A switch to feeding on cycads generates parallel accelerated evolution of toxin tolerance in two clades of Eumaeus caterpillars (Lepidoptera: Lycaenidae).

We assembled a complete reference genome of Eumaeus atala, an aposematic cycad-eating hairstreak butterfly that suffered near extinction in the United States in the last century. Based on an analysis of genomic sequences of Eumaeus and 19 representative genera, the closest relatives of Eumaeus are Theorema and Mithras We report natural history information for Eumaeus, Theorema, and Mithras Using genomic sequences for each species of Eumaeus, Theorema, and Mithras (and three outgroups), we trace the evolution of cycad feeding, coloration, gregarious behavior, and other traits. The switch to feeding on cycads and to conspicuous coloration was accompanied by little genomic change. Soon after its origin, Eumaeus split into two fast evolving lineages, instead of forming a clump of close relatives in the phylogenetic tree. Significant overlap of the fast evolving proteins in both clades indicates parallel evolution. The functions of the fast evolving proteins suggest that the caterpillars developed tolerance to cycad toxins with a range of mechanisms including autophagy of damaged cells, removal of cell debris by macrophages, and more active cell proliferation.

Genomics-based higher classification of the species-rich Hairstreaks (Lepidoptera: Lycaenidae: Eumaeini).

We propose a higher classification of the lycaenid hairstreak tribe Eumaeini - one of the youngest and most species-rich butterfly tribes - based on autosome, Lepidopteran Z sex chromosome, and mitochondrial protein-coding genes. The subtribe Neolycaenina Korb is a synonym of Callophryidina Tutt, and subtribe Tmolusina Bálint is a synonym of Strephonotina K. Johnson, Austin, Le Crom, & Salazar. Proposed names are Rhammina Prieto & Busby, new subtribe; Timaetina Busby & Prieto, new subtribe; Atlidina Martins & Duarte, new subtribe; Evenina Faynel & Grishin, new subtribe; Jantheclina Robbins & Faynel, new subtribe; Paiwarriina Lamas & Robbins, new subtribe; Cupatheclina Lamas & Grishin, new subtribe; Parrhasiina Busby & Robbins, new subtribe; Ipideclina Martins & Grishin, new subtribe; and Trichonidina Duarte & Faynel, new subtribe. Phylogenetic results from the autosome and Z sex chromosome analyses are similar. Future analyses of datasets with hundreds of terminal taxa may be more practical time-wise by focussing on the smaller number of sex chromosome sequences (2.6% of nuclear protein-coding sequences). The phylogenetic classification and biological summaries for each subtribe suggest that a variety of factors affected Eumaeini diversification. About a dozen kinds of male secondary sexual organs with frequent evolutionary gains and losses occur in Atlidina, Evenina, and Jantheclina (141 species combined). Females have been shown to use these organs to discriminate between conspecific and non-conspecific males, facilitating sympatry among close relatives. Eumaeina, Rhammina, and Timaetina (140 species combined) are overwhelmingly montane with some evidence for a higher incidence of sympatric diversification. Seven Neotropical lineages in five subtribes invaded the temperate parts of the Nearctic Region with a diversification increase in the Callophryidina (262 species). North American Satyrium and Callophrys then invaded the Palearctic at least once each, with a major species-richness increase in Satyrium. The evolution of litter feeding detritivores within Calycopidina (172 species) resulted in an increase in diversification rate compared with its flower-feeding sister lineage. Atlidina, Strephonotina, Parrhasiina, and Strymonina (562 species combined) each contain a mixture of genera that specialize on one or two caterpillar food plant families and genera that are polyphagous. These would be appropriate subtribes to assess how the breadth of caterpillar food plants and the frequency of host shifts affected diversification.

Evolutionary trade-offs between male secondary sexual traits revealed by a phylogeny of the hyperdiverse tribe Eumaeini (Lepidoptera: Lycaenidae).

