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Background Dose-adjusted EPOCH-R (etoposide, prednisone, vincristine, cyclophosphamide, doxorubicin, and rituximab) is a front-line treatment for patients with aggressive B-cell lymphomas. Bcl-2 is associated with chemoresistance due to BCL2 gene rearrangement or protein overexpression and is antagonised by venetoclax. We aimed to assess the safety of venetoclax with dose-adjusted EPOCH-R as initial therapy in aggressive B-cell lymphoma. Methods We conducted a single-arm, phase 1 study across seven treatment centres in the USA. Eligible patients were aged 18-80 years with histologically confirmed, previously untreated diffuse large B-cell lymphoma, transformed indolent non-Hodgkin lymphoma, high-grade B-cell lymphoma with double-hit or not otherwise specified, or primary mediastinal B-cell lymphoma, with Ann Arbor stage II-IV and Eastern Cooperative Oncology Group performance status of 0-2. Participants received six cycles of oral venetoclax 400 mg, 600 mg, or 800 mg once daily for 10 days per cycle with dose-adjusted EPOCH-R (one cycle every 3 weeks; baseline doses were intravenous rituximab 375 mg/m2 on day 1, intravenous etoposide 50 mg/m2 on days 1-4, oral prednisone 60 mg/m2 twice daily on days 1-5, intravenous vincristine 0·4 mg/m2 on days 1-4, intravenous cyclophosphamide 750 mg/m2 on day 5, and intravenous doxorubicin 10 mg/m2 on days 1-4). A subsequent cohort received venetoclax 600 mg once daily for 5 days per cycle. The primary endpoints were the maximum tolerated dose, dose-limiting toxicities, and the recommended phase 2 dose of venetoclax. Analyses were done per protocol. This trial is registered with ClinicalTrials.gov, NCT03036904, and enrolment is now closed. Findings Between Feb 3, 2017, and June 4, 2019, 34 patients were assessed for eligibility, and 30 were enrolled and received venetoclax with dose-adjusted EPOCH-R. The median patient age was 64·0 years (IQR 51·6-69·4). The maximum tolerated dose was 800 mg for 10 days and the established recommended phase 2 dose was 600 mg for 5 days due to tolerability for treatment duration. One (3%) of 30 patients had a dose-limiting toxicity in cycle one (grade 4 thrombocytopenia with 800 mg dose). The most common grade 3-4 adverse events were cytopenias (28 [93%] of 30 patients); febrile neutropenia occurred in 19 (63%) patients. Grade 3-4 non-haematological adverse events included hypophosphataemia (n=10), hypokalaemia (n=7), and hyperglycaemia (n=5). Serious adverse events included infection (n=7) and gastrointestinal toxicities including abdominal pain (n=3), colonic perforation (n=1), and small intestinal obstruction (n=1). There was one treatment-related death (sepsis). Overall response rate was 96·7% (95% CI 82·8-99·9); 28 (93·3% [77·9-99·2]) of 30 patients had complete response and one (3·3% [0·1-17·2]) had a partial response. Interpretation Venetoclax with dose-adjusted EPOCH-R showed an acceptable safety profile at the recommended phase 2 dose and had encouraging preliminary activity in this population at high risk of adverse outcomes, and is worthy of further study. The combination is being investigated in Alliance 051701 (NCT03984448). Funding Genentech.

Termination of antidepressant therapy often has negative consequences. Although symptoms of antidepressant withdrawal are widely recognized, the molecular processes that underlie them are not well characterized. We show that certain aspects of Gαs signaling remain suppressed after antidepressant withdrawal, even after others have reverted to baseline. Antidepressant treatment causes translocation of Gαs protein from lipid rafts to nonraft membrane regions. This results in augmented Gαs signaling, including facilitated activation of adenylyl cyclase and increased cAMP accumulation. Using CC6 or SK-N-SH cells and a lipid raft–localized cAMP sensor, we show that Gαs signaling is reduced in lipid rafts, even while signaling is enhanced elsewhere in the cell. These signaling changes mirror the changes in Gαs localization observed after antidepressant treatment. Furthermore, we show that suppression of Gαs signaling in lipid rafts persists at least 24 hours after cessation of antidepressant treatment. Gαs localization was quantified after membrane isolation and sequential detergent extraction. We show that suppression of lipid raft Gαs signaling persists for an extended time period after antidepressant withdrawal, whereas increased nonraft membrane Gαs signaling reverts partially or fully upon cessation of antidepressant treatment. Translocation of Gαs out of lipid rafts is also persistent. These events may reflect cellular adaptations to antidepressant treatment that contribute to antidepressant discontinuation syndromes and may aid in the discovery of new treatments and strategies to mitigate the symptoms of depression and antidepressant withdrawal. SIGNIFICANCE STATEMENT This work explores, for the first time, the effects of antidepressants on Gαs signaling after drug withdrawal. This provides novel insight into the cellular and molecular processes affected by antidepressant drugs and their persistence after discontinuation of treatment.

Sponges are known to produce a series of compounds with bioactivities useful for human health. This study was conducted on four sponges collected in the framework of the XXXIV Italian National Antarctic Research Program (PNRA) in November-December 2018, i.e., Mycale (Oxymycale) acerata, Haliclona (Rhizoniera) dancoi, Hemimycale topsenti, and Hemigellius pilosus. Sponge extracts were fractioned and tested against hepatocellular carcinoma (HepG2), lung carcinoma (A549), and melanoma cells (A2058), in order to screen for antiproliferative or cytotoxic activity. Two different chemical classes of compounds, belonging to mycalols and suberitenones, were identified in the active fractions. Mycalols were the most active compounds, and their mechanism of action was also investigated at the gene and protein levels in HepG2 cells. Of the differentially expressed genes, ULK1 and GALNT5 were the most down-regulated genes, while MAPK8 was one of the most up-regulated genes. These genes were previously associated with ferroptosis, a programmed cell death triggered by iron-dependent lipid peroxidation, confirmed at the protein level by the down-regulation of GPX4, a key regulator of ferroptosis, and the up-regulation of NCOA4, involved in iron homeostasis. These data suggest, for the first time, that mycalols act by triggering ferroptosis in HepG2 cells.

