Role of the Solute-Binding Protein CuaD in the Signaling and Regulating Pathway of Cellobiose and Cellulose Utilization in Ruminiclostridium cellulolyticum.

In Ruminiclostridium cellulolyticum, cellobiose is imported by the CuaABC ATP-binding cassette transporter containing the solute-binding protein (SBP) CuaA and is further degraded in the cytosol by the cellobiose phosphorylase CbpA. The genes encoding these proteins have been shown to be essential for cellobiose and cellulose utilization. Here, we show that a second SBP (CuaD), whose gene is adjacent to two genes encoding a putative two-component regulation system (CuaSR), forms a three-component system with CuaS and CuaR. Studies of mutant and recombinant strains of R. cellulolyticum have indicated that cuaD is important for the growth of strains on cellobiose and cellulose. Furthermore, the results of our RT-qPCR experiments suggest that both the three (CuaDSR)- and the two (CuaSR)-component systems are able to perceive the cellobiose signal. However, the strain producing the three-component system is more efficient in its cellobiose and cellulose utilization. As CuaD binds to CuaS, we propose an in-silico model of the complex made up of two extracellular domains of CuaS and two of CuaD. CuaD allows microorganisms to detect very low concentrations of cellobiose due to its high affinity and specificity for this disaccharide, and together with CuaSR, it triggers the expression of the cuaABC-cbpA genes involved in cellodextrins uptake.

Mesiodens: a propósito de dos casos / Mesiodens: two cases

El mesiodens es el diente supernumerario más común, localizado en la línea media maxilar entre los incisivos centrales. Su etiología es incierta y se han planteado múltiples teorías. El diagnóstico y tratamiento precoz permite la erupción espontánea de los incisivos permanentes, evitar las complicaciones asociadas, así como tratamientos posteriores más complejos. En este artículo se presentan dos casos de mesiodens de pacientes que acuden a la consulta de Pediatría de Atención Primaria (AU)

The mesiodens is the most common supernumerary tooth, located in the maxillary midline between the central incisors. Its etiology is uncertain and multiple theories have been put forward. Early diagnosis and treatment allows the spontaneous eruption of the permanent incisors, and avoids associated complications as well as more complex subsequent treatments. In this article, two cases of mesiodens in patients attending the primary care pediatric outpatient clinic are presented. (AU)

Knee Kinematics During Landing: Is It Really a Predictor of Acute Noncontact Knee Injuries in Athletes? A Systematic Review and Meta-analysis.

BACKGROUND: Although knee kinematics during landing tasks has traditionally been considered to predict noncontact knee injuries, the predictive association between noncontact knee injuries and kinematic and kinetic variables remains unclear. PURPOSE: To systematically review the association between kinematic and kinetic variables from biomechanical evaluation during landing tasks and subsequent acute noncontact knee injuries in athletes. STUDY DESIGN: Systematic review; Level of evidence, 2. METHODS: Databases used for searches were MEDLINE, LILACS, IBECS, CINAHL, SPORTDiscus, SCIELO, IME, ScienceDirect, and Cochrane from database inception to May 2020. Manual reference checks, articles published online ahead of print, and citation tracking were also considered. Eligibility criteria included prospective studies evaluating frontal and sagittal plane kinematics and kinetics of landing tasks and their association with subsequent acute noncontact knee injuries in athletes. RESULTS: A total of 13 studies met the eligibility criteria, capturing 333 acute noncontact knee injuries in 8689 participants. A meta-analysis revealed no significant effects for any kinematic and kinetic variable with regard to subsequent noncontact knee injuries. CONCLUSION: No kinetic or kinematic variables from landing tasks had a significant association with acute noncontact knee injuries. Therefore, the role and application of the landing assessment for predicting acute noncontact knee injuries are limited and unclear, particularly given the heterogeneity and risk of bias of studies to date.

Quality of Life and Dependence Degree of Chronic Patients in a Chronicity Care Model.

