MtNIP5;1, a novel Medicago truncatula boron diffusion facilitator induced under deficiency

Background: Legumes comprise important crops that offer major agronomic benefits, including the capacity of establishing symbiosis with rhizobia, fixing atmospheric N2. It has been proven that legumes are particularly susceptible to boron (B) stress, which leads to important yield penalties. Boron (B) deficiency or toxicity in plants causes the inhibition of growth and an altered development. Under such conditions, the participation of two distinct protein families (the major intrinsic protein family MIP and the Boron transporter family BOR) is required to minimize detrimental effects caused by B stress. However, in legumes, little is known about the transport mechanisms responsible for B uptake and distribution, especially under deficiency. Results: A Medicago truncatula protein, MtNIP5;1 (Medtr1g097840) (homologous to the Arabidopsis thaliana AtNIP5;1) was identified as a novel legume B transporter involved in B uptake under deficiency. Further analyses revealed that this M. truncatula aquaporin expression was boron regulated in roots, being induced under deficiency and repressed under toxicity. It localizes at the plasma membrane of root epidermal cells and in nodules, where B plays pivotal roles in symbiosis. Furthermore, the partial complementation of the nip5;1–1 A. thaliana mutant phenotype under B deficiency supports a functional role of MtNIP5;1 as a B transporter in this legume model plant. Conclusions: The results here presented support a functional role of MtNIP5;1 in B uptake under deficiency and provides new insights into B transport mechanisms in legume specie

Gene disruption of the DNA topoisomerase IB small subunit induces a non-viable phenotype in the hemoflagellate Leishmania major

<p>Abstract</p> <p>Background</p> <p>The unusual heterodimeric leishmanial DNA topoisomerase IB consists of a large subunit containing the phylogenetically conserved "core" domain, and a small subunit harboring the C-terminal region with the characteristic tyrosine residue in the active site. RNAi silencing of any of both protomers induces a non-viable phenotype in the hemoflagelate <it>Trypanosoma brucei</it>. Unfortunately, this approach is not suitable in <it>Leishmania </it>where gene replacement with an antibiotic marker is the only approach to generate lack-of-function mutants. In this work, we have successfully generated null mutants in the small subunit of the <it>L. major </it>DNA topoisomerase IB using two selection markers, each conferring resistance to hygromycin B and puromycin, respectively.</p> <p>Results</p> <p>We have successfully replaced both <it>topS </it>loci with two selection markers. However, to achieve the second transfection round, we have had to rescue the null-homozygous with an episomal vector carrying the <it>Leishmania major topS </it>gene. Phenotypic characterization of the <it>L. major </it>rescued strain and a <it>L. major </it>strain, which co-overexpresses both subunits, shows few differences in DNA relaxation and camptothecin cytotoxicity when it was compared to the wild-type strain. Studies on phosphatidylserine externalization show a poor incidence of camptothecin-induced programmed cell death in <it>L. major</it>, but an effective cell-cycle arrest occurs within the first 24 h. S-Phase delay and G₂/M reversible arrest was the main outcome at lower concentrations, but irreversible G₂arrest was detected at higher camptothecin pressure.</p> <p>Conclusion</p> <p>Results obtained in this work evidence the essentiality of the <it>topS </it>gene encoding the <it>L. major </it>DNA topoisomerase IB small subunit. Reversibility of the camptothecin effect points to the existence of effective checkpoint mechanisms in <it>Leishmania </it>parasites.</p

Trypanosomatids topoisomerase re-visited. New structural findings and role in drug discovery

