Combination of Irinotecan and Melatonin with the Natural Compounds Wogonin and Celastrol for Colon Cancer Treatment.

Colorectal cancers are one of the leading cancers worldwide and are known for their high potential for metastasis and resistance to therapy. The aim of this study was to investigate the effect of various combination therapies of irinotecan with melatonin, wogonin, and celastrol on drug-sensitive colon cancer cells (LOVO cell line) and doxorubicin-resistant colon cancer stem-like cells (LOVO/DX cell subline). Melatonin is a hormone synthesized in the pineal gland and is responsible for circadian rhythm. Wogonin and celastrol are natural compounds previously used in traditional Chinese medicine. Selected substances have immunomodulatory properties and anti-cancer potential. First, MTT and flow cytometric annexin-V apoptosis assays were performed to determine the cytotoxic effect and the induction of apoptosis. Then, the potential to inhibit cell migration was evaluated using a scratch test, and spheroid growth was measured. The results showed important cytotoxic effects of the drug combinations on both LOVO and LOVO/DX cells. All tested substances caused an increase in the percentage of apoptotic cells in the LOVO cell line and necrotic cells in the LOVO/DX cell subline. The strongest effect on the induction of cancer cell death was observed for the combination of irinotecan with celastrol (1.25 µM) or wogonin (50 µM) and for the combination of melatonin (2000 µM) with celastrol (1.25 µM) or wogonin (50 µM). Statistically significant improvements in the effect of combined therapy were found for the irinotecan (20 µM) and celastrol (1.25 µM) combination and irinotecan (20 µM) with wogonin (25 µM) in LOVO/DX cells. Minor additive effects of combined therapy were observed in LOVO cells. Inhibition of cell migration was seen in LOVO cells for all tested compounds, while only irinotecan (20 µM) and celastrol (1.25 µM) were able to inhibit LOVO/DX cell migration. Compared with single-drug therapy, a statistically significant inhibitory effect on cell migration was found for combinations of melatonin (2000 µM) with wogonin (25 µM) in LOVO/DX cells and irinotecan (5 µM) or melatonin (2000 µM) with wogonin (25 µM) in LOVO cells. Our research shows that adding melatonin, wogonin, or celastrol to standard irinotecan therapy may potentiate the anti-cancer effects of irinotecan alone in colon cancer treatment. Celastrol seems to have the greatest supporting therapy effect, especially for the treatment of aggressive types of colon cancer, by targeting cancer stem-like cells.

A New Triterpenoid Saponin from Albizia zygia Induced Apoptosis by Reduction of Mitochondrial Potential Status in Malignant Melanoma Cells.

In our ongoing research program on the proapoptotic function of saponins, two previously undescribed saponins, named zygiaosides E (1: ) and F (2: ), were isolated from the leaves of Albizia zygia. Their structures were established based on extensive analysis of 1D and 2D NMR data, HR-ESI-MS analysis, and by chemical degradation. The proapoptotic effect of zygiaoside E (1: ) was evaluated on human malignant melanoma (A375), human epidermoid cancer (A431), and normal Homo sapiens skin tissue (TE 353.SK.) cell lines by cytometric analysis. Zygiaoside E (1: ) induced apoptosis of the two human cancer cell lines (A375 and A431) in a dose-dependent manner at 1 µM but did not induce apoptosis in noncancerous skin cells (TE 353.Sk), even when treated with concentrations up to 15 µM. The underlying mechanism of the apoptosis induction activity of zygiaoside E (1: ) on the mitochondrial membrane potential status in A375 cells was further assessed by monitoring the uptake rate of DiOC6, a mitochondrial specific and voltage-dependent fluorescent dye. The number of malignant melanoma cells emitting high fluorescence levels was decreased when cells were treated with 3 or 5 µM of zygiaoside E (1: ) during either 12 or 24 h, thereby revealing a drop of mitochondrial membrane potential in A375 cells upon treatment, which indicated mitochondrial perturbation.

Cytotoxic and Pro-Apoptotic Effects of Leaves Extract of Antiaris africana Engler (Moraceae).