BACKGROUND: The complex chronic patient is a person with one or several long-term diseases, the clinical management of which are considered difficult and related to cognitive or functional impairment. The chronicity care model deeply affects the quality of life and degree of dependence. OBJECTIVES: The objective of this study was to analyse the perceived quality of life and dependence degree in complex chronic patients within a chronicity care model in the Autonomous Communities of Cantabria and the Balearic Islands (Spain). DESIGN: This was a multicentred, transversal, descriptive, and observational study on a cohort of 206 chronic patients included in a chronicity care program. METHODS: Patients' sociodemographic variables, integral valuation, nurse follow-up records, nursing outcomes classification (NOC)/nursing interventions classification (NIC), nurse diagnoses, and hospitalization data were analysed. A descriptive analysis of all data was carried out. The bivariate analysis assessed the relation between covariables and the overall scoring in European Quality of Life Scale (EuroQuol-5D), Barthel, Braden, and Chronic Patient eXperience Assessment Instrument (IEXPAC in the Spanish abbreviation). A multivariate linear regression analysis was conducted. RESULTS: The mean age was 79.4 years (standard deviation (SD) = 9.12; range: 39-94). A percentage of 79.3% of the study population shows functional impairment in one or more activities of daily life. A percentage of 83.3% of patients showed a physical dependence. There is a significant relationship between the gender and kinship degree of the caregiver (χ2 = 18.2; p = 0.001). An overall mean score of 55.38 points in EuroQuol-5D was obtained, along with a 36.87-point satisfaction with the care given in IEXPAC. The overall score correlated positively and significantly with Barthel, Braden, and IEXPAC. The dependence levels improved slightly in the observed patients, which was a very significant outcome in statistical terms (t = 2.08; p = 0.039). A percentage of 66% (R2 = 0.66) of the score variability at the Barthel index could be predicted from Braden scale scoring. CONCLUSIONS: Dependence is not only affected by the related pathology, but also by the effect on mobility and daily-life activities, which cause a worse perception of the quality of life. The health-care model based on the case management nurse is having positive effects, especially on dependence and patients with ulcer issues.

Predictive capability of the injury severity score versus the new injury severity score in the categorization of the severity of trauma patients: a cross-sectional observational study.

PURPOSE: The AIS scale is a measurement tool for single injuries. The ISS is considered the gold standard for determining the severity of injured patients, and the NISS was developed to improve the ISS with respect to loss of information, as well as to facilitate its calculation. The aim of this study was to analyse what injury severity measure, calculated according to the Abbreviated Injury Scale (AIS), 1998 and 2005 (update 2008) versions, performs better with mortality, cost and hospital length of stay healthcare indicators. METHODS: This cross-sectional observational study was carried out between February 1st 2012 and February 1st 2013. Inclusion criteria were injured patients due to external causes admitted to trauma service through the emergency department. Manual coding of all injuries was performed and ISS and NISS scores were calculated for both versions of the AIS scale. Severity was then compared to mortality (in-hospital and at 30 days), healthcare cost, and length of hospital stay. RESULTS: The index with the best predictive capability for in-hospital mortality was NISS 05 (AUC = 0.811). There was a significant increase in hospital stay and healthcare cost in the most severe patients in all indexes, except for ISS 05. CONCLUSIONS: NISS is found to be an index with higher predictive capability for in-hospital mortality and correlates better to length of hospital stay and healthcare cost.

Structure and function of the Toscana virus cap-snatching endonuclease.

Toscana virus (TOSV) is an arthropod-borne human pathogen responsible for seasonal outbreaks of fever and meningoencephalitis in the Mediterranean basin. TOSV is a segmented negative-strand RNA virus (sNSV) that belongs to the genus phlebovirus (family Phenuiviridae, order Bunyavirales), encompassing other important human pathogens such as Rift Valley fever virus (RVFV). Here, we carried out a structural and functional characterization of the TOSV cap-snatching endonuclease, an N terminal domain of the viral polymerase (L protein) that provides capped 3'OH primers for transcription. We report TOSV endonuclease crystal structures in the apo form, in complex with a di-ketoacid inhibitor (DPBA) and in an intermediate state of inhibitor release, showing details on substrate binding and active site dynamics. The structure reveals substantial folding rearrangements absent in previously reported cap-snatching endonucleases. These include the relocation of the N terminus and the appearance of new structural motifs important for transcription and replication. The enzyme shows high activity rates comparable to other His+ cap-snatching endonucleases. Moreover, the activity is dependent on conserved residues involved in metal ion and substrate binding. Altogether, these results bring new light on the structure and function of cap-snatching endonucleases and pave the way for the development of specific and broad-spectrum antivirals.

