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BACKGROUND: Forensic psychiatry is often associated with long admissions and has a high cost of care. There is little known about factors influencing length of stay (LOS), and no previous systematic review has synthesised the available data. This paper aims to identify factors influencing the LOS in forensic psychiatry hospitals to inform care and interventions that may reduce the length of admissions. METHODOLOGY: A systematic review was conducted by searching major databases, including PubMed, EMBASE and PsycInfo, from inception until May 2022. Observational studies conducted in forensic hospitals that examined associations between variables of interest and LOS were included. Following data extraction, the NewcastleâOttawa Scale was used for quality appraisal. No meta-analysis was conducted due to heterogeneity of information; a quantitative measure to assess the strength of evidence was developed and reported. RESULTS: A total of 28 studies met the inclusion criteria out of 1606 citations. A detailed quantitative synthesis was performed using robust criteria. Having committed homicide/attempted homicide, a criminal legal status with restrictions, and a diagnosis of schizophrenia-spectrum disorders were all associated with longer LOS. Higher Global Assessment of Functioning (GAF) scores were associated with a shorter LOS. CONCLUSION: High-quality research examining factors associated with LOS in forensic psychiatry is lacking, and studies are heterogeneous. No modifiable characteristics were identified, and thus, practice recommendations were not made. There is an increasing necessity to understand the factors associated with longer admissions to inform care and increase success in reintegration and rehabilitation. This paper provides recommendations for future research.
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Psiquiatría Forense , Hospitales Psiquiátricos , Tiempo de Internación , Humanos , Tiempo de Internación/estadística & datos numéricos , Trastornos Mentales/terapia , FemeninoRESUMEN
Eight cases of locally acquired, mosquito-transmitted (i.e., autochthonous) Plasmodium vivax malaria, which has not been reported in the United States since 2003, were reported to CDC from state health departments in Florida and Texas during May 18-July 17, 2023. As of August 4, 2023, case surveillance, mosquito surveillance and control activities, and public outreach and education activities continue in both states. U.S. clinicians need to consider a malaria diagnosis in patients with unexplained fever, especially in areas where autochthonous malaria has been recently reported, although the risk for autochthonous malaria in the United States remains very low. Prompt diagnosis and treatment of malaria can prevent severe disease or death and limit ongoing transmission to local Anopheles mosquitoes and other persons. Preventing mosquito bites and controlling mosquitoes at home can prevent mosquitoborne diseases, including malaria. Before traveling internationally to areas with endemic malaria, travelers should consult with a health care provider regarding recommended malaria prevention measures, including potentially taking malaria prophylaxis. Malaria is a nationally notifiable disease; continued reporting of malaria cases to jurisdictional health departments and CDC will also help ensure robust surveillance to detect and prevent autochthonous malaria in the United States.
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Brotes de Enfermedades , Malaria , Animales , Humanos , Texas/epidemiología , Florida/epidemiología , Malaria/epidemiología , Malaria/prevención & control , Personal de SaludRESUMEN
Inflammatory bowel disease (IBD) is an illness with increasing prevalence, particularly in emerging countries, which can have a substantial impact on the quality of life of the patient. The illness is rather heterogeneous with different evolution among patients. A machine learning approach is followed in this paper to identify potential genes that are related to IBD. This is done by following a Monte Carlo simulation approach. In total, 23 different machine learning techniques were tested (in addition to a base level obtained using artificial neural networks). The best model identified 74 genes selected by the algorithm as being potentially involved in IBD. IBD seems to be a polygenic illness, in which environmental factors might play an important role. Following a machine learning approach, it was possible to obtain a classification accuracy of 84.2% differentiating between patients with IBD and control cases in a large cohort of 2490 total cases. The sensitivity and specificity of the model were 82.6% and 84.4%, respectively. It was also possible to distinguish between the two main types of IBD: (1) Crohn's disease and (2) ulcerative colitis.
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Colitis Ulcerosa , Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Humanos , Calidad de Vida , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/genética , Enfermedades Inflamatorias del Intestino/epidemiología , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/genética , Colitis Ulcerosa/genética , Aprendizaje AutomáticoRESUMEN
Twenty-six triazole-based derivatives were designed for targeting both PD-L1 (programmed death receptor ligand 1) and VEGFR-2 (vascular endothelial growth factor receptor 2). These compounds were synthetized and biologically evaluated as multitarget inhibitors of VEGFR-2, PD-L1 and c-Myc proteins. The antiproliferative activity of these molecules on several tumor cell lines (HT-29, A-549, and MCF-7) and on the non-tumor cell line HEK-293 was determined. The effects on the abovementioned biological targets were evaluated for some selected compounds. Compound 23, bearing a p-chlorophenyl group, showed better results than sorafenib in regard to the downregulation of VEGFR-2 and a similar effect to BMS-8 on both PD-L1 and c-Myc proteins. The antiangiogenic and antivascular activities of chloro derivatives were also established by endothelial microtube formation assay on Matrigel®.
