Alternative translation initiation by ribosomal leaky scanning produces multiple isoforms of the Pif1 helicase.

In budding yeast, the integrity of both the nuclear and mitochondrial genomes relies on dual-targeted isoforms of the conserved Pif1 helicase, generated by alternative translation initiation (ATI) of PIF1 mRNA from two consecutive AUG codons flanking a mitochondrial targeting signal. Here, we demonstrate that ribosomal leaky scanning is the specific ATI mechanism that produces not only these, but also novel, previously uncharacterized Pif1 isoforms. Both in-frame, downstream AUGs as well as near-cognate start codons contribute to the generation of these alternative isoforms. This has crucial implications for the rational design of genuine separation-of-function alleles and provides an explanation for the suboptimal behaviour of the widely employed mitochondrial- (pif1-m1) and nuclear-deficient (pif1-m2) alleles, with mutations in the first or second AUG codon, respectively. We have taken advantage of this refined model to develop improved versions of these alleles, which will serve as valuable tools to elucidate novel functions of this helicase and to disambiguate previously described genetic interactions of PIF1 in the context of nuclear and mitochondrial genome stability.

Concurrent D-loop cleavage by Mus81 and Yen1 yields half-crossover precursors.

Homologous recombination involves the formation of branched DNA molecules that may interfere with chromosome segregation. To resolve these persistent joint molecules, cells rely on the activation of structure-selective endonucleases (SSEs) during the late stages of the cell cycle. However, the premature activation of SSEs compromises genome integrity, due to untimely processing of replication and/or recombination intermediates. Here, we used a biochemical approach to show that the budding yeast SSEs Mus81 and Yen1 possess the ability to cleave the central recombination intermediate known as the displacement loop or D-loop. Moreover, we demonstrate that, consistently with previous genetic data, the simultaneous action of Mus81 and Yen1, followed by ligation, is sufficient to recreate the formation of a half-crossover precursor in vitro. Our results provide not only mechanistic explanation for the formation of a half-crossover, but also highlight the critical importance for precise regulation of these SSEs to prevent chromosomal rearrangements.

SUMOylation modulates eIF5A activities in both yeast and pancreatic ductal adenocarcinoma cells.

BACKGROUND: The eukaryotic translation initiation protein eIF5A is a highly conserved and essential factor that plays a critical role in different physiological and pathological processes including stress response and cancer. Different proteomic studies suggest that eIF5A may be a small ubiquitin-like modifier (SUMO) substrate, but whether eIF5A is indeed SUMOylated and how relevant is this modification for eIF5A activities are still unknown. METHODS: SUMOylation was evaluated using in vitro SUMOylation assays, Histidine-tagged proteins purification from His6-SUMO2 transfected cells, and isolation of endogenously SUMOylated proteins using SUMO-binding entities (SUBES). Mutants were engineered by site-directed mutagenesis. Protein stability was measured by a cycloheximide chase assay. Protein localization was determined using immunofluorescence and cellular fractionation assays. The ability of eIF5A1 constructs to complement the growth of Saccharomyces cerevisiae strains harboring thermosensitive mutants of a yeast EIF5A homolog gene (HYP2) was analyzed. The polysome profile and the formation of stress granules in cells expressing Pab1-GFP (a stress granule marker) by immunofluorescence were determined in yeast cells subjected to heat shock. Cell growth and migration of pancreatic ductal adenocarcinoma PANC-1 cells overexpressing different eIF5A1 constructs were evaluated using crystal violet staining and transwell inserts, respectively. Statistical analysis was performed with GraphPad Software, using unpaired Student's t-test, or one-way or two-way analysis of variance (ANOVA). RESULTS: We found that eIF5A is modified by SUMO2 in vitro, in transfected cells and under endogenous conditions, revealing its physiological relevance. We identified several SUMO sites in eIF5A and found that SUMOylation modulates both the stability and the localization of eIF5A in mammalian cells. Interestingly, the SUMOylation of eIF5A responds to specific stresses, indicating that it is a regulated process. SUMOylation of eIF5A is conserved in yeast, the eIF5A SUMOylation mutants are unable to completely suppress the defects of HYP2 mutants, and SUMOylation of eIF5A is important for both stress granules formation and disassembly of polysomes induced by heat-shock. Moreover, mutation of the SUMOylation sites in eIF5A abolishes its promigratory and proproliferative activities in PANC-1 cells. CONCLUSIONS: SUMO2 conjugation to eIF5A is a stress-induced response implicated in the adaptation of yeast cells to heat-shock stress and required to promote the growth and migration of pancreatic ductal adenocarcinoma cells.