Both 3,5-diiodo-L-thyronine (3,5-T2) and 3,5,3'-triiodo-L-tyronine (T3) affect energy metabolism having mitochondria as a major target. However, the underlying mechanisms are poorly understood. Here, using a model of chemically induced hypothyroidism in male Wistar rats, we investigated the effect of administration of either 3,5-T2 or T3 on liver oxidative capacity through their influence on mitochondrial processes including: proton-leak across the mitochondrial inner membrane; complex I-, complex II- and glycerol-3-phosphate-linked respiratory pathways; respiratory complex abundance and activities as well as individual complex aggregation into supercomplexes. Background/Aims: Both 3,5-diiodo-L-thyronine (3,5-T2) and 3,5,3'-triiodo-L-tyronine (T3) affect energy metabolism having mitochondria as a major target. However, the underlying mechanisms are poorly understood. Here, using a model of chemically induced hypothyroidism in male Wistar rats, we investigated the effect of administration of either 3,5-T2 or T3 on liver oxidative capacity through their influence on mitochondrial processes including: proton-leak across the mitochondrial inner membrane; complex I-, complex II- and glycerol-3-phosphate-linked respiratory pathways; respiratory complex abundance and activities as well as individual complex aggregation into supercomplexes. Methods: Hypothyroidism was induced by propylthiouracil and iopanoic acid; 3,5-T2 and T3 were intraperitoneally administered at 25 and 15 μg/100 g BW for 1 week, respectively. Resulting alterations in mitochondrial function were studied by combining respirometry, Blue Native-PAGE followed by in-gel activity, and Western blot analyses. Results: Administration of 3,5-T2 and T3 to hypothyroid (hypo) rats enhanced mitochondrial respiration rate with only T3 effectively stimulating proton-leak (450% vs. Hypo). T3 significantly enhanced complex I (+145% vs. Hypo), complex II (+66% vs. Hypo), and glycerol-3 phosphate dehydrogenase (G3PDH)-linked oxygen consumptions (about 6- fold those obtained in Hypo), while 3,5-T2 administration selectively restored Euthyroid values of complex II- and increased G3PDH- linked respiratory pathways (+165% vs. Hypo). The mitochondrial abundance of all respiratory complexes and of G3PDH was increased by T3 administration whereas 3,5-T2 only increased complex V and G3PDH abundance. 3,5-T2 enhanced complex I and complex II in gel activities with less intensity than did T3, and T3 also enhanced the activity of all other respiratory complexes tested. In addition, only T3 enhanced individual respiratory component complex assembly into supercomplexes. Conclusions: The reported data highlight novel molecular mechanisms underlying the effect elicited by iodothyronine administration to hypothyroid rats on mitochondrial processes related to alteration in oxidative capacity in the liver. The differential effects elicited by the two iodothyronines indicate that 3,5-T2, by influencing the kinetic properties of specific mitochondrial respiratory pathways, would promote a rapid response of the organelle, while T3, by enhancing the abundance of respiratory chain component and favoring the organization of respiratory chain complex in supercomplexes, would induce a slower and prolonged response of the organelle.

Exercise under fasting conditions induces a switch to lipid metabolism, eliciting beneficial metabolic effects. Knowledge of signaling responses underlying metabolic adjustments in such conditions may help to identify therapeutic strategies. Therefore, we studied the effect of mild exercise on rats submitted to food withdrawal at thermoneutrality (28°C) for 3 days. Animals were housed at thermoneutrality rather than the standard housing temperature (22°C) to avoid beta‐adrenergic signaling responses that themselves affect metabolism and well‐being. Quantitative analysis of multi‐organ mRNA levels, myofibers, and serum metabolites shows that this protocol (a) boosts fat oxidation in muscle and liver, (b) reduces lipogenesis and increases gluconeogenesis in liver, (c) increases serum acylcarnitines (especially C4OH) and ketone bodies and the use of the latter as fuel in muscle, (d) increases Type I myofibers, and (e) is associated with an increased thyroid hormone uptake and metabolism in muscle. In addition, stool microbiome DNA analysis revealed that food withdrawal dramatically alters the presence of bacterial genera associated with ketone metabolism. Taken together, this protocol induces a drastic switch toward increased lipid and ketone metabolism compared to exercise or food withdrawal alone, which may prove beneficial and may involve local thyroid hormones, which may be regarded as exercise mimetics., Environmental temperature influences metabolic responses. The results of the present study show that the relatively mild metabolic effects of exercise at thermoneutrality are boosted when combined with food withdrawal. To our knowledge, this is the first study to reveal this, studying the whole animal's response ranging from microbiota to muscle, liver, and serum, suggesting a contributing role for thyroid hormone.

3,5-diiodo-thyronine (T2), an endogenous metabolite of thyroid hormones, exerts beneficial metabolic effects. When administered to overweight rats receiving a high fat diet (HFD), it significantly reduces body fat accumulation, which is a risk factor for the development of an inflammatory state and of related metabolic diseases. In the present study, we focused our attention on T2 actions aimed at improving the adverse effects of long-lasting HFD such as the adipocyte inflammatory response. For this purpose, three groups of rats were used throughout: i) receiving a standard diet for 14 weeks; ii) receiving a HFD for 14 weeks, and iii) receiving a HFD for 14 weeks with a simultaneous daily injection of T2 for the last 4 weeks. The results showed that T2 administration ameliorated the expression profiles of pro- and anti-inflammatory cytokines, reduced macrophage infiltration in white adipose tissue, influenced their polarization and reduced lymphocytes recruitment. Moreover, T2 improved the expression of hypoxia markers, all altered in HFD rats, and reduced angiogenesis by decreasing the pro-angiogenic miR126 expression. Additionally, T2 reduced the oxidative damage of DNA, known to be associated to the inflammatory status. This study demonstrates that T2 is able to counteract some adverse effects caused by a long-lasting HFD and to produce beneficial effects on inflammation. Irisin and SIRT1 pathway may represent a mechanism underlying the above described effects.

When administered to rats receiving a high-fat diet (HFD), 3,5-diiodo-L-thyronine (3,5-T2) [at a dose of 25 &mu, g/100 g body weight (BW)] is known to increase energy expenditure and to prevent HFD-induced adiposity. Here, we investigated which cellular and molecular processes in visceral white adipose tissue (VAT) contributed to the beneficial effect of 3,5-T2 over time (between 1 day and 4 weeks following administration). 3,5-T2 programmed the adipocyte for lipolysis by rapidly inducing hormone sensitive lipase (HSL) phosphorylation at the protein kinase A-responsive site Ser563, accompanied with glycerol release at the 1-week time-point, contributing to the partial normalization of adipocyte volume with respect to control (N) animals. After two weeks, when the adipocyte volumes of HFD-3,5-T2 rats were completely normalized to those of the controls (N), 3,5-T2 consistently induced HSL phosphorylation at Ser563, indicative of a combined effect of 3,5-T2-induced adipose lipolysis and increasing non-adipose oxidative metabolism. VAT proteome analysis after 4 weeks of treatment revealed that 3,5-T2 significantly altered the proteomic profile of HFD rats and produced a marked pro-angiogenic action. This was associated with a reduced representation of proteins involved in lipid storage or related to response to oxidative stress, and a normalization of the levels of those involved in lipogenesis-associated mitochondrial function. In conclusion, the prevention of VAT mass-gain by 3,5-T2 occurred through different molecular pathways that, together with the previously reported stimulation of resting metabolism and liver fatty acid oxidation, are associated with an anti adipogenic/lipogenic potential and positively impact on tissue health.

The role of uncoupling protein-3 (UCP3) in energy and lipid metabolism was investigated. Male wild-type (WT) and UCP3-null (KO) mice that were housed at thermoneutrality (30 &deg, C) were used as the animal model. In KO mice, the ability of skeletal muscle mitochondria to oxidize fatty acids (but not pyruvate or succinate) was reduced. At whole animal level, adult KO mice presented blunted resting metabolic rates, energy expenditure, food intake, and the use of lipids as metabolic substrates. When WT and KO mice were fed with a standard/low-fat diet for 80 days, since weaning, they showed similar weight gain and body composition. Interestingly, KO mice showed lower fat accumulation in visceral adipose tissue and higher ectopic fat accumulation in liver and skeletal muscle. When fed with a high-fat diet for 80 days, since weaning, KO mice showed enhanced energy efficiency and an increased lipid gain (thus leading to a change in body composition between the two genotypes). We conclude that UCP3 plays a role in energy and lipid homeostasis and in preserving lean tissues by lipotoxicity, in mice that were housed at thermoneutrality.

