Concurrent starch accumulation in stump and high fruit production in coffee (Coffea arabica).

In coffee, fruit production on a given shoot drops after some years of high yield, triggering pruning to induce resprouting. The timing of pruning is a crucial farmer's decision affecting yield and labour. One reason for fruit production drop could be the exhaustion of resources, particularly the non-structural carbohydrates (NSC). To test this hypothesis in a Coffea L. arabica agroforestry system, we measured the concentrations of NSC, carbon (C) and nitrogen (N) in leaves, stems and stumps of the coffee plants, 2 and 5 years after pruning. We also compared shaded vs full sun plants. For that purpose, both analytical reference and visible and near infrared reflectance spectroscopy (VNIRS) methods were used. As expected, concentrations of biochemical variables linked to photosynthesis activity (N, glucose, fructose, sucrose) decreased from leaves to stems, and then to stumps. In contrast, variables linked more closely to plant structure and reserves (total C, C:N ratio, starch concentration) were higher in long lifespan organs like stumps. Shading had little effect on most measured parameters, contrary to expectations. Concentrations of N, glucose and fructose were higher in 2-year-old organs. Conversely, starch concentration in perennial stumps was three times higher 5 years after pruning than 2 years after pruning, despite high fruit production. Therefore, the drop in fruit production occurring after 5-6 years was not due to a lack of NSC on plant scale. Starch accumulation in perennial organs concurrently to other sinks, such as fruit growth, could be considered as a 'survival' strategy, which may be a relic of the behaviour of wild coffee (a tropical shade-tolerant plant). This study confirmed that VNIRS is a promisingly rapid and cost-effective option for starch monitoring (coefficient of determination for validation, R2val = 0.91), whereas predictions were less accurate for soluble sugars, probably due to their too similar spectral signature.

Stimulation of Fibronectin Matrix Assembly by Lysine Acetylation.

Diabetic nephropathy, a devastating consequence of diabetes mellitus, is characterized by the accumulation of extracellular matrix (ECM) that disrupts the kidney's filtration apparatus. Elevated glucose levels increase the deposition of a fibronectin (FN) matrix by mesangial cells, the primary matrix-producing cells of the kidney, and also increase acetyl-CoA leading to higher levels of lysine acetylation. Here, we investigated the connection between acetylation and the ECM and show that treatment of mesangial cells with deacetylase inhibitors increases both acetylation and FN matrix assembly compared to untreated cells. The matrix effects were linked to lysine 794 (K794) in the ß1 integrin cytoplasmic domain based on studies of cells expressing acetylated (K794Q) and non-acetylated (K794R) mimetics. ß1(K794Q) cells assembled significantly more FN matrix than wildtype ß1 cells, while the non-acetylated ß1(K794R) form was inactive. We show that mutation of K794 affects FN assembly by stimulating integrin-FN binding activity and cell contractility. Wildtype and ß1(K794Q) cells but not ß1(K794R) cells further increased their FN matrix when stimulated with deacetylase inhibitors indicating that increased acetylation on other proteins is required for maximum FN assembly. Thus, lysine acetylation provides a mechanism for glucose-induced fibrosis by up-regulation of FN matrix assembly.

Elevated glucose alters global gene expression and tenascin-C alternative splicing in mesangial cells.

Mesangial cells are the major extracellular matrix (ECM)-producing cells in the kidney glomerulus and, when exposed to elevated glucose levels, they up-regulate assembly of fibronectin (FN) and other ECM proteins. Increases in glucose concentration are known to alter gene expression; here we investigated the connection between increased ECM production and changes in gene expression in mesangial cells. Comparison of mesangial cells grown in normal or high glucose conditions by RNA-sequencing showed significant expression changes in over 6000 genes and, when grouped by KEGG pathway analysis, identified the ECM-receptor interaction and focal adhesion pathways among the top 5 upregulated pathways. Of note was the significant increase in expression of tenascin-C (TN-C), a known regulator of FN matrix assembly. Mouse TN-C has multiple isoforms due to alternative splicing of 6 FNIII repeat exons. In addition to the transcriptional increase with high glucose, exon inclusion via alternative splicing was also changed resulting in production of higher molecular weight isoforms of TN-C. Mesangial cells grown in normal glucose secreted small isoforms with 1-2 variable repeats included whereas in high glucose large isoforms estimated to include 5 repeats were secreted. Unlike the smaller isoforms, the larger TN-C was not detected in the FN matrix. This change in TN-C isoforms may affect the regulation of FN matrix assembly and in this way may contribute to increased ECM accumulation under high glucose conditions.