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Antineoplásicos , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Antígeno B7-H1 , Línea Celular Tumoral , Proliferación Celular , Ensayos de Selección de Medicamentos Antitumorales , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Proteínas Proto-Oncogénicas c-myc/metabolismo , Relación Estructura-Actividad , Triazoles/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Here, we combine the use of host screening, protein crystallography and QM/MM molecular dynamics simulations to investigate how the protein structure affects iminium catalysis by biotinylated secondary amines in a model 1,4 conjugate addition reaction. Monomeric streptavidin (M-Sav) lacks a quaternary structure and the solvent-exposed reaction site resulted in poor product conversion in the model reaction with low enantio- and regioselectivities. These parameters were much improved when the tetrameric host T-Sav was used; indeed, residues at the symmetrical subunit interface were proven to be critical for catalysis through a mutagenesis study. The use of QM/MM simulations and the asymmetric dimeric variant D-Sav revealed that both Lys121 residues which are located in the hosting and neighboring subunits play a critical role in controlling the stereoselectivity and reactivity. Lastly, the D-Sav template, though providing a lower conversion than that of the symmetric tetrameric counterpart, is likely a better starting point for future protein engineering because each surrounding residue within the asymmetric scaffold can be refined for secondary amine catalysis.
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EstreptavidinaRESUMEN
BACKGROUND: Crocins are soluble apocarotenoids that mainly accumulate in the stigma tissue of Crocus sativus and provide the characteristic red color to saffron spice, in addition to being responsible for many of the medicinal properties of saffron. Crocin biosynthesis and accumulation in saffron is developmentally controlled, and the concentration of crocins increases as the stigma develops. Until now, little has been known about the molecular mechanisms governing crocin biosynthesis and accumulation. This study aimed to identify the first set of gene regulatory processes implicated in apocarotenoid biosynthesis and accumulation. RESULTS: A large-scale crocin-mediated RNA-seq analysis was performed on saffron and two other Crocus species at two early developmental stages coincident with the initiation of crocin biosynthesis and accumulation. Pairwise comparison of unigene abundance among the samples identified potential regulatory transcription factors (TFs) involved in crocin biosynthesis and accumulation. We found a total of 131 (up- and downregulated) TFs representing a broad range of TF families in the analyzed transcriptomes; by comparison with the transcriptomes from the same developmental stages from other Crocus species, a total of 11 TF were selected as candidate regulators controlling crocin biosynthesis and accumulation. CONCLUSIONS: Our study generated gene expression profiles of stigmas at two key developmental stages for apocarotenoid accumulation in three different Crocus species. Differential gene expression analyses allowed the identification of transcription factors that provide evidence of environmental and developmental control of the apocarotenoid biosynthetic pathway at the molecular level.
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Carotenoides/biosíntesis , Crocus/genética , Regulación de la Expresión Génica de las Plantas , Carotenoides/análisis , Cromatografía Líquida de Alta Presión , Dioxigenasas/genética , Dioxigenasas/metabolismo , Perfilación de la Expresión Génica , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plastidios/genética , Plastidios/metabolismo , ARN de Planta/química , ARN de Planta/metabolismo , Análisis de Secuencia de ARN , Espectrometría de Masa por Ionización de Electrospray , Factores de Transcripción/genética , Factores de Transcripción/metabolismoAsunto(s)
Antipsicóticos , Catatonia , Clozapina , Terapia Electroconvulsiva , Esquizofrenia , Humanos , Clozapina/efectos adversos , Esquizofrenia/tratamiento farmacológico , Antipsicóticos/efectos adversos , Terapia Combinada , Catatonia/tratamiento farmacológico , Esquizofrenia Catatónica/tratamiento farmacológicoRESUMEN
The origin of the catalytic power of enzymes has been a question of debate for a long time. In this regard, the possible contribution of protein dynamics in enzymatic catalysis has become one of the most controversial topics. In the present work, the hydride transfer step in the formate dehydrogenase (FDH EC 1.2.1.2) enzyme is studied by means of molecular dynamic (MD) simulations with quantum mechanics/molecular mechanics (QM/MM) potentials in order to explore any correlation between dynamics, tunnelling effects and the rate constant. The temperature dependence of the kinetic isotope effects (KIEs), which is one of the few tests that can be studied by experiments and simulations to shed light on this debate, has been computed and the results have been compared with previous experimental data. The classical mechanical free energy barrier and the number of recrossing trajectories seem to be temperature-independent while the quantum vibrational corrections and the tunnelling effects are slightly temperature-dependent over the interval of 5-45 °C. The computed primary KIEs are in very good agreement with previous experimental data, being almost temperature-independent within the standard deviations. The modest dependence on the temperature is due to just the quantum vibrational correction contribution. These results, together with the analysis of the evolution of the collective variables such as the electrostatic potential or the electric field created by the protein on the key atoms involved in the reaction, confirm that while the protein is well preorganised, some changes take place along the reaction that favour the hydride transfer and the product release. Coordinates defining these movements are, in fact, part of the real reaction coordinate.
