A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery

Background: Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand ( hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.Results: Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the beta domain of VHL and a hydroxyproline-containing HIF alpha peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFa peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.Conclusion: The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes

A Robust AFPTAS for Online Bin Packing with Polynomial Migration

In this paper we develop general LP and ILP techniques to find an approximate solution with improved objective value close to an existing solution. The task of improving an approximate solution is closely related to a classical theorem of Cook et al. in the sensitivity analysis for LPs and ILPs. This result is often applied in designing robust algorithms for online problems. We apply our new techniques to the online bin packing problem, where it is allowed to reassign a certain number of items, measured by the migration factor. The migration factor is defined by the total size of reassigned items divided by the size of the arriving item. We obtain a robust asymptotic fully polynomial time approximation scheme (AFPTAS) for the online bin packing problem with migration factor bounded by a polynomial in $\frac{1}{\epsilon}$. This answers an open question stated by Epstein and Levin in the affirmative. As a byproduct we prove an approximate variant of the sensitivity theorem by Cook at el. for linear programs

Egg yolk IgY antibodies: A therapeutic intervention against group A rotavirus in calves

Bovine group A rotavirus (RVA) is considered the major cause of diarrhea in intensively reared neonatal calves. Chicken egg yolk antibodies (IgY) are efficient in protecting neonatal calves from RVA diarrhea; however, the value of this intervention in calves once diarrhea has appeared is unclear. The aim of the present study was to evaluate the application of RVA-specific IgY as a passive treatment in those cases. The experimental groups were: G1 = RVA-specific IgY treatment; G2 = no Ab treatment; and G3 = colostrum deprived + no Ab treatment. IgY treatment significantly reduced virus shedding, diarrhea duration and severity compared to G2 and G3 calves. However, it caused a partial suppression of systemic Ab responses to RVA that could be associated with less severe diarrhea. The oral treatment with IgY for 7 days was associated with significantly higher antibody secreting cell responses in the calves compared with other groups of animals.Fil: Vega, Celina Guadalupe. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Bok, Marina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Saif, L.. Ohio State University; Estados UnidosFil: Fernandez, F.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Parreño, Gladys Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentin

Diferencias psicopatológicas en mujeres con Dependencia Emocional con y sin uso de sustancias ilegales comórbido

En este artículo se presenta un análisis clínico de las diferencias psicopatológicas entre dos grupos de pacientes, mujeres, con problemas de Dependencia Emocional con y sin consumo de sustancias ilegales comórbido. Las pacientes fueron evaluadas mediante los cuestionarios TDS100, SCL-90-R, BIS-11 y una entrevista semiestructurada adaptada de la entrevista SCID-I. Los grupos se formaron en función de si existía consumo de drogas o no en el momento de la evaluación. A través de los resultados hallados, se puede concluir que el grupo de mujeres con un consumo ocasional de drogas presenta una mayor gravedad en la escala de Acomodación medida a través del TDS-100, así como puntuaciones más altas en psicopatología comórbida (Sensibilidad Interpersonal, Ansiedad, Hostilidad, Ideación paranoide, Psicoticismo y GSI) medida por el SCL-90-R. Finalmente, el grupo con uso de drogas obtuvo puntuaciones significativamente más altas en la subescala Impulsividad Motora medida por el BIS-11. English abstract: In this article we present a clinical analysis of the psychopathological differences between women patients with Affective Dependence problems with and without comorbid substance use. The patients were assessed trough the TDS-100, SCL-90-R, BIS-11 questionnaires and a semi-structured interview adapted from the SCID-I interview. The groups were created based on the substance use at the moment of the assessment. The results reveal that the group of women with occasional use of drugs shows higher severity in the Accommodation scale measured trough the TDS-100 and in the comorbid psychopathology measured by the SCL-90R (Interpersonal Sensitivity, Anxiety, Hostility, Paranoid ideation, Psychoticism y GSI). Finally, the group with drug use obtained significantly higher scores in the Motor Impulsiveness subscale measured by the BIS-11

Surgical Options for the Refractive Correction of Keratoconus: Myth or Reality

Keratoconus provides a decrease of quality of life to the patients who suffer from it. The treatment used as well as the method to correct the refractive error of these patients may influence on the impact of the disease on their quality of life. The purpose of this review is to describe the evidence about the conservative surgical treatment for keratoconus aiming to therapeutic and refractive effect. The visual rehabilitation for keratoconic corneas requires addressing three concerns: halting the ectatic process, improving corneal shape, and minimizing the residual refractive error. Cross-linking can halt the disease progression, intrastromal corneal ring segments can improve the corneal shape and hence the visual quality and reduce the refractive error, PRK can correct mild-moderate refractive error, and intraocular lenses can correct from low to high refractive error associated with keratoconus. Any of these surgical options can be performed alone or combined with the other techniques depending on what the case requires. Although it could be considered that the surgical option for the refracto-therapeutic treatment of the keratoconus is a reality, controlled, randomized studies with larger cohorts and longer follow-up periods are needed to determine which refractive procedure and/or sequence are most suitable for each case

Reducing the data-deficiency of threatened European habitats: Spatial variation of sabellariid worm reefs and associated fauna in the Sicily Channel, Mediterranean Sea

