Reduction in inter-hemispheric connectivity in disorders of consciousness

Clinical diagnosis of disorders of consciousness (DOC) caused by brain injury poses great challenges since patients are often behaviorally unresponsive. A promising new approach towards objective DOC diagnosis may be offered by the analysis of ultra-slow (<0.1 Hz) spontaneous brain activity fluctuations measured with functional magnetic resonance imaging (fMRI) during the resting-state. Previous work has shown reduced functional connectivity within the "default network", a subset of regions known to be deactivated during engaging tasks, which correlated with the degree of consciousness impairment. However, it remains unclear whether the breakdown of connectivity is restricted to the "default network", and to what degree changes in functional connectivity can be observed at the single subject level. Here, we analyzed resting-state inter-hemispheric connectivity in three homotopic regions of interest, which could reliably be identified based on distinct anatomical landmarks, and were part of the "Extrinsic" (externally oriented, task positive) network (pre- and postcentral gyrus, and intraparietal sulcus). Resting-state fMRI data were acquired for a group of 11 healthy subjects and 8 DOC patients. At the group level, our results indicate decreased inter-hemispheric functional connectivity in subjects with impaired awareness as compared to subjects with intact awareness. Individual connectivity scores significantly correlated with the degree of consciousness. Furthermore, a single-case statistic indicated a significant deviation from the healthy sample in 5/8 patients. Importantly, of the three patients whose connectivity indices were comparable to the healthy sample, one was diagnosed as locked-in. Taken together, our results further highlight the clinical potential of resting-state connectivity analysis and might guide the way towards a connectivity measure complementing existing DOC diagnosis.     

Early events in the Fusarium verticillioides-maize interaction characterized by using a green fluorescent protein-expressing transgenic isolate

The infection of maize by Fusarium verticillioides can result in highly variable disease symptoms ranging from asymptomatic plants to severe rotting and wilting. We produced F. verticillioides green fluorescent protein-expressing transgenic isolates and used them to characterize early events in the F. verticillioides-maize interaction that may affect later symptom appearance. Plants grown in F. verticillioides-infested soil were smaller and chlorotic. The fungus colonized all of the underground parts of a plant but was found primarily in lateral roots and mesocotyl tissue. In some mesocotyl cells, conidia were produced within 14 to 21 days after infection. Intercellular mycelium was detected, but additional cells were not infected until 21 days after planting. At 25 to 30 days after planting, the mesocotyl and main roots were heavily infected, and rotting developed in these tissues. Other tissues, including the adventitious roots and the stem, appeared to be healthy and contained only a small number of hyphae. These results imply that asymptomatic systemic infection is characterized by a mode of fungal development that includes infection of certain tissues, intercellular growth of a limited number of fungal hyphae, and reproduction of the fungus in a few cells without invasion of other cells. Development of visibly rotted tissue is associated with massive production of fungal mycelium and much less organized growth.     

Overexpression of a plasma membrane aquaporin in transgenic tobacco improves plant vigor under favorable growth conditions but not under drought or salt stress

Most of the symplastic water transport in plants occurs via aquaporins, but the extent to which aquaporins contribute to plant water status under favorable growth conditions and abiotic stress is not clear. To address this issue, we constitutively overexpressed the Arabidopsis plasma membrane aquaporin, PIP1b, in transgenic tobacco plants. Under favorable growth conditions, PIP1b overexpression significantly increased plant growth rate, transpiration rate, stomatal density, and photosynthetic efficiency. By contrast, PIP1b overexpression had no beneficial effect under salt stress, whereas during drought stress it had a negative effect, causing faster wilting. Our results suggest that symplastic water transport via plasma membrane aquaporins represents a limiting factor for plant growth and vigor under favorable conditions and that even fully irrigated plants face limited water transportation. By contrast, enhanced symplastic water transport via plasma membrane aquaporins may not have any beneficial effect under salt stress, and it has a deleterious effect during drought stress.     

