Gene expression profiling of human liver transplants identifies an early transcriptional signature associated with initial poor graft function.

Liver ischemia-reperfusion injury occurring in orthotopic liver transplantation (OLT) may be responsible for early graft failure. Molecular mechanisms underlying initial poor graft function (IPGF) have been poorly documented in human. The purpose of this study was to identify the major transcriptional alterations occurring in human livers during OLT. Twenty-one RNA extracts derived from liver transplant biopsies taken after graft reperfusion were compared with 7 RNA derived from normal control livers. Three hundred seventy-one genes were significantly modulated and classified in molecular pathways relevant to liver metabolism, inflammatory response, cell proliferation and liver protection. Grafts were then subdivided into two groups based on their peak levels of serum aspartate amino transferase within 72 h after OLT (group 1, non-IPGF: 14 patients; group 2, IPGF: 7 patients). The two corresponding data sets were compared using a supervised prediction method. A new set of genes able to correctly classify 71% of the patients was defined. These genes were functionally associated with oxidative stress, inflammation and inhibition of cell proliferation. This study provides a comprehensive picture of the transcriptional events associated with human OLT and IPGF. We anticipate that such alterations provide a framework for the elucidation of the molecular mechanisms leading to IPGF.

Effect of tyrosine kinase inhibitors on stemness in normal and chronic myeloid leukemia cells.

Although tyrosine kinase inhibitors (TKIs) efficiently cure chronic myeloid leukemia (CML), they can fail to eradicate CML stem cells (CML-SCs). The mechanisms responsible for CML-SC survival need to be understood for designing therapies. Several previous studies suggest that TKIs could modulate CML-SC quiescence. Unfortunately, CML-SCs are insufficiently available. Induced pluripotent stem cells (iPSCs) offer a promising alternative. In this work, we used iPSCs derived from CML patients (Ph+). Ph+ iPSC clones expressed lower levels of stemness markers than normal iPSCs. BCR-ABL1 was found to be involved in stemness regulation and ERK1/2 to have a key role in the signaling pathway. TKIs unexpectedly promoted stemness marker expression in Ph+ iPSC clones. Imatinib also retained quiescence and induced stemness gene expression in CML-SCs. Our results suggest that TKIs might have a role in residual disease and confirm the need for a targeted therapy different from TKIs that could overcome the stemness-promoting effect caused by TKIs. Interestingly, a similar pro-stemness effect was observed in normal iPSCs and hematopoietic SCs. These findings could help to explain CML resistance mechanisms and the teratogenic side-effects of TKIs in embryonic cells.

An evolutionary view of drug-receptor interaction: the bioamine receptor family.

The large molecular diversity of receptors and their subtypes means that the pharmacologist is faced with many puzzling characterization questions. First, the molecular diversity of the receptors is deciphered only in part by a pharmacological approach, which precludes a satisfactory receptor classification based solely on pharmacological characteristics. Second, the physiological counterpart of the numerous subtypes of receptors specifically activated by single endogenous ligands remains unclear. Here, Philippe Vernier and colleagues use the example of the bioamine G protein-coupled receptors to show that many of the apparent inconsistencies that emerge from pharmacological and molecular characterizations of receptors can be better understood if the evolutionary history of the receptors is taken into account.

Sequence and regulation of European eel prolactin mRNA.

cDNA clones encoding the European eel (Anguilla anguilla L.) prolactin were isolated from a pituitary cDNA library constructed in gamma gt10, using a rainbow trout Prl cDNA fragment as a probe. Four different inserts were subcloned into the pGEM 3Z plasmid after PCR amplification. The 1082 bp-long nucleotide sequence revealed an open reading frame of 627 bp encoding a 24 amino acid-long signal peptide followed by a 185 amino acid-long mature protein. Comparison studies showed 60-70% homology with other known teleost fish prolactins and 30-45% with non-teleost fish, amphibian, reptilian, avian and mammalian prolactins. In situ hybridization studies using labelled prolactin RNA probe showed a strong signal in the rostral pars distalis of the pituitary gland. We next examined the physiological regulation of this prolactin synthesis in vivo using Northern blot analysis and prolactin cDNA probe labelled by random priming. The pituitary prolactin mRNA level was markedly decreased 3 weeks after transfer of eels from freshwater to sea water. Implants of thyroid hormones left for up to three weeks were ineffective on prolactin mRNA. Estradiol administered as implant, alone or in combination with 500 micrograms testosterone, was also unable to significantly alter the pituitary mRNA level for prolactin in the freshwater silver eels whatever the dose used (20-500 micrograms) and whatever the duration of treatment (from 4 days to 10 weeks).