Biogenic reefs, such as those produced by tube-dwelling polychaetes of the genus Sabellaria, are valuable marine habitats which are a focus of protection according to European legislation. The achievement of this goal is potentially hindered by the lack of essential empirical data, especially in the Mediterranean Sea. This study addresses some of the current knowledge gaps by quantifying and comparing multi-scale patterns of abundance and distribution of two habitat-forming species (Sabellaria alveolata and S. spinulosa) and their associated fauna along 190 km of coast on the Italian side of the Sicily Channel. While the abundance of the two sabellariids and the total number of associated taxa did not differ at any of the examined scales (from tens of centimetres to tens-100 of kilometres), the structure (composition in terms of both the identity and the relative abundance of constituting taxa) of the associated fauna and the abundance of several taxa (the polychaetes Eulalia ornata, Syllis pulvinata, S. garciai, Nereis splendida and Arabella iricolor, and the amphipods Apolochus neapolitanus, Tethylembos viguieri and Caprella acanthifera) varied among locations established ~50e100 km apart. Syllis pulvinata also showed significant variation between sites (hundreds of metres apart), analogously to the other syllid polychaetes S. armillaris and S. gracilis, the nereidid polychaete Nereis rava, and the amphipod Gammaropsis ulrici. The largest variance of S. spinulosa, of the structure of the whole associated fauna and of 56% of taxa analysed individually occurred at the scale of replicates (metres apart), while that of the dominant bio-constructor S. alveolata and of 25% of taxa occurred at the scale of sites. The remaining 19% and the total richness of taxa showed the largest variance at the scale of locations. Present findings contribute to meet a crucial requirement of any future effective protection strategy, i.e., identifying relevant scales of variation to be included in protection schemes aiming at preserving representative samples not only of target habitats and organisms, but also of the processes driving such variabilit

A yeast three-hybrid system that reconstitutes mammalian hypoxia inducible factor regulatory machinery

<p>Abstract</p> <p>Background</p> <p>Several human pathologies, including neoplasia and ischemic cardiovascular diseases, course with an unbalance between oxygen supply and demand (hypoxia). Cells within hypoxic regions respond with the induction of a specific genetic program, under the control of the Hypoxia Inducible Factor (HIF), that mediates their adaptation to the lack of oxygen. The activity of HIF is mainly regulated by the EGL-nine homolog (EGLN) enzymes that hydroxylate the alpha subunit of this transcription factor in an oxygen-dependent reaction. Hydroxylated HIF is then recognized and ubiquitinilated by the product of the tumor suppressor gene, pVHL, leading to its proteosomal degradation. Under hypoxia, the hydroxylation of HIF by the EGLNs is compromised due to the lack of oxygen, which is a reaction cosubstrate. Thus, HIF escapes degradation and drives the transcription of its target genes. Since the progression of the aforementioned pathologies might be influenced by activation of HIF-target genes, development of small molecules with the ability to interfere with the HIF-regulatory machinery is of great interest.</p> <p>Results</p> <p>Herein we describe a yeast three-hybrid system that reconstitutes mammalian HIF regulation by the EGLNs and VHL. In this system, yeast growth, under specific nutrient restrictions, is driven by the interaction between the β domain of VHL and a hydroxyproline-containing HIFα peptide. In turn, this interaction is strictly dependent on EGLN activity that hydroxylates the HIFα peptide. Importantly, this system accurately preserves the specificity of the hydroxylation reaction toward specific substrates. We propose that this system, in combination with a matched control, can be used as a simple and inexpensive assay to identify molecules that specifically modulate EGLN activity. As a proof of principle we show that two known EGLN inhibitors, dimethyloxaloylglycine (DMOG) and 6-chlor-3-hydroxychinolin-2-carbonic acid-N-carboxymethylamide (S956711), have a profound and specific effect on the yeast HIF/EGLN/VHL system.</p> <p>Conclusion</p> <p>The system described in this work accurately reconstitutes HIF regulation while preserving EGLN substrate specificity. Thus, it is a valuable tool to study HIF regulation, and particularly EGLN biochemistry, in a cellular context. In addition, we demonstrate that this system can be used to identify specific inhibitors of the EGLN enzymes.</p

Microplastics impair the feeding performance of a Mediterranean habitat-forming coral

none9siThe impact of plastic debris, and in particular of microplastics (here referred as particles smaller than 5 mm) on aquatic environments has now become a topic of raising concern. Microplastics are particularly abundant in the Mediterranean Sea, potentially exerting substantial pressures on marine organisms at different levels of organization. Ingestion of microplastics has been observed in a large number of marine species. The aim of this work is to test if microplastics produce a feeding impairment in Astroides calycularis, a shallow water, habitat-forming coral endemic to the Mediterranean Sea. Our findings suggest a lack of any avoidance mechanism allowing the polyps to discern between food items and microplastics when occurring simultaneously. Moreover, polyps spend a considerable amount of time on handling microplastic particles. As a consequence, microplastics impair the feeding efficiency in A. calycularis, since polyps may not be fully able to profit from the drifting plankton aggregations. Therefore, we suggest that microplastics can cause a reduction of fitness in A. calycularis, and presumably also in other species characterized by suspension feeding strategy.embargoed_20220123Savinelli B.; Vega Fernandez T.; Galasso N.M.; D'Anna G.; Pipitone C.; Prada F.; Zenone A.; Badalamenti F.; Musco L.Savinelli, B.; Vega Fernandez, T.; Galasso, N. M.; D'Anna, G.; Pipitone, C.; Prada, F.; Zenone, A.; Badalamenti, F.; Musco, L