Male butterflies in the hyperdiverse tribe Eumaeini possess an unusually complex and diverse repertoire of secondary sexual characteristics involved in pheromone production and dissemination. Maintaining multiple sexually selected traits is likely to be metabolically costly, potentially resulting in trade-offs in the evolution of male signals. However, a phylogenetic framework to test hypotheses regarding the evolution and maintenance of male sexual traits in Eumaeini has been lacking. Here, we infer a comprehensive, time-calibrated phylogeny from 379 loci for 187 species representing 91% of the 87 described genera. Eumaeini is a monophyletic group that originated in the late Oligocene and underwent rapid radiation in the Neotropics. We examined specimens of 818 of the 1096 described species (75%) and found that secondary sexual traits are present in males of 91% of the surveyed species. Scent pads and scent patches on the wings and brush organs associated with the genitalia were probably present in the common ancestor of Eumaeini and are widespread throughout the tribe. Brush organs and scent pads are negatively correlated across the phylogeny, exhibiting a trade-off in which lineages with brush organs are unlikely to regain scent pads and vice versa. In contrast, scent patches seem to facilitate the evolution of scent pads, although they are readily lost once scent pads have evolved. Our results illustrate the complex interplay between natural and sexual selection in the origin and maintenance of multiple male secondary sexual characteristics and highlight the potential role of sexual selection spurring diversification in this lineage.

Pharmacologic properties, metabolism, and disposition of linaclotide, a novel therapeutic peptide approved for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation.

Linaclotide, a potent guanylate cyclase C agonist, is a therapeutic peptide approved in the United States for the treatment of irritable bowel syndrome with constipation and chronic idiopathic constipation. We present for the first time the metabolism, degradation, and disposition of linaclotide in animals and humans. We examined the metabolic stability of linaclotide in conditions that mimic the gastrointestinal tract and characterized the metabolite MM-419447 (CCEYCCNPACTGC), which contributes to the pharmacologic effects of linaclotide. Systemic exposure to these active peptides is low in rats and humans, and the low systemic and portal vein concentrations of linaclotide and MM-419447 observed in the rat confirmed both peptides are minimally absorbed after oral administration. Linaclotide is stable in the acidic environment of the stomach and is converted to MM-419447 in the small intestine. The disulfide bonds of both peptides are reduced in the small intestine, where they are subsequently proteolyzed and degraded. After oral administration of linaclotide, <1% of the dose was excreted as active peptide in rat feces and a mean of 3-5% in human feces; in both cases MM-419447 was the predominant peptide recovered. MM-419447 exhibits high-affinity binding in vitro to T84 cells, resulting in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3',5'-monophosphate (cGMP). In rat models of gastrointestinal function, orally dosed MM-419447 significantly increased fluid secretion into small intestinal loops, increased intraluminal cGMP, and caused a dose-dependent acceleration in gastrointestinal transit. These results demonstrate the importance of the active metabolite in contributing to linaclotide's pharmacology.

Metopimazine is primarily metabolized by a liver amidase in humans.

Metopimazine (MPZ) is a peripherally restricted, dopamine D2 receptor antagonist used for four decades to treat acute nausea and vomiting. MPZ is currently under clinical investigation for the treatment of gastroparesis (GP). MPZ undergoes high first-pass metabolism that produces metopimazine acid (MPZA), the major circulating metabolite in humans. Despite a long history of use, the enzymes involved in the metabolism of MPZ have not been identified. Here we report a series of studies designed to identify potential MPZ metabolites in vitro, determine their clinical relevance in humans, and elucidate the enzymes responsible for their formation. The findings demonstrated that the formation of MPZA was primarily catalyzed by human liver microsomal amidase. Additionally, human liver cytosolic aldehyde oxidase (AO) catalyzes the formation of MPZA, in vitro, although to a much lesser extent. Neither cytochrome P450 enzymes nor flavin-monooxygenases (FMO) were involved in the formation MPZA, although two minor oxidative pathways were catalyzed by CYP3A4 and CYP2D6 in vitro. Analysis of plasma samples from subjects dosed 60 mg of MPZ verified that these oxidative pathways are very minor and that CYP enzyme involvement was negligible compared to microsomal amidase/hydrolase in overall MPZ metabolism in humans. The metabolism by liver amidase, an enzyme family not well defined in small molecule drug metabolism, with minimal metabolism by CYPs, differentiates this drug from current D2 antagonists used or in development for the treatment of GP.