Efficacy of moxidectin as a 1% injectable solution and a 0.1% oral drench against nasal bots, pulmonary and gastrointestinal nematodes in sheep.

Thirty ewes, 3-10 years old, known to be naturally infected with internal parasites, were allocated to three homogeneous groups of ten ewes each based on faecal nematode egg counts. The following experimental treatments were administered on Day 0: (A) moxidectin 1% injectable solution at 0.2 mg kg-1 body weight; (B) moxidectin 0.1% oral drench solution at 0.2 mg kg-1 liveweight; (C) untreated control. Faecal samples were taken on Days -7, 0, 1, 2, 3, 7, 14, 21, 28 and 35 to obtain counts of nematode eggs. One-half of the ewes in each treatment group were slaughtered 14 days after dosing, while the remainder were slaughtered 35 days after treatment to count the numbers of nasal bots, pulmonary nematodes and gastrointestinal nematodes. Moxidectin 1% injectable solution and moxidectin 0.1% oral drench solution were highly effective against gastrointestinal nematodes and against a variable infection of Dictyocaulus filiaria. Moxidectin 1% injectable solution was effective against first stage larvae of Oestrus ovis, whereas moxidectin 0.1% oral drench was ineffective.

Activity of moxidectin 1% injectable solution against first instar Hypoderma spp. in cattle and effects on antibody kinetics.

The activity of the moxidectin as an 1% w/v injectable solution on first instar Hypoderma spp. has been evaluated in sixteen naturally infested young cattle. The animals were selected on the basis of their serological status and allocated to two groups of eight animals. At the end of November, one group was treated with moxidectin at a dose rate of 0.2 mg/kg via the subcutaneous route and the non treated control calves injected with the vehicle. The serological status was assessed 1, 2, 4, 8 and 12 weeks post treatment and the presence of Hypoderma lumps determined every two weeks from February to June. A 100% efficacy of the injectable formulation was demonstrated. A progressive fall of the antibody levels was observed in the treated calves for one month following treatment, suggesting a progressive action of the test compound and a limited risk of hypersensitivity.

Persistent efficacy of topical moxidectin against Dictyocaulus viviparus and Ostertagia ostertagi.

The persistent activity of moxidectin topically administered at the dose rate of 0.5 mg kg-1 bodyweight was evaluated against experimental nematode infection in 30 calves randomly allocated to six groups. Five groups were treated on days -42, -35, -28, -21 and -14. The 6th group remained untreated as a control. On Day 0, the calves were infected experimentally with 1000 Dictyocaulus viviparus and 50,000 Ostertagia ostertagi larvae and killed 3 weeks later. The formulation of moxidectin showed excellent activity against both parasites for up to 5 weeks (> 99%). Six weeks after treatment the reduction in the number of D. viviparus was still high (> 90%). No adverse reactions to moxidectin were observed in any of the animals.

Efficacy of oral moxidectin against benzimidazole-resistant isolates of gastrointestinal nematodes in sheep.

The efficacy of orally administered moxidectin was determined against four benzimidazole-resistant nematode isolates. At the start of the trial, 30 lambs were each infected experimentally with 20,000 third stage larvae (5000 Haemonchus contortus, 7000 Teladorsagia circumcincta, 3000 Trichostrongylus colubriformis and 5000 Cooperia curticei); 28 days later they were allocated randomly to three groups of 10: one untreated group, one group treated orally with fenbendazole (5 mg/kg bodyweight) and one group treated orally with moxidectin (0.2 mg/kg). Samples of faeces were taken five and 10 days after treatment and the lambs were killed 10 days after treatment. Fenbendazole reduced the average number of nematode eggs in faeces by 95 per cent and the average number of worms by 25 to 45 per cent according to the species. The efficacy of moxidectin against these benzimidazole-resistant isolates was 100 per cent. No adverse reactions to either of the drugs were observed.

The persistence of the efficacy of injectable or oral moxidectin against Teladorsagia, Haemonchus and Trichostrongylus species in experimentally infected sheep.

