Plant YTHDF proteins are direct effectors of antiviral immunity against an N6-methyladenosine-containing RNA virus.

In virus-host interactions, nucleic acid-directed first lines of defense that allow viral clearance without compromising growth are of paramount importance. Plants use the RNA interference pathway as a basal antiviral immune system, but additional RNA-based mechanisms of defense also exist. The infectivity of a plant positive-strand RNA virus, alfalfa mosaic virus (AMV), relies on the demethylation of viral RNA by the recruitment of the cellular N6-methyladenosine (m6 A) demethylase ALKBH9B, but how demethylation of viral RNA promotes AMV infection remains unknown. Here, we show that inactivation of the Arabidopsis cytoplasmic YT521-B homology domain (YTH)-containing m6 A-binding proteins ECT2, ECT3, and ECT5 is sufficient to restore AMV infectivity in partially resistant alkbh9b mutants. We further show that the antiviral function of ECT2 is distinct from its previously demonstrated function in the promotion of primordial cell proliferation: an ect2 mutant carrying a small deletion in its intrinsically disordered region is partially compromised for antiviral defense but not for developmental functions. These results indicate that the m6 A-YTHDF axis constitutes a novel branch of basal antiviral immunity in plants.

Impact of the Potential m6A Modification Sites at the 3'UTR of Alfalfa Mosaic Virus RNA3 in the Viral Infection.

We have previously reported the presence of m6A in the AMV (Alfamovirus, Bromoviridae) genome. Interestingly, two of these putative m6A-sites are in hairpin (hp) structures in the 3'UTR of the viral RNA3. One site (2012AAACU2016) is in the loop of hpB, within the coat protein binding site 1 (CPB1), while the other (1900UGACC1904) is in the lower stem of hpE, a loop previously associated with AMV negative-strand RNA synthesis. In this work, we have performed in vivo experiments to assess the role of these two regions, containing the putative m6A-sites in the AMV cycle, by introducing compensatory point mutations to interfere with or abolish the m6A-tag of these sites. Our results suggest that the loop of hpB could be involved in viral replication/accumulation. Meanwhile, in the 1900UGACC1904 motif of the hpE, the maintenance of the adenosine residue and the lower stem hpE structure are necessary for in vivo plus-strand accumulation. These results extend our understanding of the requirements for hpE in the AMV infection cycle, indicating that both the residue identity and the base-pairing capacity in this structure are essential for viral accumulation.

Recent Acquisition of Functional m6A RNA Demethylase Domain in Orchid Ty3/Gypsy Elements.

Long terminal repeats (LTR) retrotransposons are transposable elements (TEs) representing major components of most plant genomes. The fixation of additional conserved protein domains in their genomes is considered a rare event in the course of their evolution. Such changes can bring novel functions and increase their fitness by playing a role in the regulation of their replicative cycle or by affecting their integration landscape so that the detection of new domains can in turn reveal important aspects of host-TE interactions. We have mined angiosperm genomes for the presence of additional domains in LTR retrotransposons. We report a lineage of large (25 kbp) Gypsy-type elements in the genomes of Phalaenopsis orchids that contain an additional open reading frame containing a 2-ODD domain with close similarity to those responsible for m6A RNA demethylase activity in AlkB proteins. By performing in vitro assays, we demonstrate the RNA binding capability and the demethylase activity of the Gypsy-encoded AlkB protein, suggesting it could be functional against cognate TE mRNA or any cellular RNA in planta. In line with recent literature, we propose that the fixation of an RNA demethylase in this lineage of LTR retrotransposons may reflect an important role for epitranscriptomic control in host surveillance against TEs.

The m6A RNA Demethylase ALKBH9B Plays a Critical Role for Vascular Movement of Alfalfa Mosaic Virus in Arabidopsis.

The N 6-methyladenosine (m6A) pathway has been widely described as a viral regulatory mechanism in animals. We previously reported that the capsid protein (CP) of alfalfa mosaic virus (AMV) interacts with the Arabidopsis m6A demethylase ALKBH9B regulating m6A abundance on viral RNAs (vRNAs) and systemic invasion of floral stems. Here, we analyze the involvement of other ALKBH9 proteins in AMV infection and we carry out a detailed evaluation of the infection restraint observed in alkbh9b mutant plants. Thus, via viral titer quantification experiments and in situ hybridization assays, we define the viral cycle steps that are altered by the absence of the m6A demethylase ALKBH9B in Arabidopsis. We found that ALKBH9A and ALKBH9C do not regulate the AMV cycle, so ALKBH9B activity seems to be highly specific. We also define that not only systemic movement is affected by the absence of the demethylase, but also early stages of viral infection. Moreover, our findings suggest that viral upload into the phloem could be blocked in alkbh9b plants. Overall, our results point to ALKBH9B as a possible new component of phloem transport, at least for AMV, and as a potential target to obtain virus resistance crops.

