Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus

El virus de la tristeza de los cítricos (Citrus tristeza virus; CTV) es el agente causal de unas de las enfermedades virales de los árboles cítricos más devastadoras en el mundo. CTV está restringido al floema en su huésped cítrico natural, y ha desarrollado tres proteínas supresoras de silenciamiento que actúan a nivel intra-(p23 y p20) e intercelular (p20 y p25) para superar la fuerte defensa antiviral del huésped. La interferencia de RNA, una aproximación basada en el uso de dsRNA para desencadenar el silenciamiento de RNA, ha sido utilizada ampliamente para generar plantas transgénicas resistentes a virus. Considerando el importante papel de p23, p20 y p25 en la patogénesis de CTV, hemos transformado plantas de lima Mexicana con un vector intrón-horquilla que porta la secuencia completa en versión no traducible de los genes p25, p20, p23 y el extremo 3¿-UTR de la cepa T36 de CTV, para intentar silenciar su expresión en células infectadas. Se ha observado resistencia completa a la infección viral en tres líneas transgénicas, manteniéndose todas sus propagaciones asintomáticas y libres de virus tras ser inoculadas mediante injerto con CTV-T36, tanto en el portainjertos no transgénico como directamente sobre la variedad transgénica. La acumulación de siRNA derivados del transgén fue necesaria pero no suficiente para lograr resistencia frente a CTV en las plantas. Al inocular propagaciones de las líneas transgénicas inmunes con una cepa de CTV divergente, la resistencia fue parcialmente superada, destacando la importancia de la identidad de secuencia en el mecanismo subyacente a la interferencia de RNA. Este trabajo es el primero en que se consigue resistencia completa a CTV en un huésped cítrico muy sensible, actuando simultáneamente sobre los tres supresores virales de silenciamiento mediante interferencia de RNA. La proteína p23 codificada por el virus es además un importante factor de patogenicidad. La expresión ectópica de p23 en plantas de cítricos induce aberraciones fenológicas semejantes a síntomas de CTV. Para estudiar en más detalle el papel de p23 en la patogénesis de CTV, se ha sobre-expresado en lima Mexicana el gen p23 de CTV T36 y tres versiones truncadas del mismo bajo el control del promotor 35S del virus del mosaico de la coliflor (Cauliflower mosaic virus). Solo la versión truncada, que expresa los aminoácidos del 1 al 157 (p23-¿157) indujo síntomas similares a los producidos por CTV, aunque más suaves que los inducidos por la expresión de la proteína p23 entera (209 aminoácidos), permitiendo delimitar la región responsable de la patogénesis de p23 en cítricos a un fragmento de 157 aminoácidos que incluye el dedo de zinc y los motivos básicos flanqueantes de la proteína. La actividad de p23 como supresor de silenciamiento de RNA en N. benthamiana se perdía en todos los mutantes de p23 probados, lo cual indica que la supresión de silenciamiento implica a la mayoría de las regiones de la proteína. Para profundizar más en el papel de p23 en la patogénesis, en un siguiente paso hemos restringido la expresión de transgenes derivados de p23 a células asociadas al floema de lima Mexicana mediante el uso del promotor especifico de floema del virus del moteado amarillo de la comelina (Commelina yellow mottle virus, CoYMV). Se transformó lima Mexicana con construcciones que portaban el gen p23 completo, ya sea de la cepa agresiva de CTV T36 o de la suave T317, o con un fragmento que comprende el dedo de zinc y los motivos básicos flanqueantes de la primera, todas ellas bajo el control bien del promotor de CoYMV o bien del promotor constitutivo 35S. La expresión de estas construcciones en el floema dio lugar a aberraciones semejantes a los síntomas específicos de CTV, pero no a los síntomas inespecíficos observados cuando se expresaba p23 de forma constitutiva. Por otra parte, la apariencia e intensidad de las aberraciones fenotípicas más notorias similares a síntomas inducidos por CTV generadas por la expresión específica en floema del gen p23 se relacionó positivamente con la agresividad de la cepa origen utilizada. Además, la