Molecular Modeling of Epithiospecifier and Nitrile-Specifier Proteins of Broccoli and Their Interaction with Aglycones.

Glucosinolates are secondary plant metabolites of Brassicaceae. They exert their effect after enzymatic hydrolysis to yield aglycones, which become nitriles and epithionitriles through the action of epithiospecifier (ESP) and nitrile-specifier proteins (NSP). The mechanism of action of broccoli ESP and NSP is poorly understood mainly because ESP and NSP structures have not been completely characterized and because aglycones are unstable, thus hindering experimental measurements. The aim of this work was to investigate the interaction of broccoli ESP and NSP with the aglycones derived from broccoli glucosinolates using molecular simulations. The three-dimensional structure of broccoli ESP was built based on its amino-acid sequence, and the NSP structure was constructed based on a consensus amino-acid sequence. The models obtained using Iterative Threading ASSEmbly Refinement (I-TASSER) were refined with the OPLS-AA/L all atom force field of GROMACS 5.0.7 and were validated by Veryfy3D and ERRAT. The structures were selected based on molecular dynamics simulations. Interactions between the proteins and aglycones were simulated with Autodock Vina at different pH. It was concluded that pH determines the stability of the complexes and that the aglycone derived from glucoraphanin has the highest affinity to both ESP and NSP. This agrees with the fact that glucoraphanin is the most abundant glucosinolate in broccoli florets.

Descripción de un nuevo sistema cerrado de procesamiento de grasa para la obtención de nanofat: análisis de resultados clínicos y citométricos / Description of a new closed system to process fat and to obtain nanofat: analysis of clinical and cytometric results

Antecedentes y Objetivo. En la actualidad, la grasa es una de las herramientas más importantes en la Cirugía Plástica hecho, ha cambiado el enfoque de múltiples tratamientos y protocolos en la última década. Nanofat es un nuevo procedimiento derivado de emulsionar la grasa e inyectarla en dermis indicado para mejorar la calidad de la piel especialmente en ojeras, mejillas, cuello, escote y manos. En este trabajo describimos como obtener la grasa y procesarla con un circuito cerrado que hemos desarrollado así como nuestra experiencia con la técnica nanofat. Explicamos el procedimiento, sus aplicaciones clínicas, casos y estudios del tejido procesado para evaluar su contenido en células madre. Material y Método. Entre marzo de 2013 y febrero de 2015 realizamos la técnica nanofat en 56 pacientes sanos en la piel de mejillas, surcos nasogenianos, cuello, manos, escote y en particular para corregir la coloración de las ojeras, en combinación o no con microinjertos de grasa para tratar el surco nasoyugal. Para la infiltración de nanofat utilizamos una aguja de 27G, La cantidad infiltrada vario dependiendo del área a tratar; en ojeras el promedio fue de 2 ml, en cara y cuello de 7.5 ml, y en manos de 4.8 ml por mano. Infiltramos nanofat a nivel subdermico. Evaluamos el tejido emulsionado con citometria de flujo para determinar la viabilidad de las células del producto de esta grasa emulsionada, así como la presencia de células CD34 +. Llevamos a cabo la valoración clínica mediante fotografía y escala de satisfacción de Likert a los 3, 6 y 12 meses del tratamiento. Resultados. Todos los casos mostraron mejoría significativa a partir de los 3 a 6 meses del tratamiento. No hubo complicaciones mayores, ni observamos efectos indeseables. Recogimos solo 1 complicación menor (hiperpigmentacióon inflamatoria). Con el uso de citometria de flujo, demostramos que las células CD34 + viables estaban presentes en la grasa emulsionada. Conclusiones. Nanofat parece una herramienta eficaz para el rejuvenecimiento de la piel. Observamos una mejoría importante de la calidad y la elasticidad de la piel, disminución de arrugas finas y de la pigmentación en áreas como las ojeras. El uso de un circuito cerrado evita la manipulación directa, con lo cual redujimos la exposición del tejido al aire ambiente. El mecanismo fisiológico del nanofat mediante el cual se obtiene estos resultados positivos aún no ha sido completamente dilucidado; el efecto paracrino de las células madre derivadas de tejido adiposo (ADSC) no puede descartarse como mecanismo de acción (AU)