An Institutional Approach to Fostering Inclusion and Addressing Racial Bias: Implications for Diversity in Academic Medicine.

Issue: While an increasingly diverse workforce of clinicians, researchers, and educators will be needed to address the nation's future healthcare challenges, underrepresented in medicine (UIM) perspectives remain relatively absent from academic medicine. Evidence: Prior studies have identified differential experiences within the learning environment, lack of social supports, and implicit bias in evaluations as barriers to the academic interests and successes of UIM learners. The UCSF Differences Matter initiative has shown that interventions focused on recruiting diverse academic faculty, building strong social communities, facilitating cross-cultural communication and understanding, and mitigating disparities in summative assessments can positively affect the educational experience for UIM learners and contribute to their academic success. Implications: Institution-level initiatives are needed to foster a culture of inclusion, teach cultural humility, and build a culture of trust within academic medicine. Such initiatives should aim to teach a common language to discuss diversity issues and place the responsibility of fostering inclusion on all members of the academic community. Our own institutional experience with systemic cultural reform challenges others to develop novel approaches toward fostering inclusion in academic medicine.

Porous Silicon Gas Sensors: The Role of the Layer Thickness and the Silicon Conductivity.

We studied the influences of the thickness of the porous silicon layer and the conductivity type on the porous silicon sensors response when exposed to ethanol vapor. The response was determined at room temperature (27 ∘C) in darkness using a horizontal aluminum electrode pattern. The results indicated that the intensity of the response can be directly or inversely proportional to the thickness of the porous layer depending on the conductivity type of the semiconductor material. The response of the porous sensors was similar to the metal oxide sensors. The results can be used to appropriately select the conductivity of semiconductor materials and the thickness of the porous layer for the target gas.

Effect of prepubertal nutrition on cellular apoptosis and proliferation in at term placenta of Anglo-Nubian goats.

The aim of this study was to evaluate the effect of feed restriction with or without monensin supplementation, followed by a re-feeding period on cellular apoptosis and proliferation in at term placenta of Anglo-Nubian goats. To evaluate the induction of apoptosis through the intrinsic pathway, proteins Bax and Bcl-2 were determinated. The apoptosis was related with the cell proliferation indices through Ki67 determination. The treatments were applied for 250 days and were (a) ad libitum feeding (control; n = 5); (b) restricted feeding at 70% of control (restricted; n = 7); and (c) restricted with monensin supplementation (monensin; n = 7). After treatments, all the animals were fed to support their requirements. After parturition, 27 placentas were gathered. The placental cellular structure was studied by high-resolution light microscopy and transmission electron microscopy; the cellular proliferation was determined by Ki67 index, and Bax and Bcl-2 proteins were localized by immunohistochemical analysis. Differences in cell proliferation through the Ki67 index were found in monensin group placentas. Monensin supplementation stimulated the placental cell proliferation reversing the effect of feed restriction during the peripuberal period. A significant increase of Bcl-2 in placentas of restricted group was found, and it would provide a protective effect on the placental structure. A lack of the Bcl-2 protective effect was observed in control and monensin group placentas, probably meaning that the observed apoptosis would be induced through the intrinsic signalling pathway. A balance between apoptosis and cell proliferation is necessary to maintain tissue homoeostasis during caprine placental development.

Environmental filtering drives the shape and breadth of the seed germination niche in coastal plant communities.