Elevation does not strongly influence interspecific variation in body size of small Tropical endotherms / La elevación no influencia fuertemente la variación interespecífica en el tamaño corporal de los endotermos tropicales pequeños

Introduction: Body size is an essential trait for endotherms to face the physiological requirements of cold, so there is a tendency to large body size at high altitudes and latitudes, known as Bergmann's rule. However, the validity of this ecomorphological rule to small-bodied endotherms across altitudinal gradients is poorly known. Objective: To understand the effects of environmental variation on body size, we assessed whether interspecific variation in body size of small tropical endotherms follows Bergmann's rule along tropical altitudinal gradients. Methods: We compiled data on elevational ranges and body masses for 133 species of hummingbirds of Colombia. We then assessed the association between body mass and mid-point of the altitudinal distribution using phylogenetic generalized least squares (PGLS) analyses under different evolutionary models. Results: We found a decelerating rate of evolution for body size since the Early Burst model of evolution provided a better fit to body mass data. For elevational range, we found a slow and constant rate since Pagel's lambda model provided a better fit to the mid-point of the altitudinal distribution data. Besides, phylogenetic regression analysis indicated that body mass and the altitudinal range of hummingbirds are associated through the phylogeny, with a positive but slight association (R2= 0.036). Conclusions: We found that body mass and altitude of hummingbirds are positively related, which is in agreement with expectations under Bergmann's rule. However, this association was weaker than expected for small and non-passerine birds like hummingbirds. Thus, our results suggest that environmental changes across altitudinal gradients do not strongly influence body mass in small tropical endotherms as hummingbirds.

Introducción: El tamaño corporal es un rasgo importante para determinar la respuesta de los endotermos a los requerimientos que exigen las zonas frías, por lo cual se espera una tendencia hacia el incremento del tamaño corporal al aumentar la altitud y la latitud. Sin embargo, se conoce poco acerca de la validez de esta regla ecomorfológica, conocida como la regla de Bergmann, para endotermos pequeños en gradientes altitudinales tropicales. Objetivo: Con el fin de entender los efectos de la variación ambiental sobre el tamaño corporal, se evaluó sí la variación interespecífica en la masa corporal de endotermos tropicales pequeños se ajusta a la regla de Bergmann a lo largo de gradientes de elevación. Métodos: Se compilaron datos sobre los rangos de distribución altitudinal y los tamaños corporales de 133 especies de colibríes en Colombia. Posteriormente, se evaluó la asociación entre la masa corporal y el punto medio de distribución altitudinal de los colibríes mediante análisis de mínimos cuadrados generalizados filogenéticos (PGLS) bajo diferentes modelos evolutivos. Resultados: La evolución de la masa corporal se ajustó mejor a un modelo de evolución Early Burst, mientras que el rango de elevación al modelo evolutivo lambda de Pagel; lo que indica que la tasa de evolución es desacelerada para el tamaño del cuerpo, mientras es lenta y constante para el rango de elevación. Además, el análisis de regresión filogenética indica que la masa corporal y el rango de elevación están positiva y ligeramente asociados (R2 = 0.036). Conclusiones: De acuerdo con lo esperado por la regla de Bergmann, los resultados indican que los colibríes tienden a ser más grandes a mayores altitudes. Sin embargo, esta asociación es más débil de lo esperado para aves no paseriformes de tamaño pequeño como los colibríes.Por lo tanto, los resultados sugieren que las variaciones ambientales a lo largo de gradientes de elevación no tienen una influencia fuerte sobre el tamaño corporal de endotermos pequeños como los colibríes.

Development of hybrid scaffolds with natural extracellular matrix deposited within synthetic polymeric fibers.