expresión en tejidos floemáticos del fragmento de p23 que comprende el dominio de dedo de zinc y los motivos básicos flanqueantes fue suficiente para inducir síntomas semejantes a los producidos por la infección con CTV, confirmando así que la región N-terminal delimitada por los aminoácidos 1 y 157 podría determinar, al menos en parte, la patogénesis de CTV en lima Mexicana.Citrus tristeza virus (CTV) is the causal agent of one of the most devastating viral diseases of citrus trees in the world. CTV is phloem-restricted in natural citrus hosts, and has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and inter-cellular level (p20 and p25) to overcome strong host antiviral defense in citrus. RNA interference (RNAi), an approach based on using dsRNA to trigger RNA silencing, has been widely used for generating transgenic plants resistant against viruses. Considering the important role of p23, p20 and p25 in CTV pathogenesis, we have transformed Mexican lime plants with an intron-hairpin vector carrying full untranslatable versions of genes p25, p20, p23 and the 3¿-UTR from the CTV strain T36, to attempt silencing their expression in CTV-infected cells. Complete resistance to viral infection was observed in three transgenic lines, with all their propagations remaining symptomless and virus-free after graft-inoculation with CTV-T36, either in the non-transgenic rootstock or directly in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Challenging immune transformants with a divergent CTV strain resulted in partial breakage of the resistance, stressing the importance of sequence identity in the underlying RNAi mechanism. This is the first evidence that it is possible to achieve full resistance to CTV in a highly sensitive citrus host by targeting simultaneously its three viral silencing suppressors through RNAi. The p23 protein encoded by the virus is additionally an important pathogenicity factor. Ectopic expression of p23 in transgenic citrus plants induces developmental aberrations resembling CTV symptoms. To explore in more detail the role of p23 in CTV pathogenesis, the p23 gene from CTV T36 and three truncated versions thereof under the control of the Cauliflower mosaic virus 35S promoter were used to transform Mexican lime. Only the truncated version expressing amino acids 1 to 157 (p23¿158-209) elicited CTV-like symptoms, similar to, albeit milder than, those incited by expressing the whole p23 protein (209 amino acids), thus delimiting the region responsible for p23 pathogenesis in citrus to a 157 amino acid fragment including the Zn finger and flanking basic motifs of the protein. RNA silencing suppressor activity of p23 in N. benthamiana was abolished by all mutants tested, indicating that silencing suppression involves most p23 regions. To better define the role of p23 in CTV pathogenesis, we next restricted the expression of p23-derived transgenes to phloem-associated cells in Mexican lime plants by means of using the phloem-specific promoter from Commelina yellow mottle virus (CoYMV). Constructions carrying the complete gene p23 from either the severe T36 or the mild T317 CTV strains, or a fragment comprising the zinc-finger and flanking basic motifs from the former, either under the control of the CoYMV promoter or the constitutive 35S promoter were used for genetic transformation of Mexican lime. Expression of these constructs in the phloem incited aberrations resembling CTV-specific symptoms, but not the unspecific symptoms observed when p23 was constitutively expressed. Moreover, appearance and intensity of the most notorious CTV-like phenotypic aberrations induced by the phloem-specific expression of the p23 gene were positively related with the aggressiveness of the source CTV strain used. Additionally, expression in phloem-tissues of the p23 fragment comprising the zinc-finger domain and flanking basic motifs was sufficient to induce CTV-like symptoms, corroborating that the N-terminal region (delimited by amino acids 1 and 157) determines, at least in part, CTV pathogenesis in Mexican lime.Soler Calvo, N. (2013). Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31631TESI

