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Introduction and Objective. While most isolates of peach latent mosaic viroid (PLMVd) (Hernández and Flores, PNAS USA 1992) are latent in leaves, hence its name, some incite peach mosaic (PM) or albinism (peach calico, PC). PC-inducing variants have a characteristic insertion of 12-13 nucleotides and accumulate preferentially in the symptomatic but not in the surrounding green foliar sectors. We have shown previously that two 21-nt small RNAs (PLMVd-sRNAs) containing the PC-associated insertion, guide cleavage of the mRNA encoding the chloroplastic heat-shock protein as predicted by RNA silencing (Navarro et al., Plant J. 2012), with this event likely representing the primary alteration eventually resulting in symptoms. Here we have extended these studies to PM, an issue more complex because entails identification of the corresponding molecular determinant, which is not associated with a specific insertion, and because in contrast with the well-defined PC phenotype, PM differs in type and intensity of symptoms. Material and Methods. Deep sequencing was applied to host- and PLMVd-sRNAs, RT-PCR to synthesize complete PLMVd-cDNAs, semi-quantitative RT-PCR to evaluate host mRNA levels, and RNA ligase-mediated rapid amplification of cDNA ends (RACE) to map cleavage sites in mRNAs. Results. We focused on one severe yellow mosaic (peach yellow mosaic, PYM) expressed in leaf sectors interspersed with others green. Full-length PLMVd-cDNAs from both sectors were cloned and sequenced. Two clones, differing in one nucleotide substitution, served to generate dimeric head-to-tail inserts in plasmids from which in vitro transcripts were produced and bioassayed in GF-305 peach seedlings. Whereas one of the transcripts incited an intense yellow mosaic, the other caused only mild lesions, supporting the involvement of one specific PLMVd nucleotide in PYM. Additionally, the differential nucleotide substitution: i) was preserved in the progeny of symptomatic but not in that of the adjacent green sectors, and ii) is absent in a set of PLMVd variants that replicate latently. Examination in silico of the PLMVd-sRNAs and their potential mRNAs targets of peach, whose genome is completely sequenced, has shown that one PLMVd-sRNA containing the PYM-associated substitution could guide cleavage of the mRNA coding for a thylakoid protein as predicted by RNA silencing. RACE experiments indicate that that is indeed the case and that accumulation of this mRNA is lower in symptomatic than in asymptomatic sectors. Moreover, mutants in the gene coding for this thylakoid protein exhibit yellow phenotypes. Conclusions. PLMVd variants with a single nucleotide substitution elicit a yellow mosaic by cleaving, via RNA silencing, the mRNA encoding a thylakoid protein.

Sin financiación 4.490 JCR (2017) Q1, 4/82 Obstetrics & Gynecology, 2/29 Reproductive Biology UEM

Patients with asthma need uninterrupted supplies of affordable, quality-assured essential medicines. However, access in many low- and middle-income countries (LMICs) is limited. The World Health Organization (WHO) Non-Communicable Disease (NCD) Global Action Plan 2013&ndash, 2020 sets an 80% target for essential NCD medicines&rsquo, availability. Poor access is partly due to medicines not being included on the national Essential Medicines Lists (EML) and/or National Reimbursement Lists (NRL) which guide the provision of free/subsidised medicines. We aimed to determine how many countries have essential asthma medicines on their EML and NRL, which essential asthma medicines, and whether surveys might monitor progress. A cross-sectional survey in 2013&ndash, 2015 of Global Asthma Network principal investigators generated 111/120 (93%) responses&mdash, 41 high-income countries and territories (HICs), 70 LMICs. Patients in HICs with NRL are best served (91% HICs included ICS (inhaled corticosteroids) and salbutamol). Patients in the 24 (34%) LMICs with no NRL and the 14 (30%) LMICs with an NRL, however no ICS are likely to have very poor access to affordable, quality-assured ICS. Many LMICs do not have essential asthma medicines on their EML or NRL. Technical guidance and advocacy for policy change is required. Improving access to these medicines will improve the health system&rsquo, s capacity to address NCDs.