AbstractThe Trypanosomatidae family, composed of unicellular parasites, causes severe vector-borne diseases that afflict human populations worldwide. Chagas disease, sleeping sickness, as well as different sorts of leishmaniases are amongst the most important infectious diseases produced by Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., respectively. All these infections are closely related to weak health care services in low-income populations of less developed and least economically developed countries. Search for new therapeutic targets in order to hit these pathogens is of paramount priority, as no effective vaccine is currently in use against any of these parasites. Furthermore, present-day chemotherapy comprises old-fashioned drugs full of important side effects. Besides, they are prone to produce tolerance and resistance as a consequence of their continuous use for decades. DNA topoisomerases (Top) are ubiquitous enzymes responsible for solving the torsional tensions caused during replication and transcription processes, as well as in maintaining genomic stability during DNA recombination. As the inhibition of these enzymes produces cell arrest and triggers cell death, Top inhibitors are among the most effective and most widely used drugs in both cancer and antibacterial therapies. Top relaxation and decatenation activities, which are based on a common nicking–closing cycle involving one or both DNA strands, have been pointed as a promising drug target. Specific inhibitors that bind to the interface of DNA-Top complexes can stabilize Top-mediated transient DNA breaks. In addition, important structural differences have been found between Tops from the Trypanosomatidae family members and Tops from the host. Such dissimilarities make these proteins very interesting for drug design and molecular intervention. The present review is a critical update of the last findings regarding trypanosomatid’s Tops, their new structural features, their involvement both in the physiology and virulence of these parasites, as well as their use as promising targets for drug discovery

Miltefosine and nifuratel combination: a promising therapy for the treatment of leishmania donovani visceral leishmaniasis

[EN] Visceral leishmaniasis is a neglected vector-borne tropical disease caused by Leishmania donovani and Leishmania infantum that is endemic not only in East African countries, but also in Asia, regions of South America and the Mediterranean Basin. For the pharmacological control of this disease, there is a limited number of old and, in general, poorly adherent drugs, with a multitude of adverse effects and low oral bioavailability, which favor the emergence of resistant pathogens. Pentavalent antimonials are the first-line drugs, but due to their misuse, resistant Leishmania strains have emerged worldwide. Although these drugs have saved many lives, it is recommended to reduce their use as much as possible and replace them with novel and more friendly drugs. From a commercial collection of anti-infective drugs, we have recently identified nifuratel-a nitrofurantoin used against vaginal infections-as a promising repurposing drug against a mouse model of visceral leishmaniasis. In the present work, we have tested combinations of miltefosine-the only oral drug currently used against leishmaniasis-with nifuratel in different proportions, both in axenic amastigotes from bone marrow and in intracellular amastigotes from infected Balb/c mouse spleen macrophages, finding a potent synergy in both cases. In vivo evaluation of oral miltefosine/nifuratel combinations using a bioimaging platform has revealed the potential of these combinations for the treatment of this disease.SIThis research was partially funded by MINECO; SAF2017-83575-R and PID 2020-119031RB-100 to R.M.R. E.M.-F. is contracted with EU PRIMA (PCI2022-132925); G.G. is recipient of FPI scholarship (PRE 2021 096909) supported by AE

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Changes in nutritional quality-related traits of quinoa seeds under different storage conditions

Within the context of climate change and its impact on global food security, seed storage has become key, as it ensures long-term food and next-season seed preservation. Aiming at evaluating quality-related changes in quinoa seeds over storage time, different storage temperatures (–20, 4, 12, 25, and 37°C) and humidity conditions (use of silica gel or not) were studied and different seed nutritional parameters were evaluated at different points during a year of storage. Also, to determine if these variations could be conditioned by the genotype used, two quinoa cultivars were compared. The results proved that quinoa seed quality is highly dependent on the storage temperature but is not consistently affected by the use of silica gel if the seed moisture content (SMC) is kept between 5 and 12%. Furthermore, quality can be maintained and even improved by keeping SMC lower than 12% and storage temperatures low (4°C). Under these conditions (at 4°C in hermetic packaging with or without silica gel), and after 12 months of storage, there was an increase in amino acids like isoleucine, serine, arginine, glycine, and glutamic acid and in seed viability and germination. On the contrary, quinoa seeds stored at 37°C showed an accumulation of reactive oxygen species (ROS) which was related to a lower antioxidant capacity and a reduction in the contents of essential amino acids like isoleucine, lysine, histidine, and threonine, resulting in a delayed and reduced germination capacity, and, therefore, lower seed quality. Besides, quality-related differences appeared between cultivars highlighting differences linked to the genotype. Overall, this work demonstrates that optimal storage temperatures and SMC can preserve or even improve quinoa seed nutritional quality, which in turn can impact food safety and agricultureThis work was supported by the Ministerio de Ciencia e Innovación (MICINN, Spain) (PID2019-105748RA-I00), the Comunidad de Madrid-Universidad Autónoma de Madrid (SI1/PJI/2019-00124), the CYTED (ValSe-Food 119RT0567), the FPI-UAM Fellowship Programme 2019 (to SG-R), the Ayundante de Investigación CM Programme (to IM-G), and the Ramón y Cajal Programme 2019 (to M