Background and Objective. Currently, fat is one of the most important tools in Aesthetic and Reconstructive Plastic Surgery. In fact, it has changed the approach of multiple treatments and protocols in the last decade. Nanofat is a new procedure derived from emulsifying the fat and injecting it into the dermal layer. The authors describe how to harvest the fat with a closed loop that they have developed, and their experience with nanofat technique. The procedure, clinical applications, cases reports, and tissue studies to evaluate its stemcell content will be explained. Method. Between March 2013 and February 2015, the nanofat technique was performed in 56 healthy patients. We apply this technique to the skin of cheeks, nasolabial folds, neck, hands, neckline and in particular to correct the color of dark circles in combination or not with micrograft to treat the nasal groove. For the infiltration of nanofat we used a needle of 27G, The infiltrated quantity of nanofat varied depending on the area to be treated, in the case of the dark circles the average was 2 ml, in face and neck was 7.5 ml and in hands was 4.8 ml per hand. The infiltration of nanofat was performed at the subdermal level. The emulsified tissue was evaluated with flow cytometry to determine the viability of the cells of the product of this emulsified fat, as well as the presence of CD34 + cells. The clinical assessment was done by means of photography and the Likert satisfaction scale at 3, 6 and 12 postoperative months. Results. All clinical cases showed significant improvement from 3 to 6 months after treatment. There were no major complications or undesirable effects. There was only 1 minor complication (inflammatory hyperpigmentation). With the use of flow cytometry, we have demonstrated that viable CD34+ cells were present in the emulsified fat. Conclusions. Nanofat looks like an effective tool for skin rejuvenation. We have observed a significant improvement in the quality and elasticity of the skin, decrease in fine wrinkles and pigmentation in areas such as dark circles. The use of a closed circuit avoids direct manipulation, in which the authors reduce the exposure of the tissue to ambient air. The physiological mechanism of nanofat through which these positive results are obtained has not yet been fully elucidated; the paracrine effect of stem cells derived from adipose tissue (ADSC) cannot be ruled out as a mechanism of action (AU)

Spontaneous trigeminal allodynia in rats: a model of primary headache.

Animal models are essential for studying the pathophysiology of headache disorders and as a screening tool for new therapies. Most animal models modify a normal animal in an attempt to mimic migraine symptoms. They require manipulation to activate the trigeminal nerve or dural nociceptors. At best, they are models of secondary headache. No existing model can address the fundamental question: How is a primary headache spontaneously initiated? In the process of obtaining baseline periorbital von Frey thresholds in a wild-type Sprague-Dawley rat, we discovered a rat with spontaneous episodic trigeminal allodynia (manifested by episodically changing periorbital pain threshold). Subsequent mating showed that the trait is inherited. Animals with spontaneous trigeminal allodynia allow us to study the pathophysiology of primary recurrent headache disorders. To validate this as a model for migraine, we tested the effects of clinically proven acute and preventive migraine treatments on spontaneous changes in rat periorbital sensitivity. Sumatriptan, ketorolac, and dihydroergotamine temporarily reversed the low periorbital pain thresholds. Thirty days of chronic valproic acid treatment prevented spontaneous changes in trigeminal allodynia. After discontinuation, the rats returned to their baseline of spontaneous episodic threshold changes. We also tested the effects of known chemical human migraine triggers. On days when the rats did not have allodynia and showed normal periorbital von Frey thresholds, glycerol trinitrate and calcitonin gene related peptide induced significant decreases in the periorbital pain threshold. This model can be used as a predictive model for drug development and for studies of putative biomarkers for headache diagnosis and treatment.

Cyclical expression of the Notch/Wnt regulator Nrarp requires modulation by Dll3 in somitogenesis.

Delta-like 3 (Dll3) is a divergent ligand and modulator of the Notch signaling pathway only identified so far in mammals. Null mutations of Dll3 disrupt cycling expression of Notch targets Hes1, Hes5, and Lfng, but not of Hes7. Compared with Dll1 or Notch1, the effects of Dll3 mutations are less severe for gene expression in the presomitic mesoderm, yet severe segmentation phenotypes and vertebral defects result in both human and mouse. Reasoning that Dll3 specifically disrupts key regulators of somite cycling, we carried out functional analysis to identify targets accounting for the segmental phenotype. Using microdissected embryonic tissue from somitic and presomitic mesodermal tissue, we identified new genes enriched in these tissues, including Limch1, Rhpn2, and A130022J15Rik. Surprisingly, we only identified a small number of genes disrupted by the Dll3 mutation. These include Uncx, a somite gene required for rib and vertebral patterning, and Nrarp, a regulator of Notch/Wnt signaling in zebrafish and a cycling gene in mouse. To determine the effects of Dll3 mutation on Nrarp, we characterized the cycling expression of this gene from early (8.5 dpc) to late (10.5 dpc) somitogenesis. Nrarp displays a distinct pattern of cycling phases when compared to Lfng and Axin2 (a Wnt pathway gene) at 9.5 dpc but appears to be in phase with Lfng by 10.5 dpc. Nrarp cycling appears to require Dll3 but not Lfng modulation. In Dll3 null embryos, Nrarp displayed static patterns. However, in Lfng null embryos, Nrarp appeared static at 8.5 dpc but resumed cycling expression by 9.5 and dynamic expression at 10.5 dpc stages. By contrast, in Wnt3a null embryos, Nrarp expression was completely absent in the presomitic mesoderm. Towards identifying the role of Dll3 in regulating somitogenesis, Nrarp emerges as a potentially important regulator that requires Dll3 but not Lfng for normal function.

