Glyphosate resistance by engineering the flavoenzyme glycine oxidase.

Glycine oxidase from Bacillus subtilis is a homotetrameric flavoprotein of great potential biotechnological use because it catalyzes the oxidative deamination of various amines and D-isomer of amino acids to yield the corresponding \u3b1-keto acids, ammonia/amine, and hydrogen peroxide. Glyphosate (N-phosphonomethylglycine), a broad spectrum herbicide, is an interesting synthetic amino acid: this compound inhibits 5-enolpyruvylshikimate-3-phosphate synthase in the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids in plants and certain bacteria. In recent years, transgenic crops resistant to glyphosate were mainly generated by overproducing the plant enzyme or by introducing a 5-enolpyruvylshikimate-3-phosphate synthase insensitive to this herbicide. In this work, we propose that the enzymatic oxidation of glyphosate could be an effective alternative to this important biotechnological process. To reach this goal, we used a rational design approach (together with site saturation mutagenesis) to generate a glycine oxidase variant more active on glyphosate than on the physiological substrate glycine. The glycine oxidase containing three point mutations (G51S/A54R/H244A) reaches an up to a 210-fold increase in catalytic efficiency and a 15,000-fold increase in the specificity constant (the kcat/Km ratio between glyphosate and glycine) as compared with wild-type glycine oxidase. The inspection of its three-dimensional structure shows that the \u3b12-\u3b13 loop (comprising residues 50-60 and containing two of the mutated residues) assumes a novel conformation and that the newly introduced residue Arg54 could be the key residue in stabilizing glyphosate binding and destabilizing glycine positioning in the binding site, thus increasing efficiency on the herbicide

Commercial hybrid yield of green asparagus (Asparagus officinalis L. var. altilis) processed in two spear-lengths, during the stable productivity stage, in the Province of Buenos Aires, Argentina

The perennial crop asparagus reaches maximum yield in its fifth season, exhibits marked genotype-environment interaction and requires productivity evaluation of its hybrids. To determine the behavior of green asparagus genotypes, a randomized complete block design trial (begun 16/11/11, density 23810 pl ha-1, area 1690 m2) was carried out in Azul (36°48’ S, lat. 59°51’ W, long.) within the framework of the ISHS’s Fourth International Asparagus Cultivar Trial, in which the following hybrids were harvested between 5/8/17 and 26/10/17 and evaluated for agronomic performance: ‘Italo’, ‘Vittorio’, ‘Eros’, ‘Ercole’, ‘Giove’, ‘Franco’, ‘Chino’, ‘Early-California’, ‘UC-157’, ‘Patrón’, ‘NJ-1189’, ‘NJ-1123’ and ‘NJ-1192’. Their response to pre-harvest diammonium phosphate (DAP) fertilisation was also evaluated (100 kg ha-1 vs. control). Harvested spears were cut, conditioned to two lengths (long 22 cm, short 17 cm), weighed, counted, washed and calibrated. The following characters were evaluated: total fresh commercial productivity (TFP) and that of long and short commercial spears (CFP-L and CFP-S); total commercial spear number (TSN) and that of long and short commercial spears (CSN-L and CSN-S); mean spear weight (MSW); calibre distribution (CD): (J: Jumbo; XL: Extra-Large; L: Large; M: Medium; S: Small and A: Asparagine). ANOVA-LSD test P≥0.05 was employed. DAP fertilisation raised yield by 3-10%, though not significantly. The following mean values were obtained: TFP: 8678.46 kg ha-1; TSN: 440298 spears ha-1; MSW: 20 g; CFP-L: 3109; CFP-S: 1945 kg ha-1; CSN-L: 165857 and CSN-S: 130467 spears ha-1. The following hybrids performed well: for CFP-L: ‘Vittorio’: 4379(a); ‘Franco’: 4033(ab); ‘NJ-1123’: 3849(abc); for CFP-C: ‘NJ-1123’: 2726(a); ‘Ercole’: 2483(ab); ‘Vittorio’: 2325(abc); for CSN-L: ‘NJ-1123’: 225260(a); ‘Franco’: 214161(a); ‘Vittorio’: 204358(ab); ‘Giove’: 200229(abc); for CSN-C: ‘NJ-1123’: 2725(a); ‘Ercole’: 2482(ab); ‘Vittorio’: 2325(abc); for CD: J: ‘UC-157’: 12767(a); ‘Eros’: 9882(ab); ‘NJ-1123’: 9005(abc); for XL: ‘NJ-1123’: 41124(a); ‘Franco’: 38958(ab); ‘NJ-1192’: 37644(ab); for L: ‘Ercole’: 85083(a); ‘NJ-1123’: 84700(a); ‘Franco’: 83872(a); ‘Italo’: 83095(a); for M: ‘NJ-1123’: 118198(a); ‘Franco’: 116369(a); ‘Vittorio’: 109645(ab); ‘Ercole’: 105984(ab); for S: ‘Giove’: 102748(a); ‘NJ-1123’: 80530(ab). In summary, ‘NJ-1123’ would be chosen for spear number productivity and ‘Vittorio’ and ‘Franco’ for yield.Fil: Castagnino, Ana Maria. Cresca-faa-unicen; Argentina. Asaho; Argentina. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Diaz, K. E.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Rosini, M. B.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Guisolis, Andrea Paola. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Dussi, M. C.. Universidad Nacional del Comahue; ArgentinaFil: Rogers, William John. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología; Argentin

