An engineered E.coli strain for the production of glycoglycerolipids

The glycolipid synthase MG517 from Mycoplasma genitalium catalyzes the glucosyl transfer from UDPGlc to diacylglycerol producing glycoglycerolipids (GGL) (Andrés et al., 2011). The enzyme was functional in E. coli accumulating GGL in the plasma membrane. A metabolic engineering strategy for GGL production was evaluated using this microorganism. To increase the levels of GGL precursors, UDPGlc and diacylglycerol, GalU and PlsC enzymes involved in their biosynthesis were overexpressed. Seven engineered strains were obtained containing different combinations of the mg517 with galU and plsC genes. Diacylglycerol synthesis showed to be limiting and the strain overexpressing MG517 and PlsC achieved the highest GGL yield. The new lipids were mono, di- and triglucosyldiacylglycerol with different acyl combinations in each compound. It indicates that the successive glucosyl transferase activities of MG517 have different acyl chain specificity for the acceptor substrate. GGL represented up to 6mg per g of dry weight.     

Aluminum resistance mechanisms in oat (Avena sativa L.)

Background and aims

Enhanced aluminum (Al) resistance has been observed in dicots over-expressing enzymes involved in organic acid synthesis; however, this approach for improving Al resistance has not been investigated in monocots. Among the cereals, oat (Avena sativa L.) is considered to be Al resistant, but the basis of resistance is not known.

Methods

A hydroponic assay and hematoxylin staining for Al accumulation in roots were used to evaluate Al resistance in 15 oat cultivars. Malate and citrate release from roots was measured over a 24?h period. A malate dehydrogenase gene, neMDH, from alfalfa (Medicago sativa L.) was used to transform oat.

Results

Oat seedlings were highly resistant to Al, as a concentration of 325?μM AlK(SO₄)₂ was needed to cause a 50% decrease in root growth. Most oat cultivars tested are naturally resistant to high concentrations of Al and effectively excluded Al from roots. Al-dependent release of malate and Al-independent release of citrate was observed. Al resistance was enhanced in a transgenic oat line with the highest accumulation of neMDH protein. However, overall root growth of this line was reduced and expression of neMDH in transgenic oat did not enhance malate secretion.

Conclusions

Release of malate from oat roots was associated with Al resistance, which suggests that malate plays a role in Al resistance of oat. Over-expression of alfalfa neMDH enhanced Al resistance in some lines but was not effective alone for crop improvement.     

A generalized model for overdispersed count data

Overdispersed count data are very common in ecology. The negative binomial model has been used widely to represent such data. Ecological data often vary considerably, and traditional approaches are likely to be inefficient or incorrect due to underestimation of uncertainty and poor predictive power. We propose a new statistical model to account for excessive overdisperson. It is the combination of two negative binomial models, where the first determines the number of clusters and the second the number of individuals in each cluster. Simulations show that this model often performs better than the negative binomial model. This model also fitted catch and effort data for southern bluefin tuna better than other models according to AIC. A model that explicitly and properly accounts for overdispersion should contribute to robust management and conservation for wildlife and plants.     

Genome-wide promoter methylation analysis in neuroblastoma identifies prognostic methylation biomarkers

Background

Accurate outcome prediction in neuroblastoma, which is necessary to enable the optimal choice of risk-related therapy, remains a challenge. To improve neuroblastoma patient stratification, this study aimed to identify prognostic tumor DNA methylation biomarkers.

Results

To identify genes silenced by promoter methylation, we first applied two independent genome-wide methylation screening methodologies to eight neuroblastoma cell lines. Specifically, we used re-expression profiling upon 5-aza-2''-deoxycytidine (DAC) treatment and massively parallel sequencing after capturing with a methyl-CpG-binding domain (MBD-seq). Putative methylation markers were selected from DAC-upregulated genes through a literature search and an upfront methylation-specific PCR on 20 primary neuroblastoma tumors, as well as through MBD- seq in combination with publicly available neuroblastoma tumor gene expression data. This yielded 43 candidate biomarkers that were subsequently tested by high-throughput methylation-specific PCR on an independent cohort of 89 primary neuroblastoma tumors that had been selected for risk classification and survival. Based on this analysis, methylation of KRT19, FAS, PRPH, CNR1, QPCT, HIST1H3C, ACSS3 and GRB10 was found to be associated with at least one of the classical risk factors, namely age, stage or MYCN status. Importantly, HIST1H3C and GNAS methylation was associated with overall and/or event-free survival.