Therapeutic targeting of SLC6A8 creatine transporter suppresses colon cancer progression and modulates human creatine levels.

Colorectal cancer (CRC) is a leading cause of cancer mortality. Creatine metabolism was previously shown to critically regulate colon cancer progression. We report that RGX-202, an oral small-molecule SLC6A8 transporter inhibitor, robustly inhibits creatine import in vitro and in vivo, reduces intracellular phosphocreatine and ATP levels, and induces tumor apoptosis. RGX-202 suppressed CRC growth across KRAS wild-type and KRAS mutant xenograft, syngeneic, and patient-derived xenograft (PDX) tumors. Antitumor efficacy correlated with tumoral expression of creatine kinase B. Combining RGX-202 with 5-fluorouracil or the DHODH inhibitor leflunomide caused regressions of multiple colorectal xenograft and PDX tumors of distinct mutational backgrounds. RGX-202 also perturbed creatine metabolism in patients with metastatic CRC in a phase 1 trial, mirroring pharmacodynamic effects on creatine metabolism observed in mice. This is, to our knowledge, the first demonstration of preclinical and human pharmacodynamic activity for creatine metabolism targeting in oncology, thus revealing a critical therapeutic target.

Centrin-2 is required for centriole duplication in mammalian cells.

BACKGROUND: Centrosomes are the favored microtubule-organizing framework of eukaryotic cells. Centrosomes contain a pair of centrioles that normally duplicate once during the cell cycle to give rise to two mitotic spindle poles, each containing one old and one new centriole. However, aside from their role as an anchor point for pericentriolar material and as basal bodies of flagella and cilia, the functional attributes of centrioles remain enigmatic. RESULTS: Here, using RNA interference, we demonstrate that "knockdown" of centrin-2, a protein of centrioles, results in failure of centriole duplication during the cell cycle in HeLa cells. Following inhibition of centrin-2 synthesis, the preexisting pair of centrioles separate, and functional bipolar spindles form with only one centriole at each spindle pole. Centriole dilution results from the ensuing cell division, and daughter cells are "born" with only a single centriole. Remarkably, these unicentriolar daughter cells may complete a second and even third bipolar mitosis in which spindle microtubules converge onto unusually broad spindle poles and in which cell division results in daughter cells containing either one or no centrioles at all. Cells thus denuded of the mature or both centrioles fail to undergo cytokinesis in subsequent cell cycles, give rise to multinucleate products, and finally die. CONCLUSIONS: These results demonstrate a requirement for centrin in centriole duplication and demonstrate that centrioles play a role in organizing spindle pole morphology and in the completion of cytokinesis.

Genotoxic stress leads to centrosome amplification in breast cancer cell lines that have an inactive G1/S cell cycle checkpoint.

Centrosome amplification plays a key role in the origin of chromosomal instability during cancer development and progression. In this study, breast cancer cell lines with different p53 backgrounds were used to investigate the relationship between genotoxic stress, G(1)/S cell cycle checkpoint integrity, and the development of centrosome amplification. Introduction of DNA damage in the MCF-7 cell line by treatment with hydroxyurea (HU) or daunorubicin (DR) resulted in the arrest of both G(1)/S cell cycle progression and centriole duplication. In these cells, which carry functional p53, HU treatment also led to nuclear accumulation of p53 and p21(WAF1), retinoblastoma hypophosphorylation, and downregulation of cyclin A. MCF-7 cells carrying a recombinant dominant-negative p53 mutant (vMCF-7(DNp53)) exhibited a shortened G(1) phase of the cell cycle and retained a normal centrosome phenotype. However, these cells developed amplified centrosomes following HU treatment. The MDA-MB 231 cell line, which carries mutant p53 at both alleles, showed amplified centrosomes at the outset, and developed a hyperamplified centrosome phenotype following HU treatment. In cells carrying defective p53, the development of centrosome amplification also occurred following treatment with another DNA damaging agent, DR. Taken together, these findings demonstrate that loss of p53 function alone is not sufficient to drive centrosome amplification, but plays a critical role in this process following DNA damage through abrogation of the G(1)/S cell cycle checkpoint. Furthermore, these studies have important clinical implications because they suggest that breast cancers with compromised p53 function may develop centrosome amplification and consequent chromosomal instability following treatment with genotoxic anticancer drugs.