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TT-173 is a new hemostatic agent consisting of yeast-derived microvesicles containing a modified version of recombinant human tissue factor. In the present work, the procoagulant activity of TT-173 has been evaluated for the first time in humans. This is a phase I, randomized, placebo-controlled study to evaluate the efficacy, safety, systemic absorption, and immunogenicity of TT-173 in healthy volunteers undergoing tooth extraction. Subjects received TT-173 or placebo into the alveolar cavity, just after tooth extraction. Time to clot formation, bleeding time, and adverse events were recorded. Treatment with TT-173 reduced the bleeding time and the time to clot formation. No adverse events related with product administration were reported. In the same way, neither systemic absorption nor immunogenic reaction against the product was detected. Our findings pave the way to evaluate the usefulness of this new topical hemostatic agent in more complex oral surgeries and in those patients affected with coagulation disorders that may compromise the realization of dental procedures. The new hemostatic agent TT-173 has proven efficacious and safe in healthy subjects undergoing tooth extraction supporting its further evaluation in more complex surgeries. The development of this new topical hemostatic agent could contribute to bleeding control in oral and maxillofacial surgery.

El viroide del mosaico latente del melocotonero (peach latent mosaic viroid, PLMVd) (Hernández and Flores, PNAS USA, 1992) muestra un comportamiento patogénico inusual. Mientras que, de acuerdo con su nombre, la mayoría de aislados son asintomáticos en hojas, algunos inducen mosaicos (peach mosaic, PC) o un albinismo extremo (peach calico, PM). Las variantes del PLMVd que causan PC tienen una inserción característica de 12-13 nt y se acumulan preferentemente en los sectores foliares sintomáticos y no en los verdes adyacentes. Utilizando secuenciación masiva, RT-PCR semicuantitativa y amplificación rápida de extremos de cDNA mediada por RNA ligasa (RNA ligase-mediated rapid amplification of cDNA ends, RACE), hemos mostrado recientemente que dos pequeños RNAs derivados del PLMVd (PLMVd-sRNAs) conteniendo la inserción asociada al PC guían el corte de un mRNA del hospedante como predice el silenciamiento mediado por RNA (RNA silencing) (Navarro et al., Plant J. 2012). La extensión de estos análisis a la sintomatología PM conlleva la identificación del determinante(s) molecular de la misma, una cuestión que plantea serias dificultades porque no está asociado a una inserción específica y porque, en contraste con el PC que es una alteración bien definida, el PM varía mucho en intensidad y distribución. Después de examinar distintos aislados de PLMVd-PM, hemos enfocado nuestra atención sobre uno de ellos caracterizado por un intenso mosaico amarillo (peach yellow mosaic, PYM) que se expresa en sectores foliares entremezclados con otros verdes. Partiendo de RNAs de ambos sectores se generaron por RT-PCR PLMVd-cDNAs de longitud completa que fueron clonados y secuenciados. Dos clones, difiriendo en una simple sustitución nucleotídica, se emplearon para producir insertos diméricos con orientación cabeza-cola y, de ellos, transcritos in vitro que se bioensayaron. Mientras que uno de los transcritos indujo un intenso PYM, el otro solo causó lesiones suaves, proporcionando la primera pista de la implicación de un núcleotido específico en el PYM. Además: i) amplificación por RT-PCR, clonaje y secuenciación revelaron que la sustitución nucleotídica diferencial se preserva en los sectores sintomáticos, pero no así en los verdes circundantes, y ii) la sustitución nucleotídica asociada con el PYM no está presente en una serie de variantes del PLMVd que inducen infecciones asintomáticas. Un examen in silico de los PLMVd-sRNAs y de sus posibles mRNAs dianas del hospedante (del que se conoce su genoma completo), ha mostrado que un PLMVd-sRNA con la sustitución nucleotídica asociada al PYM dirigiría el corte del mRNA que codifica una proteína tilacoidal como predice el silenciamiento mediado por RNA. Los primeros experimentos para verificar dicha predicción son consistentes con la misma: la acumulación de este mRNA es menor en sectores sintomáticos que en asintomáticos.

Introducción: El manejo de la infección todavía es un reto en el tratamiento del desnutrido grave porque la inmunosupresión y la anemia concomitante comprometen la respuesta inflamatoria. Objetivo: Comparar la respuesta inflamatoria entre niños con desnutrición aguda grave tipo marasmo y kwashiorkor y niños eutróficos con y sin anemia (grupos control), mediante la medición de los niveles de proteína C reactiva (PCR) y de las citocinas pro-inflamatorias IL-8, IL-1β, IL-6, IL-10, TNFα e IL-12. Metodología: Se realizó un estudio de corte transversal, donde se determinaron los niveles de algunos indicadores de la respuesta inflamatoria como proteína C reactiva (PCR), citocinas pro-inflamatorias como IL-8, IL-1β, IL-6, IL-10, TNF-α e IL-12p70, hemoglobina y ferritina en un grupo de 40 niños: 10 con marasmo, 10 con kwashiorkor, 10 eutróficos con anemia y 10 eutróficos sin anemia. Resultados. La PCR fue significativamente mayor en desnutridos que en eutróficos. Las concentraciones de las citocinas pro-inflamatorias fueron más altas en niños con kwashiorkor seguidos de eutróficos con anemia, marasmáticos y eutróficos sin anemia. La hemoglobina se correlacionó negativamente con los niveles de IL-8 (r=-0,409 p=0,009), IL-6 (r=-0,442 p=0,004) e IL-10 (r=-0,436 p=0,005). Conclusión. Los niños con desnutrición aguda grave producen una respuesta inflamatoria con aumento en los niveles de la PCR y de citocinas pro-inflamatorias que indican infección aun cuando no presenten signos, lo cual apoya la recomendación del protocolo de la OMS de medicarlos con antibioticos. Adicionalmente, estos resultados sugieren que las citocinas pro-inflamatorias podrian participar en la patogenesis de la anemia en pacientes con o sin desnutricion.