Several metabolic products that derive from L-thyroxine (T4) and 3,3'5-L-triiodothyronine (T3), the main thyroid hormones secreted by the thyroid gland, possess biologic activities. Among these metabolites or derivatives showing physiological actions some have received greater attention: diiodothyronines, iodothyronamines, acetic acid analogues. It is known that increased thyroid hormone (T3 and T4) levels can improve serum lipid profiles and reduce body fat. These positive effects are, however, counterbalanced by adverse effects on the heart, muscle and bone, limiting their use. In addition to the naturally occurring metabolites, thyroid hormone analogues have been developed that either have selective effects on specific tissues or bind selectively to thyroid hormone receptor (TR) isoform. Among these GC-1, KB141, KB2115, and DITPA were deeply investigated and displayed promising therapeutic results in the potential treatment of conditions such as dyslipidemias and obesity. In this review, we summarize the current knowledge of metabolites and analogues of T4 and T3 with reference to their possible clinical application in the treatment of human diseases.

The conversion of white adipose cells into beige adipose cells is known as browning, a process affecting energy metabolism. It has been shown that 3,5 diiodo-l-thyronine (T2), an endogenous metabolite of thyroid hormones, stimulates energy expenditure and a reduction in fat mass. In light of the above, the purpose of this study was to test whether in an animal model of fat accumulation, T2 has the potential to activate a browning process and to explore the underlying mechanism. Three groups of rats were used: (i) receiving a standard diet for 14 weeks, (ii) receiving a high-fat diet (HFD) for 14 weeks, and (iii) receiving a high fat diet for 10 weeks and being subsequently treated for four weeks with an HFD together with the administration of T2. We showed that T2 was able to induce a browning in the white adipose tissue of T2-treated rats. We also showed that some miRNA (miR133a and miR196a) and MAP kinase 6 were involved in this process. These results indicate that, among others, the browning may be another cellular/molecular mechanism by which T2 exerts its beneficial effects of contrast to overweight and of reduction of fat mass in rats subjected to HFD.

Background and Purpose: Triptans are 5-HT1B/1D receptor agonists (that also display 5-HT1F receptor affinity) with antimigraine action, contraindicated in patients with coronary artery disease due to their vasoconstrictor properties. Conversely, lasmiditan was developed as an antimigraine 5-HT1F receptor agonist. To assess the selectivity and cardiovascular effects of lasmiditan, we investigated the binding, functional activity, and in vitro/in vivo vascular effects of lasmiditan and compared it to sumatriptan. Experimental Approach: Binding and second messenger activity assays of lasmiditan and other serotoninergic agonists were performed for human 5-HT1A, 5-HT1B, 5-HT1D, 5-ht1E, 5-HT1F, 5-HT2A, 5-HT2B, and 5-HT7 receptors, and the results were correlated with their potency to constrict isolated human coronary arteries (HCAs). Furthermore, concentration–response curves to lasmiditan and sumatriptan were performed in proximal and distal HCA, internal mammary, and middle meningeal arteries. Finally, anaesthetized female beagle dogs received i.v. infusions of lasmiditan or sumatriptan in escalating cumulative doses, and carotid and coronary artery diameters were measured. Key Results: Lasmiditan showed high selectivity for 5-HT1F receptors. Moreover, the functional potency of the analysed compounds to inhibit cAMP increase through 5-HT1B receptor activation positively correlated with their potency to contract HCA. In isolated human arteries, sumatriptan, but not lasmiditan, induced contractions. Likewise, in vivo, sumatriptan decreased coronary and carotid artery diameters at clinically relevant doses, while lasmiditan was devoid of vasoconstrictor activity at all doses tested. Conclusions and Implications: Lasmiditan is a selective 5-HT1F receptor agonist devoid of vasoconstrictor activity. This may represent a cardiovascular safety advantage when compared to the triptans.

A single base mutation in the Gα(i2) protein (G184S) renders this Gα subunit insensitive to the negative modulatory effects of Regulator of G-protein Signaling (RGS) proteins. Mice expressing this RGS insensitive (RGSi) variant of Gα(i2) (RGSi Gα(i2)) display a spontaneous antidepressant-like phenotype that is reversible by treatment with the 5-HT1A receptor (5-HT1AR) antagonist WAY100635. Here we test the hypothesis that increased activity of 5-HT1ARs in the hippocampus of RGSi Gα(i2) knock-in mice is responsible for the expression of the observed antidepressant-like behavior. We administered the 5-HT1AR antagonist WAY100635 or the agonist 8-OH-DPAT via bilateral intra-hippocampal infusion cannulae and evaluated antidepressant-like behavior using the tail suspension test (TST). WAY100635 reversed the antidepressant-like phenotype of the RGSi Gα(i2) knock-in mice and 8-OH-DPAT produced an antidepressant-like response in wild type mice that was blocked by systemic WAY100635. Furthermore, intra-hippocampal infusion of the RGS4/19 inhibitor CCG-203769 produced an antidepressant-like effect in female mice. Ex-vivo slice recording confirmed the 5-HT1AR-mediated decrease in hippocampal CA1 pyramidal neuron excitability was enhanced in the RGSi Gα(i2) knock-in mice. There was no change in hippocampal 5-HT1AR expression as measured by ligand binding or in the ability of 8-OH-DPAT to activate G-protein as measured in the [(35)S]GTPγS binding assay. The findings demonstrate that RGS protein control of hippocampal 5-HT1AR signaling is necessary and sufficient to account for the antidepressant-like phenotype in the RGSi Gα(i2) knock-in mice and that RGS proteins highly expressed in the hippocampus should be investigated as targets for novel antidepressant therapies.

Hyperlipidemic state-associated perturbations in the network of factors controlling mitochondrial functions, i.e., morphogenesis machinery and metabolic sensor proteins, produce metabolic inflexibility, insulin resistance and reduced oxidative capacity in skeletal muscle. Moreover, intramyocellular lipid (IMCL) accumulation leads to tissue damage and inflammation. The administration of the naturally occurring metabolite 3,5-diiodo-L-thyronine (T2) with thyromimetic actions to high fat diet (HFD)-fed rats exerts a systemic hypolipidemic effect, which produces a lack of IMCL accumulation, a shift toward glycolytic fibers and amelioration of insulin sensitivity in gastrocnemius muscle. In this study, an integrated approach combining large-scale expression profile and functional analyses was used to characterize the response of skeletal muscle mitochondria to 12 during a HFD regimen. Long-term 12 administration to HDF rats induced a glycolytic phenotype of gastrocnemius muscle as well as an adaptation of mitochondria to the fiber type, with a decreased representation of enzymes involved in mitochondrial oxidative metabolism. At the same time, T2 stimulated the activity of individual respiratory complex I, IV and V. Moreover, 12 prevented the HFD-associated increase in the expression of peroxisome proliferative activated receptor gamma coactivator-1 alpha and dynamin-1-like protein as well as mitochondrial morphological aberrations, favoring the appearance of tubular and tethered organelles in the intermyofibrillar regions. Remarkably, 12 reverted the HDF-associated expression pattern of proinflammatory factors, such as p65 subunit of NF-kB, and increased the fiber-specific immunoreactivity of adipose differentiation related protein in lipid droplets. All together, these results further support a role of T2 in counteracting in vivo some of the HFD-induced impairment in structural/metabolic features of skeletal muscle by impacting the mitochondrial phenotype.

Evidence indicates that many forms of fructose‐induced metabolic disturbance are associated with oxidative stress and mitochondrial dysfunction. Mitochondria are prominent targets of oxidative damage; however, it is not clear whether mitochondrial DNA (mtDNA) damage and/or its lack of repair are events involved in metabolic disease resulting from a fructose‐rich diet. In the present study, we evaluated the degree of oxidative damage to liver mtDNA and its repair, in addition to the state of oxidative stress and antioxidant defense in the liver of rats fed a high‐fructose diet. We used male rats feeding on a high‐fructose or control diet for eight weeks. Our results showed an increase in mtDNA damage in the liver of rats fed a high‐fructose diet and this damage, as evaluated by the expression of DNA polymerase γ, was not repaired; in addition, the mtDNA copy number was found to be significantly reduced. A reduction in the mtDNA copy number is indicative of impaired mitochondrial biogenesis, as is the finding of a reduction in the expression of genes involved in mitochondrial biogenesis. In conclusion, a fructose‐rich diet leads to mitochondrial and mtDNA damage, which consequently may have a role in liver dysfunction and metabolic diseases.