Male secondary sexual structures and the systematics of the Thereus oppia species group (Lepidoptera, Lycaenidae, Eumaeini).

The Thereus oppia species group includes species with and without a scent pad, which is a histologically and morphologically characterized male secondary sexual structure on the dorsal surface of the forewing. To assess the hypothesis that these structures are lost evolutionarily, but not regained (Dollo's Law), the taxonomy of this species group is revised. Thereus lomalarga sp. n., and Thereus brocki sp. n., are described. Diagnostic traits, especially male secondary structures, within the Thereus oppia species group are illustrated. Distributional and biological information is summarized for each species. Three species have been reared, and the caterpillars eat Loranthaceae. An inferred phylogeny is consistent with the hypothesis that scent pads in the Thereus oppia species group have been lost evolutionarily twice (in allopatry), and not re-gained.

Metabolism and disposition of MM-433593, a selective FAAH-1 inhibitor, in monkeys.

MM-433593 is a highly potent and selective inhibitor of fatty acid amide hydrolase-1 (FAAH-1) with potential utility as an orally administered treatment of pain, inflammation, and other disorders. In this study, we investigated the metabolism and pharmacokinetics of MM-433593 in monkeys, and compared plasma and urine metabolites of this compound to the in vitro metabolites produced by monkey hepatocytes. Intravenous administration of MM-433593 to cynomolgus monkeys produced a rapid distribution phase and slower elimination phase with a mean systemic clearance rate of 8-11 mL/min/kg. Absolute oral bioavailability was determined to be 14-21% with maximum plasma concentrations reached ∼3 h (T max) following a 10 mg/kg oral dose. The average terminal half-life of MM-433593 was 17-20 h, and there were no qualitative sex differences in the metabolite profile of MM-433593. The major site of metabolism was oxidation of the methyl group at the five position of the indole ring, which was confirmed by chromatography and mass spectrometry comparison to a synthesized authentic standard. This metabolite was further oxidized to the corresponding carboxylic acid and/or conjugated with sulfate, glucuronide, or glutathione. In all, 18 metabolites were found in plasma and urine. In vitro incubations of MM-433593 with monkey hepatocytes yielded 13 metabolites, all of which were found in vivo, indicating a good correlation between the in vitro and in vivo metabolism data. A comprehensive pathway for the metabolism of MM-433593 is proposed, including a plausible, five-step biotransformation for the formation of N-acetylcysteine conjugate metabolite (M18) from the hydroxylated parent (M5).

Matrix detachment and proteasomal inhibitors diminish Sulf-2 expression in breast cancer cell lines and mouse xenografts.

Sulfatase 2 (Sulf-2) has been previously shown to be upregulated in breast cancer. Sulf-2 removes sulfate moieties on heparan sulfate proteoglycans which in turn modulate heparin binding growth factor signaling. Here we report that matrix detachment resulted in decreased Sulf-2 expression in breast cancer cells and increased cleavage of poly ADP-ribose polymerase. Silencing of Sulf-2 promotes matrix detachment induced cell death in MCF10DCIS cells. In an attempt to identify Sulf-2 specific inhibitor, we found that proteasomal inhibitors such as MG132, Lactacystin and Bortezomib treatment abolished Sulf-2 expression in multiple breast cancer cell lines. Additionally, we show that Bortezomib treatment of MCF10DCIS cell xenografts in mouse mammary fat pads significantly reduced tumor size, caused massive apoptosis and more importantly reduced Sulf-2 levels in vivo. Finally, our immunohistochemistry analysis of Sulf-2 expression in cohort of patient derived breast tumors indicates that Sulf-2 is significantly upregulated in autologous metastatic lesions compared to primary tumors (p < 0.037, Pearson correlation, Chi-Square analysis). In all, our data suggest that Sulf-2 might play an important role in breast cancer progression from ductal carcinoma in situ into an invasive ductal carcinoma potentially by resisting cell death.