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Formiato Deshidrogenasas/metabolismo , Isótopos/química , Temperatura , Formiato Deshidrogenasas/química , CinéticaRESUMEN
There has been growing interest in performing organocatalysis within a supramolecular system as a means of controlling reaction reactivity and stereoselectivity. Here, a protein is used as a host for iminium catalysis. A pyrrolidine moiety is covalently linked to biotin and introduced to the protein host streptavidin for organocatalytic activity. Whereas in traditional systems stereoselectivity is largely controlled by the substituents added to the organocatalyst, enantiomeric enrichment by the reported supramolecular system is completely controlled by the host. Also, the yield of the model reaction increases over 10-fold when streptavidin is included. A 1.1â Å crystal structure of the protein-catalyst complex and molecular simulations of a key intermediate reveal the chiral scaffold surrounding the organocatalytic reaction site. This work illustrates that proteins can be an excellent supramolecular host for driving stereoselective secondary amine organocatalysis.
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Iminas/química , Estreptavidina/química , Sitios de Unión , Biotina/química , Biotina/metabolismo , Biotinilación , Catálisis , Cromatografía de Gases y Espectrometría de Masas , Enlace de Hidrógeno , Conformación Molecular , Simulación de Dinámica Molecular , Estereoisomerismo , Estreptavidina/metabolismoRESUMEN
The carotenoid cleavage dioxygenase 2, a new member of the CCD family, catalyzes the conversion of zeaxanthin into crocetin-dialdehyde in Crocus. CCD2 is expressed in flowers, being responsible for the yellow, orange and red colorations displayed by tepals and stigma. Three CsCCD2 genes were identified in Crocus sativus, the longest contains ten exons and the shorter is a truncated copy with no introns and which lacks one exon sequence. Analysis of RNA-seq datasets of three developmental stages of saffron stigma allowed the determination of alternative splicing in CsCCD2, being intron retention (IR) the prevalent form of alternative splicing in CsCCD2. Further, high IR was observed in tissues that do not accumulate crocetin. The analysis of one CsCCD2 promoter showed cis-regulatory motifs involved in the response to light, temperature, and circadian regulation. The light and circadian regulation are common elements shared with the previously characterized CsLycB2a promoter, and these shared common cis-acting elements may represent binding sites for transcription factors responsible for co-regulation of these genes during the development of the stigma in saffron. A daily coordinated rhythmic regulation for CsCCD2 and CsLycB2a was observed, with higher levels of mRNA occurring at low temperatures during darkness, confirming the results obtained in the in silico promoter analysis. In addition, to the light and temperature dependent regulation of CsCCD2 expression, the apocarotenoid ß-cyclocitral up-regulated CsCCD2 expression and could acts as a mediator of chromoplast-to-nucleus signalling, coordinating the expression of CsCCD2 with the developmental state of the chromoplast in the developing stigma.