The persistence of the efficacy of moxidectin was evaluated against experimental gastrointestinal nematode infections in 55 lambs randomly allocated to 11 equal groups and infected on day 0. Moxidectin 1 per cent injectable solution was administered at a dose rate of 0.2 mg moxidectin/kg bodyweight to five of the groups on days -42, -35, -28, -21 and -14; five other groups were treated with moxidectin 0.1 per cent oral drench at the same dose rate on days -35, -28, -21, -14 and -7, and the 11th group remained untreated as a control. The lambs were infected experimentally with 8000 Teladorsagia circumcincta, 2000 Haemonchus contortus and 10,000 Trichostrongylus colubriformis infective larvae and killed three weeks later. Both formulations of moxidectin showed excellent activity against T circumcincta and H contortus with almost 100 per cent efficacy against the abomasal parasites for up to 35 days after treatment. The efficacy of moxidectin 1 per cent injectable against T colubriformis was much higher (> 99 per cent) than that of the oral drench and it was highly effective up to 21 days after treatment, and gave a moderate reduction in worm burden for up to 35 days after treatment. No adverse reactions to moxidectin were observed in any of the animals.

Efficacy of moxidectin pour-on against nematode infections in cattle.

Three groups of eight calves, naturally infected with gastrointestinal nematodes and artificially infected with Dictyocaulus viviparus were used to evaluate the efficacy of moxidectin pour-on at dose rates of 0.35 mg/kg and 0.5 mg/kg bodyweight. With both doses the efficacy was 100 per cent against adult D viviparus, Trichostrongylus axei, Ostertagia species and Nematodirus helvetianus. It was more than 99 per cent against Ostertagia and Nematodirus species fourth stage larvae. A small number of Cooperia species were found after treatment, and for this parasite, the efficacy of moxidectin ranged from 97.6 per cent against the larval stages to 98.8 per cent against the adults. No adverse reactions to the moxidectin treatment were observed.

[Evolution of monoamine receptors and the origin of motivational and emotional systems in vertebrates]. / L'évolution des récepteurs des monoamines et l'émergence des systèmes motivationnels et émotionnels chez les vertébrés.

The evolving vertebrate nervous system was accompanied by major gene duplication events generating novel organs and a sympathetic system. Vertebrate neural pathways synthesizing catecholamine neurotransmitters (dopamine and noradrenaline), were subsequently recruited to process increased information demands by mediating psychomotor functions such as selective attention/predictive reward and emotional drive via the activation of multiple G-protein linked catecholamine receptor subtypes. Here we show that the evolution of these receptor-mediated events were similarly driven by forces of gene duplication, at the cephalochordate/vertebrate transition. In the cephalochordate Amphioxus, a sister group to vertebrates, a single catecholamine receptor gene was found, which based on molecular phylogeny and functional analysis formed a monophyletic group with both vertebrate dopamine D1 and beta adrenergic receptor classes. In addition, the presence of dopamine but not of noradrenaline was assayed in Amphioxus. In contrast, two distinct genes homologous to jawed vertebrate dopamine D1 and beta adrenergic receptor genes were extant in representatives of the earliest craniates, lamprey and hagfish, paralleling high dopamine and noradrenaline content throughout the brain. These data suggest that a D1/beta receptor gene duplication was required to elaborate novel catecholamine psychomotor adaptive responses and that a noradrenergic system specifically emerged at the origin of vertebrate evolution.

MiR-210 promotes a hypoxic phenotype and increases radioresistance in human lung cancer cell lines.