Review of the species level taxonomy of the neotropical butterfly genus Oenomaus (Lycaenidae, Theclinae, Eumaeini).

Seven new species of the Neotropical hairstreak genus Oenomaus are described: Oenomaus mancha Busby & Faynel, sp. n. (type locality Ecuador); Oenomaus gwenish Robbins & Faynel, sp. n. (type locality Panama); Oenomaus lea Faynel & Robbins, sp. n. (type locality Ecuador); Oenomaus myrteana Busby, Robbins & Faynel, sp. n. (type locality Ecuador); Oenomaus mentirosa Faynel & Robbins, sp. n. (type locality Peru); Oenomaus andi Busby & Faynel, sp. n. (type locality Ecuador) and Oenomaus moseri Robbins & Faynel, sp. n. (type locality Brazil, Santa Catarina). For each new Oenomaus species, we present diagnostic characters and notes on its habitat and biology. We illustrate adults, genitalia, and distribution. New distributional and biological data are presented for 21 previously described Oenomaus species. Oenomaus melleus guyanensis Faynel, 2008 is treated as a new synonym of Oenomaus melleus melleus (Druce, 1907). Females are described and associated with males for ten species using a variety of factors, including mitochondrial COI DNA "barcode" sequences. We summarize the reasons why the number of recognized Oenomaus species has grown in the past decade from one species to 28 species. Finally, we overview the habitats that Oenomaus species occupy and note that the agricultural pest on Annonaceae, Oenomaus ortygnus, is the only Oenomaus species that regularly occurs in greatly disturbed habitats.

CCN1, a candidate target for zoledronic acid treatment in breast cancer.

CCN1, also known as CYR61, is a survival and proangiogenic factor overexpressed in about 30% of invasive breast carcinomas, and particularly in triple-negative breast carcinomas (TNBC). CCN1 expression in breast cancer promotes tumorigenicity, metastasis, antihormone, and chemoresistance. TNBCs often develop bone metastasis, thus the vast majority of patients receive bisphosphonate treatment as a companion to chemotherapy. Zoledronic acid (ZOL), a bisphosphonate currently in use, inhibits bone resorption, prevents development of new osteolytic lesions induced by tumor metastasis, and has a direct antitumor activity in breast cancer cells and tumors. We have shown that ZOL inhibits anchorage independent growth as well as branching and morphogenesis in CCN1 overexpressing cells. However, the mechanism is not yet well understood. In this study, we investigate the effect of ZOL in breast cancer cells with high and undetectable CCN1 expression levels. We show that CCN1-expressing cells are more sensitive to ZOL, that ZOL induces downregulation of the CCN1 promoter activity and CCN1 protein expression in a dose-dependent manner, and that ZOL is associated with a decrease in phosphorylated Akt and translocation of FOXO3a, a negative regulator of CCN1 expression, to the nucleus. Deletion of the FOXO3a binding site in the CCN1 promoter prevents ZOL inhibition of the CCN1 promoter activity showing that FOXO3a transcriptional activation is necessary for ZOL to induce CCN1 inhibition. This study provides evidence that ZOL targets the proangiogenic factor (CCN1) through FOXO3a and reveals a new mechanism of ZOL action in breast cancer cells.

Linaclotide, through activation of guanylate cyclase C, acts locally in the gastrointestinal tract to elicit enhanced intestinal secretion and transit.