Antiaris africana Engler leaves have been used in Senegalese folk medicine to treat breast cancer. The present study aimed to investigate the anticancer potential of Antiaris africana Engler leaves using several human cancer cell lines. The leaves of Antiaris africana Engler were extracted in parallel with water or 70% ethanol and each extract divided into three parts by successive liquid-liquid extraction with ethyl acetate and butanol. The phytochemical components of the active extract were investigated using ultra-performance liquid chromatography-diode array detector-quadrupole time-of-flight tandem mass spectrometry (UPLC-DAD-QTOF-MS/MS). The cytotoxic and cytostatic effects of each extract, as well as their fractions, were evaluated in vitro via flow and image cytometry on different human cancer phenotypes, such as breast (MCF-7), pancreas (AsPC-1), colon (SW-620) and acute monocytic leukemia (THP-1). Both hydro-alcoholic and aqueous extracts induced strong apoptosis in MCF-7 cells. The water fraction of the hydro-alcoholic extract was found to be the most active, suppressing the cell growth of MCF-7 in a dose-dependent manner. The half maximum effective concentration (EC50) of this fraction was 64.6 ± 13.7 µg/mL for MCF-7, with equivalent values for all tested phenotypes. In parallel, the apoptotic induction by this fraction resulted in a EC50 of 63.5 ± 1.8 µg/mL for MCF-7, with again equivalent values for all other cellular tested phenotypes. Analysis of this fraction by UPLC-DAD-QTOF-MS/MS led to the identification of hydroxycinnamates as major components, one rutin isomer, and three cardiac glycosides previously isolated from seeds and bark of Antiaris africana Engler and described as cytotoxic in human cancer models. These results provide supportive data for the use of Antiaris africana Engler leaves in Senegal.

Triple dehydrofluorination as a route to amidine-functionalized, aromatic phosphorus heterocycles.

An unexpected route to hitherto unknown amidine-functionalized phosphinines has been developed that is rapid and simple. Starting from primary amines and CF3-substituted λ3,σ2-phosphinines, a cascade of dehydrofluorination reactions leads selectively to ortho-amidinephosphinines. DFT calculations reveal that this unusual transformation can take place via a series of nucleophilic attacks at the electrophilic, low-coordinate phosphorus atom.

Analysis of hyperforin (St. John's wort) action at TRPC6 channel leads to the development of a new class of antidepressant drugs.

St. John's wort is an herb, long used in folk medicine for the treatment of mild depression. Its antidepressant constituent, hyperforin, has properties such as chemical instability and induction of drug-drug interactions that preclude its use for individual pharmacotherapies. Here we identify the transient receptor potential canonical 6 channel (TRPC6) as a druggable target to control anxious and depressive behavior and as a requirement for hyperforin antidepressant action. We demonstrate that TRPC6 deficiency in mice not only results in anxious and depressive behavior, but also reduces excitability of hippocampal CA1 pyramidal neurons and dentate gyrus granule cells. Using electrophysiology and targeted mutagenesis, we show that hyperforin activates the channel via a specific binding motif at TRPC6. We performed an analysis of hyperforin action to develop a new antidepressant drug that uses the same TRPC6 target mechanism for its antidepressant action. We synthesized the hyperforin analog Hyp13, which shows similar binding to TRPC6 and recapitulates TRPC6-dependent anxiolytic and antidepressant effects in mice. Hyp13 does not activate pregnan-X-receptor (PXR) and thereby loses the potential to induce drug-drug interactions. This may provide a new approach to develop better treatments for depression, since depression remains one of the most treatment-resistant mental disorders, warranting the development of effective drugs based on naturally occurring compounds.

Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis.

Haematophagous leeches express a broad variety of secretory proteins in their salivary glands, among them are hirudins and hirudin-like factors. Here, we describe the identification, molecular and initial functional characterization of Tandem-Hirudin (TH), a novel salivary gland derived factor identified in the Asian medicinal leech, Hirudinaria manillensis. In contrast to the typical structure of hirudins, TH comprises two globular domains arranged in a tandem-like orientation and lacks the elongated C-terminal tail. Similar structures of thrombin inhibitors have so far been identified only in kissing bugs and ticks. Expression of TH was performed in both cell-based and cell-free bacterial systems. A subsequent functional characterization revealed no evidence for a thrombin-inhibitory potency of TH.

Life without blood: Molecular and functional analysis of hirudins and hirudin-like factors of the Asian non-hematophagous leech Whitmania pigra.