Productivity of male green asparagus genotypes (Asparagus officinalis var. altilis L.) in their seventh year

El espárrago es una hortaliza no tradicional perenne, la que puede ingresar a los sistemas productivos como una alternativa de diversificación productiva, siendo necesario evaluar el comportamiento de distintos genotipos para cada zona productiva en particular. Con el objetivo de determinar la productividad de genotipos masculinos italianos se efectuó un ensayo (22/11/2006) mediante plantines (P) grandes (PG) y chicos (PCH). Se evaluó en el séptimo año de cosecha: productividad fresca total (PFT) y comercial (PFC), turiones totales (NTT) y comerciales (NTC), calibres (J: Jumbo; XL: Extra-Large; L: Large; M: Medium; S: Small y A: Asparagina), defectos (DE: espigados; P: daño plagas; y OD: otros defectos). Se efectuaron 32 cosechas en el período del 01/09/2014-14/11/2014. Se realizó análisis de la varianza ANOVA-LSD test (P≥0.05). No se encontraron diferencias significativas en P.Como resultado de las 32 cosechas realizadas se logró una productividad promedio de 14,5 t.ha-1 y comercial de primera calidad de 5,4 t.ha-1. En de turiones se lograron 553.241 totales y 252.420 comerciales de primera calidad, siendo el peso promedio por turión de 21 g. Respecto del tamaño de plantines, no se encontraron diferencias significativas. Los genotipos italianos superaron al testigo americano destacándose en PFT Eros y en PFC: Ercole, Eros, Italo y H-668. En turiones producidos se lograron diferencias significativas entre híbridos: en NTT se destacaron Zeno, Eros, Ercole y H-668; mientras que en NTC, Ercole. En distribución de calibres se destacaron, en J: Italo, UC-157, H-668, Zeno y Eros; en XL: Eros, Italo, Zeno y H-668; en L: Eros, Italo, Ercole y H 668, UC-157 y Zeno. En M: Ercole; Eros y H-668 y UC-157. En calibres S: UC-157 y Ercole, mientras en A: UC-157 y Ercole. Los híbridos masculinos, productivamente representan una alternativa valiosa para las características productivas de la Provincia de Buenos Aires.Asparagus is a non-traditional perennial vegetable crop for which evaluation of the performance of different genotypes is required. With the aim of determining the productivity of a set of all-male genotypes from Italy, a trial was planted on 22/11/06 using large (PG) and small (PCH) seedlings. The following characters were evaluated: total (PFT) and commercial (PFC) fresh production, total (NTT) and commercial (NTC) spear number, calibre distribution (J: Jumbo; XL: Extra-Large; L: Large; M: Medium; S: Small and A: Asparagina), defects (DE: opened bracts; P: plague damage; and OD: other defects). Thirty-two harvests were carried out between 1/9/14 and 14/11/14. Data were analysed by ANOVA-LSD (P≥0.05). No significant differences were found between the two seedling sizes. From the thirty-two harvests, a mean productivity of 14.5t.ha-1 was obtained, with a commercial prime quality productivity of 5.4 t.ha-1 . A total of 553,241 spears were obtained, of which 252,420 were of commercial prime quality, with an average weight of 21 g per spear. Regarding seedling size, no significant differences were found. The Italian genotypes performed better than the control genotype from the USA, with Eros outstanding for PFT, and Ercole, Eros, Italo and H-668 for PFC. For spears produced, significant differences were observed between hybrids: for NTT, Zeno, Eros, Ercole and H-668 stood out, while for NTC, Ercole. Regarding calibre distribution, Italo, UC-157, H-668, Zeno and Eros stood out for J; Eros, Italo, Zeno and H-668 for XL; Eros, Italo, Ercole and H-668, UC-157 and Zeno for L; Ercole, Eros and H-668, and UC-157 for M; UC157 and Ercole for S; and UC-157 and Ercole for A. In conclusion, all male hybrids represent a valuable production alternative.Fil: Romero, F.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Castagnino, Ana Maria. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; Argentina. Pontificia Universidad Católica Argentina; ArgentinaFil: Diaz, K. E.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Guisolis, Andrea Paola. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Rosini, M. B.. Universidad Nacional del Centro de la Provincia de Buenos Aires. Facultad de Agronomía. Centro Regional de Estudios Sistémico de Cadenas Agroalimentarias; ArgentinaFil: Rogers, William John. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Cientifico Tecnolológico Mar del Plata. Instituto de Investigaciones en Biodiversidad y Biotecnología. Laboratorio de Biología Funcional y Biotecnología; Argentin