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Carotenoides/metabolismo , Crocus/metabolismo , Dioxigenasas/metabolismo , Intrones/fisiología , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Carotenoides/biosíntesis , Dioxigenasas/genética , Dioxigenasas/fisiología , Regulación de la Expresión Génica de las Plantas/fisiología , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiología , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Vitamina A/análogos & derivados , Zeaxantinas/metabolismoRESUMEN
CYP19A1 aromatase is a member of the Cytochrome P450 family of hemeproteins, and is the enzyme responsible for the final step of the androgens conversion into the corresponding estrogens, via a three-step oxidative process. For this reason, the inhibition of this enzyme plays an important role in the treatment of hormone-dependent breast cancer. The first catalytic subcycle, corresponding to the hydroxilation of androstenedione, has been proposed to occur through a first hydrogen abstraction and a subsequent oxygen rebound step. In present work, we have studied the mechanism of the first catalytic subcycle by means of hybrid quantum mechanics/molecular mechanics methods. The inclusion of the protein flexibility has been achieved by means of Free Energy Perturbation techniques, giving rise to a free energy of activation for the hydrogen abstraction step of 13.5 kcal/mol. The subsequent oxygen rebound step, characterized by a small free energy barrier (1.5 kcal/mol), leads to the hydroxylated products through a highly exergonic reaction. In addition, an analysis of the primary deuterium kinetic isotopic effects, calculated for the hydrogen abstraction step, reveals values (â¼10) overpassing the semiclassical limit for the CH, indicating the presence of a substantial tunnel effect. Finally, a decomposition analysis of the interaction energy for the substrate and cofactor in the active site is also discussed. According to our results, the role of the enzymatic environment consists of a transition state stabilization by means of dispersive and polarization effects.
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Androstenodiona/metabolismo , Aromatasa/metabolismo , Androstenodiona/química , Aromatasa/química , Neoplasias de la Mama/enzimología , Dominio Catalítico , Femenino , Humanos , Hidroxilación , Simulación de Dinámica Molecular , Oxígeno/metabolismo , Teoría Cuántica , TermodinámicaRESUMEN
Dihydroxyacetone (DHA) kinase from Citrobacter freundii provides an easy entry for the preparation of DHA phosphate; a very important C3 building block in nature. To modify the phosphoryl donor specificity of this enzyme from ATP to inorganic polyphosphate (poly-P); a directed evolution program has been initiated. In the first cycle of evolution, the native enzyme was subjected to one round of error-prone PCR (EP-PCR) followed directly (without selection) by a round of DNA shuffling. Although the wild-type DHAK did not show activity with poly-P, after screening, sixteen mutant clones showed an activity with poly-phosphate as phosphoryl donor statistically significant. The most active mutant presented a single mutation (Glu526Lys) located in a flexible loop near of the active center. Interestingly, our theoretical studies, based on molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) optimizations, suggest that this mutation has an effect on the binding of the poly-P favoring a more adequate position in the active center for the reaction to take place.
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Adenosina Trifosfato/química , Modelos Moleculares , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Polifosfatos/química , Adenosina Trifosfato/metabolismo , Sitios de Unión , Biblioteca de Genes , Conformación Molecular , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Polifosfatos/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Background: The COVID-19 pandemic resulted in an abrupt transition from in-person to online learning in Spring 2020. Objective: The purpose of this study was to identify the impact of the transition on undergraduates during the period following the campus closure. Method: 131 psychology undergraduate students completed an online survey of how the COVID-19 closure had impacted their academics, online learning environment, and traumatic stress symptoms (using the Posttraumatic Stress Disorder Checklist for the DSM-5). Results: Most participants reported increased academic difficulty compared to before closure. Approximately 30% reported elevated traumatic stress symptoms. Greater traumatic stress was associated with greater difficulty completing assignments, more limited access to the internet and quiet places to study, and greater sibling-care responsibilities. Conclusions: The acute transition to online instruction posed academic and emotional challenges to many students, especially those from environments with competing demands or less access to academic supports. Follow-up evaluation is needed to determine whether these difficulties have persisted in subsequent semesters of online instruction. Teaching Implication: Instructors should anticipate the emotional and academic needs of students who are relatively unfamiliar with online instruction and consider ways to minimize negative environmental impacts and increase access to mental health resources.
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Staphylococcus aureus is the main etiological agent of mastitis in small ruminants worldwide. This disease has a difficult cure and possible relapse, leading to significant economic losses in production, milk quality and livestock. This study performed comparative genomic analyses between 73 S. aureus genomes from different hosts (human, bovine, pig and others). This work isolated and sequenced 12 of these genomes from ovine. This study contributes to the knowledge of genomic specialization and the role of specific genes in establishing infection in ovine mastitis-associated S. aureus. The genomes of S. aureus isolated from sheep maintained a higher representation when grouped with clonal complexes 130 and 133. The genomes showed high genetic similarity, the species pan-genome consisting of 4200 genes (central = 2008, accessory = 1559 and unique = 634). Among these, 277 unique genes were related to the genomes isolated from sheep, with 39.6 % as hypothetical proteins, 6.4 % as phages, 6.4 % as toxins, 2.9 % as transporters, and 44.7 % as related to other proteins. Furthermore, at the pathogen level, they showed 80 genes associated with virulence factors and 19 with antibiotic resistance shared in almost all isolates. Although S. aureus isolated from ovine showed susceptibility to antimicrobials in vitro, ten genes were predicted to be associated with antibiotic inactivation and efflux pump, suggesting resistance to gentamicin and penicillin. This work may contribute to identifying genes acquired by horizontal transfer and their role in host adaptation, virulence, bacterial resistance, and characterization of strains affecting ovine.