A small library of antiplasmodial methoxy-thiazinoquinones, rationally designed on the model of the previously identified hit 1, has been prepared by a simple and inexpensive procedure. The synthetic derivatives have been subjected to in vitro pharmacological screening, including antiplasmodial and toxicity assays. These studies afforded a new lead candidate, compound 9, endowed with higher antiplasmodial potency compared to 1, a good selectivity index when tested against a panel of mammalian cells, no toxicity against RBCs, a synergistic antiplasmodial action in combination with dihydroartemisinin, and a promising inhibitory activity on stage V gametocyte growth. Computational studies provided useful insights into the structural requirements needed for the antiplasmodial activity of thiazinoquinone compounds and on their putative mechanism of action.

Microtubule targeting drugs like taxanes, vinca alkaloids, and epothilones are widely-used and effective chemotherapeutic agents that target the dynamic instability of microtubules and inhibit spindle functioning. However, these drugs have limitations associated with their production, solubility, efficacy and unwanted toxicities, thus driving the need to identify novel antimitotic drugs that can be used as anticancer agents. We have discovered and characterized the Microtubins (Microtubule inhibitors), a novel class of small synthetic compounds, which target tubulin to inhibit microtubule polymerization, arrest cancer cells predominantly in mitosis, activate the spindle assembly checkpoint and trigger an apoptotic cell death. Importantly, the Microtubins do not compete for the known vinca or colchicine binding sites. Additionally, through chemical synthesis and structure-activity relationship studies, we have determined that specific modifications to the Microtubin phenyl ring can activate or inhibit its bioactivity. Combined, these data define the Microtubins as a novel class of compounds that inhibit cancer cell proliferation by perturbing microtubule polymerization and they could be used to develop novel cancer therapeutics.

Staphylococcus aureus is a leading cause of hospital-acquired infections in the United States and is a major health concern as methicillin-resistant S. aureus (MRSA) and other antibiotic resistant strains are common. Compounds that inhibit the S. aureus sortase (SrtA) cysteine transpeptidase may function as potent anti-infective agents as this enzyme attaches virulence factors to the bacterial cell wall. While a variety of SrtA inhibitors have been discovered, the vast majority of these small molecules have not been optimized using structure-based approaches. Here we have used NMR spectroscopy to determine the molecular basis through which pyridazinone-based small molecules inhibit SrtA. These inhibitors covalently modify the active cysteine thiol and partially mimic the natural substrate of SrtA by inducing the closure of an active site loop. Computational and synthetic chemistry methods led to second generation analogs that are ~70-fold more potent than the lead molecule. These optimized molecules exhibit broad-spectrum activity against other types of class A sortases, have reduced cytotoxicity and impair SrtA-mediated protein display on S. aureus cell surface. Our work shows that pyridazinone analogs are attractive candidates for further development into anti-infective agents, and highlights the utility of employing NMR spectroscopy and solubility-optimized small molecules in structure-based drug discovery.

Introduction: Patients (pts) with aggressive B-cell lymphomas who are relapsed or refractory to frontline therapy are typically treated with regimens including R-ICE (rituximab, ifosfamide, carboplatin, etoposide) followed by autologous stem cell transplant (ASCT) in responding pts. A minority of pts are cured with this approach, and the majority have poor outcomes. Selinexor is an oral, selective inhibitor of nuclear export, which activates tumor suppressor proteins and blocks translation of proteins that can contribute to chemoresistance, including MYC and BCL2. We conducted a phase I trial of selinexor plus R-ICE in pts with relapsed/refractory aggressive B-cell lymphomas. Methods: This phase I study used a 3+3 design with dose expansion. Eligible pts had diffuse large B-cell lymphoma (DLBCL), double hit lymphoma (DHL), and indolent lymphoma transformed to DLBCL. Treatment with 1 prior regimen administered with curative intent was required. Central nervous system (CNS) involvement was excluded. A separate cohort enrolled pts with Richter's transformation (RT) from chronic lymphocytic leukemia; there was no requirement with regard to prior therapies for this group. Selinexor was initially dosed at 40 mg (DL-1), 60 mg (DL1) and 80 mg (DL2) on days (d) -5, -3, 1, 3, and 5 and R-ICE on d1-3 of a 21-d cycle. Because of CNS toxicity thought to be primarily related to ifosfamide, the protocol was amended so that R-ICE was given on d1-3 and selinexor following completion of ifosfamide on d3, 5, and 7 of each cycle. After 2 cycles, responding pts were eligible to receive SCT or chimeric antigen receptor (CAR) T-cell therapy at discretion of treating physician. Dose limiting toxicity (DLT) period was cycle 1 and included grade (gr) 4 febrile neutropenia; gr 4 neutrophils for ≥7d; gr 4 platelets for ≥10d; gr 3 nausea, vomiting, diarrhea, or fatigue lasting >3d; or gr ≥3 non-hematologic toxicities except alopecia, fatigue or electrolyte abnormalities correctable with supportive care. Results: 22 pts enrolled with median age 67 (range 34-79). 64% were male and 36% female. Stage was III-IV in 72%. IPI was intermediate to high risk in 77%. Diagnosis was DLBCL NOS in 12, DHL in 4, transformed indolent lymphoma in 2, primary mediastinal large B-cell lymphoma in 1, and RT in 3. 7 pts were treated on the initial dosing schedule. In the first group at DL1, 0/3 pts had DLT. At DL2, 2/2 pts had DLT (gr 3 altered mental status, AMS). In the second group at DL1, 1/2 pts had DLT (gr 3 AMS). The dosing schedule was then modified as noted above. 12 DLBCL pts were treated on the modified dosing schedule. At DL1, 2/3 pts had DLT (gr 5 sepsis, gr 4 platelets ≥10d). At DL-1, 1/6 pts had DLT (gr 3 abdominal infection). Selinexor 40 mg was declared the recommended phase 2 dose (RP2D) and 3 pts were enrolled in an expansion. 3 pts with RT were treated on the modified dosing schedule; 2 received selinexor and neither had DLT at DL1. Median number of cycles of selinexor plus R-ICE was 2 (range 1-3). Most common gr 3-4 toxicities were cytopenias and hyponatremia. Most common non-hematologic toxicities of all grades were hyponatremia, fatigue, transaminitis, and nausea. Of 21 pts who received selinexor plus R-ICE, objective response rate (ORR) was 71% with 7 complete responses (CR), 8 partial responses (PR), and 3 stable disease (SD). 5 underwent SCT (3 autologous, 2 allogeneic) and 8 received CAR T-cells. Those who underwent transplant have not required additional therapy to date. 4 complete responders did not receive ASCT in part related to difficulty mobilizing stem cells; 3 have not required additional therapy with follow up of 26, 46, and 46 months. At the RP2D in the DLBCL cohort (n=9), ORR was 78% with 4 CR, 3 PR, and 2 SD. Conclusions: We report the first data on the combination of selinexor, which is now FDA-approved as a single agent in relapsed/refractory DLBCL, and chemotherapy in pts with aggressive B-cell lymphomas. We identified a dosing schedule of selinexor with R-ICE (40 mg on d3, 5, and 7) that is worthy of further study based on initial efficacy, with attention to CNS toxicity and impact on stem cell collection. Disclosures Rutherford: Karyopharm: Consultancy, Research Funding; Regeneron: Research Funding; Seattle Genetics: Consultancy; LAM Therapeutics: Research Funding; Kite: Consultancy; Juno: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; Dova: Consultancy; Genentech/Roche: Research Funding; Heron: Consultancy. Allan:Acerta, Genentech, Abbvie, Sunesis, Ascentage, Pharmacyclics, Janssen, AstraZeneca, BeiGene: Consultancy; Celgene, Genentech, Janssen, TG Therapeutics: Research Funding; Abbvie, Janssen, AstraZeneca, Pharmacyclics: Honoraria. Ruan:Celgene: Consultancy, Research Funding; AstraZeneca: Consultancy, Research Funding; Pharmacyclics: Research Funding; Seattle Genetics: Research Funding; Kite Pharma: Consultancy; Juno: Consultancy; BMS: Consultancy, Research Funding. Furman:Oncotarget: Consultancy; Loxo Oncology: Consultancy; Verastem: Consultancy; Beigene: Consultancy; AstraZeneca: Consultancy, Research Funding; Acerta: Consultancy; Abbvie: Consultancy; Genentech: Consultancy; Incyte: Consultancy; Janssen: Consultancy, Speakers Bureau; TG Therapeutics: Consultancy, Research Funding; Sunesis: Consultancy; Pharmacyclics: Consultancy. Cerchietti:BMS: Research Funding. Gergis:Incyte: Speakers Bureau; Merck: Speakers Bureau; Astellas: Consultancy, Speakers Bureau; Kite: Speakers Bureau; Mesoblast: Other: Ad Board; Jazz: Other: Ad board, Speakers Bureau. Leonard:Regeneron: Consultancy; BMS/Celgene: Consultancy; Roche/Genentech: Consultancy; Epizyme: Consultancy; Bayer: Consultancy; Gilead/Kite: Consultancy; Karyopharm: Consultancy; GenMab: Consultancy; ADC Therapeutics: Consultancy; AstraZeneca: Consultancy; MEI Pharma: Consultancy; Sutro: Consultancy; Miltenyi: Consultancy. Martin:Bayer: Consultancy; Beigene: Consultancy; Kite: Consultancy; Incyte: Consultancy; Cellectar: Consultancy; Morphosys: Consultancy; Regeneron: Consultancy; Sandoz: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy, Research Funding; Teneobio: Consultancy; I-MAB: Consultancy.