Several years ago, two cherry small circular RNAs (cscRNA1 and cscRNA2) and 10-12 double stranded RNAs (dsRNAs) of presumable viral origin were identified in sweet and sour cherry plants grown in Italy. These RNAs were found to be closely associated to a disease termed cherry chlorotic rusty spot (CCRS). The Italian CCRS disease is symptomatologically very similar to the Amasya cherry disease (ACD) described previously in Turkey, and both diseases have been closely associated with similar mycelium-like structures and with double-stranded (ds) RNAs of mycoviruses from the genera Chrysovirus, Partitivirus and Totivirus. More recently it has been realized that cherry trees from Spain affected by cherry leaf scorch (CLS), a fungal disease reported to be caused by Apiognomonia erythrostoma, family Gnomoniaceae (order Diaporthales), show a very similar symptomatology (translucent-chlorotic leaf spots evolving into rusty areas). However, although CLSand CCRS-affected trees have been associated with similar mycelia, fungal fructifications, fungal genes and mycoviral dsRNAs sharing high sequence similarity ? further supporting a close relationship between these two disorders the involvement of A. erythrostoma in CCRS has not been conclusively shown (Carrieri et al., J. Plant Pathol. 2012). Here we report that a small viroid-like RNA similar to cscRNA1 associated with CCRS is also present in CLS-affected trees, thus providing an additional link between them. Comparisons between cscRNAs from CLS and CCRS isolates have shown several common features, including sequence identity (83%), a quasi rod-like conformation with short bifurcations at both termini, and the presence of hammerhead ribozymes in both polarity strands. However, the CLS isolate lacks the recombinant molecules of smaller size (cscRNA2). Although the biological nature of cscRNAs remain to be conclusively shown, the identification of at least cscRNA1 in different cherry cultivars and geographic areas (Spain and Italy) in close association with the same mycoviral dsRNAs, strongly support the satellite nature of the cscRNAs, which could thus be the first mycovirus satellite RNAs.

Viroid RNAs are targeted by dicer-like (DCLs) enzymes, the activity of which generates viroid-derived small RNAs (vd-sRNAs) of 21-24 nucleotides (nt) in the infected tissues, thus supplying a convincing evidence of a first host anti-viroid barrier based on RNA silencing. Whether a second RNA silencing barrier, based on the RNA-induced silencing complex (RISC), may also target for degradation viroid RNAs remains controversial. The RISC core is always formed by an Argonaute (AGO) protein, which after loading endogenous or viral sRNAs, guides the complex to target for inactivation complementary RNAs (or DNAs). Using RNA immunoprecipitation with a specific antibody against the endogenous AGO1 of Nicotiana benthamiana we showed that, when infected by potato spindle tuber viroid (PSTVd), this protein is also loaded with vd-sRNAs. Moreover, agroexpression in N. benthamiana infected by PSTVd of epitope-tagged versions of nine of the ten AGOs from Arabidopsis thaliana, followed by immunoprecipitation analyses of the agroinfiltrated halos, revealed that AGO1, AGO2 and AGO3 preferentially associated with vd-sRNAs of 21 and 22 nt, while AGO4, AGO5 and AGO9 additionally bound those of 24 nt. In contrast, AGO6, AGO7 and AGO10 did not load vd-sRNAs. Deep-sequencing analysis revealed that the endogenous AGO1 of N. benthamiana and the agroexpressed AGO1, AGO2, AGO4 and AGO5 bound vd-sRNAs according to their size and 5'-terminal nucleotide, with AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Interestingly, agroexpression of AGO1, AGO2, AGO4 and AGO5 in PSTVd-infected tissue attenuated the PSTVd level, suggesting RISC-targeting of the viroid genomic RNAs or their precursors and thus supporting a role of RISC in anti-viroid defense. Moreover, PSTVd RNAs with 5'-termini compatible with degradation mediated by AGO-loaded vd-sRNA have been identified and characterized by 5'-RML-RACE in infected N. benthamiana. Further degradome analysis should provide a description of how the viroid genomic and host RNAs are targeted by AGO-loaded vd-sRNAs, thus supplying a broader view of the biological functions of vd-sRNAs.

