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Root NO 3 À efflux to the outer medium is a component of NO 3 À net uptake and can even overcome influx upon various stresses. Its role and molecular basis are unknown. Following a functional biochemical approach, NAXT1 (for NITRATE EXCRETION TRANSPORTER1) was identified by mass spectrometry in the plasma membrane (PM) of Arabidopsis thaliana suspension cells, a localization confirmed using a NAXT1-Green Fluorescent Protein fusion protein. NAXT1 belongs to a subclass of seven NAXT members from the large NITRATE TRANSPORTER1/PEPTIDE TRANSPORTER family and is mainly expressed in the cortex of mature roots. The passive NO 3 À transport activity (K m ¼ 5 mM) in isolated root PM, electrically coupled to the ATPdependant H þ -pumping activity, is inhibited by anti-NAXT antibodies. In standard culture conditions, NO 3 À contents were altered in plants expressing NAXT-interfering RNAs but not in naxt1 mutant plants. Upon acid load, unidirectional root NO 3 À efflux markedly increased in wild-type plants, leading to a prolonged NO 3 À excretion regime concomitant with a decrease in root NO 3 À content. In vivo and in vitro mutant phenotypes revealed that this response is mediated by NAXT1, whose expression is upregulated at the posttranscriptional level. Strong medium acidification generated a similar response. In vitro, the passive efflux of NO 3 À (but not of Cl À ) was strongly impaired in naxt1 mutant PM. This identification of NO 3 À efflux transporters at the PM of plant cells opens the way to molecular studies of the physiological role of NO 3 À efflux in stressed or unstressed plants.

ObjectiveAlthough counting of circulating tumour cells (CTC) has attracted a broad interest as potential markers of tumour progression and treatment response, the lack of functional characterisation of these cells had become a bottleneck in taking these observations to the clinic. Our objective was to culture these cells in order to understand them and exploit their therapeutic potential to the full.DesignHere, hypothesising that some CTC potentially have cancer stem cell (CSC) phenotype, we generated several CTC lines from the blood of patients with advanced metastatic colorectal cancer (CRC) based on their self-renewal abilities. Multiple standard tests were then employed to characterise these cells.ResultsOur CTC lines self-renew, express CSC markers and have multilineage differentiation ability, both in vitro and in vivo. Patient-derived CTC lines are tumorigenic in subcutaneous xenografts and are also able to colonise the liver after intrasplenic injection. RNA sequencing analyses strikingly demonstrate that drug metabolising pathways represent the most upregulated feature among CTC lines in comparison with primary CRC cells grown under similar conditions. This result is corroborated by the high resistance of the CTC lines to conventional cytotoxic compounds.ConclusionsTaken together, our results directly demonstrate the existence of patient-derived colorectal CTCs that bear all the functional attributes of CSCs. The CTC culture model described here is simple and takes <1 month from blood collection to drug testing, therefore, routine clinical application could facilitate access to personalised medicine.Clinical Trial RegistrationClinicalTrial.gov NCT01577511.

In most plants, NO(3)(-) constitutes the major source of nitrogen, and its assimilation into amino acids is mainly achieved in shoots. Furthermore, recent reports have revealed that reduction of NO(3)(-) translocation from roots to shoots is involved in plant acclimation to abiotic stress. NPF2.3, a member of the NAXT (nitrate excretion transporter) sub-group of the NRT1/PTR family (NPF) from Arabidopsis, is expressed in root pericycle cells, where it is targeted to the plasma membrane. Transport assays using NPF2.3-enriched Lactococcus lactis membranes showed that this protein is endowed with NO(3)(-) transport activity, displaying a strong selectivity for NO(3)(-) against Cl(-). In response to salt stress, NO(3)(-) translocation to shoots is reduced, at least partly because expression of the root stele NO(3)(-) transporter gene NPF7.3 is decreased. In contrast, NPF2.3 expression was maintained under these conditions. A loss-of-function mutation in NPF2.3 resulted in decreased root-to-shoot NO(3)(-) translocation and reduced shoot NO(3)(-) content in plants grown under salt stress. Also, the mutant displayed impaired shoot biomass production when plants were grown under mild salt stress. These mutant phenotypes were dependent on the presence of Na(+) in the external medium. Our data indicate that NPF2.3 is a constitutively expressed transporter whose contribution to NO(3)(-) translocation to the shoots is quantitatively and physiologically significant under salinity.