Opioid drugs are the gold standard for the management of pain, but their use is severely limited by dangerous and unpleasant side effects. All clinically available opioid analgesics bind to and activate the mu-opioid receptor (MOR), a heterotrimeric G-protein-coupled receptor, to produce analgesia. The activity of these receptors is modulated by a family of intracellular RGS proteins or regulators of G-protein signaling proteins, characterized by the presence of a conserved RGS Homology (RH) domain. These proteins act as negative regulators of G-protein signaling by serving as GTPase accelerating proteins or GAPS to switch off signaling by both the Gα and βγ subunits of heterotrimeric G-proteins. Consequently, knockdown or knockout of RGS protein activity enhances signaling downstream of MOR. In this review we discuss current knowledge of how this activity, across the different families of RGS proteins, modulates MOR activity, as well as activity of other members of the opioid receptor family, and so pain and analgesia in animal models, with particular emphasis on RGS4 and RGS9 families. We discuss inhibition of RGS proteins with small molecule inhibitors that bind to sensitive cysteine moieties in the RH domain and the potential for targeting this family of intracellular proteins as adjuncts to provide an opioid sparing effect or as standalone analgesics by promoting the activity of endogenous opioid peptides. Overall, we conclude that RGS proteins may be a novel drug target to provide analgesia with reduced opioid-like side effects, but that much basic work is needed to define the roles for specific RGS proteins, particularly in chronic pain, as well as a need to develop newer inhibitors.

Purpose: Inspiring high partial pressure of oxygen (FiO2 > 60%) for a prolonged duration can lead to lung damage termed pulmonary oxygen toxicity (PO2T). While current practice is to limit oxygen exposure, there are clinical and military scenarios where higher FiO2 levels and partial pressures of oxygen are required. The purpose of this study is to develop a non-invasive breath-based biomarker to detect PO2T prior to the onset of clinical symptoms. Methods: Male Yorkshire swine (20-30 kg) were placed into custom airtight runs and randomized to air (20.9% FiO2, n=12) or oxygen (>95% FiO2, n=10) for 72 hours. Breath samples, arterial blood gases, and vital signs were assessed every 12 hours. After 72 hours of exposure, animals were euthanized and the lungs processed for histology and wet-dry ratios. Results: Swine exposed to hyperoxia developed pulmonary injury consistent with PO2T. Histology of oxygen-exposed swine showed pulmonary lymphatic congestion, epithelial sloughing, and neutrophil transmigration. Pulmonary injury was also evidenced by increased interstitial edema and a decreased PaO2/FiO2 ratio in the oxygen group when compared to the air control group. Breath volatile organic compound (VOC) sample analysis identified six VOCs that were combined into an algorithm which generated a breath score predicting PO2T with a ROC/AUC curve of 0.72 defined as a decrement of PaO2/FiO2 ratio less than 350 mmHg. Conclusions: Exposing swine to 72 hours of hyperoxia induced a pulmonary injury consistent with human clinical endpoints of PO2T. VOC analysis identified six VOCs in exhaled breath that preceded PO2T. Results show promise that a simple, non-invasive breath test could potentially predict the risk of pulmonary injury in humans exposed to high partial pressures of oxygen.

Targeting the leukemia proliferation cycle has been a successful approach to developing antileukemic therapies. However, drug screening efforts to identify novel antileukemic agents have been hampered by the lack of a suitable high-throughput screening platform for suspension cells that does not rely on flow cytometry analyses. We report the development of a novel leukemia cell-based high-throughput chemical screening platform for the discovery of cell cycle phase specific inhibitors that utilizes chemical cell cycle profiling. We have used this approach to analyze the cell cycle response of acute lymphoblastic leukemia CCRF-CEM cells to each of 181,420 drug-like compounds. This approach yielded cell cycle phase specific inhibitors of leukemia cell proliferation. Further analyses of the top G2-phase and M-phase inhibitors identified the leukemia specific inhibitor 1 (Leusin-1). Leusin-1 arrests cells in G2-phase and triggers an apoptotic cell death. Most importantly, Leusin-1 was more active in acute lymphoblastic leukemia cells than other types of leukemias, non-blood cancers, or normal cells and represents a lead molecule for developing antileukemic drugs.

