Metformin acts in the gut and induces gut-liver crosstalk.

Metformin is the most prescribed drug for DM2, but its site and mechanism of action are still not well established. Here, we investigated the effects of metformin on basolateral intestinal glucose uptake (BIGU), and its consequences on hepatic glucose production (HGP). In diabetic patients and mice, the primary site of metformin action was the gut, increasing BIGU, evaluated through PET-CT. In mice and CaCo2 cells, this increase in BIGU resulted from an increase in GLUT1 and GLUT2, secondary to ATF4 and AMPK. In hyperglycemia, metformin increased the lactate (reducing pH and bicarbonate in portal vein) and acetate production in the gut, modulating liver pyruvate carboxylase, MPC1/2, and FBP1, establishing a gut-liver crosstalk that reduces HGP. In normoglycemia, metformin-induced increases in BIGU is accompanied by hypoglycemia in the portal vein, generating a counter-regulatory mechanism that avoids reductions or even increases HGP. In summary, metformin increases BIGU and through gut-liver crosstalk influences HGP.

Regioselection Switch in Nucleophilic Addition to Isoquinolinequinones: Mechanism and Origin of the Regioselectivity in the Total Synthesis of Ellipticine.

Ellipticine was synthesized in six steps and 20% global yield starting from the readily available 2,5-dimethoxy isoquinoline. Unprecedented regioselective control of the nucleophilic attack on the isoquinoline-5,8-dione is first described. Investigation of the possible pathways of this transformation through density functional theory calculations reveals unexpected N-oxide assistance in cascade tautomerizations, which was crucial for directing the nucleophilic attack and hastening the overall process. Using this strategy, we prepared the aniline-isoquinolinedione adduct and submitted it to an intramolecular double C-H cross-coupling activation to furnish ellipticinequinone, which gave ellipticine after a MeLi addition/BH3 reduction sequence.

HER2 aptamer-conjugated iron oxide nanoparticles with PDMAEMA-b-PMPC coating for breast cancer cell identification.

Aim: To synthesize HER2 aptamer-conjugated iron oxide nanoparticles with a coating of poly(2-(dimethylamino) ethyl methacrylate)-poly(2-methacryloyloxyethylphosphorylcholine) block copolymer (IONPPPs). Methods: Characterization covered molecular structure, chemical composition, thermal stability, magnetic characteristics, aptamer interaction, crystalline nature and microscopic features. Subsequent investigations focused on IONPPPs for in vitro cancer cell identification. Results: Results demonstrated high biocompatibility of the diblock copolymer with no significant toxicity up to 150 µg/ml. The facile coating process yielded the IONPP complex, featuring a 13.27 nm metal core and a 3.10 nm polymer coating. Functionalized with a HER2-targeting DNA aptamer, IONPPP enhanced recognition in HER2-amplified SKBR3 cells via magnetization separation. Conclusion: These findings underscore IONPPP's potential in cancer research and clinical applications, showcasing diagnostic efficacy and HER2 protein targeting in a proof-of-concept approach.

Metabolomics by NMR Combined with Machine Learning to Predict Neoadjuvant Chemotherapy Response for Breast Cancer.

Neoadjuvant chemotherapy (NACT) is offered to patients with operable or inoperable breast cancer (BC) to downstage the disease. Clinical responses to NACT may vary depending on a few known clinical and biological features, but the diversity of responses to NACT is not fully understood. In this study, 80 women had their metabolite profiles of pre-treatment sera analyzed for potential NACT response biomarker candidates in combination with immunohistochemical parameters using Nuclear Magnetic Resonance (NMR). Sixty-four percent of the patients were resistant to chemotherapy. NMR, hormonal receptors (HR), human epidermal growth factor receptor 2 (HER2), and the nuclear protein Ki67 were combined through machine learning (ML) to predict the response to NACT. Metabolites such as leucine, formate, valine, and proline, along with hormone receptor status, were discriminants of response to NACT. The glyoxylate and dicarboxylate metabolism was found to be involved in the resistance to NACT. We obtained an accuracy in excess of 80% for the prediction of response to NACT combining metabolomic and tumor profile data. Our results suggest that NMR data can substantially enhance the prediction of response to NACT when used in combination with already known response prediction factors.