Mapping of Functional Subdomains in the atALKBH9B m6A-Demethylase Required for Its Binding to the Viral RNA and to the Coat Protein of Alfalfa Mosaic Virus.

N 6-methyladenosine (m6A) modification is a dynamically regulated RNA modification that impacts many cellular processes and pathways. This epitranscriptomic methylation relies on the participation of RNA methyltransferases (referred to as "writers") and demethylases (referred to as "erasers"), respectively. We previously demonstrated that the Arabidopsis thaliana protein atALKBH9B showed m6A-demethylase activity and interacted with the coat protein (CP) of alfalfa mosaic virus (AMV), causing a profound impact on the viral infection cycle. To dissect the functional activity of atALKBH9B in AMV infection, we performed a protein-mapping analysis to identify the putative domains required for regulating this process. In this context, the mutational analysis of the protein revealed that the residues between 427 and 467 positions are critical for in vitro binding to the AMV RNA. The atALKBH9B amino acid sequence showed intrinsically disordered regions (IDRs) located at the N-terminal part delimiting the internal AlkB-like domain and at the C-terminal part. We identified an RNA binding domain containing an RGxxxRGG motif that overlaps with the C-terminal IDR. Moreover, bimolecular fluorescent experiments allowed us to determine that residues located between 387 and 427 are critical for the interaction with the AMV CP, which should be critical for modulating the viral infection process. Finally, we observed that atALKBH9B deletions of either N-terminal 20 residues or the C-terminal's last 40 amino acids impede their accumulation in siRNA bodies. The involvement of the regions responsible for RNA and viral CP binding and those required for its localization in stress granules in the viral cycle is discussed.

Principales causas de infertilidad en parejas atendidas en consulta municipal, Policlínico Guillermo Tejas Silva de Las Tunas / Main causes of infertility in couples treated in municipal consultation, Guillermo Tejas Silva de Las Tunas Polyclinical Hospital

Teniendo en cuenta que la infertilidad en parejas constituye un problema de Salud Pública en Cuba y ante la necesidad de determinar sus principales causas en parejas infértiles atendidas en consulta municipal de infertilidad Policlínico Guillermo Tejas Silva de la ciudad de Las Tunas. Se realizó un estudio descriptivo de corte transversal desde septiembre de 2017 a mayo del 2018, con un universo de 88 parejas, los datos fueron obtenidos de las historias clínicas. Las principa les causas de infertilidad femenina encontradas fueron los trastornos ovulatorios seguido por el factor tubárico, mientras que en la masculina las testiculares. Predominaron las edades de 29-35 años en mujeres y >35 años en hombres. Según la historia obstétrica anterior prevalecieron las gestaciones previas y abortos espontáneos. La mayoría de las parejas fueron a consulta por una infertilidad secundaria, sin embargo, prevaleció la causa mixta (50%), con una duración invo- luntaria de 4 a 6 años (40,91%). Los hallazgos encontrados en este estudio coinciden de forma general con la literatura científica; pero resultan significativos para contribuir a la solución de los problemas relacionados con la infertilidad en parejas de este municipio tunero

Infertility in couples constitutes a Public Health problem in Cuba. It is evident the need to deter- mine its main causes in infertile couples treated at the municipal infertility consultation at Guillermo Tejas Silva Polyclinic Hospital in the city of Las Tunas. A descriptive cross-sectional study was conducted from September 2017 to May 2018, with a universe of 88 couples, the data were obtained from medical records. The main causes of female infertility found were ovulatory disorders followed by the tubal factor, while in the male, there were testicular ones. The ages of 29-35 years in women and> 35 years in men predominated. According to the previous obstetric history, previous pregnancies and spontaneous abortions prevailed. Most of the couples were consulted for secondary infertility, however, the mixed cause prevailed (50%), with an involun- tary duration of 4 to 6 years (40.91%). The findings coincided with the scientific literature; but they are significant to contribute to the solution of problems related to infertility in couples in this municipality of Las Tunas.

A sensitive and rapid RNA silencing suppressor activity assay based on alfalfa mosaic virus expression vector.