Over 30 years of research has demonstrated that 3,5-diiodo-L-thyronine (3,5-T2), an endogenous metabolite of thyroid hormones, exhibits interesting metabolic activities. In rodent models, exogenously administered 3,5-T2 rapidly increases resting metabolic rate and elicits short-term beneficial hypolipidemic effects; however, very few studies have evaluated the effects of endogenous and exogenous T2 in humans. Further analyses on larger cohorts are needed to determine whether 3,5-T2 is a potent additional modulator of energy metabolism. In addition, while several lines of evidence suggest that 3,5-T2 mainly acts through Thyroid hormone receptors (THRs)- independent ways, with mitochondria as a likely cellular target, THRs-mediated actions have also been described. The detailed cellular and molecular mechanisms through which 3,5-T2 elicits a multiplicity of actions remains unknown. Here, we provide an overview of the most recent literature on 3,5-T2 bioactivity with a particular focus on short-term and long-term effects, describing data obtained through in vivo and in vitro approaches in both mammalian and non-mammalian species. Over 30 years of research has demonstrated that 3,5-diiodo-L-thyronine (3,5-T2), an endogenous metabolite of thyroid hormones, exhibits interesting metabolic activities. In rodent models, exogenously administered 3,5-T2 rapidly increases resting metabolic rate and elicits short-term beneficial hypolipidemic effects; however, very few studies have evaluated the effects of endogenous and exogenous T2 in humans. Further analyses on larger cohorts are needed to determine whether 3,5-T2 is a potent additional modulator of energy metabolism. In addition, while several lines of evidence suggest that 3,5-T2 mainly acts through Thyroid hormone receptors (THRs)-independent ways, with mitochondria as a likely cellular target, THRs-mediated actions have also been described. The detailed cellular and molecular mechanisms through which 3,5-T2 elicits a multiplicity of actions remains unknown. Here, we provide an overview of the most recent literature on 3,5-T2 bioactivity with a particular focus on short-term and long-term effects, describing data obtained through in vivo and in vitro approaches in both mammalian and non-mammalian species.

3,3′,5-triiodo-L-thyronine (T3) improves hepatic lipid accumulation by increasing lipid catabolism but it also increases lipogenesis, which at first glance appears contradictory. Recent studies have shown that 3,5-diiodothyronine (T2), a natural thyroid hormone derivative, also has the capacity to stimulate hepatic lipid catabolism, however, little is known about its possible effects on lipogenic gene expression. Because genes classically involved in hepatic lipogenesis such as SPOT14, acetyl-CoA-carboxylase (ACC), and fatty acid synthase (FAS) contain thyroid hormone response elements (TREs), we studied their transcriptional regulation, focusing on TRE-mediated effects of T3 compared to T2 in rats receiving high-fat diet (HFD) for 1 week. HFD rats showed a marked lipid accumulation in the liver, which was significantly reduced upon simultaneous administration of either T3 or T2 with the diet. When administered to HFD rats, T2, in contrast with T3, markedly downregulated the expression of the above-mentioned genes. T2 downregulated expression of the transcription factors carbohydrate-response element-binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) involved in activation of transcription of these genes, which explains the suppressed expression of their target genes involved in lipogenesis. T3, however, did not repress expression of the TRE-containing ChREBP gene but repressed SREBP-1c expression. Despite suppression of SREBP-1c expression by T3 (which can be explained by the presence of nTRE in its promoter), the target genes were not suppressed, but normalized to HFD reference levels or even upregulated (ACC), partly due to the presence of TREs on the promoters of these genes and partly to the lack of suppression of ChREBP. Thus, T2 and T3 probably act by different molecular mechanisms to achieve inhibition of hepatic lipid accumulation.

The full absolute configuration assignment of phosphoeleganin (1), a recently discovered marine-derived phosphorylated polyketide with protein tyrosine phosphatase 1B inhibitory activity, was achieved. It was based on the synthesis of model diasteroisomeric compounds of the C-8–C-12 segment portion of phosphoeleganin, chiral derivatization methods, and application of the universal NMR database concept.

Chronic overnutrition and modern life-styles are causing a worldwide epidemic of obesity and associated comorbidities, which is creating a demand to identify underlying biological mechanisms and to devise effective treatments. In rats receiving a high-fat diet (HFD), we analyzed the effects of a 4-wk administration of a novel functional analog of iodothyronines, TRC150094 (TRC). HFD-TRC rats exhibited increased energy expenditure (+24% vs. HFD rats; P

Peroxisome proliferator-activated receptors (PPARs), which are known to regulate lipid homeostasis, are tightly controlled by nutrient availability, and they control nutrient handling. In this paper, we focus on how nutrients control the expression and action of PPARs and how cellular signaling events regulate the action of PPARs in metabolically active tissues (e.g., liver, skeletal muscle, heart, and white adipose tissue). We address the structure and function of the PPARs, and their interaction with other nuclear receptors, including PPAR cross-talk. We further discuss the roles played by different kinase pathways, including the extracellular signal-regulated kinases/mitogen-activated protein kinase (ERK MAPK), AMP-activated protein kinase (AMPK), Akt/protein kinase B (Akt/PKB), and the NAD+-regulated protein deacetylase SIRT1, serving to control the activity of the PPARs themselves as well as that of a key nutrient-related PPAR coactivator, PPARγcoactivator-1α(PGC-1α). We also highlight how currently applied nutrigenomic strategies will increase our understanding on how nutrients regulate metabolic homeostasis through PPAR signaling.

8003 Background: Dose-adjusted (DA) EPOCH-R is a frontline treatment for aggressive B-cell lymphomas. Bcl-2 is associated with chemoresistance due to BCL2 gene rearrangement or protein overexpression in lymphomas and is antagonized by BH3 mimetic venetoclax (ven). We conducted a phase I study combining ven with DA-EPOCH-R in aggressive B-cell lymphomas. Methods: This phase 1 study used Bayesian optimal interval design with dose expansion. Eligible patients (pts) were 18 years with newly diagnosed diffuse large B-cell (DLBCL), primary mediastinal, and high grade B-cell lymphoma (HGBCL) with double hit (DHL) or not otherwise specified (NOS). Ven was dosed at 400 mg (DL1), 600 mg (DL2), and 800 mg (DL3) daily for 10 days with 6 cycles of DA-EPOCH-R. A subsequent cohort received ven 600 mg daily for 5 days (DL2B). Toxicities were graded by CTCAE v4.0 and response assessed by Lugano criteria. Dose limiting toxicity (DLT) period was cycle 1 and primarily included grade (gr) ≥3 neutropenia on cycle 2 day 1, gr ≥4 febrile neutropenia/thrombocytopenia, and gr ≥3 nausea, vomiting, diarrhea despite supportive care. Results: 30 pts enrolled with median age 64 (24-79), and 50% female. Ann Arbor stage was III-IV in 23. IPI was high risk in > 50%. Diagnosis was DHL (15), DLBCL NOS (13), and HGBCL NOS (2). 18 had MYC and 14 had BCL2 rearrangements. Bcl-2 was expressed ≥50% by IHC in 21/26 with data. There were no DLTs in DL1 (3 pts) or DL2 (9 pts). 1/6 had DLT in DL3 (gr 4 thrombocytopenia). Ven dose reductions occurred in subsequent cycles in 4 (2 in DL2; 2 in DL3). Of 18 in DL1-3, EPOCH was escalated above level 1 in 1 and de-escalated below level 1 in 7. Because of delays and ven dose reductions in DL2-3 due in part to cytopenias, infections and GI toxicities, we accrued DL2B. In DL2B, 0/12 pts had DLTs or ven dose reductions. EPOCH was escalated above level 1 in 4 and de-escalated below level 1 in 3. 1 died of sepsis during cycle 3. Most common gr 3-4 toxicities across all dose levels were cytopenias; febrile neutropenia occurred in 57%. Most common non-hematologic toxicities of all grades were hypocalcemia, nausea, diarrhea, hypokalemia and fatigue. ORR (N = 30) ITT was 97% with 27 (90%) complete and 2 (7%) partial responses; 1 was not evaluable. Follow up is ongoing. Of 15 DHL, ORR and CRR were 93% and 80%. Conclusions: We identified ven 600 mg for 5 days per cycle as RP2D with DA-EPOCH-R. DL2B was well tolerated and required no ven dose reductions. Further efficacy and safety is being evaluated in Alliance 51701, DA-EPOCH-R/R-CHOP in DH/double expressor lymphomas, using the dosing regimen defined by this study. Clinical trial information: NCT03036904 .