Fluoxetine Inhibits Enterovirus Replication by Targeting the Viral 2C Protein in a Stereospecific Manner.

Enteroviruses (family Picornaviridae) comprise a large group of human pathogens against which no licensed antiviral therapy exists. Drug-repurposing screens uncovered the FDA-approved drug fluoxetine as a replication inhibitor of enterovirus B and D species. Fluoxetine likely targets the nonstructural viral protein 2C, but detailed mode-of-action studies are missing because structural information on 2C of fluoxetine-sensitive enteroviruses is lacking. We here show that broad-spectrum anti-enteroviral activity of fluoxetine is stereospecific concomitant with binding to recombinant 2C. (S)-Fluoxetine inhibits with a 5-fold lower 50% effective concentration (EC50) than racemic fluoxetine. Using a homology model of 2C of the fluoxetine-sensitive enterovirus coxsackievirus B3 (CVB3) based upon a recently elucidated structure of a fluoxetine-insensitive enterovirus, we predicted stable binding of (S)-fluoxetine. Structure-guided mutations disrupted binding and rendered coxsackievirus B3 (CVB3) resistant to fluoxetine. The study provides new insights into the anti-enteroviral mode-of-action of fluoxetine. Importantly, using only (S)-fluoxetine would allow for lower dosing in patients, thereby likely reducing side effects.

Comparison of injury severity scores (ISS) obtained by manual coding versus "Two-step conversion" from ICD-9-CM.

BACKGROUND: The International Classification of Diseases (ICD) is the standard diagnostic tool for classifying and coding diseases and injuries. The Abbreviated Injury Scale (AIS) is the most widely used injury severity scoring system. Although manual coding is considered the gold standard, it is sometimes unavailable or impractical. There have been many prior attempts to develop programs for the automated conversion of ICD rubrics into AIS codes. OBJECTIVE: To convert ICD, Ninth Revision, Clinical Modification (ICD-9-CM) codes into AIS 2005 (update 2008) codes via a derived map using a two-step process and, subsequently, to compare Injury Severity Score (ISS) resulting from said conversion with manually coded ISS values. METHODS: A cross-sectional retrospective study was designed in which medical records at the Hospital Universitario Marqués de Valdecilla of Cantabria (HUMV) and the Complejo Hospitalario of Navarra (CHN), both in Spain, were reviewed. Coding of injuries using AIS 2005 (update 2008) version was done manually by a certified AIS specialist and ISS values were calculated. ICD-9-CM codes were automatically converted into ISS values by another certified AIS specialist in a two-step process. ISS scores obtained from manual coding were compared to those obtained through this conversion process. RESULTS: The comparison of obtained through conversion versus manual ISS resulted in 396 concordant pairs (70.2%); the analysis of values according to ISS categories (ISS<9, ISS 9-15, ISS 16-24, ISS>24) showed 493 concordant pairs (87.4%). Regarding the criterion of "major trauma" patient (i.e., ISS> 15), 538 matching pairs (95.2%) were obtained. The conversion process resulted in underestimation of ISS in 112 cases (19.9%) and conversion was not possible in 136 cases (19%) for different reasons. CONCLUSIONS: The process used in this study has proven to be a useful tool for selecting patients who meet the ISS>15 criterion for "major trauma". Further research is needed to improve the conversion process.

Deciphering the Nucleotide and RNA Binding Selectivity of the Mayaro Virus Macro Domain.

Mayaro virus (MAYV) is a member of Togaviridae family, which also includes Chikungunya virus as a notorious member. MAYV recently emerged in urban areas of the Americas, and this emergence emphasized the current paucity of knowledge about its replication cycle. The macro domain (MD) of MAYV belongs to the N-terminal region of its non-structural protein 3, part of the replication complex. Here, we report the first structural and dynamical characterization of a previously unexplored Alphavirus MD investigated through high-resolution NMR spectroscopy, along with data on its ligand selectivity and binding properties. The structural analysis of MAYV MD reveals a typical "macro" (ßßαßßαßαßα) fold for this polypeptide, while NMR-driven interaction studies provide in-depth insights into MAYV MD-ligand adducts. NMR data in concert with thermodynamics and biochemical studies provide convincing experimental evidence for preferential binding of adenosine diphosphate ribose (ADP-r) and adenine-rich RNAs to MAYV MD, thus shedding light on the structure-function relationship of a previously unexplored viral MD. The emerging differences with any other related MD are expected to enlighten distinct functions.