Background and Aims: A phylogenetic comparative analysis of the seed germination niche was conducted in coastal plant communities of western Europe. Two hypotheses were tested, that (1) the germination niche shape (i.e. the preference for a set of germination cues as opposed to another) would differ between beaches and cliffs to prevent seedling emergence in the less favourable season (winter and summer, respectively); and (2) the germination niche breadth (i.e. the amplitude of germination cues) would be narrower in the seawards communities, where environmental filtering is stronger. Methods: Seeds of 30 specialist species of coastal plant communities were collected in natural populations of northern Spain. Their germination was measured in six laboratory treatments based on field temperatures. Germination niche shape was estimated as the best germination temperature. Germination niche breadth was calculated using Pielou's evenness index. Differences between plant communities in their germination niche shape and breadth were tested using phylogenetic generalized least squares regression (PGLS). Key Results: Germination niche shape differed between communities, being warm-cued in beaches (best germination temperature = 20 °C) and cold-cued in cliffs (14 °C). Germination niche was narrowest in seawards beaches (Pielou's index = 0·89) and broadest in landwards beaches (0·99). Cliffs had an intermediate germination niche breadth (0·95). The relationship between niche and plant community had a positive phylogenetic signal for shape (Pagel's λ = 0·64) and a negative one for breadth (Pagel's λ = -1·71). Conclusion: Environmental filters shape the germination niche to prevent emergence in the season of highest threat for seedling establishment. The germination niche breadth is narrower in the communities with stronger environmental filters, but only in beaches. This study provides empirical support to a community-level generalization of the hypotheses about the environmental drivers of the germination niche. It highlights the role of germination traits in community assembly.

Traumatic Shoulder Dislocation Among Adolescents: Hill-Sachs Lesion Volume and Recurrent Instability.

BACKGROUND: Accurate assessment of Hill-Sachs lesions (HSLs) and their relationship to the glenoid track is essential to optimizing management following traumatic shoulder dislocation. The purpose of this study was to measure the size of HSLs by a novel method with magnetic resonance (MR) arthrography and to investigate the relationship between lesion size and instability history in adolescents (below 19 y of age) with a history of traumatic dislocation. METHODS: All shoulder MR arthrograms queried to identify HSLs over a 4-year period were retrospectively reviewed and independently evaluated by 2 blinded musculoskeletal radiologists. HSLs were evaluated on axial T1-weighted fat-saturated images. For each consecutive image slice in which the HSL was identified, a freehand region of interest was constructed along the compression fracture cavity. Region of interest sum was multiplied by image slice thickness (and gap between slices if present) to generate a total HSL volume (mm). Subject chart review was then performed to acquire demographic data, including shoulder dislocation history. RESULTS: A total of 30 patients met inclusion criteria and included 22 boys (mean age, 16.3 y) and 8 girls (mean age, 16.4 y). Four patients had a history of 1 traumatic dislocation, 12 had a history of 2, and 14 had a history of ≥3. The average size of the HSL for all patients was 3.8 mm (range, 0.52 to 11 mm). There was no significant difference in HSL volume between the 21 skeletally immature (3.7 mm) and 9 skeletally mature (4.2 mm) patients (P=0.67). Patients with a history of 1, 2, or 3+ dislocations had an average HSL volume of 1.3, 3.7, and 4.7 mm, respectively. The measurement method revealed excellent interreader reliability (P=0.00). There was a statistically significant difference between dislocation history group mean HSL volumes (P=0.019), as well as a statistically significant difference between the number of dislocations and lesion volume (P=0.011). CONCLUSIONS: HSLs can be effectively measured in adolescent patients using MR arthrography and patients with larger HSLs have more recurrent instability episodes, potentially meriting greater and earlier attention to the defect. LEVEL OF EVIDENCE: Prognostic/diagnostic study level IV.

Management of opioid-dependent patients: comparison of the cost associated with use of buprenorphine/naloxone or methadone, and their interactions with concomitant treatments for infectious or psychiatric comorbidities.