Antiparasitic effect of synthetic aromathecins on Leishmania infantum

[EN} Background Canine leishmaniasis is a zoonotic disease caused by Leishmania infantum, being the dogs one of the major reservoirs of human visceral leishmaniasis. DNA topology is a consolidated target for drug discovery. In this regard, topoisomerase IB – one of the enzymes controlling DNA topology – has been poisoned by hundreds of compounds that increase DNA fragility and cell death. Aromathecins are novel molecules with a multiheterocyclic ring scaffold that have higher stability than camptothecins. Results Aromathecins showed strong activity against both forms of L. infantum parasites, free-living promastigotes and intra-macrophagic amastigotes harbored in ex vivo splenic explant cultures obtained from infected BALB/c mice. However, they prevented the relaxation activity of leishmanial topoisomerase IB weakly, which suggests that the inhibition of topoisomerase IB partially explains the antileishmanial effect of these compounds. The effect of aromathecins was also studied against a strain resistant to camptothecin, and results suggested that the trafficking of these compounds is not through the ABCG6 transporter. Conclusions Aromathecins are promising novel compounds against canine leishmaniasis that can circumvent potential resistances based on drug efflux pumps.SIThis research had the financial support of: Ministerio de Economía y Competitividad (MINECO, AEI, FEDER, UE) [MINECO: AGL2016–79813-C2-1R, SAF2017–83575-R]. Junta de Castilla y León cofinanced by FEDER UE [LE020P17, Grant UIC108]. The funding body does not participate in the design of the study; collection, analysis and interpretation of data and in writing the manuscript

Axenic interspecies and intraclonal hybrid formation in Leishmania: Successful crossings between visceral and cutaneous strains

[EN] Neglected Tropical Diseases (NTDs) represent a serious threat to humans, especially for those living in poor or developing countries. Leishmanianiosis is considered a zoonotic NTD transmitted by the bite of female phlebotomine sandflies, and is manifested mainly as a visceral form (caused by L. infantum and L. donovani) and a cutaneous form (caused by many species including L. major, L. tropica and L. braziliensis). Although it is now known that sexual reproduction occurs in these parasites, more studies are necessary to characterize the ability of Leishmania to generate hybrids, which may represent an important mechanism for virulence, drug resistance or adaptation to the host immune system. Therefore, several experiments were conducted to generate either intraclonal or interspecies hybrids in vitro. Results demonstrated that hybrids can be formed even with outcrosses between parasites causing visceral and cutaneous forms of the disease. Characterization of hybrids in terms of ploidy, kDNA content, growth rate and infection capacity provide important information about sexual reproduction in these parasites.SI: C.G.C (LE255-16) and B.D.A (LE208-17) are recipients of Junta de Castilla y Leo´n (JCyL) and European Social Found (ESF)’s Fellowships Scheme for Doctoral Training Programs. This research was funded by MINECO; SAF2017- 83575-R to RMR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscrip

Antileishmanial activity of terpenylquinones on Leishmania infantum and their effects on Leishmania topoisomerase IB