Assessment of the long-acting ivermectin formulation in sheep: Further insight into potential pharmacokinetic interactions.

The aim of the current study was to evaluate the in vivo pharmacokinetic of ivermectin (IVM) after the administration of a long-acting (LA) formulation to sheep and its impact on potential drug-drug interactions. The work included the evaluation of the comparative plasma profiles of IVM administered at a single therapeutic dose (200 µg/kg) and as LA formulation at 630 µg/kg. Additionally, IVM was measured in different gastrointestinal tissues at 15 days posttreatment with both IVM formulations. The impact of the long-lasting and enhanced IVM exposure on the disposition kinetics of abamectin (ABM) was also assessed. Plasma (IVM and ABM) and gastrointestinal (IVM) concentrations were analyzed by HPLC with fluorescent detection. In plasma, the calculated Cmax and AUC0-t values of the IVM-LA formulation were 1.47- and 3.35-fold higher compared with IVM 1% formulation, respectively. The T1/2ab and Tmax collected after administration of the LA formulation were 2- and 3.5-fold longer than those observed after administration of IVM 1% formulation, respectively. Significantly higher IVM concentrations were measured in the intestine mucosal tissues and luminal contents with the LA formulation, and in the liver, the increase was 7-fold higher than conventional formulation. There was no drug interaction between IVM and ABM after the single administration of ABM at 15 days post-administration of the IVM LA formulation. The characterization of the kinetic behavior of the LA formulation to sheep and its potential influence on drug-drug interactions is a further contribution to the field.

Classification of the severe trauma patient with the Abbreviated Injury Scale: degree of correlation between versions 98 and 2005 (2008 update). / Grado de correlación entre las versiones 98 y 2005 (actualización 2008) de la Abbreviated Injury Scale (AIS) en la categorización del paciente traumatológico grave.

OBJECTIVES: To explore differences in severity classifications according to 2 versions of the Abbreviated Injury Scale (AIS): version 2005 (the 2008 update) and the earlier version 98. To determine whether possible differences might have an impact on identifying severe trauma patients. MATERIAL AND METHODS: Descriptive study and cross-sectional analysis of a case series of patients admitted to two spanish hospitals with out-of-hospital injuries between February 2012 and February 2013. For each patient we calculated the Injury Severity Score (ISS), the New Injury Severity Score (NISS), and the AIS scores according to versions 98 and 2005. RESULTS: The sample included 699 cases. The mean Severity (SD) age of patients was 52.7 (29.2) years, and 388 (55.5%) were males. Version 98 of the AIS correlated more strongly with both the ISS (2.6%) and the NISS (2.9%). CONCLUSION: The 2008 update of the AIS (version 2005) classified fewer trauma patients than version 98 at the severity levels indicated by the ISS and NISS.

OBJETIVO: Estudiar si existen diferencias en la asignación de gravedad entre las versiones 98 y 2005 ­actualización 2008­ de la escala Abbreviated Injury Scale (AIS) y determinar si estas posibles diferencias podrían tener repercusión en la definición de paciente traumatológico grave. METODO: Estudio descriptivo de una serie de casos con análisis transversal que incluyó a pacientes ingresados por lesiones debidas a causas externas en dos hospitales españoles, llevado a cabo entre febrero de 2012 y febrero de 2013. Se calculó el Injury Severity Score (ISS) y el New Injury Severity Score (NISS) de cada uno de los casos con ambas versiones de la escala AIS. RESULTADOS: La muestra estuvo compuesta por 699 casos, con una edad media de 52,7 (DE 29,2) años, de los cuales 388 (55,5%) fueron varones. Se obtuvo una mayor clasificación de pacientes graves con la versión AIS 98, tanto para el ISS (2,6%) como el NISS (2,9%). CONCLUSIONES: La versión AIS 2005 ­actualización 2008­ clasifica un menor número de pacientes como graves en comparación con la versión AIS 98.