A major challenge of tissue engineering is to generate materials that combine bioactivity with stability in a form that captures the robust nature of native tissues. Here we describe a procedure to fabricate a novel hybrid extracellular matrix (ECM)-synthetic scaffold biomaterial by cell-mediated deposition of ECM within an electrospun fiber mat. Synthetic polymer fiber mats were fabricated using poly(desamino tyrosyl-tyrosine carbonate) (PDTEC) co-spun with poly(ethylene glycol) (PEG) used as a sacrificial polymer. PEG removal increased the overall mat porosity and produced a mat with a layered structure that could be peeled into separate sheets of about 50 µm in thickness. Individual layers had pore sizes and wettability that facilitated cell infiltration over the depth of the scaffold. Confocal microscopy showed the formation of a highly interpenetrated network of cells, fibronectin fibrils, and synthetic fibers mimicking a complex ECM as observed within tissues. Decellularization did not perturb the structure of the matrix or the fiber mat. The resulting hybrid ECM-scaffold promoted cell adhesion and spreading and stimulated new ECM assembly by stem cells and tumor cells. These results identify a new technique for fabricating highly porous synthetic fibrous scaffolds and an approach to supplement them with natural biomimetic cues. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2162-2170, 2017.

Collaboration of fibronectin matrix with other extracellular signals in morphogenesis and differentiation.

Tissue formation and cell differentiation depend on a properly assembled extracellular matrix (ECM). Fibronectin is a key constituent of the pericellular ECM, forming essential connections between cell surface integrin receptors and structural components of the ECM. Recent studies using vertebrate models, conditional gene knockouts, tissue explants, and cell culture systems have identified developmental processes that depend on fibronectin and its receptor α5ß1 integrin. We describe requirements for fibronectin matrix in the cardiovascular system, somite and precartilage development, and epithelial-mesenchymal transition. Information about molecular mechanisms shows the importance of fibronectin and integrins during tissue morphogenesis and cell differentiation, as well as their cooperation with growth factors to mediate changes in cell behaviors.

Inhibition of Notch signaling enhances transdifferentiation of the esophageal squamous epithelium towards a Barrett's-like metaplasia via KLF4.

Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.

A common p53 mutation (R175H) activates c-Met receptor tyrosine kinase to enhance tumor cell invasion.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive forms of human cancer with poor prognosis due to late diagnosis and metastasis. Common genomic alterations in ESCC include p53 mutation, p120ctn inactivation, and overexpression of oncogenes such as cyclin D1, EGFR, and c-Met. Using esophageal epithelial cells transformed by the overexpression of EGFR and p53(R175H), we find novel evidence of a functional link between p53(R175H) and the c-Met receptor tyrosine kinase to mediate tumor cell invasion. Increased c-Met receptor activation was observed upon p53(R175H) expression and enhanced further upon subsequent EGFR overexpression. We inhibited c-Met phosphorylation, resulting in diminished invasion of the genetically transformed primary esophageal epithelial cells (EPC-hTERT-EGFR-p53(R175H)), suggesting that the mechanism of increased invasiveness upon EGFR and p53(R175H) expression may be the result of increased c-Met activation. These results suggest that the use of therapeutics directed at c-Met in ESCC and other squamous cell cancers.

Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.

This protocol describes the isolation and characterization of mouse and human esophageal epithelial cells and the application of 3D organotypic culture (OTC), a form of tissue engineering. This model system permits the interrogation of mechanisms underlying epithelial-stromal interactions. We provide guidelines for isolating and cultivating several sources of epithelial cells and fibroblasts, as well as genetic manipulation of these cell types, as a prelude to their integration into OTC. The protocol includes a number of important applications, including histology, immunohistochemistry/immunofluorescence, genetic modification of epithelial cells and fibroblasts with retroviral and lentiviral vectors for overexpression of genes or RNA interference strategies, confocal imaging, laser capture microdissection, RNA microarrays of individual cellular compartments and protein-based assays. The OTC (3D) culture protocol takes 15 d to perform.