BACKGROUND: Several leech species of the genera Hirudo, Hirudinaria, and Whitmania are widely used in traditional Chinese medicine (TCM) for the oral treatment of disorders associated with blood stasis. Among them, the non-hematophagous leech Whitmania pigra expresses a variety of components that have the potential to act on the vertebrate blood coagulation system. OBJECTIVE: Whether the thrombin inhibitor hirudin, probably the most prominent leech-derived anticoagulant, is actually present in Whitmania pigra, is still a matter of debate. To answer that open question was the aim of the study. METHODS: We identified several putative hirudin-encoding sequences in transcriptome data of Whitmania pigra. Upon gene synthesis and molecular cloning the respective recombinant proteins were expressed in Escherichia coli, purified, processed, and eventually functionally characterized for thrombin-inhibitory potencies in coagulation assays. RESULTS: We were successful in the identification and functional characterization of several putative hirudins in Whitmania pigra. Some, but not all, of these factors are indeed thrombin inhibitors. Whitmania pigra hence expresses both hirudins (factors that inhibit thrombin) and hirudin-like factors (that do not or only very weakly inhibit thrombin). Furthermore, we revealed the exon/intron structures of the corresponding genes. Coding sequences of some putative hirudins of Whitmania pigra were present also in transcriptome datasets of Hirudo nipponia, a hematophagous leech that is likewise used in TCM. CONCLUSIONS: Based on both structural and functional data we provide very strong evidence for the expression of hirudins in Whitmania pigra. This is the first description of hirudins in a non-hematophagous leech.

Expression of cardiovascular-related microRNAs is altered in L-arginine:glycine amidinotransferase deficient mice.

In humans and mice, L-arginine:glycine amidinotransferase (AGAT) and its metabolites homoarginine (hArg) and creatine have been linked to cardiovascular disease (CVD), specifically myocardial infarction (MI) and heart failure (HF). The underlying molecular and regulatory mechanisms, however, remain unclear. To identify potential pathways of cardiac AGAT metabolism, we sequenced microRNA (miRNA) in left ventricles of wild-type (wt) compared to AGAT-deficient (AGAT-/-) mice. Using literature search and validation by qPCR, we identified eight significantly regulated miRNAs in AGAT-/- mice linked to atherosclerosis, MI and HF: miR-30b, miR-31, miR-130a, miR-135a, miR-148a, miR-204, miR-298, and let-7i. Analysis of Gene Expression Omnibus (GEO) data confirmed deregulation of these miRNAs in mouse models of MI and HF. Quantification of miRNA expression by qPCR in AGAT-/- mice supplemented with creatine or hArg revealed that miR-30b, miR-31, miR-130a, miR-148a, and miR-204 were regulated by creatine, while miR-135a and miR-298 showed a trend of regulation by hArg. Finally, bioinformatics-based target prediction showed that numerous AGAT-dependent genes previously linked to CVD are likely to be regulated by the identified miRNAs. Taken together, AGAT deficiency and hArg/creatine supplementation are associated with cardiac miRNA expression which may influence cardiac (dys)function and CVD.

Cannabis sativa Extract Induces Apoptosis in Human Pancreatic 3D Cancer Models: Importance of Major Antioxidant Molecules Present Therein.

In recent years, interest in Cannabis sativa L. has been rising, as legislation is moving in the right direction. This plant has been known and used for thousands of years for its many active ingredients that lead to various therapeutic effects (pain management, anti-inflammatory, antioxidant, etc.). In this report, our objective was to optimize a method for the extraction of cannabinoids from a clone of Cannabis sativa L. #138 resulting from an agronomic test (LaFleur, Angers, FR). Thus, we wished to identify compounds with anticancer activity on human pancreatic tumor cell lines. Three static maceration procedures, with different extraction parameters, were compared based on their median inhibitory concentration (IC50) values and cannabinoid extraction yield. As CBD emerged as the molecule responsible for inducing apoptosis in the human pancreatic cancer cell line, a CBD-rich cannabis strain remains attractive for therapeutic applications. Additionally, while gemcitabine, a gold standard drug in the treatment of pancreatic cancer, only triggers cell cycle arrest in G0/G1, CBD also activates the cell signaling cascade to lead to programmed cell death. Our results emphasize the potential of natural products issued from medicinal hemp for pancreatic cancer therapy, as they lead to an accumulation of intracellular superoxide ions, affect the mitochondrial membrane potential, induce G1 cell cycle arrest, and ultimately drive the pancreatic cancer cell to lethal apoptosis.