Grado de correlación entre las versiones 98 y 2005 (actualización 2008) de la Abbreviated Injury Scale (AIS) en la categorización del paciente traumatológico grave / Classification of the severe trauma patient with the Abbreviated Injury Scale: degree of correlation between versions 98 and 2005 (2008 update)

Objetivos. Estudiar si existen diferencias en la asignación de gravedad entre las versiones 98 y 2005 -actualización 2008- de la escala Abbreviated Injury Scale (AIS) y determinar si estas posibles diferencias podrían tener repercusión en la definición de paciente traumatológico grave. Método. Estudio descriptivo de una serie de casos con análisis transversal que incluyó a pacientes ingresados por lesiones debidas a causas externas en dos hospitales españoles, llevado a cabo entre febrero de 2012 y febrero de 2013. Se calculó el Injury Severity Score (ISS) y el New Injury Severity Score (NISS) de cada uno de los casos con ambas versiones de la escala AIS. Resultados. La muestra estuvo compuesta por 699 casos, con una edad media de 52,7 (DE 29,2) años, de los cuales 388 (55,5%) fueron varones. Se obtuvo una mayor clasificación de pacientes graves con la versión AIS 98, tanto para el ISS (2,6%) como el NISS (2,9%). Conclusiones. La versión AIS 2005 -actualización 2008- clasifica un menor número de pacientes como graves en comparación con la versión AIS 98 (AU)

Objectives. To explore differences in severity classifications according to 2 versions of the Abbreviated Injury Scale (AIS): version 2005 (the 2008 update) and the earlier version 98. To determine whether possible differences might have an impact on identifying severe trauma patients. Methods. Descriptive study and cross-sectional analysis of a case series of patients admitted to two Spanish hospitals with out-of-hospital injuries between February 2012 and February 2013. For each patient we calculated the Injury Severity Score (ISS), the New Injury Severity Score (NISS), and the AIS scores according to versions 98 and 2005. Results. The sample included 699 cases. The mean Severity (SD) age of patients was 52.7 (29.2) years, and 388 (55.5%) were males. Version 98 of the AIS correlated more strongly with both the ISS (2.6%) and the NISS (2.9%). Conclusion. The 2008 update of the AIS (version 2005) classified fewer trauma patients than version 98 at the severity levels indicated by the ISS and NISS (AU)

A seven-gene cluster in Ruminiclostridium cellulolyticum is essential for signalization, uptake and catabolism of the degradation products of cellulose hydrolysis.

BACKGROUND: Like a number of anaerobic and cellulolytic Gram-positive bacteria, the model microorganism Ruminiclostridium cellulolyticum produces extracellular multi-enzymatic complexes called cellulosomes, which efficiently degrade the crystalline cellulose. Action of the complexes on cellulose releases cellobiose and longer cellodextrins but to date, little is known about the transport and utilization of the produced cellodextrins in the bacterium. A better understanding of the uptake systems and fermentation of sugars derived from cellulose could have a major impact in the field of biofuels production. RESULTS: We characterized a putative ABC transporter devoted to cellodextrins uptake, and a cellobiose phosphorylase (CbpA) in R. cellulolyticum. The genes encoding the components of the ABC transporter (a binding protein CuaA and two integral membrane proteins) and CbpA are expressed as a polycistronic transcriptional unit induced in the presence of cellobiose. Upstream, another polycistronic transcriptional unit encodes a two-component system (sensor and regulator), and a second binding protein CuaD, and is constitutively expressed. The products might form a three-component system inducing the expression of cuaABC and cbpA since we showed that CuaR is able to recognize the region upstream of cuaA. Biochemical analysis showed that CbpA is a strict cellobiose phosphorylase inactive on longer cellodextrins; CuaA binds to all cellodextrins (G2-G5) tested, whereas CuaD is specific to cellobiose and presents a higher affinity to this sugar. This results are in agreement with their function in transport and signalization, respectively. Characterization of a cuaD mutant, and its derivatives, indicated that the ABC transporter and CbpA are essential for growth on cellobiose and cellulose. CONCLUSIONS: For the first time in a Gram-positive strain, we identified a three-component system and a conjugated ABC transporter/cellobiose phosphorylase system which was shown to be essential for the growth of the model cellulolytic bacterium R. cellulolyticum on cellobiose and cellulose. This efficient and energy-saving system of transport and phosphorolysis appears to be the major cellobiose utilization pathway in R. cellulolyticum, and seems well adapted to cellulolytic life-style strain. It represents a new way to enable engineered strains to utilize cellodextrins for the production of biofuels or chemicals of interest from cellulose.

