Expression of CsSCL1 and Rooting Response in Chestnut Leaves Are Dependent on the Auxin Polar Transport and the Ontogenetic Origin of the Tissues.

The mechanisms underlying the de novo regeneration of adventitious roots are still poorly understood, particularly in trees. We developed a system for studying adventitious rooting (AR) at physiological and molecular levels using leaves excised from chestnut microshoots of the same genotype but with two distinct ontogenetic origins that differ in rooting competence. Leaves were treated with auxin and N-1-naphthyl-phthalamic acid (NPA), an inhibitor of auxin polar transport (PAT). The physiological effects were investigated by recording rooting rates and the number and quality of the roots. Molecular responses were examined by localizing and monitoring the changes in the expression of CsSCL1, an auxin-inducible gene in juvenile and mature shoots during AR. The rooting response of leaves was ontogenetic-stage dependent and similar to that of the donor microshoots. Initiation of root primordia and root development were inhibited by application of NPA, although its effect depended on the timing of application. CsSCL1 was upregulated by auxin only in rooting-competent leaves during the novo root organogenesis, and the expression was reduced by NPA. The inhibitory effect on gene expression was detected during the reprograming of rooting competent cells towards root initials in response to auxin, indicating that PAT-mediated upregulation of CsSCL1 is required in the initial steps of AR in chestnut leaves. The localized expression of CsSCL1 in the quiescent center (QC) also suggests a role for this gene in the maintenance of meristematic competence and root radial patterning.

Degeneration pattern in somatic embryos of Pinus sylvestris L.

Somatic embryos can be used for propagating forest trees vegetatively, which is of great importance for capturing the genetic gain in breeding programs. However, many economically important Pinus species are difficult or impossible to propagate via somatic embryogenesis. In order to get a better understanding of the difficulties to propagate Pinus species via somatic embryogenesis, we are studying the developmental pathway of somatic embryos in different cell lines. In a previous study, we showed that the morphology of early somatic embryos in Scots pine (Pinus sylvestris) differs between cell lines giving rise to normal or abnormal cotyledonary embryos. In this study, we have compared the proliferation and degeneration pattern of early and late embryos in a normal and abnormal cell line. In both cell lines, a high frequency of the embryos degenerated. Among the degenerating embryos, two main degeneration patterns could be distinguished. In the normal cell line, the embryos degenerated similar to how the subordinate embryos are degraded in the seed. In the abnormal cell line, the degeneration of the embryos resulted in a continuous loop of embryo degeneration and differentiation of new embryos. We observed a similar degeneration pattern when embryogenic tissue was initiated from megagametophytes containing zygotic embryos at the stage of cleavage polyembryony. Based on our results, we suggest that the degeneration pattern in abnormal cell lines starts during initiation of embryogenic cultures.

The Role of Morbid Obesity in the Promotion of Metabolic Disruptions and Non-Alcoholic Steatohepatitis by Helicobacter Pylori.

BACKGROUND: Helicobacter pylory (HP) infection has been associated to an increased rate of type 2 diabetes (T2D) and liver disease through its effect on insulin resistance and systemic inflammation. However, results are inconstant and no studies exist in morbidly obese patients, in which both insulin resistance and inflammation coexist. MATERIAL AND METHODS: Cross-sectional study to evaluate the relationship between HP infection and alterations in carbohydrate metabolism, lipid profile, inflammation markers, and liver disease in patients awaiting for bariatric surgery. HP infection was histologically assessed in gastric antrum biopsy from 416 subjects. Liver biopsy was also available in 93 subjects. RESULTS: Both impaired fasting glucose and T2D were similar when comparing subjects with and without HP infection (24.2% vs. 22%, p = 0.290 and 29.4% vs. 29.1%, p = 0.916, respectively), with no differences between groups in the HOMA-IR, lipid profile neither inflammatory parameters. However, HP infection was higher among subjects with a BMI ≥ 40.0 kg/m2 in comparison with lower degrees of obesity (71.7% vs. 60.0%, p = 0.041). In addition, subjects without HP infection showed higher degrees of steatosis (44.1±26.4% vs. 32.0±20.7%, p = 0.038), as well as a lower prevalence of non-alcoholic steatohepatitis (9.3% vs. 30.7%, p = 0.023). CONCLUSIONS: In patients with morbid obesity, HP infection does not seem to be associated with abnormal carbohydrate metabolism. In addition, less advanced degrees of non-alcoholic fatty disease were observed. We suggest that low-grade inflammation that accompanies obesity mitigates the diabetogenic effect of HP, so the presence of obesity should be considered in studies that evaluate the HP metabolic effects.