Grapevine-infecting viroids do not induce symptoms, except for Grapevine yellow speckle viroid-1 (GYSVd-1) and Grapevine yellow speckle viroid-2 (GYSVd-2), the agents of yellow speckle (YS), a disease characterized by yellow spots or flecks scattered on the leaf blade. The association of these viroids with Grapevine fanleaf virus (GFLV) is thought to elicit vein banding (VB), a syndrome characterized by chrome yellow flecks localized along the main veins and progressing into the interveinal areas of affected vines. The occurrence of these diseases and their causal agents was investigated in north-west Iran with a survey in which 137 vines were tested by multiplex RT-PCR for the presence of the five known grapevines viroids. GYSVd-1, GYSVd-2, Australian grapevine viroid (AGVd) and Hop stunt viroid (HSVd) were detected in 91%, 64%, 95%, and 100% of the tested samples, respectively, whereas Citrus exocortis viroid (CEVd) was not found. Combinations of three and four different viroids were present in most plants (88%) whereas GFLV was found in 50 samples (37%). The Iranian isolates of GYSVd-1, GYSVd-2, HSVd and AGVd showed minor molecular changes compared with the respective reference strains from grapevine. VB occurred in 22 vines infected by GYSVd-1, GYSVd-2 and GFLV, whereas YS symptoms, which occurred in 10% of the tested plants, were always shown by vines infected by GYSVd-1 and/or GYSVd-2. These findings are in line with the notion that assigns to GYSVd-1 and GYSVd-2 a role in the induction of YS and to both viroids and GFLV the genesis of VB.

Viroids, like RNA viruses, propagate in their hosts as populations of closely-related sequence variants (quasi-species). When assayed individually, such variants often incite distinct phenotypes but their stability along time may vary because viroids, like chrysanthemum chlorotic mottle viroid (CChMVd), display a hight mutation rate [2] that rapidly generates heterogeneous populations. The behavior of these populations is essentially determined by interference among individual components, which in viroids must occur at the RNA level. During host colonization virus populations suffer bottlenecks imposed by the host, the vector, and the territorial behavior (superinfection exclusion) of the infecting viruses, which limit the number of genomes entering and replicating in individual cells [3]. Some reports suggest that bottlenecks also operate during viroid infection, but such issue has not been further addressed. Here we have examined the evolution in time and space of two natural CChMVd variants: one containing a UUUC tetraloop strictly associated with symptoms and the other with a GAAA tetraloop and asymptomatic [1]. Inoculation with the latter induced no phenotype, with only variants with the same tetraloop in the progeny analyzed by RT-PCR, cloning and sequencing. Conversely, early symptoms incited by the first variant consisted in chlorotic and green sectors. RT-PCR, cloning and sequencing of the progeny, and deep sequencing of the viroid-derived RNAs generated by RNA silencing, showed that variants with the UUUC tetraloop predominate in chlorotic sectors, while they coexist in green sectors with others having one or two mutations in the pathogenic determinant. Cloning and bioassay of these mutant variants resulted in asymptomatic infections (with the UUUC tetraloop absent in the progeny), showing that a single substitution in the tetraloop annuls pathogenicity, a finding consistent with involvement of an RNA silencing mechanism. Therefore, pathogenic and non-pathogenic CChMVd variants exhibit distinct evolutionary patterns. The very high mutation rate of CChMVd, combined with the ability of different variants to preferentially colonize some leaf sectors and exclude other variants, may account for the observed results.

Mulberry mosaic dwarf (MMD) disease was first recorded in 1957 and has long been endemic in China. A new viroid-like RNA was detected from mulberry trees affected by MMD and its full nucleotide sequence was determined (1). This mulberry small circular RNA (mscRNA), consisting of 356 nt, does not share significant sequence homology with other viroids and viroid-like satellite RNAs. Here we show that (+) strand of mcRNA contains a hammerhead ribozyme, while the (-) strand contains a hairpin ribozyme, resembling viroid-like satellite RNAs of nepoviruses. mscRNA transcripts of (+) and (-) strands self-cleaved in vitro as predicted by the hammerhead and hairpin structures, respectively, generating fragments with the expected 5' terminus. Circular forms of mscRNA, purified from plant extracts, and dimeric transcripts of both polarities, generated in vitro, were slash-inoculated in mulberry, hop, tomato and cucumber seedlings. Northern-blot hybridization did not detect mscRNA in the inoculated plants so far, suggesting that this circular RNA is not endowed of autonomous replication. These data, taking also into consideration that a nepovirus has been recently reported in mulberry in China (2), strongly suggest that mscRNA could be a novel viroid-like satellite RNA of a nepovirus. This hypothesis is supported by the identification, by next generation sequencing, of both mscRNA and a nepovirus in the same mulberry host. Moreover, a close association of mscRNA with MMD disease was excluded on the bases of a field survey carried out on mulberry trees affected or not affected by the disease. This data are in agreement with our recent results showing that MMD is closely associated with a new geminivirus (3).