Assessment of liver slices for research on metabolic drug-drug interactions in cattle.

1. Precision-cut liver slices (PCLS) from food-producing animals have not been extensively used to study xenobiotic metabolism, and thus information on this field of research is sparse. 2. The aims of the present work were to further validate the technique of production and culture of bovine PCLS and to characterize the metabolic interaction between the anthelmintic albendazole (ABZ) and the flavin-monooxygenase (FMO) inhibitor methimazole (MTZ). 3. Nine steers were used as donors. PCLS were produced and incubated under two methods: a dynamic organ culture (DOC) incubator and a well-plate (WP) system. 4. Tissue viability, assessed through both structural and functional markers, was preserved throughout 12 h of incubation. ABZ was metabolized to its (+) and (-) albendazole sulfoxide stereoisomers (ABZSO) in bovine PCLS. The interaction between ABZ and MTZ resulted in a reduction (p < 0.001) in the rates of appearance of (+) ABZSO. Conversely, in presence of MTZ, the rates of appearance of (-) ABZSO increased under both systems (p < 0.05). 5. Both culture systems were suitable for assessing the interaction between ABZ and MTZ. 6. Overall, the results presented herein show that PCLS are a useful and reliable tool for short-term studies on metabolic drug-drug interactions in the bovine species.

Mechanisms involved in xyloglucan catabolism by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum.

Xyloglucan, a ubiquitous highly branched plant polysaccharide, was found to be rapidly degraded and metabolized by the cellulosome-producing bacterium Ruminiclostridium cellulolyticum. Our study shows that at least four cellulosomal enzymes displaying either endo- or exoxyloglucanase activities, achieve the extracellular degradation of xyloglucan into 4-glucosyl backbone xyloglucan oligosaccharides. The released oligosaccharides (composed of up to 9 monosaccharides) are subsequently imported by a highly specific ATP-binding cassette transporter (ABC-transporter), the expression of the corresponding genes being strongly induced by xyloglucan. This polysaccharide also triggers the synthesis of cytoplasmic ß-galactosidase, α-xylosidase, and ß-glucosidase that act sequentially to convert the imported oligosaccharides into galactose, xylose, glucose and unexpectedly cellobiose. Thus R. cellulolyticum has developed an energy-saving strategy to metabolize this hemicellulosic polysaccharide that relies on the action of the extracellular cellulosomes, a highly specialized ABC-transporter, and cytoplasmic enzymes acting in a specific order. This strategy appears to be widespread among cellulosome-producing mesophilic bacteria which display highly similar gene clusters encoding the cytosolic enzymes and the ABC-transporter.

Screening of a Library of FDA-Approved Drugs Identifies Several Enterovirus Replication Inhibitors That Target Viral Protein 2C.

Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired.

Insights into the Hendra virus NTAIL-XD complex: Evidence for a parallel organization of the helical MoRE at the XD surface stabilized by a combination of hydrophobic and polar interactions.

The Hendra virus is a member of the Henipavirus genus within the Paramyxoviridae family. The nucleoprotein, which consists of a structured core and of a C-terminal intrinsically disordered domain (N(TAIL)), encapsidates the viral genome within a helical nucleocapsid. N(TAIL) partly protrudes from the surface of the nucleocapsid being thus capable of interacting with the C-terminal X domain (XD) of the viral phosphoprotein. Interaction with XD implies a molecular recognition element (MoRE) that is located within N(TAIL) residues 470-490, and that undergoes α-helical folding. The MoRE has been proposed to be embedded in the hydrophobic groove delimited by helices α2 and α3 of XD, although experimental data could not discriminate between a parallel and an antiparallel orientation of the MoRE. Previous studies also showed that if the binding interface is enriched in hydrophobic residues, charged residues located close to the interface might play a role in complex formation. Here, we targeted for site directed mutagenesis two acidic and two basic residues within XD and N(TAIL). ITC studies showed that electrostatics plays a crucial role in complex formation and pointed a parallel orientation of the MoRE as more likely. Further support for a parallel orientation was afforded by SAXS studies that made use of two chimeric constructs in which XD and the MoRE were covalently linked to each other. Altogether, these studies unveiled the multiparametric nature of the interactions established within this complex and contribute to shed light onto the molecular features of protein interfaces involving intrinsically disordered regions.