Plant viruses express RNA silencing suppressor (RSS) proteins to counteract plant defence mechanisms. Here, we describe a method to assess the RSS activity based on an alfalfa mosaic virus (AMV) RNA 3 expression vector and transgenic Nicotiana tabacum plants that express the P1 and P2 subunits of the AMV replicase (P12 plants). Inoculation of P12 plants with different AMV RNA 3 constructs expressing different HC-Pro mutants that differ in their RSS capabilities, revealed a perfect correlation between necrotic lesions on inoculated leaves and RSS activity. Protoplast analysis showed that the RSS activity correlated with the accumulation of the AMV RNAs. A direct comparison between three RSS activity assays and the AMV-P12 system revealed that the coat protein of carnation mottle virus displays RSS activity with the four assays and reduced the accumulation of the siRNAs.

Arabidopsis m6A demethylase activity modulates viral infection of a plant virus and the m6A abundance in its genomic RNAs.

N6-methyladenosine (m6A) is an internal, reversible nucleotide modification that constitutes an important regulatory mechanism in RNA biology. Unlike mammals and yeast, no component of the m6A cellular machinery has been described in plants at present. m6A has been identified in the genomic RNAs of diverse mammalian viruses and, additionally, viral infection was found to be modulated by the abundance of m6A in viral RNAs. Here we show that the Arabidopsis thaliana protein atALKBH9B (At2g17970) is a demethylase that removes m6A from single-stranded RNA molecules in vitro. atALKBH9B accumulates in cytoplasmic granules, which colocalize with siRNA bodies and associate with P bodies, suggesting that atALKBH9B m6A demethylase activity could be linked to mRNA silencing and/or mRNA decay processes. Moreover, we identified the presence of m6A in the genomes of two members of the Bromoviridae family, alfalfa mosaic virus (AMV) and cucumber mosaic virus (CMV). The demethylation activity of atALKBH9B affected the infectivity of AMV but not of CMV, correlating with the ability of atALKBH9B to interact (or not) with their coat proteins. Suppression of atALKBH9B increased the relative abundance of m6A in the AMV genome, impairing the systemic invasion of the plant, while not having any effect on CMV infection. Our findings suggest that, as recently found in animal viruses, m6A modification may represent a plant regulatory strategy to control cytoplasmic-replicating RNA viruses.

Effect of headless compression screw on construct stability for centre of rotation and angulation-based levelling osteotomy. Biomechanical testing in flexion and torsion.

OBJECTIVE: To compare the biomechanical properties of bone and implant constructs when used for the centre of rotation and angulation (CORA) based levelling osteotomy, with and without implantation of a trans-osteotomy headless compression screw tested under three-point flexural and torsional forces; thereby determining the contribution of a trans-osteotomy headless compression screw with regards to stability of the construct. METHODS: Experimental biomechanical study utilizing 12 pairs of cadaveric canine tibias. Using the CORA based levelling osteotomy (CBLO) procedure, the osteotomy was stabilized with either a standard non-locking CBLO bone plate augmented with a headless compression screw (HCS) or a CBLO bone plate alone. Tibial constructs were mechanically tested in three-point craniocaudal flexural testing or in torsion. RESULTS: In three-point flexural testing, the difference between the two constructs was not significant. In torsion, the difference in the angle of failure between constructs with a HCS (48.46°) and constructs without a HCS (81.65°) was significant (p = 0.036). Maximum torque achieved by constructs with a HCS (21.7 Nm) was greater than those without (18.7 Nm) (p = 0.056). Stiffness differences between both groups in torsion and bending were not significant. Use of a HCS did increase the stability of the CBLO construct in torsional testing, but not in flexural testing.

Single-lumen and multi-lumen poly(ethylene glycol) nerve conduits fabricated by stereolithography for peripheral nerve regeneration in vivo.

BACKGROUND: The use of nerve conduits to facilitate nerve regrowth after peripheral nerve injury is limited to defects less than 3 cm. The purpose of this study is to determine the capability of novel single and multi-lumen poly(ethylene glycol) (PEG) conduits manufactured by stereolithography to promote peripheral nerve regeneration. MATERIALS AND METHODS: Eight Sprague Dawley rats with sharp transection injuries of the sciatic nerve were randomly assigned to receive single-lumen or multi-lumen PEG conduits to bridge a 10-mm gap. Sciatic nerve and conduit samples were harvested after 5 weeks, and axon number, myelin thickness, fiber diameter, and g-ratio were analyzed. The contralateral intact nerve was also harvested for comparison. RESULTS: Partial nerve regeneration was observed in three out of four single-lumen conduits and one out of four multi-lumen conduits. Axon number in the single-lumen regenerated nerve approached that of the contralateral intact nerve at 4,492 ± 2,810.0 and 6,080 ± 627.9 fibers/mm(2), respectively. The percentage of small fibers was greater in the single-lumen conduit compared with the intact nerve, whereas myelin thickness and g-ratio were consistently greater in the autologous nerve. Axon regrowth through the multi-lumen conduits was severely limited. CONCLUSION: Single-lumen stereolithography-manufactured PEG nerve conduits promote nerve regeneration, with regenerating axon numbers approaching that of normal nerve. Multi-lumen conduits demonstrated significantly less nerve regeneration, possibly due to physical properties of the conduit inhibiting growth. Further studies are necessary to compare the efficacy of the two conduits for functional recovery and to elucidate the reasons underlying their differences in nerve regeneration potential.