Identification and characterization of the anti-SARS-CoV-2 activity of cationic amphiphilic steroidal compounds.

The ongoing COVID-19 pandemic caused a significant loss of human lives and a worldwide decline in quality of life. Treatment of COVID-19 patients is challenging, and specific treatments to reduce COVID-19 aggravation and mortality are still necessary. Here, we describe the discovery of a novel class of epiandrosterone steroidal compounds with cationic amphiphilic properties that present antiviral activity against SARS-CoV-2 in the low micromolar range. Compounds were identified in screening campaigns using a cytopathic effect-based assay in Vero CCL81 cells, followed by hit compound validation and characterization. Compounds LNB167 and LNB169 were selected due to their ability to reduce the levels of infectious viral progeny and viral RNA levels in Vero CCL81, HEK293, and HuH7.5 cell lines. Mechanistic studies in Vero CCL81 cells indicated that LNB167 and LNB169 inhibited the initial phase of viral replication through mechanisms involving modulation of membrane lipids and cholesterol in host cells. Selection of viral variants resistant to steroidal compound treatment revealed single mutations on transmembrane, lipid membrane-interacting Spike and Envelope proteins. Finally, in vivo testing using the hACE2 transgenic mouse model indicated that SARS-CoV-2 infection could not be ameliorated by LNB167 treatment. We conclude that anti-SARS-CoV-2 activities of steroidal compounds LNB167 and LNB169 are likely host-targeted, consistent with the properties of cationic amphiphilic compounds that modulate host cell lipid biology. Although effective in vitro, protective effects were cell-type specific and did not translate to protection in vivo, indicating that subversion of lipid membrane physiology is an important, yet complex mechanism involved in SARS-CoV-2 replication and pathogenesis.

A role for focal adhesion kinase in cardiac mitochondrial biogenesis induced by mechanical stress.

We studied the implication of focal adhesion kinase (FAK) in cardiac mitochondrial biogenesis induced by mechanical stress. Prolonged stretching (2-12 h) of neonatal rat ventricular myocytes (NRVM) upregulated the main components of mitochondrial transcription cascade [peroxisome proliferator-activated receptor coactivator-1 (PGC-1α), nuclear respiratory factor (NRF-1), and mitochondrial transcription factor A]. Concomitantly, prolonged stretching enhanced mitochondrial biogenesis [copy number of mitochondrial DNA (mtDNA), content of the subunit IV of cytochrome oxidase, and mitochondrial staining-green fluorescence intensity of Mitotracker green] and induced the hypertrophic growth (cell size and atrial natriuretic peptide transcripts) of NRVM. Furthermore, the stretching of NRVM enhanced phosphorylation, nuclear localization, and association of FAK with PGC-1α. Recombinant FAK COOH-terminal, but not the NH(2)-terminal or kinase domain, precipitated PGC-1α from nuclear extracts of NRVM. Depletion of FAK by RNA interference suppressed the upregulation of PGC-1α and NRF-1 and markedly attenuated the enhanced mitochondrial biogenesis and hypertrophic growth of stretched NRVM. In the context of energy metabolism, FAK depletion became manifest by a reduction of ATP levels in stretched NRVM. Complementary studies in adult mice left ventricle demonstrated that pressure overload upregulated PGC-1α, NRF-1, and mtDNA. In vivo FAK silencing transiently attenuated the upregulation of PGC-1α, NRF-1, and mtDNA, as well as the left ventricular hypertrophy induced by pressure overload. In conclusion, activation of FAK signaling seems to be important for conferring enhanced mitochondrial biogenesis coupled to the hypertrophic growth of cardiomyocytes in response to mechanical stress, via control of mitochondrial transcription cascade.

Electrospun Nanofibrous Architectures of Thrombin-Loaded Poly(ethylene oxide) for Faster in Vivo Wound Clotting.