[EN] Leishmania is the aethiological agent responsible for the visceral leishmaniasis, a serious parasite-borne disease widely spread all over the World. The emergence of resistant strains makes classical treatments less effective; therefore, new and better drugs are necessary. Naphthoquinones are interesting compounds for which many pharmacological properties have been described, including leishmanicidal activity. This work shows the antileishmanial effect of two series of terpenyl-1,4-naphthoquinones (NQ) and 1,4-anthraquinones (AQ) obtained from natural terpenoids, such as myrcene and myrceocommunic acid. They were evaluated both in vitro and ex vivo against the transgenic iRFP-Leishmania infantum strain and also tested on liver HepG2 cells to determine their selectivity indexes. The results indicated that NQ derivatives showed better antileishmanial activity than AQ analogues, and among them, compounds with a diacetylated hydroquinone moiety provided better results than their corresponding quinones. Regarding the terpenic precursor, compounds obtained from the monoterpenoid myrcene displayed good antiparasitic efficiency and low cytotoxicity for mammalian cells, whereas those derived from the diterpenoid showed better antileishmanial activity without selectivity. In order to explore their mechanism of action, all the compounds have been tested as potential inhibitors of Leishmania type IB DNA topoisomerases, but only some compounds that displayed the quinone ring were able to inhibit the recombinant enzyme in vitro. This fact together with the docking studies performed on LTopIB suggested the existence of another mechanism of action, alternative or complementary to LTopIB inhibition. In silico druglikeness and ADME evaluation of the best leishmanicidal compounds has shown good predictable druggabilitySIFinancial support came from Spanish MINECO (CTQ2015-68175-R, AGL2016-79813-C2-1-R, AGL2016-79813-C2-2-R and SAF2017-83575-R), ISCIII-RICET Network (RD12/0018/0002) and Consejería de Educación de la Junta de Castilla y León (LE020P17) co-financed by the Fondo Social Europeo of the European Union (FEDER-EU). P. G. J. acknowledges funding by Fundación Salamanca Ciudad de Cultura y Saberes (’‘Programme for attracting scientific talent to Salamanca’‘

Quinoa plant architecture: A key factor determining plant productivity and seed quality under long-term drought

Chenopodium quinoa (quinoa) is an underutilized crop proposed as key to achieving food security within the current climatic context, where water scarcity in rainfed areas is becoming more frequent and severe, especially in Mediterranean regions. Thus, aiming at deepening our knowledge regarding the impact of water limitation on the quinoa seed nutritional quality, seeds obtained from primary and secondary panicles of four different European-adapted cultivars (F14, F15, F16, and Titicaca) growing under full irrigation and water-limiting conditions, were here analyzed. A set of parameters were evaluated in this work, including agronomical (such as yield, seed weight, seed area, and seed germination and viability rates) and nutritional (including the seed proximate composition, mineral content, and antioxidants) traits. Our results indicate that the morphological changes associated with drought stress affect secondary panicles’ seed yield. This phenomenon was generally associated with an improvement in the nutritional quality of those seeds. However, cultivars such as F16, despite keeping total seed yield under low water availability, showed drought's detrimental effect on the seed nutritional quality. In contrast, cultivars like F15 and Titicaca reduced their seed yield under water-limiting conditions but increased their protein, iron, copper, calcium, manganese, and zinc contents, especially in secondary panicles. Therefore, the dichotomy between seed quantity and quality has to be considered in this crop under water stress scenarios, highlighting differences in sink strength along the plant panicles determining seed nutritional qualityThe authors gratefully acknowledge the financial support received from the Ministerio de Ciencia e Innovacion ´ (MICINN, Spain) and the Agencia Estatal de Investigacion ´ (PID2019-105748RA-I00 AEI/10.13039/ 501100011033), the CYTED (ValSe-Food 119RT0567), the FPI UAM Fellowship Programme 2019 (to SG-R), and the Ramon ´ y Cajal Programme 2019 (to MR