Imagen social de la enfermería / Social image of nursing

Se realizó un estudio descriptivo de corte transversal en la población del municipio Caibarién durante el año 2000. El universo de estudio quedó constituido por 32 600 habitantes mayores de 15 años. Se realizó un muestreo aleatorio estratificado y se consideró como estratos el sexo y lugar de residencia (por Consejos Populares), la muestra se constituyó con 1 630 personas encuestadas con el objetivo de identificar la imagen social de la enfermería en el municipio. Se estudiaron las variables edad, sexo, opinión acerca del sexo que deben tener los profesionales de enfermería, consideración sobre los cuidados recibidos, actividades que debe realizar este personal, así como imagen que les merece, y status social del personal de enfermería. El 48,9 (por ciento) de la población encuestada pertenece al sexo masculino y el 51,1 al femenino; el grupo etáreo más representado fue el de 20 a 29 años, el 93,5 (por ciento) opinó que el personal de enfermería puede ser de uno y otro sexos; el 91,8 (por ciento) consideró haber sido atendido adecuadamente y un gran porcentaje opina que la profesión de enfermería le merece respeto y admiración. Se concluye que la sociedad concede a la enfermería un elevado status ya que siempre recibe importantes aportes de estos profesionales(AU)

He/she was carried out a descriptive study of traverse court in the population of the municipality Caibarién during the year 2000. The study universe was constituted by 32 600 inhabitants bigger than 15 years. He/she was carried out a stratified aleatory sampling and it was considered as strata the sex and residence place (for Popular Council), the sample was constituted with 1 630 people interviewed with the objective of identifying the social image of the infirmary in the municipality. They were studied the variable age, sex, opinion about the sex that you/they should have the infirmary professionals, consideration on the received cares, activities that this personnel should carry out, as well as image that deserves them, and the infirmary personnel's social status. The 48,9 (percent) of the interviewed population it belongs at the masculine sex and the 51,1 to the feminine one; the group more represented etáreo went the one from 20 to 29 years, the 93,5 (percent) he/she said that the infirmary personnel can be of one and another sexes; the 91,8 (percent) it considered to have been assisted appropriately and a great percentage says that the infirmary profession deserves him respect and admiration. You concludes that the society grants to the infirmary since a high status he/she always receives these professionals' important contributions(AU)

Imagen social de la enfermería / Social image of nursing

Se realizó un estudio descriptivo de corte transversal en la población del municipio Caibarién durante el año 2000. El universo de estudio quedó constituido por 32 600 habitantes mayores de 15 años. Se realizó un muestreo aleatorio estratificado y se consideró como estratos el sexo y lugar de residencia (por Consejos Populares), la muestra se constituyó con 1 630 personas encuestadas con el objetivo de identificar la imagen social de la enfermería en el municipio. Se estudiaron las variables edad, sexo, opinión acerca del sexo que deben tener los profesionales de enfermería, consideración sobre los cuidados recibidos, actividades que debe realizar este personal, así como imagen que les merece, y status social del personal de enfermería. El 48,9 (por ciento) de la población encuestada pertenece al sexo masculino y el 51,1 al femenino; el grupo etáreo más representado fue el de 20 a 29 años, el 93,5 (por ciento) opinó que el personal de enfermería puede ser de uno y otro sexos; el 91,8 (por ciento) consideró haber sido atendido adecuadamente y un gran porcentaje opina que la profesión de enfermería le merece respeto y admiración. Se concluye que la sociedad concede a la enfermería un elevado status ya que siempre recibe importantes aportes de estos profesionales(AU)

Dolor abdominal recurrente / Recurrent abdominal pain

Se ha sugerido que entre el 10 y 18 por ciento de los niños en edad escolar (5-15 años) han padecido el síndrome conocido como dolor Dolor Abdominal Recurrente (DAR), siendo causa frecuente de consulta al pediatra y al gastroenterólogo infantil. Este se define como la presencia de 3 o más episodios dolorosos que limitan las actividades y funciones del niño y ocurre durante un lapso mayor de tres meses antes de la consulta. Es más frecuente en las niñas y tiene alta incidencia durante la adolescencia, entre los 11 y los 15 años. Existen más de 100 trastornos somáticos que pueden producir DAR o relacionarse con éste, como son las disfunciones gastroenterológicas, padecimientos ginecológicos, urológicos, neurológicos, metabólicos, inflamatorios e infecciosos que varían desde enfermedades orgánicas inequívocas hasta disfunciones leves