Correction : Chaparro et al. Incidence, Clinical Characteristics and Management of Inflammatory Bowel Disease in Spain: Large-Scale Epidemiological Study. J. Clin. Med. 2021, 10, 2885

The authors wish to make the following corrections to this paper [...]

Effectiveness of an intervention for improving drug prescription in primary care patients with multimorbidity and polypharmacy:Study protocol of a cluster randomized clinical trial (Multi-PAP project)

This study was funded by the Fondo de Investigaciones Sanitarias ISCIII (Grant Numbers PI15/00276, PI15/00572, PI15/00996), REDISSEC (Project Numbers RD12/0001/0012, RD16/0001/0005), and the European Regional Development Fund ("A way to build Europe").Background: Multimorbidity is associated with negative effects both on people's health and on healthcare systems. A key problem linked to multimorbidity is polypharmacy, which in turn is associated with increased risk of partly preventable adverse effects, including mortality. The Ariadne principles describe a model of care based on a thorough assessment of diseases, treatments (and potential interactions), clinical status, context and preferences of patients with multimorbidity, with the aim of prioritizing and sharing realistic treatment goals that guide an individualized management. The aim of this study is to evaluate the effectiveness of a complex intervention that implements the Ariadne principles in a population of young-old patients with multimorbidity and polypharmacy. The intervention seeks to improve the appropriateness of prescribing in primary care (PC), as measured by the medication appropriateness index (MAI) score at 6 and 12months, as compared with usual care. Methods/Design: Design:pragmatic cluster randomized clinical trial. Unit of randomization: family physician (FP). Unit of analysis: patient. Scope: PC health centres in three autonomous communities: Aragon, Madrid, and Andalusia (Spain). Population: patients aged 65-74years with multimorbidity (≥3 chronic diseases) and polypharmacy (≥5 drugs prescribed in ≥3months). Sample size: n=400 (200 per study arm). Intervention: complex intervention based on the implementation of the Ariadne principles with two components: (1) FP training and (2) FP-patient interview. Outcomes: MAI score, health services use, quality of life (Euroqol 5D-5L), pharmacotherapy and adherence to treatment (Morisky-Green, Haynes-Sackett), and clinical and socio-demographic variables. Statistical analysis: primary outcome is the difference in MAI score between T0 and T1 and corresponding 95% confidence interval. Adjustment for confounding factors will be performed by multilevel analysis. All analyses will be carried out in accordance with the intention-to-treat principle. Discussion: It is essential to provide evidence concerning interventions on PC patients with polypharmacy and multimorbidity, conducted in the context of routine clinical practice, and involving young-old patients with significant potential for preventing negative health outcomes. Trial registration: Clinicaltrials.gov, NCT02866799Publisher PDFPeer reviewe

Multidifferential study of identified charged hadron distributions in ZZ-tagged jets in proton-proton collisions at s=\sqrt{s}=13 TeV

Jet fragmentation functions are measured for the first time in proton-proton collisions for charged pions, kaons, and protons within jets recoiling against a $Z$ boson. The charged-hadron distributions are studied longitudinally and transversely to the jet direction for jets with transverse momentum 20 $< p_{\textrm{T}} < 100$ GeV and in the pseudorapidity range $2.5 < \eta < 4$. The data sample was collected with the LHCb experiment at a center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 1.64 fb$^{-1}$. Triple differential distributions as a function of the hadron longitudinal momentum fraction, hadron transverse momentum, and jet transverse momentum are also measured for the first time. This helps constrain transverse-momentum-dependent fragmentation functions. Differences in the shapes and magnitudes of the measured distributions for the different hadron species provide insights into the hadronization process for jets predominantly initiated by light quarks.Comment: All figures and tables, along with machine-readable versions and any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-013.html (LHCb public pages

Study of the B−→Λc+Λˉc−K−B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-} decay

The decay $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ is studied in proton-proton collisions at a center-of-mass energy of $\sqrt{s}=13$ TeV using data corresponding to an integrated luminosity of 5 $\mathrm{fb}^{-1}$ collected by the LHCb experiment. In the $\Lambda_{c}^+ K^{-}$ system, the $\Xi_{c}(2930)^{0}$ state observed at the BaBar and Belle experiments is resolved into two narrower states, $\Xi_{c}(2923)^{0}$ and $\Xi_{c}(2939)^{0}$, whose masses and widths are measured to be $$m(\Xi_{c}(2923)^{0}) = 2924.5 \pm 0.4 \pm 1.1 \,\mathrm{MeV}, \\ m(\Xi_{c}(2939)^{0}) = 2938.5 \pm 0.9 \pm 2.3 \,\mathrm{MeV}, \\ \Gamma(\Xi_{c}(2923)^{0}) = \phantom{000}4.8 \pm 0.9 \pm 1.5 \,\mathrm{MeV},\\ \Gamma(\Xi_{c}(2939)^{0}) = \phantom{00}11.0 \pm 1.9 \pm 7.5 \,\mathrm{MeV},$$ where the first uncertainties are statistical and the second systematic. The results are consistent with a previous LHCb measurement using a prompt $\Lambda_{c}^{+} K^{-}$ sample. Evidence of a new $\Xi_{c}(2880)^{0}$ state is found with a local significance of $3.8\,\sigma$, whose mass and width are measured to be $2881.8 \pm 3.1 \pm 8.5\,\mathrm{MeV}$ and $12.4 \pm 5.3 \pm 5.8 \,\mathrm{MeV}$, respectively. In addition, evidence of a new decay mode $\Xi_{c}(2790)^{0} \to \Lambda_{c}^{+} K^{-}$ is found with a significance of $3.7\,\sigma$. The relative branching fraction of $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ with respect to the $B^{-} \to D^{+} D^{-} K^{-}$ decay is measured to be $2.36 \pm 0.11 \pm 0.22 \pm 0.25$, where the first uncertainty is statistical, the second systematic and the third originates from the branching fractions of charm hadron decays.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-028.html (LHCb public pages