The extension of π-conjugation of polycyclic aromatic hydrocarbons (PAHs) via alkyne benzannulation reactions has become an increasingly utilized tool over the past few years. This short review will highlight recent work of alkyne benzannulations in the context of large nanographene as well as graphene nanoribbon synthesis along with a brief discussion of the interesting physical properties these molecules display.

Ketamine produces rapid and robust antidepressant effects in depressed patients within hours of administration, often when traditional antidepressant compounds have failed to alleviate symptoms. We hypothesized that ketamine would translocate Gα(s) from lipid rafts to non-raft microdomains, similarly to other antidepressants but with a distinct, abbreviated treatment duration. C6 glioma cells were treated with 10 μM ketamine for 15 min, which translocated Gα(s) from lipid raft domains to non-raft domains. Other NMDA antagonist did not translocate Gα(s) from lipid raft to non-raft domains. The ketamine-induced Gα(s) plasma membrane redistribution allows increased functional coupling of Gα(s) and adenylyl cyclase to increase intracellular cyclic adenosine monophosphate (cAMP). Moreover, increased intracellular cAMP increased phosphorylation of cAMP response element-binding protein (CREB), which, in turn, increased BDNF expression. The ketamine-induced increase in intracellular cAMP persisted after knocking out the NMDA receptor indicating an NMDA receptor-independent effect. Furthermore, 10 μM of the ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) also induced Gα(s) redistribution and increased cAMP. These results reveal a novel antidepressant mechanism mediated by acute ketamine treatment that may contribute to ketamine’s powerful antidepressant effect. They also suggest that the translocation of Gαs from lipid rafts is a reliable hallmark of antidepressant action that might be exploited for diagnosis or drug development.

This paper presents preliminary results about Turin's air quality before, during and after the realization of the infrastructure projects for the Turin 2006 XX Winter Olympic Games. We compared the 3-year in-operam (work in progress) period (i.e. 2003-2005, when all infrastructures needed for the organization were built) with the periods before (ante-operam) and after (post-operam): 2000-2002 and 2006-2008, respectively. In particular, we analyzed the concentrations of the primary pollutants (nitrogen dioxide, carbon monoxide and fine particulate matter, PM10) in Turin and Milan. In this way, we could use the measurements from Milan as a control dataset for comparison with the atmospheric pollution conditions in Turin. We found that infrastructural work for the Olympic Games in Turin affected NO2 and PM10 atmospheric concentrations, determining a peak in the average values during the in-operam period (probably due to caterpillar tractor emissions and excavation). This pattern did not emerge from the Milan data, where a decreasing trend can be seen between the ante-operam and the post-operam periods. On the other hand, a negative effect on CO levels was not observed: the decreasing trend, more evident in Turin compared to Milan, can be linked to the expansion during the same period of limited traffic areas created to facilitate the infrastructural work.

We investigated the mechanism by which 3,5-diiodo-l-thyronine (T2) affects skeletal muscle mitochondrial bioenergetic parameters following its acute administration to hypothyroid rats. One hour after injection, T2 increased both coupled and uncoupled respiration rates by +27% and +42%, respectively. Top-down elasticity analysis revealed that these effects were the result of increases in the substrate oxidation and mitochondrial uncoupling. Discriminating between proton-leak and redox-slip processes, we identified an increased mitochondrial proton conductance as the "pathway" underlying the effect of T2 on mitochondrial uncoupling. As a whole, these results may provide a mechanism by which T2 rapidly affects energy metabolism in hypothyroid rats. © 2007 Federation of European Biochemical Societies.

A novel functional iodothyronine analogue, TRC150094, which has a much lower potency toward thyroid hormone receptor (α1/β1) activation than triiodothyronine, has been shown to be effective at reducing adiposity in rats simultaneously receiving a high-fat diet (HFD). Here, by combining metabolic, functional and proteomic analysis, we studied how the hepatic and skeletal muscle phenotypes might respond to TRC150094 treatment in HFD-fed overweight rats. Drug treatment increased both the liver and skeletal muscle mitochondrial oxidative capacities without altering mitochondrial efficiency. Coherently, in terms of individual respiratory in-gel activity, blue-native analysis revealed an increased activity of complex V in the liver and of complexes II and V in tibialis muscle in TCR150094-treated animals. Subsequently, the identification of differentially expressed proteins and the analysis of their interrelations gave an integrated view of the phenotypic/metabolic adaptations occurring in the liver and muscle proteomes during drug treatment. TRC150094 significantly altered the expression of several proteins involved in key liver metabolic pathways, including amino acid and nitrogen metabolism, and fructose and mannose metabolism. The canonical pathways most strongly influenced by TRC150094 in tibialis muscle included glycolysis and gluconeogenesis, amino acid, fructose and mannose metabolism, and cell signaling. The phenotypic/metabolic influence of TRC150094 on the liver and skeletal muscle of HFD-fed overweight rats suggests the potential clinical application of this iodothyronine analogue in ameliorating metabolic risk parameters altered by diet regimens. © 2012 The Royal Society of Chemistry.

The worldwide prevalence of obesity-associated pathologies, including type 2 diabetes, requires thorough investigation of mechanisms and interventions. Recent studies have highlighted thyroid hormone analogs and derivatives as potential agents able to counteract such pathologies. In this study, in rats receiving a high-fat diet (HFD), we analyzed the effects of a 4-wk daily administration of a naturally occurring iodothyronine, 3,5-diiodo-L-thyronine (T2), on the gastrocnemius muscle metabolic/structural phenotype and insulin signaling. The HFD-induced increases in muscle levels of fatty acid translocase (3-fold; P

OBJECTIVE High-fat diets (HFDs) are known to induce insulin resistance. Previously, we showed that 3,5-diiodothyronine (T2), concomitantly administered to rats on a 4-week HFD, prevented gain in body weight and adipose mass. Here we investigated whether and how T2 prevented HFD-induced insulin resistance. RESEARCH DESIGN AND METHODS We investigated the biochemical targets of T2 related to lipid and glucose homeostasis over time using various techniques, including genomic and proteomic profiling, immunoblotting, transient transfection, and enzyme activity analysis. RESULTS Here we show that, in rats, HFD feeding induced insulin resistance (as expected), whereas T2 administration prevented its onset. T2 did so by rapidly stimulating hepatic fatty acid oxidation, decreasing hepatic triglyceride levels, and improving the serum lipid profile, while at the same time sparing skeletal muscle from fat accumulation. At the mechanistic level, 1) transfection studies show that T2 does not act via thyroid hormone receptor β; 2) AMP-activated protein kinase is not involved in triggering the effects of T2; 3) in HFD rats, T2 rapidly increases hepatic nuclear sirtuin 1 (SIRT1) activity; 4) in an in vitro assay, T2 directly activates SIRT1; and 5) the SIRT1 targets peroxisome proliferator–activated receptor (PPAR)-γ coactivator (PGC-1α) and sterol regulatory element–binding protein (SREBP)-1c are deacetylated with concomitant upregulation of genes involved in mitochondrial biogenesis and downregulation of lipogenic genes, and PPARα/δ-induced genes are upregulated, whereas genes involved in hepatic gluconeogenesis are downregulated. Proteomic analysis of the hepatic protein profile supported these changes. CONCLUSIONS T2, by activating SIRT1, triggers a cascade of events resulting in improvement of the serum lipid profile, prevention of fat accumulation, and, finally, prevention of diet-induced insulin resistance.