Tannin extract from maritime pine bark exhibits anticancer properties by targeting the epigenetic UHRF1/DNMT1 tandem leading to the re-expression of TP73.

Maritime pine bark is a rich source of polyphenolic compounds and is commonly employed as a herbal supplement worldwide. This study was designed to check the potential of maritime pine tannin extract (MPTE) in anticancer therapy and to determine the underlying mechanism of action. Our results showed that MPTE, containing procyanidin oligomers and lanostane type terpenoids, has an inhibitory effect on cancer cell proliferation through cell cycle arrest in the G2/M phase. Treatment with MPTE also induced apoptosis in a concentration-dependent manner in human cancer cell lines (HeLa and U2OS), as evidenced by the enhanced activation of caspase 3 and the cleavage of PARP along with the downregulation of the antiapoptotic protein Bcl-2. Interestingly, human non-cancerous fibroblasts are much less sensitive to MPTE, suggesting that it preferentially targets cancer cells. MPTE played a pro-oxidant role in cancer cells and promoted the expression of the p73 tumor suppressor gene in p53-deficient cells. It also downregulated the protooncogenic proteins UHRF1 and DNMT1, mediators of the DNA methylation machinery, and reduced the global methylation levels in HeLa cells. Overall, our results show that maritime pine tannin extract can play a favorable role in cancer treatment, and can be further explored by the pharmaceutical industry.

Short breaks at school: effects of a physical activity and a mindfulness intervention on children's attention, reading comprehension, and self-esteem.

BACKGROUND: Although breaks are essential to restoring cognitive and psychological conditions for learning, short breaks within school lessons are not established and the specificity of effects has not often been investigated. Therefore, the effects of a physical activity (Study 1) and a mindfulness intervention (Study 2) were investigated. PROCEDURE: By an intervention-control group design, the effects of daily 10-min physical activity (Study 1: N = 162, 4th grade) and mindfulness breaks (Study 2: N = 79, 5th grade) were implemented within regular school lessons over a 2-week time period to research the impact on attention, reading comprehension, and self-esteem. RESULTS: In the physical activity intervention children's attention improved (attention-processing speed: p < .004, ηp2 = .05, attention-performance: p < .025, ηp2 = .03), and in the mindfulness intervention reading comprehension improved (p < .012, ηp2 = .08) compared to the controls. Results further indicated that self-esteem moderated the relationship between groups and attention improvement in study 1. CONCLUSION: Classroom-based short physical and mindfulness breaks could support attention and reading comprehension, which are known to support overall academic success.

Short tail stories: the hirudin-like factors HLF6 and HLF7 of the Asian medicinal leech, Hirudinaria manillensis.

The leech-derived hirudins and hirudin-like factors (HLFs) share a common molecule structure: a short N-terminus, a central globular domain, and an elongated C-terminal tail. All parts are important for function. HLF6 and HLF7 were identified in the Asian medicinal leech, Hirudinaria manillensis. The genes of both factors encode putative splice variants that differ in length and composition of their respective C-terminal tails. In either case, the tails are considerably shorter compared to hirudins. Here we describe the functional analyses of the natural splice variants and of synthetic variants that comprise an altered N-terminus and/or a modified central globular domain. All natural splice variants of HLF6 and HLF7 display no detectable thrombin-inhibitory potency. In contrast, some synthetic variants effectively inhibit thrombin, even with tails as short as six amino acid residues in length. Our data indicate that size and composition of the C-terminal tail of hirudins and HLFs can vary in a great extent, yet the full protein may still retain the ability to inhibit thrombin.

Proteomic analysis reveals brain Rab35 as a potential biomarker of mitragynine withdrawal in rats.

Mitragyna speciosa, also known as kratom, has been used for mitigating the severity of opioid withdrawal in humans. Its main indole alkaloid, mitragynine, has been considered as a pharmacotherapy for pain conditions and opioid replacement therapy. However, at high doses, chronic mitragynine may also have an addiction potential. The effects of chronic action of mitragynine in the brain are still unknown. The present study developed a mitragynine withdrawal model in rats and used it for a proteomic analysis of mitragynine withdrawal effects. Mitragynine (30 mg/kg, i.p.) was administered daily over a period of 14 days and then withdrawn. A proteomic analysis revealed that from a total of 1524 proteins identified, 31 proteins were upregulated, and 3 proteins were downregulated in the mitragynine withdrawal model. The Rab35 protein expression increased most profoundly in the mitragynine withdrawal group as compared to vehicle group. Therefore, it is proposed that Rab35 in the brain might be considered as a potential biomarker during mitragynine withdrawal and might be valuable target protein in developing new pharmacotherapies in the future.

