RESUMEN
Response surface methodology (RSM) is a widely used mathematical and statistical technique for modeling and optimizing the process for the extraction of bioactive compounds. This review explains the optimization approach through the use of experimental design and empirical models for response prediction and the utilization of the desirability function for multiple response optimization. This paper also reviews recent studies on the application of RSM to optimize bioactive compound extraction processes such as conventional solvent extraction, microwave-assisted extraction, supercritical fluid extraction, and ultrasound-assisted extraction. Finally, the challenges associated with the use of RSM and the efforts made to improve RSM in the extraction process are also highlighted. Overall, this review informs many aspects of RSM that are occasionally ignored or insufficiently discussed with regard to the optimization of bioactive compound extraction processes, and it summarizes significant applications where RSM proved suitable. © 2022 Society of Chemical Industry.
Asunto(s)
Fraccionamiento Químico , Cromatografía con Fluido Supercrítico , Fraccionamiento Químico/métodos , Extractos Vegetales/química , MicroondasRESUMEN
Vertebrate appendage patterning is programmed by Hox-TALE factor-bound regulatory elements. However, it remains unclear which cell lineages are commissioned by Hox-TALE factors to generate regional specific patterns and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis.
Asunto(s)
Tipificación del Cuerpo , Extremidades/embriología , Proteínas de Homeodominio/metabolismo , Osteogénesis , Animales , Secuencia de Bases , Sitios de Unión/genética , Tipificación del Cuerpo/genética , Linaje de la Célula , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Elementos de Facilitación Genéticos , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Genoma , Proteínas de Homeodominio/genética , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Modelos Biológicos , Motivos de Nucleótidos/genética , Osteogénesis/genética , Unión ProteicaRESUMEN
Disruption of presumptive enhancers downstream of the human SHOX gene (hSHOX) is a frequent cause of the zeugopodal limb defects characteristic of Léri-Weill dyschondrosteosis (LWD). The closely related mouse Shox2 gene (mShox2) is also required for limb development, but in the more proximal stylopodium. In this study, we used transgenic mice in a comparative approach to characterize enhancer sequences in the hSHOX and mShox2 genomic regions. Among conserved noncoding elements (CNEs) that function as enhancers in vertebrate genomes, those that are maintained near paralogous genes are of particular interest given their ancient origins. Therefore, we first analyzed the regulatory potential of a genomic region containing one such duplicated CNE (dCNE) downstream of mShox2 and hSHOX. We identified a strong limb enhancer directly adjacent to the mShox2 dCNE that recapitulates the expression pattern of the endogenous gene. Interestingly, this enhancer requires sequences only conserved in the mammalian lineage in order to drive strong limb expression, whereas the more deeply conserved sequences of the dCNE function as a neural enhancer. Similarly, we found that a conserved element downstream of hSHOX (CNE9) also functions as a neural enhancer in transgenic mice. However, when the CNE9 transgenic construct was enlarged to include adjacent, non-conserved sequences frequently deleted in LWD patients, the transgene drove expression in the zeugopodium of the limbs. Therefore, both hSHOX and mShox2 limb enhancers are coupled to distinct neural enhancers. This is the first report demonstrating the activity of cis-regulatory elements from the hSHOX and mShox2 genomic regions in mammalian embryos.