Effects of sardine-enriched diet on metabolic control, inflammation and gut microbiota in drug-naïve patients with type 2 diabetes: a pilot randomized trial.

BACKGROUND: Nutrition therapy is the cornerstone of treating diabetes mellitus. The inclusion of fish (particularly oily fish) at least two times per week is recommended by current international dietary guidelines for type 2 diabetes. In contrast to a large number of human studies examining the effects of oily fish on different cardiovascular risk factors, little research on this topic is available in patients with type 2 diabetes. The aims of this pilot study were to investigate the effects of a sardine-enriched diet on metabolic control, adiponectin, inflammatory markers, erythrocyte membrane fatty acid (EMFA) composition, and gut microbiota in drug-naïve patients with type 2 diabetes. METHODS: 35 drug-naïve patients with type 2 diabetes were randomized to follow either a type 2 diabetes standard diet (control group: CG), or a standard diet enriched with 100 g of sardines 5 days a week (sardine group: SG) for 6 months. Anthropometric, dietary information, fasting glycated hemoglobin, glucose, insulin, adiponectin, inflammatory markers, EMFA and specific bacterial strains were determined before and after intervention. RESULTS: There were no significant differences in glycemic control between groups at the end of the study. Both groups decreased plasma insulin (SG: -35.3%, P = 0.01, CG: -22.6%, P = 0.02) and homeostasis model of assessment--insulin resistance (HOMA-IR) (SG: -39.2%, P = 0.007, CG: -21.8%, P = 0.04) at 6-months from baseline. However only SG increased adiponectin in plasma compared to baseline level (+40.7%, P = 0.04). The omega-3 index increased 2.6% in the SG compared to 0.6% in the CG (P = 0.001). Both dietary interventions decreased phylum Firmicutes (SG and CG: P = 0.04) and increased E. coli concentrations (SG: P = 0.01, CG: P = 0.03) at the end of the study from baseline, whereas SG decreased Firmicutes/Bacteroidetes ratio (P = 0.04) and increased Bacteroides-Prevotella (P = 0.004) compared to baseline. CONCLUSIONS: Although enriching diet with 100 g of sardines 5 days a week during 6 months to a type 2 diabetes standard diet seems to have neutral effects on glycemic control in drug-naïve patients with type 2 diabetes, this nutritional intervention could have beneficial effects on cardiovascular risk. Furthermore, both dietary interventions decreased HOMA-IR and altered gut microbiota composition of drug-naïve patients with type 2 diabetes. TRIAL REGISTRATION: Trial number and name of the registry: NCT02294526, ClinicalTrials.gov.

Utilidad de la plasmaféresis en el tratamiento preoperatorio de la tirotoxicosis / Usefulness of preoperative plasmapheresis in patients with thyrotoxicosis

No disponible

Effect of glycemic control on nocturnal arterial oxygen saturation: a case-control study in type 2 diabetic patients.