Wound healing materials to prevent blood loss are crucial during emergency medical treatment because uncontrolled bleeding can lead to patient death. Herein, bioabsorbable fibrous architectures of thrombin-loaded poly(ethylene oxide)-PEO/thrombin-are conceptualized and accomplished via electrospinning for faster wound clotting. Membranes with average fiber diameters ranging from 188 to 264 nm are achieved, where the active thrombin is entrapped within the nanofibers. The results of in vitro and in vivo wound healing activity tests revealed that when the nanofibers with thrombin-loaded capacity are in contact with the wound, the presence of water in the skin or blood catalyzes the degradation of the membranes, thus releasing thrombin. Thrombin then accelerates the wound clotting process. In contrast to other hemostatic materials, PEO/thrombin nanofibers do not require mechanical removal after application, and the viscoelastic nature of such biomaterials enables their conformation to a variety of wound topographies. Remarkably, PEO/thrombin membranes are promising functional materials and their use is a powerful strategy for hemostatic treatment, ranging from simple first aid and sealing to a wound to small surgical procedures.

Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors.

Xyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.

Using a Fragment-Based Approach to Identify Alternative Chemical Scaffolds Targeting Dihydrofolate Reductase from Mycobacterium tuberculosis.

Dihydrofolate reductase (DHFR), a key enzyme involved in folate metabolism, is a widely explored target in the treatment of cancer, immune diseases, bacteria, and protozoa infections. Although several antifolates have proved successful in the treatment of infectious diseases, they have been underexplored to combat tuberculosis, despite the essentiality of M. tuberculosis DHFR (MtDHFR). Herein, we describe an integrated fragment-based drug discovery approach to target MtDHFR that has identified hits with scaffolds not yet explored in any previous drug design campaign for this enzyme. The application of a SAR by catalog strategy of an in house library for one of the identified fragments has led to a series of molecules that bind to MtDHFR with low micromolar affinities. Crystal structures of MtDHFR in complex with compounds of this series demonstrated a novel binding mode that considerably differs from other DHFR antifolates, thus opening perspectives for the development of relevant MtDHFR inhibitors.

GLS2 is protumorigenic in breast cancers.

Many types of cancers have a well-established dependence on glutamine metabolism to support survival and growth, a process linked to glutaminase 1 (GLS) isoforms. Conversely, GLS2 variants often have tumor-suppressing activity. Triple-negative (TN) breast cancer (testing negative for estrogen, progesterone, and Her2 receptors) has elevated GLS protein levels and reportedly depends on exogenous glutamine and GLS activity for survival. Despite having high GLS levels, we verified that several breast cancer cells (including TN cells) express endogenous GLS2, defying its role as a bona fide tumor suppressor. Moreover, ectopic GLS2 expression rescued cell proliferation, TCA anaplerosis, redox balance, and mitochondrial function after GLS inhibition by the small molecule currently in clinical trials CB-839 or GLS knockdown of GLS-dependent cell lines. In several cell lines, GLS2 knockdown decreased cell proliferation and glutamine-linked metabolic phenotypes. Strikingly, long-term treatment of TN cells with another GLS-exclusive inhibitor bis-2'-(5-phenylacetamide-1,3,4-thiadiazol-2-yl)ethyl sulfide (BPTES) selected for a drug-resistant population with increased endogenous GLS2 and restored proliferative capacity. GLS2 was linked to enhanced in vitro cell migration and invasion, mesenchymal markers (through the ERK-ZEB1-vimentin axis under certain conditions) and in vivo lung metastasis. Of concern, GLS2 amplification or overexpression is linked to an overall, disease-free and distant metastasis-free worse survival prognosis in breast cancer. Altogether, these data establish an unforeseen role of GLS2 in sustaining tumor proliferation and underlying metastasis in breast cancer and provide an initial framework for exploring GLS2 as a novel therapeutic target.

Taurine supplementation enhances nutrient-induced insulin secretion in pancreatic mice islets.

