stabilized by both peptides as compared to an untreated time-control series. Other parameters such as LVP, CF, heart rate, or total activation time were not effected by either of the peptides. In a second protocol, rectangular pulses were delivered to the back wall and the propagation velocity was determined longitudinal and transversal to the fiber axis. We found an increase in both longitudinal and transversal conduction velocity. Using a commercial PKC assay on HeLa-Cx43 cells we found that 50 nM AAP10 and 50 nM ZP123 increased activity by 99 +/- 6% and 146 +/- 54%, respectively. The PKC activation induced by either of these compounds was completely blocked using the selective PKCalpha inhibitor GCP54345. We conclude that AAP10 and ZP123 have similar effects in vitro, but the superior enzymatic stability of ZP123 makes this compound the preferred substance for in vivo studies of antiarrhythmic peptides.

1 This study reports on the pharmacological characterization of ZP120, a novel ligand of the nociceptin/orphanin FQ (N/OFQ) peptide receptor, NOP. ZP120 is a structure inducing probes modified NOP ligand: Zealand Pharma proprietary SIP technology was used to increase the enzymatic stability and half-life of peptide. 2 In vitro, ZP120 mimicked the inhibitory effects of N/OFQ in the electrically stimulated mouse vas deferens, showing however higher potency (pEC(50) 8.88 vs 7.74), lower maximal effects (E(max) 69+/-5% vs 91+/-2%), and slower onset of action. Like N/OFQ, the effects of ZP120 were not modified by 1 micro M naloxone, but they were antagonized by the NOP receptor selective antagonist J-113397 (pA(2) 7.80 vs ZP120, 7.81 vs N/OFQ). 3 In vivo, ZP120 mimicked the effects of N/OFQ, producing pronociceptive effects in the tail withdrawal assay and decreased locomotor activity after i.c.v., but not after i.v. administration in mice. ZP120 elicited similar maximal effects as N/OFQ, but it was about 10 fold more potent and its effects lasted longer. 4 In conclusion, the novel NOP receptor ligand ZP120 is a highly potent and selective partial agonist of the NOP receptor with prolonged effects in vivo.

The model prodrug D-Asp(OBzl)-Ala has previously been shown to have affinity and to be transported by the oligopeptide transporter PepT1 expressed in Caco-2 cells. The main objective of the present study was to investigate the aqueous stability of D-Asp(OBzl)-Ala and its in vitro metabolism in different gastrointestinal media arising from rats and humans, as well as in human plasma. The second major aim of the study was to evaluate our previous study in Caco-2 cell culture, by determining the effective intestinal permeability (Peff) of D-Asp(OBzl)-Ala in situ using the single-pass rat perfusion model. The aqueous stability studies show water, general buffer, as well as specific acid and base catalysis of D-Asp(OBzl)-Ala. The degradation of the model prodrug was independent of ionic strength. The half-lives in rat jejunal fluid and homogenate were >3 h. In human gastric and intestinal fluids, the half-lives were >3 h and 2.3+/-0. 03 h, respectively. Using the rat single-pass perfusion technique, the effective jejunal permeability (Peff) of D-Asp(OBzl)-Ala was determined to be high (1.29+/-0.5.10-4 cm/s). The 32 times higher Peff value found in the perfusion model compared to Caco-2 cells is most likely due to a higher functional expression of the oligopeptide transporter. Rat jejuna Peff was reduced by approximately 50% in the presence of well known oligopeptide transporter substrates, such as Gly-Sar and cephalexin. It may be that D-Asp(OBzl)-Ala is primarily absorbed intact by the rat jejunal oligopeptide transporter, since the stability in the intestinal homogenate and fluids was rather high (t1/2>2.3 h).

Four N-terminal 4-imidazolidinone prodrugs of Leu-enkephalin are prepared and characterized. Their enzymatic and chemical stability are assessed using high-performance liquid chromatography. The prodrug derivatives are shown to degrade stoichiometrically to Leu-enkephalin in phosphate buffer [t1/2 (0.05 M phosphate buffer without KCl): acetone prodrug (II) 930 min; cyclopentanone prodrug (III): 216 min; cyclohexanone prodrug (IV): 432 min; 4-methylcyclohexanone prodrug (V): 792 min]. Furthermore, the prodrugs are shown to afford global stabilization of the Leu-enkephalin molecule towards the enzymes, aminopeptidase N and angiotensin converting enzyme, primarily responsible for degradation of Leu-enkephalin at the blood-brain barrier and in plasma. Therefore, the 4-imidazolidinones, being metabolic stable and bioreversible, may be suitable prodrug candidates for delivery of Leu-enkephalin to important target areas such as the brain, if given intravenously.

A new heterobifunctional cross-linking reagent, 1,2,3-thiadiazole-4-carboxylic acid, for the photochemical conjugation of peptides to proteins is described. The title compound can be coupled directly to a protected peptide resin during solid-phase peptide synthesis (SPPS) using standard coupling procedures. The probe is stable to TFA deprotection/cleavage mixtures containing ethanedithiol commonly used in Fmoc-SPPS. Furthermore, tritium may easily be introduced into the thiadiazole ring by base-catalyzed hydrogen-exchange. Upon irradiation at 245-300 nm, parent 1,2,3-thiadiazole rapidly eliminates N2, generating very reactive thioketene which reacts with amines to give a thioamide in high yield, even when the photolysis is carried out in hydroxylic solvents. In order to investigate the potential of the title compound as a heterobifunctional cross-linking reagent a model study with angiotensin II (AII) was conducted. The photoreactive peptide N2-4-carbonyl-1,2,3-thiadiazole-AII (TDA-AII) was synthesized by Fmoc-SPPS and conjugated to bovine serum albumin (BSA) by photolysis at 245 and 300 nm. By use of a capture competition ELISA, the C-terminal Pro-Phe epitope of photoconjugated AII with the sequence DRVYIHPF was shown to bind specifically to antiAII antibodies (anti-AII abs), although antibodies against both the C- and N-terminal epitopes were present in the assay. A dipeptide His-Leu carboxy-extension form of AII, angiotensin I (AI), only bound to anti-AII abs at 100-200 times higher concentrations, showing that the C-terminal epitope was blocked by the dipeptide.

During solid-phase peptide synthesis of homo-oligopeptides containing leucine or alanine using the Fmoc strategy, we have observed ineffective N-alpha-deprotection with piperidine in a sequence-dependent manner. Incomplete deprotection was found to be associated with subsequent slow or incomplete amino acid coupling. Optimization of the deprotection step was carried out by varying the experimental conditions e.g. deprotection time, temperature, solvents and addition of chaotropes. Coupling and deprotection steps have been investigated using color monitoring, as well as FAB MS and HPLC for product analysis. The phenomena of difficult coupling and deprotection steps in the investigated systems have been demonstrated to have the same physical chemical origins, beta-sheet formation.