BACKGROUND: There is growing evidence suggesting a deleterious effect of type 2 diabetes on lung function and sleep breathing. The aim of this case-control study was to evaluate whether or not an improvement in glycemic control could arrest or ameliorate nocturnal hypoxemia in type 2 diabetes. METHODS: Thirty patients with type 2 diabetes with HbA1c ≥8% (cases) and 10 non-diabetic subjects were analyzed. Controls were closely matched to cases by age, gender, body mass index, and neck circumference. The nocturnal oxygen desaturation index (ODI) was calculated at baseline and 5 days after blood glucose improvement with pharmacological intervention. Four different oxygen desaturation thresholds (reductions in SaO2 ≥ 3%, 4%, 6%, and 8%) as indicators of hypoxemia severity (ODI-3%, ODI-4%, ODI-6%, ODI-8%) were used. RESULTS: At baseline, patients with diabetes showed a higher number of ODI-3%, ODI-4%, and ODI-6% in comparison with controls. A significant reduction in ODI-3% (29.7 [4.8-107.9] events per hour at baseline versus 24.6 [3.1-97.7] e/h at discharge, P < 0.001), ODI-4% (21.7 [1.6-79.3] versus 14.7 [0.3- 79.4], P = 0.003), ODI-6% (9.3 [0.3-71.8] versus 4.0 [0.0-73.7], P = 0.001), and ODI-8% (4.1 [0.0-64.3] versus 1.1 [0.0-69.8], P = 0.033) was observed in patients with diabetes after 5 days of follow-up. However, no changes in ODI events were observed in non-diabetic patients after the same period. No significant changes in weight were observed in either group. CONCLUSIONS: Glycemic control improvement significantly reduces the increased number of nocturnal oxygen desaturations that exist in type 2 diabetes. Although the mechanisms are not yet fully understood, the rapid effect without changes in body weight suggests a central mechanism involving respiratory center output.

Chestnut, European (Castanea sativa).

Development of a system for direct transfer of antifungal candidate genes into European chestnut (Castanea sativa) would provide an alternative approach to conventional breeding for production of chestnut trees that are tolerant to ink disease caused by Phytophthora spp. Overexpression of genes encoding PR proteins (such as thaumatin-like proteins), which display antifungal activity, may represent an important advance in control of the disease. We have used a chestnut thaumatin-like protein gene (CsTL1) isolated from European chestnut cotyledons and have achieved overexpression of the gene in chestnut somatic embryogenic lines used as target material. We have also acclimatized the transgenic plants and grown them on in the greenhouse. Here, we describe the various steps of the process, from the induction of somatic embryogenesis to the production of transgenic plants.

Alteraciones del metabolismo hidrocarbonado en la acromegalia / Abnormalities of carbohydrate metabolism in acromegaly

Fundamento y objetivo: Hasta un 50% de los pacientes con acromegalia presentan alteraciones en el metabolismo hidrocarbonado (AMHC). La evolución natural de la enfermedad y las distintas alternativas terapéuticas impactan de forma diferente en esta predisposición. El objetivo de este trabajo fue valorar la prevalencia, las características de los pacientes y el efecto de los distintos tratamientos en la AMHC en los pacientes acromegálicos de nuestro centro. Pacientes y método: Se realizó un estudio transversal que incluyó a 55 pacientes con acromegalia. Se analizaron: edad, sexo, índice de masa corporal (IMC), factor de crecimiento insulínico tipo 1 (IGF-1), tamaño tumoral, tratamientos, y presencia de diabetes mellitus (DM) y grado de control metabólico inicial y tras las distintas alternativas terapéuticas. Resultados: De los 55 pacientes estudiados, el 54% eran varones, con una edad media (DE) de 50 (17) años y un IMC de 27,9 (3,8) kg/m2. Las AMHC estaban presentes en el 50,9% (n = 28) (DM en el 24% y glucosa basal alterada en el 27%). Los pacientes con DM no presentaban diferencias en el IMC, la edad ni el IGF-1 inicial respecto a los que no tenían DM. Sin embargo, presentaban más macroadenomas. En los pacientes diabéticos, la hemoglobina glucosilada (HbA1c) descendió después de la cirugía de 7,6 a 6,7% y después de los análogos de la somatostatina de 7,1 a 6,6%, pero solo con pegvisomant hemos observado una reducción significativa de HbA1c: del 9,8 al 5,6% (p < 0,05). Es más, solo pegvisomant ha permitido disminuir la intensidad del tratamiento hipoglucemiante. Conclusiones. La prevalencia de AMHC supera al 50% de los casos y se correlaciona con el tamaño tumoral. No hemos observado diferencias en el control glucémico en los pacientes tratados con las diferentes alternativas terapéuticas, excepto en el grupo que recibió pegvisomant, que logró una mejoría del mismo, junto con una reducción del tratamiento hipoglucemiante (AU)