BACKGROUND: Taurine (TAU), a naturally occurring sulfur-containing amino acid, is found at high concentrations in plasma and mammalian tissues and regulates osmolarity, ion channel activity, and glucose homeostasis. Several reports have shown that physiological plasma TAU levels seem to be important for adequate beta (beta)-cell function and insulin action, since low concentrations of TAU in the plasma have been reported in the pre-diabetic and diabetic states. METHODS: Glucose tolerance and insulin sensitivity were investigated in mice supplemented with 2% (w/v) TAU in their drinking water for 30 days, as well as the insulin secretion from isolated islets stimulated by glucose or L-leucine. RESULTS: TAU-supplemented mice demonstrated improved glucose tolerance and higher insulin sensitivity, compared to controls (CTL). In addition, their islets secreted more insulin in response to high concentrations of glucose and L-leucine. L-[U-(14)C]leucine oxidation was higher in TAU than in CTL islets, whereas D-[U-(14)C]glucose oxidation, ATP levels, glucose transporter (GLUT) 2 and glucokinase (GCK) protein expressions were similar in both types of islets. The L-type beta(2) subunit voltage-sensitive Ca(2+) channel protein, as well as (45)Ca uptake, were significantly higher in TAU-supplemented than CTL islets. In addition, islets from TAU-supplemented mice secreted more glucagon than CTL islets at low glucose. CONCLUSIONS: TAU supplementation improves glucose tolerance and insulin sensitivity in mice, as well as insulin secretion from isolated islets. The latter effect seems to be, at least in part, dependent on a better Ca(2+) handling by the islets.

Synthesis and testing of novel alternative oxidase (AOX) inhibitors with antifungal activity against Moniliophthora perniciosa (Stahel), the causal agent of witches' broom disease of cocoa, and other phytopathogens.

BACKGROUND: Moniliophthora perniciosa (Stahel) Aime & Phillips-Mora is the causal agent of witches' broom disease (WBD) of cocoa (Theobroma cacao L.) and a threat to the chocolate industry. The membrane-bound enzyme alternative oxidase (AOX) is critical for M. perniciosa virulence and resistance to fungicides, which has also been observed in other phytopathogens. Notably AOX is an escape mechanism from strobilurins and other respiration inhibitors, making AOX a promising target for controlling WBD and other fungal diseases. RESULTS: We present the first study aimed at developing novel fungal AOX inhibitors. N-Phenylbenzamide (NPD) derivatives were screened in the model yeast Pichia pastoris through oxygen consumption and growth measurements. The most promising AOX inhibitor (NPD 7j-41) was further characterized and displayed better activity than the classical AOX inhibitor SHAM in vitro against filamentous fugal phytopathogens, such as M. perniciosa, Sclerotinia sclerotiorum and Venturia pirina. We demonstrate that 7j-41 inhibits M. perniciosa spore germination and prevents WBD symptom appearance in infected plants. Finally, a structural model of P. pastoris AOX was created and used in ligand structure-activity relationships analyses. CONCLUSION: We present novel fungal AOX inhibitors with antifungal activity against relevant phytopathogens. We envisage the development of novel antifungal agents to secure food production. © 2018 Society of Chemical Industry.

Inhibition of UCP2 expression reverses diet-induced diabetes mellitus by effects on both insulin secretion and action.

Recent characterization of the ability of uncoupling protein 2 (UCP2) to reduce ATP production and inhibit insulin secretion by pancreatic beta-cells has placed this mitochondrial protein as a candidate target for therapeutics in diabetes mellitus. In the present study we evaluate the effects of short-term treatment of two animal models of type 2 diabetes mellitus with an antisense oligonucleotide to UCP2. In both models, Swiss mice (made obese and diabetic by a hyperlipidic diet) and ob/ob mice, the treatment resulted in a significant improvement in the hyperglycemic syndrome. This effect was due not only to an improvement of insulin secretion, but also to improved peripheral insulin action. In isolated pancreatic islets, the partial inhibition of UCP2 increased ATP content, followed by increased glucose-stimulated insulin secretion. This was not accompanied by increased expression of enzymes involved in protection against oxidative stress. The evaluation of insulin action in peripheral tissues revealed that the inhibition of UCP2 expression significantly improved insulin signal transduction in adipose tissue. In conclusion, short-term inhibition of UCP2 expression ameliorates the hyperglycemic syndrome in two distinct animal models of obesity and diabetes. Metabolic improvement is due to a combined effect on insulin-producing pancreatic islets and in at least one peripheral tissue that acts as a target for insulin.