Deletion of p120-catenin results in a tumor microenvironment with inflammation and cancer that establishes it as a tumor suppressor gene.

p120-catenin (p120ctn) interacts with E-cadherin, but to our knowledge, no formal proof that p120ctn functions as a bona fide tumor suppressor gene has emerged to date. We report herein that p120ctn loss leads to tumor development in mice. We have generated a conditional knockout model of p120ctn whereby mice develop preneoplastic and neoplastic lesions in the oral cavity, esophagus, and squamous forestomach. Tumor-derived cells secrete granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNFα). The tumors contain significant desmoplasia and immune cell infiltration. Immature myeloid cells comprise a significant percentage of the immune cells present and likely participate in fostering a favorable tumor microenvironment, including the activation of fibroblasts.

Transformation by Tribbles homolog 2 (Trib2) requires both the Trib2 kinase domain and COP1 binding.

Tribbles homolog 2 (Trib2) is a pseudokinase that induces acute myelogenous leukemia (AML) in mice and is highly expressed in a subset of human AML. Trib2 has 3 distinct regions, a proline-rich N-terminus, a serine/threonine kinase homology domain, and a C-terminal constitutive photomorphogenesis 1 (COP1)-binding domain. We performed a structure-function analysis of Trib2 using in vitro and in vivo assays. The N-terminus was not required for Trib2-induced AML. Deletion or mutation of the COP1-binding site abrogated the ability of Trib2 to degrade CCAAT/enhancer-binding protein-α (C/EBP-α), block granulocytic differentiation, and to induce AML in vivo. Furthermore, COP1 knockdown inhibited the ability of Trib2 to degrade C/EBP-α, showing that it is important for mediating Trib2 activity. We also show that the Trib2 kinase domain is essential for its function. Trib2 contains variant catalytic loop sequences, compared with conventional kinases, that we show are necessary for Trib2 activity. The kinase domain mutants bind, but cannot efficiently degrade, C/EBP-α. Together, our data demonstrate that Trib2 can bind both COP1 and C/EBP-α, leading to degradation of C/EBP-α. Identification of the functional regions of Trib2 that are essential to its oncogenic role provides the basis for developing inhibitors that will block Trib functions in cancer.

Differential ability of Tribbles family members to promote degradation of C/EBPalpha and induce acute myelogenous leukemia.

Trib1, Trib2, and Trib3 are mammalian homologs of Tribbles, an evolutionarily conserved Drosophila protein family that mediates protein degradation. Tribbles proteins function as adapters to recruit E3 ubiquitin ligases and enhance ubiquitylation of the target protein to promote its degradation. Increased Trib1 and Trib2 mRNA expression occurs in human myeloid leukemia and induces acute myeloid leukemia in mice, whereas Trib3 has not been associated with leukemia. Given the high degree of structural conservation among Tribbles family members, we directly compared the 3 mammalian Tribbles in hematopoietic cells by reconstituting mice with hematopoietic stem cells retrovirally expressing these proteins. All mice receiving Trib1 or Trib2 transduced hematopoietic stem cells developed acute myeloid leukemia, whereas Trib3 mice did not. Our previous data indicated that Trib2-mediated degradation of the transcription factor, CCAAT/enhancer-binding protein-alpha (C/EBPalpha), is important for leukemogenesis. Similar to Trib2, Trib1 induced C/EBPalpha degradation and inhibited its function. In contrast, Trib3 failed to inactivate or promote efficient degradation of C/EBPalpha. These data reveal that the 3 Tribbles homologs differ in their ability to promote degradation of C/EBPalpha, which account for their differential ability to induce leukemia.

Epidermal growth factor receptor and mutant p53 expand an esophageal cellular subpopulation capable of epithelial-to-mesenchymal transition through ZEB transcription factors.