Introduction: RECIPE study was designed to assess the impact of dietary supplementation with fermented milk enriched with plant sterols (FMPS) on the nutritional behavior, lifestyle and the joint therapeutic responsibility of hypercholesterolemic patients. Methods: Observational, prospective, multicenter, international study, was conducted to assess dietary habits and other lifestyle components in hypercholesterolemic patients in whom the general practitioner indicated FMPS supplementation. Nutritional, lifestyle and clinical data were collected at the initial visit and after a 4 months follow-up. The biochemical and anthropometric data were retrieved from medical records obtained at baseline and 4 months. Results: We report the results of Spain. Two hundred and one physicians who provided valid data of 1044 patients participated. The addition of FMPS was associated with improved overall nutritional index, being adequate initially in 28.2% of patients, and increasing up to 75.2% at the end of the study (P< 001). This nutritional change was associated with an improvement in the lipid profile and anthropometric data. Patients more adherent to therapy generally achieved a better result in all parameters compared to non-compliant ones. Conclusions: The addition of FMPS to a diet designed to reduce the LDL enhances the patient's attitude regarding changes in lifestyle, leading to better overall control of dyslipidemia and anthropometric improvement. © 2012 Elsevier España, S.L. and SEA.

10.1371/journal.pone.0103634 The first arrivals of hominin populations into Eurasia during the Early Pleistocene are currently considered to have occurred as short and poorly dated biological dispersions. Questions as to the tempo and mode of these early prehistoric settlements have given rise to debates concerning the taxonomic significance of the lithic assemblages, as trace fossils, and the geographical distribution of the technological traditions found in the Lower Palaeolithic record. Here, we report on the Barranc de la Boella site which has yielded a lithic assemblage dating to ~1 million years ago that includes large cutting tools (LCT). We argue that distinct technological traditions coexisted in the Iberian archaeological repertoires of the late Early Pleistocene age in a similar way to the earliest sub-Saharan African artefact assemblages. These differences between stone tool assemblages may be attributed to the different chronologies of hominin dispersal events. The archaeological record of Barranc de la Boella completes the geographical distribution of LCT assemblages across southern Eurasia during the EMPT (Early-Middle Pleistocene Transition, circa 942 to 641 kyr). Up to now, chronology of the earliest European LCT assemblages is based on the abundant Palaeolithic record found in terrace river sequences which have been dated to the end of the EMPT and later. However, the findings at Barranc de la Boella suggest that early LCT lithic assemblages appeared in the SW of Europe during earlier hominin dispersal episodes before the definitive colonization of temperate Eurasia took place.

The JAK2V617F allele burden has been identified as a risk factor for vascular events and myelofibrotic transformation in polycythemia vera (PV) and essential thrombocythemia (ET). However, all previous studies have evaluated a single time point JAK2V617F measurement. Therefore, the frequency and the clinical significance of changes in the JAK2V617F mutant load occurring during the disease evolution remain unknown. In the present study, JAK2V617F monitoring was performed during the follow-up of 347 patients (PV = 163, ET = 184). According to their JAK2V617F evolutionary patterns, patients were stratified as stable 50% (n = 261), stable =50% (n = 52), progressive increase (n = 24) and unexplained decrease (n = 10). After a 2,453 person-years follow-up, a total of 59 thrombotic events, 16 major hemorrhages, and 27 cases of myelofibrotic transformations were registered. At multivariate analyses, patients with a persistently high (=50%) or unsteady JAK2V617F load during follow-up had an increased risk of myelofibrotic transformation (Incidence rate ratio [IRR]: 20.7, 95% CI: 6.5-65.4; P 0.001) and a trend for a higher incidence of thrombosis (IRR: 1.7, 1-3.3; P = 0.05) than patients with a stable allele burden below 50%. In conclusion, JAK2V617F monitoring could be useful in patients with PV and ET for predicting disease's complications, especially myelofibrotic transformation.