The objective was to estimate the annual interaction management cost of agonist opioid treatment (AOT) for opioid-dependent (OD) patients with buprenorphine-naloxone (Suboxone®) (B/N) or methadone associated with concomitant treatments for infectious (HIV) or psychiatric comorbidities. A costs analysis model was developed to calculate the associated cost of AOT and interaction management. The AOT cost included pharmaceutical costs, drug preparation, distribution and dispensing, based on intake regimen (healthcare center or take-home) and type and frequency of dispensing (healthcare center or pharmacy), and medical visits. The cost of methadone also included single-dose bottles, monthly costs of custody at pharmacy, urine toxicology drug screenings and nursing visits. Potential interactions between AOT and concomitant treatments (antivirals, antibacterials/antifungals, antipsychotics, anxiolytics, antidepressant and anticonvulsants), were identified to determine the additional use of healthcare resources for each interaction management. The annual cost per patient of AOT was €1,525.97 for B/N and €1,467.29 for methadone. The average annual cost per patient of interaction management was €257.07 (infectious comorbidities), €114.03 (psychiatric comorbidities) and €185.55 (double comorbidity) with methadone and €7.90 with B/N in psychiatric comorbidities. Total annual costs of B/N were €1,525.97, €1,533.87 and €1,533.87 compared to €1,724.35, €1,581.32 and €1,652.84 for methadone per patient with infectious, psychiatric or double comorbidity respectively.Compared to methadone, the total cost per patient with OD was lower with B/N (€47.45-€198.38 per year). This is due to the differences in interaction management costs associated with the concomitant treatment of infectious and/or psychiatric comorbidities.

El objetivo fue estimar en pacientes con dependencia a opiáceos (DO), el coste anual del manejo de interacciones del tratamiento sustitutivo con buprenorfina/naloxona (Suboxone®) (B/N) o metadona, asociado con tratamientos concomitantes por comorbilidades infecciosas (VIH) o psiquiátricas. Se realizó un análisis de costes (€, 2013), del tratamiento sustitutivo y del manejo de interacciones. El coste del tratamiento de B/N incluyó costes farmacológicos, elaboración, distribución y dispensación, en función del régimen de administración (centro asistencial o domiciliaria) y del tipo y frecuencia de dispensación (centro asistencial o farmacia), y visitas al especialista para prescripción. El coste de tratamiento con metadona incluyó, además, frascos monodosis, coste de custodia en farmacia, determinación en orina y visitas a enfermería. Se identificaron las interacciones para determinar los recursos sanitarios adicionales consumidos por la administración conjunta del tratamiento sustitutivo y concomitante (antirretrovirales, bactericidas/antifúngicos, antipsicóticos, ansiolíticos, antidepresivos y anticonvulsivos). El coste anual/paciente estimado del tratamiento sustitutivo fue de 1.525,97€ (B/N) y 1.467,29€ (metadona). El coste promedio anual/paciente estimado del manejo de interacciones fue de 257,07€ (infecciosas), 114,03€ (psiquiátricas) y 185,55€ (ambas) con metadona, y de 7,90€ con B/N por comorbilidades psiquiátricas. El coste total anual/paciente estimado de B/N fue 1.525,97€, 1.533,87€ y 1.533,87€ comparado con 1.724,35€, 1.581,32€ y 1.652,84€ de metadona, en pacientes que presentan comorbilidad infecciosa, psiquiátrica o ambas, respectivamente. Comparado con metadona, el coste total por paciente con DO de B/N fue menor (47,45€-198,38€ anuales) derivado de la diferencia del coste por manejo de interacciones del tratamiento concomitante de las comorbilidades infecciosas y/o psiquiátricas.

Beclin-mediated Autophagy Drives Dorsal Longitudinal Flight Muscle Histolysis in the Variable Field Cricket, Gryllus lineaticeps.

Flight muscle histolysis is a widespread strategy used by insects to break down functional flight muscle and modulate the energetic costs associated with flight muscle use and maintenance. The variable field cricket, Gryllus lineaticeps, undergoes histolysis during their transition between dispersal flight and reproduction. Despite the importance of histolysis on insect reproduction and fitness, the molecular mechanisms driving this flight muscle breakdown are not well understood. Here, we show that beclin-mediated autophagy, a conserved lysosomal-dependent degradation process, drives breakdown of dorsal longitudinal flight muscle in female flight capable G. lineaticeps. We found that female G. lineaticeps activate autophagy in their dorsal longitudinal flight muscle (DLM), but to a greater extent than the neighboring dorsoventral flight muscle (DVM) during histolysis. RNA interference knockdown of beclin, a gene which encodes a critical autophagy initiation protein, delayed DLM histolysis, but did not affect DVM histolysis. This suggests that crickets selectively activate autophagy to break down the DLMs, while maintaining DVM function for other fitness-relevant activities such as walking. Overall, we confirmed that autophagy is a critical pathway used to remodel flight muscle cells during flight muscle histolysis, providing novel insights into the mechanisms underlying a major life history transition between dispersal and reproduction.