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Mastitis Bovina , Infecciones Estafilocócicas , Femenino , Animales , Bovinos , Ovinos/genética , Humanos , Porcinos , Factores de Virulencia/genética , Staphylococcus aureus/genética , Adaptación al Huésped , Infecciones Estafilocócicas/genética , Infecciones Estafilocócicas/veterinaria , Infecciones Estafilocócicas/microbiología , Rumiantes/genética , Genómica , Secuencias Repetitivas Esparcidas , Mastitis Bovina/genética , Mastitis Bovina/microbiologíaRESUMEN
Mounting a balanced and robust humoral immune response is of utmost importance for reducing the infectivity of Trypanosoma cruzi. While the role of such a response in controlling the infection is well known, there is a lack of tools that can be used to quickly evaluate it. We developed a serum parasite inhibition assay (to evaluate changes in the parasite infection after exposing infective T. cruzi trypomastigotes to serum samples from infected patients). It is based on Vero cells as the hosts and the Tulahuen ß-galactosidase parasite strain, genetically engineered to be quantifiable by spectrophotometry. In parallel, we developed an in-house ELISA to correlate the anti-T. cruzi antibody titres of the clinical samples with their observed anti-parasitic effect in the serum parasite inhibition assay. Serum samples from chronically T. cruzi-infected patients significantly inhibited parasite invasion in a titre-dependant manner, regardless of the patient's clinical status, compared to samples from the non-infected controls. In addition, there was a clear correlation between the reactivity of the samples to the whole-parasite lysates by ELISA and the inhibitory effect. The results of this work confirm the previously described anti-parasitic effect of the serum of individuals exposed to T. cruzi and present a framework for its large-scale evaluation in further studies. The serum parasite inhibition assay represents a reproducible way to evaluate the intensity and anti-parasitic effect of humoral responses against T. cruzi, which could be applied to the evaluation of candidate antigens/epitopes in the design of Chagas disease vaccine candidates.
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A theoretical study on the alkaline hydrolysis of paraoxon, one of the most popular organophosphorus pesticides, in aqueous solution and in the active site of Pseudomonas diminuta phosphotriesterase (PTE) is presented. Simulations by means of hybrid quantum mechanics/molecular mechanics (QM/MM) potentials show that the hydrolysis of paraoxon takes place through an A(N)D(N) or associative mechanism both in solution and in the active site of PTE. The results correctly reproduce the magnitude of the activation free energies and can be used to rationalize the observed kinetic isotope effects (KIEs) for the hydrolysis of paraoxon in both media. Enzymatic hydrolysis of O,O-diethyl p-chlorophenyl phosphate, a phosphotriester having a leaving group with higher pK(a) than paraoxon, was also simulated. Hydrolysis of this phosphotriester by PTE follows a A(N)+D(N) mechanism with a pentacoordinate intermediate. Moreover, the leaving group of this new substrate coordinates to one of the zinc ions of the bimetallic active site in order to stabilize the large negative charge developed on the oxygen atom of the leaving group when the P-O bond is broken in the products state. To accommodate this new ligand in the coordination shell, carbamylated Lys169 must be displaced from one zinc ion to the other, which in turn affects the acidity of Asp301, a residue originally bound to the second zinc ion. This ability to displace some of the ligands of the coordination shell of the zinc centers would explain the promiscuity of this enzyme, which is capable of catalyzing hydrolysis of different substrate by means of different mechanisms.
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Modelos Teóricos , Paraoxon/farmacología , Hidrolasas de Triéster Fosfórico/metabolismo , Pseudomonas/enzimología , Catálisis , Dominio Catalítico , Hidrólisis , Paraoxon/química , Pseudomonas/metabolismo , Soluciones , Zinc/químicaRESUMEN
Lateral pterygoid muscle dystonia is characterized by mandibular displacement towards the opposite side of the affected muscle. It may be associated with functional disorders affecting speech, swallowing, chewing and facial symmetry. Injection with botulinum toxin is recognized as the most effective treatment. Locating the lower head of the lateral pterygoid muscle for the injection is not difficult using electromyographic guidance; however, location of the upper head is more complicated, even with electromyography. We report a case of lateral pterygoid muscle dystonia in which precise injection of the upper head was achieved with the aid of arthroscopy.