Measurement of the ratios of branching fractions R(D∗)\mathcal{R}(D^{*}) and R(D0)\mathcal{R}(D^{0})

The ratios of branching fractions $\mathcal{R}(D^{*})\equiv\mathcal{B}(\bar{B}\to D^{*}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(\bar{B}\to D^{*}\mu^{-}\bar{\nu}_{\mu})$ and $\mathcal{R}(D^{0})\equiv\mathcal{B}(B^{-}\to D^{0}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(B^{-}\to D^{0}\mu^{-}\bar{\nu}_{\mu})$ are measured, assuming isospin symmetry, using a sample of proton-proton collision data corresponding to 3.0 fb${ }^{-1}$ of integrated luminosity recorded by the LHCb experiment during 2011 and 2012. The tau lepton is identified in the decay mode $\tau^{-}\to\mu^{-}\nu_{\tau}\bar{\nu}_{\mu}$. The measured values are $\mathcal{R}(D^{*})=0.281\pm0.018\pm0.024$ and $\mathcal{R}(D^{0})=0.441\pm0.060\pm0.066$, where the first uncertainty is statistical and the second is systematic. The correlation between these measurements is $\rho=-0.43$. Results are consistent with the current average of these quantities and are at a combined 1.9 standard deviations from the predictions based on lepton flavor universality in the Standard Model.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-039.html (LHCb public pages

From social integration to diversity management: the role of Latvia's municipalities

Bakalaura darba ”No sabiedrības integrācijas uz dažādības vadības politiku: pašvaldību loma Latvijā” mērķis ir izvērtēt Latvijas novadu pašvaldību sabiedrības integrācijas politiku un tajā iespējamās dažādības vadības politikas iestrādnes. Autore izvirza hipotēzes: 1)Latvijas novadu pašvaldībās tiek izstrādātas un īstenotas vietējās integrācijas programmas; 2) Latvijas novadu pašvaldības domes darbā izmanto dažādības vadības politikas iestrādnes. Darba rezultātā tika izpētīta sabiedrības integrācijas un dažādības vadības politika Latvijā gan nacionālā, gan reģionālā līmenī. Autore izdarīja secinājumu, ka Latvijas sabiedrības integrācijas politiku varētu uzlabot ar dažādības vadības politikas iestrādnēm. Atslēgvārdi: integrācija, dažādība, politika, pašvaldības, LatvijaThe aim of the Bachelor's thesis " From Social Integration to Diversity Management: the role of Latvia's Municipalities" is to assess Social Integration policy and Diversity Management potential in the municipalities of Latvia The hypotheses of Bachelor’s thesis are: 1) There are designed and implemented local integration programs in the municipalities of Latvia; 2) Latvia’s municipal councils use Diversity Management policies. In the result of Bachelor’s thesis was analyzed both Social Integration and Diversity Management Policy in Latvia in both national and regional levels. The author concluded that Social Integration policy in Latvia could be improved with including Diversity Management policies on it. Keywords: integration, diversity, politics, municipalities, Latvi

Low-density lipoprotein receptor-related protein 1 deficiency in cardiomyocytes reduces susceptibility to insulin resistance and obesity