Linaclotide is a first-in-class, orally administered 14-amino acid peptide that is in development for the treatment of irritable bowel syndrome with constipation and chronic constipation. We have characterized the solution structure of linaclotide, the in vitro binding and agonist activity to guanylate cyclase C receptors, the stability of linaclotide under conditions mimicking the gastric environment, oral bioavailability, and the pharmacodynamic effects in rat models of gastrointestinal transit and intestinal secretion. Nuclear magnetic resonance spectroscopy analysis determined that the molecular structure of linaclotide is stabilized by three intramolecular disulfide bridges. Linaclotide exhibited high affinity and pH-independent binding (K(i): 1.23-1.64 nM) to guanylate cyclase C receptors on human colon carcinoma T84 cells and concomitantly, linaclotide binding resulted in a significant, concentration-dependent accumulation of intracellular cyclic guanosine-3', 5'-monophosphate (cGMP) (EC50:99 nM). Linaclotide was stable after 3 h incubation in simulated gastric fluid (pH 1) and similarly, was completely resistant to hydrolysis by pepsin. Pharmacokinetic analysis of linaclotide showed very low oral bioavailability (0.1%). Orally administered linaclotide elicited a significant, dose-dependent increase in gastrointestinal transit rates in rats at doses of ≥5 µg/kg. Exposure of surgically ligated small intestinal loops to linaclotide induced a significant increase in fluid secretion, accompanied by a significant increase in intraluminal cGMP levels. These results suggest that the guanylate cyclase C agonist linaclotide elicits potent pharmacological responses locally in the gastrointestinal tract, and that orally administered guanylate cyclase C agonists may be capable of improving bowel habits in patients suffering from irritable bowel syndrome with constipation and chronic constipation.

Linaclotide is a potent and selective guanylate cyclase C agonist that elicits pharmacological effects locally in the gastrointestinal tract.

AIMS: Linaclotide is an orally administered 14-amino acid peptide being developed for the treatment of constipation-predominant irritable bowel syndrome (IBS-C) and chronic constipation. We determined the stability of linaclotide in the intestine, measured the oral bioavailability, and investigated whether the pharmacodynamic effects elicited in rodent models of gastrointestinal function are mechanistically linked to the activation of intestinal guanylate cyclase C (GC-C). MAIN METHODS: Linaclotide binding to intestinal mucosal membranes was assessed in competitive binding assays. Stability and oral bioavailability of linaclotide were measured in small intestinal fluid and serum, respectively, and models of gastrointestinal function were conducted using wild type (wt) and GC-C null mice. KEY FINDINGS: Linaclotide inhibited in vitro [(125)I]-STa binding to intestinal mucosal membranes from wt mice in a concentration-dependent manner. In contrast, [(125)I]-STa binding to these membranes from GC-C null mice was significantly decreased. After incubation in vitro in jejunal fluid for 30 min, linaclotide was completely degraded. Pharmacokinetic analysis showed very low oral bioavailability (0.10%). In intestinal secretion and transit models, linaclotide exhibited significant pharmacological effects in wt, but not in GC-C null mice: induction of increased fluid secretion into surgically ligated jejunal loops was accompanied by the secretion of elevated levels of cyclic guanosine-3',5'-monophosphate and accelerated gastrointestinal transit. SIGNIFICANCE: Linaclotide is a potent and selective GC-C agonist that elicits pharmacological effects locally in the gastrointestinal tract. This pharmacological profile suggests that orally administered linaclotide may be capable of improving the abdominal symptoms and bowel habits of patients suffering from IBS-C and chronic constipation.

Amplified centrosomes in breast cancer: a potential indicator of tumor aggressiveness.

Molecular mechanisms leading to genomic instability and phenotypic variation during tumor development and progression are poorly understood. Such instability represents a major problem in the management of breast cancer because of its contribution to more aggressive phenotypes as well as chemoresistance. In this study we analyzed breast carcinomas and tumor-derived cell lines to determine the relationship between centrosome amplification and established prognostic factors. Our results show that centrosome amplification can arise independent of ER or p53 status and is a common feature of aneuploid breast tumors. Centrosome amplification is associated with mitotic spindle abnormalities in breast carcinomas and thus may contribute to genomic instability and the development of more aggressive phenotypes during tumor progression.