The resistance of hypoxic cells to radiotherapy and chemotherapy is a major problem in the treatment of cancer. Recently, an additional mode of hypoxia-inducible factor (HIF)-dependent transcriptional regulation, involving modulation of a specific set of micro RNAs (miRNAs), including miR-210, has emerged. We have recently shown that HIF-1 induction of miR-210 also stabilizes HIF-1 through a positive regulatory loop. Therefore, we hypothesized that by stabilizing HIF-1 in normoxia, miR-210 may protect cancer cells from radiation. We developed a non-small cell lung carcinoma (NSCLC)-derived cell line (A549) stably expressing miR-210 (pmiR-210) or a control miRNA (pmiR-Ctl). The miR-210-expressing cells showed a significant stabilization of HIF-1 associated with mitochondrial defects and a glycolytic phenotype. Cells were subjected to radiation levels ranging from 0 to 10 Gy in normoxia and hypoxia. Cells expressing miR-210 in normoxia had the same level of radioresistance as control cells in hypoxia. Under hypoxia, pmiR-210 cells showed a low mortality rate owing to a decrease in apoptosis, with an ability to grow even at 10 Gy. This miR-210 phenotype was reproduced in another NSCLC cell line (H1975) and in HeLa cells. We have established that radioresistance was independent of p53 and cell cycle status. In addition, we have shown that genomic double-strand breaks (DSBs) foci disappear faster in pmiR-210 than in pmiR-Ctl cells, suggesting that miR-210 expression promotes a more efficient DSB repair. Finally, HIF-1 invalidation in pmiR-210 cells removed the radioresistant phenotype, showing that this mechanism is dependent on HIF-1. In conclusion, miR-210 appears to be a component of the radioresistance of hypoxic cancer cells. Given the high stability of most miRNAs, this advantage could be used by tumor cells in conditions where reoxygenation has occurred and suggests that strategies targeting miR-210 could enhance tumor radiosensitization.

miR-210 is overexpressed in late stages of lung cancer and mediates mitochondrial alterations associated with modulation of HIF-1 activity.

Following the identification of a set of hypoxia-regulated microRNAs (miRNAs), recent studies have highlighted the importance of miR-210 and of its transcriptional regulation by the transcription factor hypoxia-inducible factor-1 (HIF-1). We report here that miR-210 is overexpressed at late stages of non-small cell lung cancer. Expression of miR-210 in lung adenocarcinoma A549 cells caused an alteration of cell viability associated with induction of caspase-3/7 activity. miR-210 induced a loss of mitochondrial membrane potential and the apparition of an aberrant mitochondrial phenotype. The expression profiling of cells overexpressing miR-210 revealed a specific signature characterized by enrichment for transcripts related to 'cell death' and 'mitochondrial dysfunction', including several subunits of the electron transport chain (ETC) complexes I and II. The transcript coding for one of these ETC components, SDHD, subunit D of succinate dehydrogenase complex (SDH), was validated as a bona fide miR-210 target. Moreover, SDHD knockdown mimicked miR-210-mediated mitochondrial alterations. Finally, miR-210-dependent targeting of SDHD was able to activate HIF-1, in line with previous studies linking loss-of-function SDH mutations to HIF-1 activation. miR-210 can thus regulate mitochondrial function by targeting key ETC component genes with important consequences on cell metabolism, survival and modulation of HIF-1 activity. These observations help explain contradictory data regarding miR-210 expression and its putative function in solid tumors.

Early emergence of three dopamine D1 receptor subtypes in vertebrates. Molecular phylogenetic, pharmacological, and functional criteria defining D1A, D1B, and D1C receptors in European eel Anguilla anguilla.

The existence of dopamine D1C and D1D receptors in Xenopus and chicken, respectively, challenged the established duality (D1A and D1B) of the dopamine D1 receptor class in vertebrates. To ascertain the molecular diversity of this gene family in early diverging vertebrates, we isolated four receptor-encoding sequences from the European eel Anguilla anguilla. Molecular phylogeny assigned two receptor sequences (D1A1 and D1A2) to the D1A subtype, and a third receptor to the D1B subtype. Additional sequence was orthologous to the Xenopus D1C receptor and to several other previously unclassified fish D1-like receptors. When expressed in COS-7 cells, eel D1A and D1B receptors display affinity profiles for dopaminergic ligands similar to those of other known vertebrate homologues. The D1C receptor exhibits pharmacological characteristics virtually identical to its Xenopus homologue. Functionally, while all eel D1 receptors stimulate adenylate cyclase, the eel D1B receptor exhibits greater constitutive activity than either D1A or D1C receptors. Semiquantitative reverse transcription-polymerase chain reaction reveals the differential distribution of D1A1, D1A2, D1B, and D1C receptor mRNA within the hypothalamic-pituitary axis of the eel brain. Taken together, these data suggest that the D1A, D1B, and D1C receptors arose prior to the evolutionary divergence of fish and tetrapods and exhibit molecular, pharmacological, and functional attributes that unambiguously allow for their classification as distinct D1 receptor subtypes in the vertebrate phylum.