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Toxinas Botulínicas Tipo A/administración & dosificación , Distonía/tratamiento farmacológico , Fármacos Neuromusculares/administración & dosificación , Músculos Pterigoideos , Artroscopía , Electromiografía , Femenino , Humanos , Persona de Mediana EdadRESUMEN
Neglected tropical diseases are infectious diseases that impose high morbidity and mortality rates over 1.5 billion people worldwide. Originally restricted to tropical and subtropical regions, changing climate conditions have increased their potential to emerge elsewhere. Control of their impact suffers from shortages like poor epidemiological surveillance or irregular drug distribution, and some NTDs still lack of appropriate diagnostics and/or efficient therapeutics. For these, availability of vaccines to prevent new infections, or the worsening of those already established, would mean a major breakthrough. However, only dengue and rabies count with approved vaccines at present. Herein, we review the state-of-the-art of vaccination strategies for NTDs, setting the focus on third generation vaccines and the concept of reverse vaccinology. Its capability to address pathogens´ biological complexity, likely contributing to save developmental costs is discussed. The use of computational tools is a fundamental aid to analyze increasingly large datasets aimed at designing vaccine candidates with the highest, possibly, opportunities to succeed. Ultimately, we identify and analyze those studies that took an in silico approach to find vaccine candidates, and experimentally assessed their immunogenicity and/or protection capabilities.
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A theoretical study of the protein dynamic effects on the hydride transfer between the formate anion and nicotinamide adenine dinucleotide (NAD(+)), catalyzed by formate dehydrogenase (FDH), is presented in this paper. The analysis of free downhill molecular dynamic trajectories, performed in the enzyme and compared with the reaction in aqueous solution, has allowed the study of the dynamic coupling between the reacting fragments and the protein or the solvent water molecules, as well as an estimation of the dynamic effect contribution to the catalytic effect from calculation of the transmission coefficient in the enzyme and in solution. The obtained transmission coefficients for the enzyme and in solution were 0.46±0.04 and 0.20±0.03, respectively. These values represent a contribution to catalysis of 0.5 kcal mol(-1), which, although small, is not negligible keeping in mind the low efficiency of FDH. The analysis of the reactive trajectories also reveals how the relative movements of some amino acids, mainly His332 and Arg284, precede and promote the chemical reaction. In spite of these movements, the time-dependent evolution of the electric field created by the enzyme on the key atoms of the reaction reveals a permanent field, which reduces the work required to reach the transition state, with a concomitant polarization of the cofactor. Finally, application of Grote-Hynes theory has allowed the identification of the modes responsible for the substrate-environment coupling, showing how some protein motions take place simultaneously with the reaction. Thus, the equilibrium approach would provide, in this case, an overestimation of the catalyzed rate constant.
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Formiato Deshidrogenasas/metabolismo , NAD/metabolismo , Algoritmos , Catálisis , Cinética , Modelos Teóricos , Estructura Molecular , Resonancia Magnética Nuclear BiomolecularRESUMEN
Colored apocarotenoids accumulate at high concentrations in few plant species, where display a role in attraction of pollinators and seed dispersers. Among these apocarotenoids, crocins accumulate at high concentrations in the stigma of saffron and are responsible for the organoleptic and medicinal properties of this spice. Phytoene synthase and Orange protein are key for carotenoid biosynthesis and accumulation. We previously isolated four phytoene synthase genes from saffron with differential roles in carotenoid and apocarotenoid biosynthesis. However, the implications of Orange genes in the regulation of apocarotenoid accumulation are unknown. Here, we have identified two Orange genes from saffron, with different expression patterns. CsOr-a was mainly expressed in vegetative tissues and was induced by light and repressed by heat stress. Both CsOr-a and CsOr-b were expressed in stigmas but showed a different profile during the development of this tissue. The interactions of CsOr-a and CsOr-b were tested with all the four phytoene synthase proteins from saffron and with CsCCD2. None interactions were detected with CCD2 neither with the phytoene synthase 2, involved in apocarotenoid biosynthesis in saffron. The obtained results provide evidence of different mechanisms regulating the phytoene synthase enzymes in saffron by Orange for carotenoid and apocarotenoid accumulation in saffron.