The effects of chronic mitragynine (Kratom) exposure on the EEG in rats.

Mitragynine is the main alkaloid isolated from the leaves of Mitragyna speciosa Korth (Kratom). Kratom has been widely used to relieve pain and opioid withdrawal symptoms in humans but may also cause memory deficits. Here we investigated the changes in brain electroencephalogram (EEG) activity after acute and chronic exposure to mitragynine in freely moving rats. Vehicle, morphine (5 mg/kg) or mitragynine (1, 5 and 10 mg/kg) were administered for 28 days, and EEG activity was repeatedly recorded from the frontal cortex, neocortex and hippocampus. Repeated exposure to mitragynine increased delta, but decreased alpha powers in both cortical regions. It further decreased delta power in the hippocampus. These findings suggest that acute and chronic mitragynine can have profound effects on EEG activity, which may underlie effects on behavioral activity and cognition, particularly learning and memory function.

Cross-reinstatement of mitragynine and morphine place preference in rats.

Kratom is a medicinal plant that exhibits promising results as an opiate substitute. However, there is little information regarding the abuse profile of its main psychoactive constituent, mitragynine (MG), particularly in relapse to drug abuse. Using the place conditioning procedure as a model of relapse, this study aims to evaluate the ability of MG to induce conditioned place preference (CPP) reinstatement in rats. To evaluate the cross-reinstatement effects, MG and morphine were injected to rats that previously extinguished a morphine- or MG-induced CPP. Following a CPP acquisition induced by either MG (10 and 30 mg/kg, i.p.) or morphine (10 mg/kg, i.p.), rats were subjected to repeated CPP extinction sessions. A low dose priming injection of MG or morphine produced a reinstatement of the previously extinguished CPP. In the second experiment of this study, a priming injection of morphine (1, 3 and 10 mg/kg, i.p.) dose-dependently reinstated an MG-induced CPP. Likewise, a priming injection of MG (3, 10 and 30 mg/kg, i.p.) was able to dose-dependently reinstate a morphine-induced CPP. The present study demonstrates a cross-reinstatement effect between MG and morphine, thereby suggesting a similar interaction in their rewarding motivational properties. The findings from this study also suggesting that a priming exposure to kratom and an opioid may cause relapse for a previously abused drug.

Anti-Proliferative and Apoptosis-Inducing Activity of Acacia Modesta and Opuntia Monocantha Extracts on HeLa Cells.

BACKGROUND: Cancer is one of the leading causes of death in the world. Numerous phytochemicals from plants have shown antineoplastic effects via programmed cell death (apoptosis). The aim of this study was to evaluate the effect of anti-proliferative and apoptosis-inducing activity of Acacia modesta and Opuntia monocantha against HeLa cells. METHODS: To estimate anti-proliferative activity of the plants against HeLa cells, ethanol solvent was used for the extraction. For the evaluation of anti-proliferative effects, MTT assay was performed with 100, 200, and 400 µg/mL dose. The antioxidant assays including glutathione reductase (GSH), superoxide dismutase (SOD) and catalase were performed. Moreover, enzyme linked immunosorbent assay (ELISA) was performed. Furthermore, immunocytometry P53 and flow cytometry were also carried out to assess the apoptosis in HeLa cell. RESULTS: MTT assay showed that the groups treated with Opuntia monocantha and Acacia modest have less level of toxicity as compared to untreated groups. Antioxidant assays confirmed that GSH, SPD and, catalase activities were quite decreased in treated groups as compared to untreated groups. Similarly, ELISA and apoptosis p53 have shown more pronounced apoptosis effect in treated groups as compared to untreated groups. CONCLUSION: Based on above findings, treatment of HeLa cells with these plant extracts induced apoptosis, restricts proliferation, and enhances the anti-oxidative index in post treated cells.
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Biological Activities of Aqueous Extracts from Carob Plant (Ceratonia siliqua L.) by Antioxidant, Analgesic and Proapoptotic Properties Evaluation.