Altres ajuts: Fundació La Marató TV3, projectes 201521-10 87/C/2016Background: Low-density lipoprotein receptor-related protein 1 (LRP1) plays a key role in fatty acid metabolism and glucose homeostasis. In the context of dyslipemia, LRP1 is upregulated in the heart. Our aim was to evaluate the impact of cardiomyocyte LRP1 deficiency on high fat diet (HFD)-induced cardiac and metabolic alterations, and to explore the potential mechanisms involved. Methods: We used TnT-iCre transgenic mice with thoroughly tested suitability to delete genes exclusively in cardiomyocytes to generate an experimental mouse model with conditional Lrp1 deficiency in cardiomyocytes (TNT-iCre+-LRP1flox/flox). Findings: Mice with Lrp1-deficient cardiomyocytes (cm-Lrp1−/−) have a normal cardiac function combined with a favorable metabolic phenotype against HFD-induced glucose intolerance and obesity. Glucose intolerance protection was linked to higher hepatic fatty acid oxidation (FAO), lower liver steatosis and increased whole-body energy expenditure. Proteomic studies of the heart revealed decreased levels of cardiac pro-atrial natriuretic peptide (pro-ANP), which was parallel to higher ANP circulating levels. cm-Lrp1−/− mice showed ANP signalingactivation that was linked to increased fatty acid (FA) uptake and increased AMPK/ ACC phosphorylation in the liver. Natriuretic peptide receptor A (NPR-A) antagonist completely abolished ANP signaling and metabolic protection in cm-Lrp1−/− mice. Conclusions: These results indicate that an ANP-dependent axis controlled by cardiac LRP1 levels modulates AMPK activity in the liver, energy homeostasis and whole-body metabolism

Efficacy of Bromhexine versus Standard of Care in Reducing Viral Load in Patients with Mild-to-Moderate COVID-19 Disease Attended in Primary Care: A Randomized Open-Label Trial

A 28-day randomized open-label multicenter study was conducted to assess the efficacy of bromhexine plus standard of care (SOC) (n = 98) vs. SOC alone (n = 93) in 191 outpatients with mild-to-moderate COVID-19 in the primary health care setting. Bromhexine three daily doses of 10 mL (48 mg/day) were administered for seven days. The primary efficacy endpoint was the reduction of viral load estimated as the cycle thresholds (Ct) to detect ORF1ab, N Protein, and S Protein genes by RT-qPCR in saliva samples on day 4 as compared with baseline. Ct values of the three genes increased from baseline throughout days 4 to 14 (p < 0.001) but significant differences between the study groups were not found. Differences in the percentages of patients with low, medium, and high viral loads at 4, 7, and 14 days were not found either. In summary, treatment with bromhexine plus SCO was associated with a viral load reduction of ORF1ab, N Protein, and S Protein genes at day 4, which was not significantly different than similar viral load reductions observed with SOC alone. The present findings do not seem to favor the use of bromhexine as an antiviral in patients with COVID-19

Associated factors to serious infections in a large cohort of juvenile-onset systemic lupus erythematosus from Lupus Registry (RELESSER).

To assess the incidence of serious infection (SI) and associated factors in a large juvenile-onset systemic lupus erythematosus (jSLE) retrospective cohort. All patients in the Spanish Rheumatology Society Lupus Registry (RELESSER) who meet ≥4 ACR-97 SLE criteria and disease onset A total of 353 jSLE patients were included: 88.7% female, 14.3 years (± 2.9) of age at diagnosis, 16.0 years (± 9.3) of disease duration and 31.5 years (±10.5) at end of follow-up. A total of 104 (29.5%) patients suffered 205 SI (1, 55.8%; 2-5, 38.4%; and ≥6, 5.8%). Incidence rate was 3.7 (95%CI: 3.2-4.2) SI per 100 patient years. Respiratory location and bacterial infections were the most frequent. Higher number of SLE classification criteria, SLICC/ACR DI score and immunosuppressants use were associated to the presence of SI. Associated factors to shorter time to first infection were higher number of SLE criteria, splenectomy and immunosuppressants use. The risk of SI in jSLE patients is significant and higher than aSLE. It is associated to higher number of SLE criteria, damage accrual, some immunosuppressants and splenectomy