The electrochemical response of four natural cytotoxic thiazinoquinones isolated from the Aplidium species was studied using conventional solution-phase and solid-state techniques, based on the voltammetry of immobilized particles methodology. The interaction with O-2 and electrochemically generated reactive oxygen species (ROS) was electrochemically monitored. At the same time, a molecular modeling study including density functional theory (DFT) calculations was performed in order to analyze the conformational and electronic properties of the natural thiazinoquinones, as well as those of their reduced intermediates. The obtained electrochemical and computational results were analyzed and correlated to cytotoxic activity of these compounds, highlighting some features possibly related to their mechanism of action.

A thermokinetic model for the reaction rates of solids measured in constant heating rate differential thermal analysis is derived from heat transfer and chemical kinetics. An explicit result is obtained for the maximum reaction rate in a constant heating rate experiment in terms of the thermal properties of the solid, the activation energy of the thermal process, the heat of reaction, the sample mass and the heating rate in the test. The theoretical predictions compare well with numerical simulations and experimental data for polyoxymethylene and polystyrene obtained by two different thermal analysis methods using three different instruments over a wide range of sample mass and heating rate. Based on these results, an accuracy criterion for thermal analysis measurements is proposed that is consistent with recommended practices.

We defined the transcriptomic and proteomic profiles of rat ageing skeletal muscle using a combined cDNA array, 2D- and Blue native-PAGE approach. This was allowed to obtain an overview of the interrelated events leading to the transcriptome/proteome/mitoproteome changes likely to underlie the structural/metabolic features of aged skeletal muscle. The main differences were found in genes/proteins related to energy metabolism, mitochondrial pathways, myofibrillar filaments, and detoxification. Concerning the abundance of mitochondrial OXPHOS complexes as well as their supramolecular organization and activity, mitochondria from old rats, when compared with those from young rats, contained significantly lower amounts of complex I (NADH:ubiquinone oxidoreductase), V (FoF1-ATP synthase), and III (ubiquinol:cytochrome c oxidoreductase). The same mitochondria contained a significantly larger amount of complex II (succinate:ubiquinone oxidoreductase), but an unchanged amount of complex IV (cytochrome c oxidase, COX). When comparing the supercomplex profiles between young and old muscle mitochondria, the densitometric analysis revealed that lighter supercomplexes were significantly reduced in older mitochondria, and that in the older group the major supercomplex bands were those representing heavier supercomplexes, likely suggesting a compensatory mechanism that, in ageing muscle, is functionally directed towards substrate channeling and catalytic enhancement advantaging the respirosome. © 2009 Elsevier B.V. All rights reserved.

Polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDDs/Fs) were analyzed in samples of the emissions from a secondary aluminum smelter (ALS) and soil samples around the plant. The purpose was to estimate the impact of the emissions on the surrounding environment. PCDD/F soil concentrations were higher in the proximity of the plant, exceeding the limit adopted in Italy in soils for green areas and residential uses and the upper limit of several reference concentrations. The most contaminated sites were less than 500 m from the plant and the dioxin concentration with the distance from the ALS. Principal component analysis (PCA) showed that emissions from the ALS were the source of PCDD/F contamination in the soils closest to the plant. Multivariate data analyses such as PCA are therefore useful to identify sources of emission causing contamination.

Context: Hesperidin, a citrus flavonoid, and its metabolite hesperetin may have vascular actions relevant to their health benefits. Molecular and physiological mechanisms of hesperetin actions are unknown. Objective: We tested whether hesperetin stimulates production of nitric oxide (NO) from vascular endothelium and evaluated endothelial function in subjects with metabolic syndrome on oral hesperidin therapy. Design, Setting, and Interventions: Cellular mechanisms of action of hesperetin were evaluated in bovine aortic endothelial cells (BAEC) in primary culture. A randomized, placebo-controlled, double-blind, crossover trial examined whether oral hesperidin administration (500 mg once daily for 3 wk) improves endothelial function in individuals with metabolic syndrome (n = 24). Main Outcome Measure: We measured the difference in brachial artery flow-mediated dilation between placebo and hesperidin treatment periods. Results: Treatment of BAEC with hesperetin acutely stimulated phosphorylation of Src, Akt, AMP kinase, and endothelial NO synthase to produce NO; this required generation of H2O2. Increased adhesion of monocytes to BAEC and expression of vascular cell adhesion molecule-1 in response to TNF-α treatment was reduced by pretreatment with hesperetin. In the clinical study, when compared with placebo, hesperidin treatment increased flow-mediated dilation (10.26 ± 1.19 vs. 7.78 ± 0.76%; P = 0.02) and reduced concentrations of circulating inflammatory biomarkers (high-sensitivity C-reactive protein, serum amyloid A protein, soluble E-selectin). Conclusions: Novel mechanisms for hesperetin action in endothelial cells inform effects of oral hesperidin treatment to improve endothelial dysfunction and reduce circulating markers of inflammation in our exploratory clinical trial. Hesperetin has vasculoprotective actions that may explain beneficial cardiovascular effects of citrus consumption.

T(3) regulates energy metabolism by stimulating metabolic rate and decreasing metabolic efficiency. The discovery of mitochondrial uncoupling protein 3 (UCP3), its homology to UCP1, and regulation by T(3) rendered it a possible molecular determinant of the action of T(3) on energy metabolism, but data are controversial. This controversy may in part be attributable to discrepancies observed between the regulation by T(3) of UCP3 expression in rats, humans, and mice. To clarify this issue, we studied 1) the induction kinetics of the UCP3 gene by T(3) in rat skeletal muscle, 2) the influence of fatty acids, and 3) the structure and regulation of the various UCP3 promoters by T(3). Within 8 h of single-dose T(3) administration, hypothyroid rats showed a rise in serum fatty acid levels concomitant with a rapid increase in UCP3 expression in gastrocnemius muscle, followed by inductions of peroxisome proliferator activated receptor delta (PPARdelta) (within 24 h) and PPAR target gene expression (after 24 h). This T(3)-induced early UCP3 expression depended on fatty acid-PPAR signaling because depleting serum fatty acid levels abolished its expression, restorable by administration of the PPARdelta agonist L165,041 (4-[3-(4-acetyl-3-hydroxy-2-propylphenoxy)propoxy]phenoxy]acetic acid). In transfected rat L6 myoblasts, only the rat UCP3 promoter positively responded to T(3) and L165,041 together in the presence of MyoD, thyroid hormone receptor beta1 (TRbeta1), PPARdelta, or PPARdelta plus the TR dimerization partner retinoid X receptor alpha. All promoters share a response element common to TR and PPAR (TRE 1), but the observed species differences may be attributable to different localizations of the MyoD response element, which in the rat maps to exon 1.

Purpose: Pulmonary oxygen toxicity (PO2T) is a concern for any patient or warfighter exposed to oxygen enriched atmospheres for an extended amount of time. While current practice is to limit increased oxygen exposure; there are clinical and operational scenarios where higher oxygen levels must be used and PO2T becomes a concern. The purpose of the current study is to develop a non-invasive breath exhalate based biomarker which can detect PO2T prior to clinical manifestations of the disease. Methods: Male Yorkshire swine (20-30 kg) were placed into custom plexi-glass runs and randomized to air (n=10) or 100% oxygen (n=10) for 72 hours. Breath exhalate samples, arterial blood gas samples, and vital signs were assessed every 12 hours. At the end of the 72 hour exposure animals were euthanized and the lungs were processed for histological injury and wet/dry ratios. Results: Swine exposed to the oxygen enriched atmosphere developed a variable pulmonary injury consistent with PO2T. Histologically there was evid...