Background and objective: Carbohydrate metabolism (CHM) is impaired in over 50% of acromegalic patients. Natural history of acromegaly and treatment modalities may impact in a different way on CHM. We assessed CHM alterations in acromegaly and their relationship with clinical features and treatment options. Patients and method: Retrospective study with 55 patients with acromegaly. Age, sex, body mass index (BMI), tumor size, insulin growth factor type 1 (IGF-1) levels and the presence of impaired fasting glucose (IFG) or diabetes mellitus (DM) were analyzed before and after surgery or medical treatment. Results: There were 30 men and 25 women. Mean age was 50 17 years and mean BMI was 27.9 3.8 Kg/ m2. Impaired CHM was found in 50.9% (n = 28) (DM in 27% and IFG in 24%). In diabetic patients, we found no differences in age, sex, BMI and IGF-1 levels between IFG/DM and patients without CHM impairment. However, IFG/DM patients had macroadenomas more commonly. In diabetic patients, glycosylated hemoglobin (HbA1c) decreased after surgery from 7.6 to 6.7% and after somatostatin analogues from 7.1 to 6.6%; in patients on pegvisomant we observed a significant reduction of HbA1c: from 9.8 to 5.6% (P < .005). Furthermore, only in the pegvisomant group, insulin and/or oral agents had to be lowered. Conclusions: Up to 50% of patients with active acromegaly have CHM impairment which correlates with tumor size. Only pegvisomant is associated with significant improvement in glycemic control and a reduction in hypoglycemic treatment (AU)

[Abnormalities of carbohydrate metabolism in acromegaly]. / Alteraciones del metabolismo hidrocarbonado en la acromegalia.

BACKGROUND AND OBJECTIVE: Carbohydrate metabolism (CHM) is impaired in over 50% of acromegalic patients. Natural history of acromegaly and treatment modalities may impact in a different way on CHM. We assessed CHM alterations in acromegaly and their relationship with clinical features and treatment options. PATIENTS AND METHOD: Retrospective study with 55 patients with acromegaly. Age, sex, body mass index (BMI), tumor size, insulin growth factor type 1 (IGF-1) levels and the presence of impaired fasting glucose (IFG) or diabetes mellitus (DM) were analyzed before and after surgery or medical treatment. RESULTS: There were 30 men and 25 women. Mean age was 50 ± 17 years and mean BMI was 27.9 ± 3.8 Kg/m(2). Impaired CHM was found in 50.9% (n = 28) (DM in 27% and IFG in 24%). In diabetic patients, we found no differences in age, sex, BMI and IGF-1 levels between IFG/DM and patients without CHM impairment. However, IFG/DM patients had macroadenomas more commonly. In diabetic patients, glycosylated hemoglobin (HbA1c) decreased after surgery from 7.6 to 6.7% and after somatostatin analogues from 7.1 to 6.6%; in patients on pegvisomant we observed a significant reduction of HbA1c: from 9.8 to 5.6% (P < .005). Furthermore, only in the pegvisomant group, insulin and/or oral agents had to be lowered. CONCLUSIONS: Up to 50% of patients with active acromegaly have CHM impairment which correlates with tumor size. Only pegvisomant is associated with significant improvement in glycemic control and a reduction in hypoglycemic treatment.

Variables involved in the discordance between HbA1c and fructosamine: the glycation gap revisited.

AIMS: Glycation gap (GG) is defined as the difference between the measured level of HbA1c and the level that would be predicted from its regression on the fructosamine level. The aims of the study were: 1) To determine the reproducibility and consistency of GC; 2) To discover factors related to GG value. Given that metformin might increase glucose transport through the erythrocyte membrane, this treatment was also considered in the analyses of the results. METHODS: GG was calculated in two blood samples separated 30.6 (SD 7.3) weeks, obtained in 508 type 2 diabetic patients. The following variables were considered: HbA1c, fructosamine, glucose, creatinine, hematological parameters and treatment with metformin. Multivariate and logistic regression analyses were performed to explore the variables independently related to CG. RESULTS: GG was reproducible and consistent over time. Creatinine, mean corpuscular hemoglobin concentration (MCHC), and glycemia (inverse relationship); and HbA1c and treatment with metformin (direct relationship) were independently related to GG. Patients treated with metformin showed higher HbA1c values, despite similar fructosamine concentrations, than patients not treated with the drug. CONCLUSIONS: GG is independently related to serum levels of creatinine, MCHC and treatment with metformin. The spurious effect of metformin on Hb glycation could have serious clinical implications and should be considered when interpreting the results of clinical trials.