Cólico y llanto / Colic and excessive crying

El cólico del lactante es una molestia frecuente, por la cual el infante es llevado por primera vez al médico. Es un síndrome en el que lactantes sanos con crecimiento y desarrollo normales, habitualmente menores de tres meses de edad, tienen episodios de irritabilidad, llanto excesivo e inconsolable sin razón aparente. Es un problema que afecta a todas las clases sociales, con una prevalencia entre un 10 a un 2 por ciento de los lactantes. El inicio puede ser en cualquier momento después del nacimiento; suele comenzar a las 2-3 semanas de vida, alcanza su pico máximo entre las 6-8 semanas y la mayoria mejora a los meses de edad

Interleuquina 1 en niños desnutridos / Interleukin 1 in undernutrition children

La interleuquina-1 (IL-1) es un medidor soluble con propíedades inmunoreguladoras, inflamatorias, anorexígenas y antianabólicas, secretado principalmente por macrófagos activados. Evaluamos los niveles séricos de IL-1alfa en 54 lactantes y preescolares con desnutrición leve, moderada o grave con o sin infección clínica asociada y en 40 cintroles eutróficos de edad, sexo, raza y condición socioeconómica comparables, mediante ensayo inmunoenzimático con un nivel inferior de detección de 37.5 pg/ml. El porcentaje de sueros con niveles detectables de la citoquina fue similar en el grupo total de niños desnutridos y en los eutróficos. Sin embargo, cuando se analizaron los valores promedio de IL-1 alfa en los sueros con niveles detectables de la citoquina se observó que: 1) El nivel de IL-1 alfa fué significativamente mayor en los desnutridos totales que en los eutróficos. 2) Cuando el grupo de niños desnutridos se subdividió de acuerdo a la severidad del déficit nutricional, cada subgrupo (desnutridos leves, moderados o graves) mostró una media de IL-1 alfa mayor a la del grupo control. No hubo diferencias en los nivles promedios de IL-1 alfa en sueros de desnutridos con infección clínica asociada o sin infección aparente. Se concluye que en la desnutrición primaria persiste la capacidad para sintetizar IL-1 alfa y 2) que los valores aumentados de IL-1 alfa observados en desnutridos serías causados por la alta prevalencia de infecciones clínicas asociadas. A su vez, los niveles elevados de IL-1 alfa en los desnutridos sin infección clínica asociada serían determinados por infecciones latentes, y/o traslocación de bacterias y endotoxina través de la mucosa intestinal atrófica

Frecuencia de Campylobacter jejuni en un grupo de lactantes venezolanos con diarrea aguda / Frequency of Campylobacter jejuni and other pathogenic agents in a group of Venezuelan infants with acute diarrhea

Con el propósito de contribuir a mejorar el conocimiento sobre la epidemiología de la diarrea infantil por Campylobacter jejuni en Venezuela, se estudió la frecuencia de este y otros agentes patógenos en un grupo de 196 niños menores de 2 años con síndrome diarreico atendidos en dos hospitales de Caracas (76 por ciento ambulatorios y 24 por ciento hospitalizados) y en un grupo testigo. De todos los niños se obtuvieron muestras fecales para efectuar el aislamiento y la caracterización de los agentes patógenos. Se estableció además una correlación entre el agente causal aislado y las manifestaciones clínicas... (AU)

Frecuencia de Campylobacter jejuni en un grupo de lactantes venezolanos con diarrea aguda / Frequency of Campylobacter jejuni and other pathogenic agents in a group of Venezuelan infants with acute diarrhea

Con el proposito de contribuir a mejorar el conocimiento sobre la epidemiología de la diarrea infantil por Campylobacter jejuni en Venezuela, se estudio la frecuencia de este y otros agentes patogenos en un grupo de 196 ninos menores de 2 anos con sindrome diarreico atendidos en dos hospitales de Caracas (76 por ciento ambulatorios y 24 por ciento hospitalizados) y en un grupo testigo. De todos los ninos se obtuvieron muestras fecales para efectuar el aislamiento y la caracterizacion de los agentes patogenos. Se establecio ademas una correlacion entre el agente causal aislado y las manifestaciones clinicas...