A flexible extension of the Drosophila ultrabithorax homeodomain defines a novel Hox/PBC interaction mode.

The patterning function of Hox proteins relies on assembling protein complexes with PBC proteins, which often involves a protein motif found in most Hox proteins, the so-called Hexapeptide (HX). Hox/PBC complexes likely gained functional diversity by acquiring additional modes of interaction. Here, we structurally characterize the first HX alternative interaction mode based on the paralogue-specific UbdA motif and further functionally validate structure-based predictions. The UbdA motif folds as a flexible extension of the homeodomain recognition helix and defines Hox/PBC contacts that occur, compared with those mediated by the HX motif, on the opposing side of the DNA double helix. This provides a new molecular facet to Hox/PBC complex assembly and suggests possible mechanisms for the diversification of Hox protein function.

Gene expression and enzyme function of two cytochrome P450 3A isoenzymes in rat and cattle precision cut liver slices.

1. Precision-cut liver slices are one of the in vitro models used in studies concerning xenobiotic metabolism. Sparse information on this field is actually available for cattle and other veterinary species. 2. The aim of the current work was to study the effect of dexamethasone (DEX) on the gene expression and function of CYP3A23 (in rat), CYP3A28 (in cattle) and the transcriptional factors involved in their regulation. 3. DEX (at 100 µM) up-regulated CYP3A23 mRNA (3.2-fold, p = 0.028) in rat liver slices after 12 h culture, whereas the gene expression profiles of transcriptional factors involved in CYP3A regulation were unaffected. A CYP3A-dependent enzyme activity (triacetyl-oleandomycin N-demethylase) increased 3.4-fold (p < 0.05) in rat liver slices cultured in the presence of DEX. 4. The protocol used for rat liver slices was used as reference to study the expression of a CYP3A isoenzyme in cattle liver slices. Oppositely, DEX did neither affect the gene expression profile of CYP3A28 nor the CYP3A activity tested in cattle liver slices. 5. The data reported here are a further contribution to demonstrate the usefulness of liver slices as an in vitro tool for studies on the expression and function of xenobiotic metabolizing enzymes in cattle and in other ruminant species.

Effects of Sublethal Exposure to a Glyphosate-Based Herbicide Formulation on Metabolic Activities of Different Xenobiotic-Metabolizing Enzymes in Rats.

The activities of different xenobiotic-metabolizing enzymes in liver subcellular fractions from Wistar rats exposed to a glyphosate (GLP)-based herbicide (Roundup full II) were evaluated in this work. Exposure to the herbicide triggered protective mechanisms against oxidative stress (increased glutathione peroxidase activity and total glutathione levels). Liver microsomes from both male and female rats exposed to the herbicide had lower (45%-54%, P < 0.01) hepatic cytochrome P450 (CYP) levels compared to their respective control animals. In female rats, the hepatic 7-ethoxycoumarin O-deethylase (a general CYP-dependent enzyme activity) was 57% higher (P < 0.05) in herbicide-exposed compared to control animals. Conversely, this enzyme activity was 58% lower (P < 0.05) in male rats receiving the herbicide. Lower (P < 0.05) 7-ethoxyresorufin O-deethlyase (EROD, CYP1A1/2 dependent) and oleandomycin triacetate (TAO) N-demethylase (CYP3A dependent) enzyme activities were observed in liver microsomes from exposed male rats. Conversely, in females receiving the herbicide, EROD increased (123%-168%, P < 0.05), whereas TAO N-demethylase did not change. A higher (158%-179%, P < 0.01) benzyloxyresorufin O-debenzylase (a CYP2B-dependent enzyme activity) activity was only observed in herbicide-exposed female rats. In herbicide-exposed rats, the hepatic S-oxidation of methimazole (flavin monooxygenase dependent) was 49% to 62% lower (P < 0.001), whereas the carbonyl reduction of menadione (a cytosolic carbonyl reductase-dependent activity) was higher (P < 0.05). Exposure to the herbicide had no effects on enzymatic activities dependent on carboxylesterases, glutathione transferases, and uridinediphospho-glucuronosyltransferases. This research demonstrated certain biochemical modifications after exposure to a GLP-based herbicide. Such modifications may affect the metabolic fate of different endobiotic and xenobiotic substances. The pharmacotoxicological significance of these findings remains to be clarified.