Conclusions

This study combines two genome-wide methylation discovery methodologies and is the most extensive validation study in neuroblastoma performed thus far. We identified several novel prognostic DNA methylation markers and provide a basis for the development of a DNA methylation-based prognostic classifier in neuroblastoma.     

The crystal structure of the dimeric colicin M immunity protein displays a 3D domain swap

Bacteriocins are proteins secreted by many bacterial cells to kill related bacteria of the same niche. To avoid their own suicide through reuptake of secreted bacteriocins, these bacteria protect themselves by co-expression of immunity proteins in the compartment of colicin destination. In Escherichia coli the colicin M (Cma) is inactivated by the interaction with the Cma immunity protein (Cmi). We have crystallized and solved the structure of Cmi at a resolution of 1.95? by the recently developed ab initio phasing program ARCIMBOLDO. The monomeric structure of the mature 10kDa protein comprises a long N-terminal α-helix and a four-stranded C-terminal β-sheet. Dimerization of this fold is mediated by an extended interface of hydrogen bond interactions between the α-helix and the four-stranded β-sheet of the symmetry related molecule. Two intermolecular disulfide bridges covalently connect this dimer to further lock this complex. The Cmi protein resembles an example of a 3D domain swapping being stalled through physical linkage. The dimer is a highly charged complex with a significant surplus of negative charges presumably responsible for interactions with Cma. Dimerization of Cmi was also demonstrated to occur in vivo. Although the Cmi-Cma complex is unique among bacteria, the general fold of Cmi is representative for a class of YebF-like proteins which are known to be secreted into the external medium by some Gram-negative bacteria.     

Study of the volatile compounds produced by Debaryomyces hansenii NRRL Y-7426 during the fermentation of detoxified concentrated distilled grape marc hemicellulosic hydrolysates

The volatile compounds produced by Debaryomyces hansenii NRRL Y-7426 during the fermentation of detoxified concentrated distilled grape marc hemicellulosic hydrolysates was analysed by GC?CMS. Thirty-five compounds corresponding to ten groups of volatile compounds: terpenes, higher alcohols, C₆ alcohols, aldehydes, volatile acids, acetates, ethyl esters, volatile phenols, sulphur compounds and hydrocarbons were identified. The supplementation with commercial nutrients increased the concentration of 2-phenylethanol, a commercial flavour and fragrance compound, with a rose-like odour, employed in cosmetics and food industries; and other positive compounds to the aroma such as terpenes and ethyl esters. Non-supplemented media produced the highest content in higher alcohols, volatile acids, sulphur compounds and in the majority of hydrocarbons detected, meanwhile supplementation with vinasses hardly produced volatile compounds. Only four volatile compounds contributed directly to the aroma according to the OAVs values higher than 1. Finally, a PCA analysis allowed accounting for 100?% of the variance.     

Identification of Mutations in TMEM5 and ISPD as a Cause of Severe Cobblestone Lissencephaly

Cobblestone lissencephaly is a peculiar brain malformation with characteristic radiological anomalies. It is defined as cortical dysplasia that results when neuroglial overmigration into the arachnoid space forms an extracortical layer that produces agyria and/or a “cobblestone” brain surface and ventricular enlargement. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal-recessive diseases characterized by cerebral, ocular, and muscular deficits. These include Walker-Warburg syndrome, muscle-eye-brain disease, and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN, and FKRP identified these diseases as alpha-dystroglycanopathies. Our exhaustive screening of these six genes, in a cohort of 90 fetal cases, led to the identification of a mutation in only 53% of the families, suggesting that other genes might also be involved. We therefore decided to perform a genome-wide study in two multiplex families. This allowed us to identify two additional genes: TMEM5 and ISPD. Because TMEM has a glycosyltransferase domain and ISPD has an isoprenoid synthase domain characteristic of nucleotide diP-sugar transferases, these two proteins are thought to be involved in the glycosylation of dystroglycan. Further screening of 40 families with cobblestone lissencephaly identified nonsense and frameshift mutations in another four unrelated cases for each gene, increasing the mutational rate to 64% in our cohort. All these cases displayed a severe phenotype of cobblestone lissencephaly A. TMEM5 mutations were frequently associated with gonadal dysgenesis and neural tube defects, and ISPD mutations were frequently associated with brain vascular anomalies.