Genomic analysis reveals the molecular basis for capsule loss in the group B Streptococcus population

The human and bovine bacterial pathogen Streptococcus agalactiae (Group B Streptococcus, GBS) expresses a thick polysaccharide capsule that constitutes a major virulence factor and vaccine target. GBS can be classified into ten distinct serotypes differing in the chemical composition of their capsular polysaccharide. However, non-typeable strains that do not react with anti-capsular sera are frequently isolated from colonized and infected humans and cattle. To gain a comprehensive insight into the molecular basis for the loss of capsule expression in GBS, a collection of well-characterized non-typeable strains was investigated by genome sequencing. Genome based phylogenetic analysis extended to a wide population of sequenced strains confirmed the recently observed high clonality among GBS lineages mainly containing human strains, and revealed a much higher degree of diversity in the bovine population. Remarkably, non-typeable strains were equally distributed in all lineages. A number of distinct mutations in the cps operon were identified that were apparently responsible for inactivation of capsule synthesis. The most frequent genetic alterations were point mutations leading to stop codons in the cps genes, and the main target was found to be cpsE encoding the portal glycosyl trasferase of capsule biosynthesis. Complementation of strains carrying missense mutations in cpsE with a wild-type gene restored capsule expression allowing the identification of amino acid residues essential for enzyme activity

Endogenous aldehyde accumulation generates genotoxicity and exhaled biomarkers in esophageal adenocarcinoma.

Volatile aldehydes are enriched in esophageal adenocarcinoma (EAC) patients' breath and could improve early diagnosis, however the mechanisms of their production are unknown. Here, we show that weak aldehyde detoxification characterizes EAC, which is sufficient to cause endogenous aldehyde accumulation in vitro. Two aldehyde groups are significantly enriched in EAC biopsies and adjacent tissue: (i) short-chain alkanals, and (ii) medium-chain alkanals, including decanal. The short-chain alkanals form DNA-adducts, which demonstrates genotoxicity and confirms inadequate detoxification. Metformin, a putative aldehyde scavenger, reduces this toxicity. Tissue and breath concentrations of the medium-chain alkanal decanal are correlated, and increased decanal is linked to reduced ALDH3A2 expression, TP53 deletion, and adverse clinical features. Thus, we present a model for increased exhaled aldehydes based on endogenous accumulation from reduced detoxification, which also causes therapeutically actionable genotoxicity. These results support EAC early diagnosis trials using exhaled aldehyde analysis