Unsaturated fatty acids as high-affinity ligands of the C-terminal Per-ARNT-Sim domain from the Hypoxia-inducible factor 3α.

Hypoxia-inducible transcription factors (HIF) form heterodimeric complexes that mediate cell responses to hypoxia. The oxygen-dependent stability and activity of the HIF-α subunits is traditionally associated to post-translational modifications such as hydroxylation, acetylation, ubiquitination, and phosphorylation. Here we report novel evidence showing that unsaturated fatty acids are naturally occurring, non-covalent structural ligands of HIF-3α, thus providing the initial framework for exploring its exceptional role as a lipid sensor under hypoxia.

Potent cardioprotective effect of the 4-anilinoquinazoline derivative PD153035: involvement of mitochondrial K(ATP) channel activation.

BACKGROUND: The aim of the present study was to evaluate the protective effects of the 4-anilinoquinazoline derivative PD153035 on cardiac ischemia/reperfusion and mitochondrial function. METHODOLOGY/PRINCIPAL FINDINGS: Perfused rat hearts and cardiac HL-1 cells were used to determine cardioprotective effects of PD153035. Isolated rat heart mitochondria were studied to uncover mechanisms of cardioprotection. Nanomolar doses of PD153035 strongly protect against heart and cardiomyocyte damage induced by ischemia/reperfusion and cyanide/aglycemia. PD153035 did not alter oxidative phosphorylation, nor directly prevent Ca(2+) induced mitochondrial membrane permeability transition. The protective effect of PD153035 on HL-1 cells was also independent of AKT phosphorylation state. Interestingly, PD153035 activated K(+) transport in isolated mitochondria, in a manner prevented by ATP and 5-hydroxydecanoate, inhibitors of mitochondrial ATP-sensitive K(+) channels (mitoK(ATP)). 5-Hydroxydecanoate also inhibited the cardioprotective effect of PD153035 in cardiac HL-1 cells, demonstrating that this protection is dependent on mitoK(ATP) activation. CONCLUSIONS/SIGNIFICANCE: We conclude that PD153035 is a potent cardioprotective compound and acts in a mechanism involving mitoK(ATP) activation.

MEF2C silencing attenuates load-induced left ventricular hypertrophy by modulating mTOR/S6K pathway in mice.

BACKGROUND: The activation of the members of the myocyte enhancer factor-2 family (MEF2A, B, C and D) of transcription factors promotes cardiac hypertrophy and failure. However, the role of its individual components in the pathogenesis of cardiac hypertrophy remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated whether MEF2C plays a role in mediating the left ventricular hypertrophy by pressure overload in mice. The knockdown of myocardial MEF2C induced by specific small interfering RNA (siRNA) has been shown to attenuate hypertrophy, interstitial fibrosis and the rise of ANP levels in aortic banded mice. We detected that the depletion of MEF2C also results in lowered levels of both PGC-1alpha and mitochondrial DNA in the overloaded left ventricle, associated with enhanced AMP:ATP ratio. Additionally, MEF2C depletion was accompanied by defective activation of S6K in response to pressure overload. Treatment with the amino acid leucine stimulated S6K and suppressed the attenuation of left ventricular hypertrophy and fibrosis in the aforementioned aortic banded mice. CONCLUSION/SIGNIFICANCE: These findings represent new evidences that MEF2C depletion attenuates the hypertrophic responses to mechanical stress and highlight the potential of MEF2C to be a target for new therapies to cardiac hypertrophy and failure.