Genetic transformation of European chestnut somatic embryos with a native thaumatin-like protein (CsTL1) gene isolated from Castanea sativa seeds.

The availability of a system for direct transfer of antifungal candidate genes into European chestnut (Castanea sativa Mill.) would offer an alternative approach to conventional breeding for production of chestnut trees tolerant to ink disease caused by Phytophthora spp. For the first time, a chestnut thaumatin-like protein gene (CsTL1), isolated from chestnut cotyledons, has been overexpressed in three chestnut somatic embryogenic lines. Transformation experiments have been performed using an Agrobacterium tumefaciens Smith and Townsend vector harboring the neomycin phosphotransferase (NPTII) selectable and the green fluorescent protein (EGFP) reporter genes. The transformation efficiency, determined on the basis of the fluorescence of surviving explants, was clearly genotype dependent and ranged from 32.5% in the CI-9 line to 7.1% in the CI-3 line. A total of 126 independent transformed lines were obtained. The presence and integration of chestnut CsTL1 in genomic DNA was confirmed by polymerase chain reaction (PCR) and Southern blot analyses. Quantitative real-time PCR revealed that CsTL1 expression was up to 13.5-fold higher in a transgenic line compared with its corresponding untransformed line. In only one of the 11 transformed lines tested, expression of the CsTL1 was lower than the control. The remaining 115 transformed lines were successfully subjected to cryopreservation. Embryo proliferation was achieved in all of the transgenic lines regenerated and the transformed lines showed a higher mean number of cotyledonary stage embryos and total number of embryos per embryo clump than their corresponding untransformed lines. Transgenic plants were regenerated after maturation and germination of transformed somatic embryos. Furthermore, due to the low plantlet conversion achieved, axillary shoot proliferation cultures were established from partially germinated embryos (only shoot development), which were multiplied and rooted according to procedures already established. Transgenic plants were acclimatized and grown in a greenhouse. No phenotypic differences were found with control plants, suggesting no potential cytotoxic effects of the green fluorescent protein. The results reported in the present work could be considered as a first step toward the production of fungal-disease tolerant cisgenic chestnut plants.

Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor.

Somatic embryogenesis is used for vegetative propagation of conifers. Embryogenic cultures can be established from zygotic embryos; however, the embryogenic potential decreases during germination. In Arabidopsis, LEAFY COTYLEDON (LEC) genes are expressed during the embryonic stage, and must be repressed to allow germination. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) causes de-repression of LEC genes. ABSCISIC ACID3 (ABI3) and its Zea mays ortholog VIVIPAROUS1 (VP1) act together with the LEC genes to promote embryo maturation. In this study, we have asked the question whether TSA treatment in a conifer affects the embryogenic potential and the expression of embryogenesis-related genes. We isolated two conifer LEC1-type HAP3 genes, HAP3A and HAP3B, from Picea abies and Pinus sylvestris. A comparative phylogenetic analysis of plant HAP3 genes suggests that HAP3A and HAP3B are paralogous genes originating from a duplication event in the conifer lineage. The expression of HAP3A is high, in both somatic and zygotic embryos, during early embryo development, but decreases during late embryogeny. In contrast, the expression of VP1 is initially low but increases during late embryogeny. After exposure to TSA, germinating somatic embryos of P. abies maintain the competence to differentiate embryogenic tissue, and simultaneously the germination progression is partially inhibited. Furthermore, when embryogenic cultures of P. abies are exposed to TSA during embryo maturation, the maturation process is arrested and the expression levels of PaHAP3A and PaVP1 are maintained, suggesting a possible link between chromatin structure and expression of embryogenesis-related genes in conifers.

Characterization and expression of a Pinus radiata putative ortholog to the Arabidopsis SHORT-ROOT gene.