Neddylation orchestrates the complex transcriptional and posttranscriptional program that drives Schwann cell myelination.

Myelination is essential for neuronal function and health. In peripheral nerves, >100 causative mutations have been identified that cause Charcot-Marie-Tooth disease, a disorder that can affect myelin sheaths. Among these, a number of mutations are related to essential targets of the posttranslational modification neddylation, although how these lead to myelin defects is unclear. Here, we demonstrate that inhibiting neddylation leads to a notable absence of peripheral myelin and axonal loss both in developing and regenerating mouse nerves. Our data indicate that neddylation exerts a global influence on the complex transcriptional and posttranscriptional program by simultaneously regulating the expression and function of multiple essential myelination signals, including the master transcription factor EGR2 and the negative regulators c-Jun and Sox2, and inducing global secondary changes in downstream pathways, including the mTOR and YAP/TAZ signaling pathways. This places neddylation as a critical regulator of myelination and delineates the potential pathogenic mechanisms involved in CMT mutations related to neddylation.

A local dormancy cline is related to the seed maturation environment, population genetic composition and climate.

BACKGROUND AND AIMS: Seed dormancy varies within species in response to climate, both in the long term (through ecotypes or clines) and in the short term (through the influence of the seed maturation environment). Disentangling both processes is crucial to understand plant adaptation to environmental changes. In this study, the local patterns of seed dormancy were investigated in a narrow endemic species, Centaurium somedanum, in order to determine the influence of the seed maturation environment, population genetic composition and climate. METHODS: Laboratory germination experiments were performed to measure dormancy in (1) seeds collected from different wild populations along a local altitudinal gradient and (2) seeds of a subsequent generation produced in a common garden. The genetic composition of the original populations was characterized using intersimple sequence repeat (ISSR) PCR and principal co-ordinate analysis (PCoA), and its correlation with the dormancy patterns of both generations was analysed. The effect of the local climate on dormancy was also modelled. KEY RESULTS: An altitudinal dormancy cline was found in the wild populations, which was maintained by the plants grown in the common garden. However, seeds from the common garden responded better to stratification, and their release from dormancy was more intense. The patterns of dormancy variation were correlated with genetic composition, whereas lower temperature and summer precipitation at the population sites predicted higher dormancy in the seeds of both generations. CONCLUSIONS: The dormancy cline in C. somedanum is related to a local climatic gradient and also corresponds to genetic differentiation among populations. This cline is further affected by the weather conditions during seed maturation, which influence the receptiveness to dormancy-breaking factors. These results show that dormancy is influenced by both long-and short-term climatic variation. Such processes at such a reduced spatial scale highlight the potential of plants to adapt to fast environmental changes.

The utility of an online discussion board for reflective writing in an emergency medicine rotation.

Background: Narrative analysis and reflection have been found to support professional identity formation (PIF) and resilience among medical students. In the emergency department, students have used reflective practice to process challenging clinical experiences, such as ethical dilemmas or moral distress. An online discussion board, however, has not been described as a curricular component of emergency medicine (EM) rotations. The objective of this educational innovation was to support medical students in an EM clinical rotation via an online discussion board for reflecting on and debriefing clinical experiences with faculty and peers. Methods: Fifty-two medical students enrolled in the pass/fail EM elective between May 13, 2019, and October 30, 2020. Each cohort of six students took part in a cohort-specific discussion using the Canvas learning management system. Students were encouraged to post about any observations, reflections, or emotions after their shifts. Faculty course directors responded to each post using concepts of debriefing, coaching, and trauma-informed teaching. Results: Over 18 months, 49 of 52 (94%) students participated in the discussion board. Of 346 total posts, half were by students, and the other half were faculty responses. Students posted 3.27 times each, on average. Students rarely raised questions about scientific knowledge content, fact-based aspects of patient care, or specific skills. Rather, they often posted about intensely affective reactions to experiences that left them with complex emotions. Upon review of posts by the course directors, the majority (54%) of students' posts contained a range of affective responses. Students appreciated faculty responses and supported each other in their written responses to peers. Conclusions: An online discussion board can be used successfully for asynchronous reflective practice to debrief clinical experiences during an EM rotation, if designed incorporating faculty and peer support using trauma-informed teaching principles to bolster well-being and PIF.