Bile duct proliferation in Jag1/fringe heterozygous mice identifies candidate modifiers of the Alagille syndrome hepatic phenotype.

UNLABELLED: Alagille syndrome (AGS) is a heterogeneous developmental disorder associated with bile duct paucity and various organ anomalies. The syndrome is caused by mutations in JAG1, which encodes a ligand in the Notch signaling pathway, in the majority of cases and mutations in the NOTCH2 receptor gene in less than 1% of patients. Although a wide array of JAG1 mutations have been identified in the AGS population, these mutational variants have not accounted for the wide phenotypic variability observed in patients with this syndrome. The Fringe genes encode glycosyltransferases, which modify Notch and alter ligand-receptor affinity. In this study, we analyzed double heterozygous mouse models to examine the Fringe genes as potential modifiers of the Notch-mediated hepatic phenotype observed in AGS. We generated mice that were haploinsufficient for both Jag1 and one of three paralogous Fringe genes: Lunatic (Lfng), Radical (Rfng), and Manic (Mfng). Adult Jag1(+/-)Lfng(+/-) and Jag1(+/-)Rfng(+/-) mouse livers exhibited widespread bile duct proliferation beginning at 5 weeks of age and persisting up to 1 year. The Jag1(+/-)Mfng(+/-) livers showed a subtle, yet significant increase in bile duct numbers and bile duct to portal tract ratios. These abnormalities were not observed in the newborn period. Despite the portal tract expansion by bile ducts, fibrosis was not increased and epithelial to mesenchymal transition was not shown in the affected portal tracts. CONCLUSION: Mice heterozygous for mutations in Jag1 and the Fringe genes display striking bile duct proliferation, which is not apparent at birth. These findings suggest that the Fringe genes may regulate postnatal bile duct growth and remodeling, and serve as candidate modifiers of the hepatic phenotype in AGS.

Dll3 and Notch1 genetic interactions model axial segmental and craniofacial malformations of human birth defects.

Mutations in the Notch1 receptor and delta-like 3 (Dll3) ligand cause global disruptions in axial segmental patterning. Genetic interactions between members of the notch pathway have previously been shown to cause patterning defects not observed in single gene disruptions. We examined Dll3-Notch1 compound mouse mutants to screen for potential gene interactions. While mice heterozygous at either locus appeared normal, 30% of Dll3-Notch1 double heterozygous animals exhibited localized, segmental anomalies similar to human congenital vertebral defects. Unexpectedly, double heterozygous mice also displayed statistically significant reduction of mandibular height and decreased length of the [corrected] maxillary hard palate. Examination of somite-stage embryos and perinatal anatomy and histology did not reveal any organ defects, so we used microarray-based analysis of Dll3 and Notch1 mutant embryos to identify gene targets that may be involved in notch-regulated segmental or craniofacial development. Thus, Dll3-Notch1 double heterozygous mice model human congenital scoliosis and craniofacial disorders.

Identification of oscillatory genes in somitogenesis from functional genomic analysis of a human mesenchymal stem cell model.

During somitogenesis, oscillatory expression of genes in the notch and wnt signaling pathways plays a key role in regulating segmentation. These oscillations in expression levels are elements of a species-specific developmental mechanism. To date, the periodicity and components of the human clock remain unstudied. Here we show that a human mesenchymal stem/stromal cell (MSC) model can be induced to display oscillatory gene expression. We observed that the known cycling gene HES1 oscillated with a 5 h period consistent with available data on the rate of somitogenesis in humans. We also observed cycling of Hes1 expression in mouse C2C12 myoblasts with a period of 2 h, consistent with previous in vitro and embryonic studies. Furthermore, we used microarray and quantitative PCR (Q-PCR) analysis to identify additional genes that display oscillatory expression both in vitro and in mouse embryos. We confirmed oscillatory expression of the notch pathway gene Maml3 and the wnt pathway gene Nkd2 by whole mount in situ hybridization analysis and Q-PCR. Expression patterns of these genes were disrupted in Wnt3a(tm1Amc) mutants but not in Dll3(pu) mutants. Our results demonstrate that human and mouse in vitro models can recapitulate oscillatory expression observed in embryo and that a number of genes in multiple developmental pathways display dynamic expression in vitro.