Transforming growth factor-beta (TGF-beta) is a potent inducer of epithelial to mesenchymal transition (EMT). However, it remains elusive about which molecular mechanisms determine the cellular capacity to undergo EMT in response to TGF-beta. We have found that both epidermal growth factor receptor (EGFR) overexpression and mutant p53 tumor suppressor genes contribute to the enrichment of an EMT-competent cellular subpopulation among telomerase-immortalized human esophageal epithelial cells during malignant transformation. EGFR overexpression triggers oncogene-induced senescence, accompanied by the induction of cyclin-dependent kinase inhibitors p15(INK4B), p16(INK4A), and p21. Interestingly, a subpopulation of cells emerges by negating senescence without loss of EGFR overexpression. Such cell populations express increased levels of zinc finger E-box binding (ZEB) transcription factors ZEB1 and ZEB2, and undergo EMT on TGF-beta stimulation. Enrichment of EMT-competent cells was more evident in the presence of p53 mutation, which diminished EGFR-induced senescence. RNA interference directed against ZEB resulted in the induction of p15(INK4B) and p16(INK4A), reactivating the EGFR-dependent senescence program. Importantly, TGF-beta-mediated EMT did not take place when cellular senescence programs were activated by either ZEB knockdown or the activation of wild-type p53 function. Thus, senescence checkpoint functions activated by EGFR and p53 may be evaded through the induction of ZEB, thereby allowing the expansion of an EMT-competent unique cellular subpopulation, providing novel mechanistic insights into the role of ZEB in esophageal carcinogenesis.

A comparison of two maxillary molar distalizing appliances with the distal jet.

AIMS: Previous studies on maxillary molar distalization have usually concentrated on only one appliance and featured small sample sizes. The purpose of this retrospective study was two-fold: (1) to determine the skeletal, dental, and soft tissue effects of 3 molar distalization appliances, 2 of which do not depend upon patient compliance (ie, distal jet and Greenfield molar distalizing appliance) and 1 that does (ie, sagittal appliance combined with cervical headgear); and (2) to determine differences in treatment effects among the 3 appliances. METHODS: Pretreatment and post-distalization cephalometric radiographs were obtained for each appliance (14 females and 11 males for the distal jet; 12 females and 13 males for the Greenfield molar distalizing appliance; and 17 females and 13 males for the sagittal appliance with headgear). RESULTS: Pretreatment to transition evaluation showed significant distal movement of the first molars for the distal jet (3.4 mm), the Greenfield molar distalizing appliance (3.9 mm), and the sagittal appliance with headgear (2.1 mm). Distal tipping of the first molar was seen in all samples, but significantly more so in the Greenfield molar distalizing appliance (6.5 degrees +/- 6.6) and the sagittal appliance with headgear (13.5 degrees +/- 8. 1) than in the distal jet (3.2 degrees +/- 2.8). CONCLUSIONS: Maxillary molar distalization was effective using the distal jet, the Greenfield molar distalizing appliance, and the sagittal appliance with headgear, but better control of molar bodily movement was reported with the distal jet.

Estudio de la sensibilización al dinitroclorobenceno en un grupo de pacientes con enfermedad de Hodgkin / Dinitrochlorobencene hypersensitivity in a group of patients with Hodgkin's disease

Se presentan los resultados de la prueba de sensibilización al dinitroclorobenceno en un grupo de 103 pacientes con enfermedad de Hodgkin no tratados. Encontramos que fue posible sensibilizar al 80,6% de los pacientes, independientemente de la edad, el tipo histológico de la lesión, la extensión de la enfermedad y respuesta cutánea o antígenos de memoria convencionales. Sin embargo, la reactividad al dinitroclorobenceno apareció asociada a los conteos relativos de linfocitos con receptor para eritrocitos de carnero

Estudio de la sensibilización al dinitroclorobenceno en un grupo de pacientes con enfermedad de Hodgkin / Dinitrochlorobencene hypersensitivity in a group of patients with Hodgkin's disease

Se presentan los resultados de la prueba de sensibilización al dinitroclorobenceno en un grupo de 103 pacientes con enfermedad de Hodgkin no tratados. Encontramos que fue posible sensibilizar al 80,6

de los pacientes, independientemente de la edad, el tipo histológico de la lesión, la extensión de la enfermedad y respuesta cutánea o antígenos de memoria convencionales. Sin embargo, la reactividad al dinitroclorobenceno apareció asociada a los conteos relativos de linfocitos con receptor para eritrocitos de carnero (AU)