Addressing Medicine's Dark Matter.

In the 20th century, the models used to predict the motion of heavenly bodies did not match observation. Investigating this incongruity led to the discovery of dark matter-the most abundant substance in the universe. In medicine, despite years of using a data-hungry approach, our models have been limited in their ability to predict population health outcomes-that is, our observations also do not meet our expectations. We believe this phenomenon represents medicine's "dark matter"- the features with have a tremendous effect on clinical outcomes that we cannot directly observe yet. Advancing the information science of health care systems will thus require unique solutions and a humble approach that acknowledges its limitations. Dark matter changed the way the scientific community understood the universe; what might medicine learn from what it cannot yet see?

Hydrothermal treatment as a complementary tool to control the invasive Pampas grass (Cortaderia selloana).

The rapid spread of invasive Pampas grass (PG) is having not only ecosystems impact, but also significant economic and social effects. The tonnes of bulky waste from the plant disposal require proper treatment to avoid seed dispersal, greenhouse gas emissions and landscape damage. In the pursuit of zero-waste management, hydrothermal treatment (HT) appears as a challenging alternative. The possibility of mobile HT systems offers an alternative to accomplish on-site both the PG waste management and the application of the resulting by-products within a circular framework. As a first step, this research shows that, without a prior drying step, the hydrothermal treatment at 100-230 °C under autogenous water vapor pressure for only 30 min allows safe seeds inertization, while a stable carbon-enriched solid and an aqueous stream are generated. Prolonging the process for 2 h has no profitable effects. As the reaction temperature increases, the PG residue is converted into a material with 49-58 wt% of carbon, 41-32 wt% of oxygen and 3-4 wt% of ash. The pH (~6.3), low electrical conductivity (1.21-0.86 dS/m), high carbon content, open porosity (5-8 m2/g) and improved performance in seed germination and in the early growth test suggest the potential of HT-solids derived at 100-120 °C as amendment to sequester carbon in the soil and improve its physico-biological properties. The phytotoxicity detected in the peat/lignite-like solids obtained at 200-230 °C limits its application in soil, but calorific values of 22-24 MJ/kg indicate their suitability as CO2-neutral fuel. The agrochemical analysis of the liquid by-products indicates poor value on their own, but their use supplemented with compost may be an option.

A critical race theory curriculum for emergency medicine learners.

OBJECTIVES: We set out to develop and implement a critical race theory (CRT) curriculum to address an identified gap in emergency medicine education. Sessions explored concepts of CRT and issues of racism as they relate to the clinical and extraclinical environments. METHODS: We developed a series of five virtual workshop sessions in 2019 that were held over Zoom in June and July 2020 in the setting of the COVID-19 pandemic. Eight learners completed the curriculum. Prior to each session, learners were provided presession materials including podcasts, recorded lectures, and readings. Thought-provoking questions were also provided with presession materials to facilitate discussion during sessions. Materials were curated to provide foundational knowledge on CRT and U.S. history as well as local history of the San Francisco Bay Area. RESULTS: Participants found the curriculum useful, reported increased familiarity with CRT, and were more likely to have an analytic framework for topics of race and racism. Participants also reported that their perspective had been changed after completing the curriculum. CONCLUSIONS: Our curriculum promoted effective engagement with topics of race and racism by learners. Opt-in participation contributed to an engaged cohort and the small cohort size encouraged participation by all learners. Semistructured facilitation allowed participants to guide conversations to their own topics of interest while also addressing specific topics at hand. Independent guided presession work allowed participants to gather knowledge at their own pace prior to each session, which likely contributed to more active and in-depth participation.