Evidence for sub-haplogroup h5 of mitochondrial DNA as a risk factor for late onset Alzheimer's disease

BACKGROUND: Alzheimer's Disease (AD) is the most common neurodegenerative disease and the leading cause of dementia among senile subjects. It has been proposed that AD can be caused by defects in mitochondrial oxidative phosphorylation. Given the fundamental contribution of the mitochondrial genome (mtDNA) for the respiratory chain, there have been a number of studies investigating the association between mtDNA inherited variants and multifactorial diseases, however no general consensus has been reached yet on the correlation between mtDNA haplogroups and AD. METHODOLOGY/PRINCIPAL FINDINGS: We applied for the first time a high resolution analysis (sequencing of displacement loop and restriction analysis of specific markers in the coding region of mtDNA) to investigate the possible association between mtDNA-inherited sequence variation and AD in 936 AD patients and 776 cognitively assessed normal controls from central and northern Italy. Among over 40 mtDNA sub-haplogroups analysed, we found that sub-haplogroup H5 is a risk factor for AD (OR=1.85, 95% CI:1.04-3.23) in particular for females (OR=2.19, 95% CI:1.06-4.51) and independently from the APOE genotype. Multivariate logistic regression revealed an interaction between H5 and age. When the whole sample is considered, the H5a subgroup of molecules, harboring the 4336 transition in the tRNAGln gene, already associated to AD in early studies, was about threefold more represented in AD patients than in controls (2.0% vs 0.8%; p=0.031), and it might account for the increased frequency of H5 in AD patients (4.2% vs 2.3%). The complete re-sequencing of the 56 mtDNAs belonging to H5 revealed that AD patients showed a trend towards a higher number (p=0.052) of sporadic mutations in tRNA and rRNA genes when compared with controls. CONCLUSIONS: Our results indicate that high resolution analysis of inherited mtDNA sequence variation can help in identifying both ancient polymorphisms defining sub-haplogroups and the accumulation of sporadic mutations associated with complex traits such as AD

“Topological Significance” Analysis of Gene Expression and Proteomic Profiles from Prostate Cancer Cells Reveals Key Mechanisms of Androgen Response

The problem of prostate cancer progression to androgen independence has been extensively studied. Several studies systematically analyzed gene expression profiles in the context of biological networks and pathways, uncovering novel aspects of prostate cancer. Despite significant research efforts, the mechanisms underlying tumor progression are poorly understood. We applied a novel approach to reconstruct system-wide molecular events following stimulation of LNCaP prostate cancer cells with synthetic androgen and to identify potential mechanisms of androgen-independent progression of prostate cancer.We have performed concurrent measurements of gene expression and protein levels following the treatment using microarrays and iTRAQ proteomics. Sets of up-regulated genes and proteins were analyzed using our novel concept of "topological significance". This method combines high-throughput molecular data with the global network of protein interactions to identify nodes which occupy significant network positions with respect to differentially expressed genes or proteins. Our analysis identified the network of growth factor regulation of cell cycle as the main response module for androgen treatment in LNCap cells. We show that the majority of signaling nodes in this network occupy significant positions with respect to the observed gene expression and proteomic profiles elicited by androgen stimulus. Our results further indicate that growth factor signaling probably represents a "second phase" response, not directly dependent on the initial androgen stimulus.We conclude that in prostate cancer cells the proliferative signals are likely to be transmitted from multiple growth factor receptors by a multitude of signaling pathways converging on several key regulators of cell proliferation such as c-Myc, Cyclin D and CREB1. Moreover, these pathways are not isolated but constitute an interconnected network module containing many alternative routes from inputs to outputs. If the whole network is involved, a precisely formulated combination therapy may be required to fight the tumor growth effectively