We characterized a Pinus radiata D. Don putative ortholog to the Arabidopsis thaliana (L.) Heynh. SHORT--ROOT gene (AtSHR) and analyzed its expression in different organs during vegetative development and in response to exogenous auxin during adventitious rooting. The predicted protein sequence contained domains characteristic of the GRAS protein family and showed a strong similarity to the SHORT--ROOT (SHR) proteins. Quantitative reverse transcriptase--polymerase chain reaction (qRT-PCR) and in situ hybridization showed that the gene is predominantly expressed in roots, root primordia and in the cambial region of hypocotyl cuttings. Increased mRNA levels were observed, independently of the presence or absence of exogenous auxin, in the cambial region and rooting competent cells of hypocotyl cuttings within the first 24 h of adventitious rooting, before the activation of cell divisions and the organization of the adventitious root meristem. The expression pattern in organs and during adventitious rooting was similar to that of a Pinus radiata SCARECROW-LIKE (PrSCL1) gene, except that PrSCL1 is induced in response to exogenous auxin. Results suggest that the Pinus radiata SHORT-ROOT (PrSHR) gene has a role in root meristem formation and maintenance and in the cambial region of hypocotyl cuttings.

Cryopreservation of embryogenic cultures from mature Quercus suber trees using vitrification.

Recent progress in somatic embryogenesis from selected mature trees of Quercus suber, has led to a demand for maintenance of a large number of selected embryogenic lines. To facilitate the management of this material a protocol for the long-term storage of this germplasm should be defined. This study reports on the use of a simple vitrification procedure for the successful cryopreservation of three cork oak embryogenic lines. High embryo recovery levels (88-93 percent) were obtained by first preculturing 2-4 mg clumps of two or three globular embryos on semisolid medium containing 0.3 M sucrose for three days, followed by incubation in PVS2 vitrification solution at 0 degree C for 60 min before direct immersion in liquid nitrogen. The mean number of embryos produced per explant was significantly greater for cryostored embryos than for untreated stock cultures, but the productivity of the latter was recovered in subsequent subcultures of the material produced by cryostored embryos. The germination and plant regeneration rates achieved by cultures derived from cryostored embryos, around 60 percent, were similar to those of non-cryopreserved stock cultures.

Maturation and germination of oak somatic embryos originated from leaf and stem explants: RAPD markers for genetic analysis of regenerants.

Experiments were performed to determine the influence of maturation medium carbohydrate content on the rates of germination and plantlet conversion (root and shoot growth) of somatic embryos from four embryogenic lines derived from leaf or internode explants of Quercus robur L. seedlings. The conversion rate was favoured by high carbohydrate content as long as the maturation medium contained at least 2% sucrose, which was necessary for healthy embryo development. Given this, sorbitol and mannitol favoured the conversion rate more efficiently than sucrose, the highest rate, 32%, being achieved by medium with 6% sorbitol and 3% sucrose. Maturation treatment did not affect the root or shoot lengths of converted embryos. In supplementary experiments, 2 weeks of gibberellic acid treatment between maturation and germination treatments did not improve germination rates, but did reduce root length and the number of leaves per regenerated plantlet. In the four embryogenic lines tested, plant recovery rate was enhanced by inclusion of benzyladenine into the germination medium following culture of the embryos on maturation medium with 6% sorbitol and 2-3% sucrose. In embryogenic systems it is important to assess the uniformity of the regenerants. Random amplified polymorphic DNA (RAPD) analysis using 32 arbitrary oligonucleotide primers was performed to study variability in DNA sequences within and between four embryogenic lines. No intraclonal nor interclonal polymorphism was detected between embryogenic lines originating from different types of explant from the same seedling, but every one of the primers detected enough polymorphism among clones originating from different plants to allow these three origins to be distinguished. No differences in DNA sequences between regenerated plantlets and their somatic embryos of origin were detected, but a nodular callus line that had lost its embryogenic capacity was found to be mutant with respect to three other clones originating from the same plantlet. This study shows that high carbohydrate levels in the maturation medium significantly increase plant conversion of oak somatic embryos, which exhibit no variation in DNA sequences when proliferated by secondary embryogenesis.