Making Merit Just in Medical School Admissions.

Medical school education must better align with patient care needs for a rapidly changing population. One challenge is to eliminate bias in merit-based admissions to more equitably review candidates with the structural competency skills desperately needed to promote public health and health equity. Aligning merit-based admissions approaches with holistic admissions approaches and equitable candidate evaluation will simultaneously support learners and improve patient care.

Holliday Junction Resolution.

Holliday junctions are four-way DNA structures that may arise during meiotic recombination, double-strand break repair, or postreplicative repair by the reciprocal exchange of single strands between two DNA molecules. Given their ability to effectively bridge two sister chromatids or homologous chromosomes, cells have implemented various pathways to ensure their timely removal. One of them is the nucleolytic processing of the Holliday junctions by specialized structure-selective endonucleases termed resolvases, which sever the connection between the linked molecules. These Holliday junction resolvases are essential tools of the DNA damage repair machinery to ensure accurate chromosomal segregation, whose activities can be modulated by posttranslational modifications like phosphorylation. Here, we describe a protocol to purify S. cerevisiae Yen1 resolvase in two different phosphorylation states (high and low) and to set up a biochemical assay to compare their ability to process a synthetic, oligonucleotide-based Holliday junction structures.

RNA Helicase DDX3: A Double-Edged Sword for Viral Replication and Immune Signaling.

DDX3 is a cellular ATP-dependent RNA helicase involved in different aspects of RNA metabolism ranging from transcription to translation and therefore, DDX3 participates in the regulation of key cellular processes including cell cycle progression, apoptosis, cancer and the antiviral immune response leading to type-I interferon production. DDX3 has also been described as an essential cellular factor for the replication of different viruses, including important human threats such HIV-1 or HCV, and different small molecules targeting DDX3 activity have been developed. Indeed, increasing evidence suggests that DDX3 can be considered not only a promising but also a viable target for anticancer and antiviral treatments. In this review, we summarize distinct functional aspects of DDX3 focusing on its participation as a double-edged sword in the host immune response and in the replication cycle of different viruses.

Soluble Interleukin-6 Receptor Regulates Interleukin-6-Dependent Vascular Remodeling in Long-Distance Runners.

Little is known about the effects of training load on exercise-induced plasma increase of interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6R) and their relationship with vascular remodeling. We sought to evaluate the role of sIL 6R as a regulator of IL-6-induced vascular remodeling. Forty-four male marathon runners were recruited and allocated into two groups: low-training (LT, <100 km/week) and high-training (HT, ≥100 km/week), 22 athletes per group. Twenty-one sedentary participants were used as reference. IL-6, sIL-6R and sgp130 levels were measured in plasma samples obtained before and immediately after finishing a marathon (42.2-km). Aortic diameter was measured by echocardiography. The inhibitory effect of sIL-6R on IL-6-induced VSMC migration was assessed using cultured A7r5 VSMCs. Basal plasma IL-6 and sIL-6R levels were similar among sedentary and athlete groups. Plasma IL-6 and sIL-6R levels were elevated after the marathon, and HT athletes had higher post-race plasma sIL-6R, but not IL-6, level than LT athletes. No changes in sgp130 plasma levels were found in LT and HT groups before and after running the marathon. Athletes had a more dilated ascending aorta and aortic root than sedentary participants with no differences between HT and LT athletes. However, a positive correlation between ascending aorta diameter and plasma IL-6 levels corrected by training load and years of training was observed. IL-6 could be responsible for aorta dilation because IL-6 stimulated VSMC migration in vitro, an effect that is inhibited by sIL-6R. However, IL-6 did not modify cell proliferation, collagen type I and contractile protein of VSMC. Our results suggest that exercise induces vascular remodeling. A possible association with IL-6 is proposed. Because sIL-6R inhibits IL-6-induced VSMC migration, a possible mechanism to regulate IL-6-dependent VSMC migration is also proposed.