The present work describes the volatile compounds profile and phytochemical content of Ceratonia siliqua L. Fifty different components have been identified. Among them, three constituents are shared i.e., 2-methlybutanoic acid, methyl hexanoate and limonene by different common carob preparations: pulp decoction (PD), seeds decoction (SD) and Rob, a sweet syrup extracted from the pulp of the carob pod. Each extract exhibits different volatile aromatic emission profiles. The antioxidant activity of the extracts was evaluated using three methods, DPPH, ABTS and FRAP, producing a dose-dependent response. The IC50, when determined by FRAP, gave the lowest values (0.66 ± 0.01, 0.73 ± 0.05 and 0.55 ± 0.00 mg/mL PD, SD and Rob, respectively). The nociception essay, after intraperitoneal injection of acetic acid in mice, demonstrated that Rob, pulp and seeds decoction extracts showed an efficient inhibition of writhes over time, with persistence over 30 min. The SD decoction revealed the highest efficacy in decreasing the writhing reflex (90.3 ± 1.2%; p < 0.001). Furthermore, the proapoptotic activity of SD against three human cell line, THP-1, MCF-7 and LOVO, evaluated by flow cytometry, showed a significantly stronger proapoptotic activity on colon cancer (LOVO) than on the other cell lines, a phenomenon known as phenotypic selectivity.

[Metacognitive and Mentalization Based Concepts and Treatment Models: Implications for Child and Adolescent Psychiatry]. / Metakognitive und mentalisierungsbasierte Konzepte und Behandlungsmodelle: Implikationen für die Kinder- und Jugendpsychiatrie.

Metacognitive and Mentalization Based Concepts and Treatment Models: Implications for Child and Adolescent Psychiatry The aim of this article is to provide a survey of metacognitive and mentalization-based therapeutic approaches and possible applications in the field of child and adolescent psychiatry. Firstly areas of metacognition are described and the concept of mentalization is introduced. Next, the treatments Mentalization Based Treatment (MBT), Metacognitive Interpersonal Therapy (MIT) and Metacognitive Therapy (MCT) are presented. Treatment models, theoretical backgrounds, the most important treatment steps, and typical interventions are explained. The treatment modalities are discussed concerning their conceptual similarities and differences as well as their applications in child and adolescent psychiatry. Finally the treatments are subsumed under the aspect of therapeutic change processes. An outlook for an integrative approach matching the level of metacognitive competency is given.

The hirudin-like factors HLF3 and HLF4-hidden hirudins of European medicinal leeches.

The hirudin-like factors 3 (HLF3) and 4 (HLF4) belong to a new class of leech-derived factors and are present in specimens of the three European medicinal leeches, Hirudo medicinalis, Hirudo verbana, and Hirudo orientalis, respectively. Here we describe the functional analysis of natural and synthetic variants of HLF3 and HLF4. Whereas the natural variants display only very low or no detectable anti-coagulatory activities, modifications within the N-termini in combination with an exchange of the central globular domain have the potency to greatly enhance the inhibitory effects of respective HLF3 and HLF4 variants on blood coagulation. Our results support previous observations on the crucial importance of all parts (both the N- and C-termini as well as the central globular domains) of hirudin and HLF molecules for thrombin inhibition.

Analysis of L-arginine:glycine amidinotransferase-, creatine- and homoarginine-dependent gene regulation in the murine heart.

L-arginine:glycine amidinotransferase (AGAT) and its metabolites creatine and homoarginine (HA) have been linked to cardiovascular pathologies in both human and murine studies, but the underlying molecular mechanisms are poorly understood. Here, we report the first analysis of heart transcriptome variation using microarrays in an AGAT-deficient (AGAT-/-) mouse model to evaluate AGAT-, creatine- and HA-dependent gene regulation. Our data revealed significant differences of gene expression between AGAT-/- and wild-type (WT) mice, affecting cardiac energy metabolism (Fbp2, Ucp2), cardiac hypertrophy and fibrosis (Nppa, Ctgf), immune response (Fgl2), and the conduction system of the heart (Dsc2, Ehd4, Hcn2, Hcn4, Scn4a, Scn4b). All of these genes being expressed on WT level in creatine-supplemented mice. Using in silico analysis based on the GEO database we found that most of these candidate genes (Ctgf, Dsc2, Fbp2, Fgl2, Hcn2, Nppa)  revealed significant alterations in a WT mouse model of myocardial infarction underlining a pathophysiological relationship between AGAT metabolism and cardiovascular disease.