Isolation of a premycorrhizal infection (pmi2) mutant of tomato,resistant to arbuscular mycorrhizal fungal colonization

Arbuscular mycorrhizae (AM) represent an ancient symbiosis between mycorrhizal fungi and plant roots which co-evolved to exhibit a finely tuned, multistage interaction that assists plant growth. Direct screening efforts for Myc- plant mutants resulted in the identification of a tomato (Lycopersicon esculentum L. cv. Micro-Tom) mutant, M20, which was impaired in its ability to support the premycorrhizal infection (pmi) stages. The Myc- phenotype of the M20 mutant was a single Mendelian recessive trait, stable for nine generations, and nonallelic to a previously identified M161 pmi mutant. The M20 mutant was resistant to infection by isolated AM spores and colonized roots. Formation of Glomus intraradices appressoria on M20 roots was normal, as on wild-type (WT) plants, but in significantly reduced numbers. A significant reduction in spore germination was observed in vitro in the presence of M20 exudates relative to WT. Our results indicate that this new mutant shares similar physiological characteristics with the M161 pmi mutant, but has a more suppressive Myc- phenotype response.     

Bioreactor cultivation enhances the efficiency of human embryoid body (hEB) formation and differentiation

The promise of human embryonic stem cells (hESCs) to provide an unlimited supply of cells for cell therapy and tissue engineering depends on the availability of a controllable bioprocess for their expansion and differentiation. We describe for the first time the formation of differentiating human embryoid bodies (hEBs) in rotating bioreactors to try and control their agglomeration. The efficacy of the dynamic process compared to static cultivation in Petri dishes was analyzed with respect to the yield of hEB formation and differentiation. Quantitative analyses of hEBs, DNA and protein contents, and viable cell concentration, as measures for culture cellularity and scale-up, revealed 3-fold enhancement in generation of hEBs compared to the static culture. Other metabolic indices such as glucose consumption, lactic acid production, and pH pointed to efficient cell expansion and differentiation in the dynamic cultures. The type of rotating vessel had a significant impact on the process of hEB formation and agglomeration. In the slow turning lateral vessel (STLV), hEBs were smaller in size and no large necrotic centers were seen, even after 1-month cultivation. In the high aspect rotating vessel (HARV), hEB agglomeration was massive. The appearance of representative tissues derived from the three germ layers as well as primitive neuronal tube organization, blood vessel formation, and specific-endocrine secretion indicated that the initial developmental events are not altered in the dynamically formed hEBs. Collectively, our study defines the culture conditions in which control over the aggregation of differentiating hESCs is obtained, thus enabling scaleable cell production for clinical and industrial applications.     

The ywad gene from Bacillus subtilis encodes a double-zinc aminopeptidase

The yet uncharacterized ywad gene from Bacillus subtilis has been cloned and overexpressed in Escherichia coli. The gene product (BSAP) was purified and shown to be an aminopeptidase. The activity of BSAP was optimal at pH 8.4, the enzyme was stable for 20 min at 80 degrees C and its activity was not affected by serine protease and aspartic protease inhibitors, but was completely diminished by the Zn-chelator 1,10-phenanthroline. ZnCl2 was able to restore activity, and the binding stoichiometry of zinc to apo-BSAP indicated two Zn ions per protein molecule. BSAP exhibited high preference toward p-nitroanilide derived Arg, Lys, and Leu synthetic substrates resulting in kcat/Km values of 1-5 x 10(1) s(-1) mM(-1).     

Selective separation of cis-trans geometrical isomers of beta-carotene via CO2 supercritical fluid extraction

We investigated a novel method for the selective separation of beta-carotene isomers from a freeze-dried powder of the algae Dunaliella bardawil using supercritical fluid extraction. The separation method relies on the different dissolution rate of the 9Z and all-E isomers of beta-carotene in SC-CO(2). At first, the equilibrium solubility of the two isomers in SC-CO(2) was determined at the extraction conditions of 44.8 MPa and 40 degrees C. The solubility of the 9Z isomer was found to be nearly 4 times higher than that of the all-E isomer (1.92 x 10(-5) g all-E isomer/g CO(2) compared to 7.64 x 10(-5) g 9Z isomer/g CO(2)). When supercritical fluid extraction was applied to a carotenoid concentrate from the algae (29 wt% beta-carotene) or a freeze-dried powder of the algae (3.1% beta-carotene), a selective separation of the 9Z/all-E isomers of beta-carotene was obtained. Thirty-nine percent recovery of beta-carotene with 80% purity of 9Z isomer was achieved at the initial stages of extraction (40 mL CO(2)). The extraction rate of beta-carotene from the freeze-dried algae powder was slower than that from the carotenoid concentrate, resulting in a reduction in the recovery and purity of the 9Z isomer. This indicates that even at the initial stage of the extraction the internal mass resistance is significant. Isomer purity and recovery could be enhanced upon grinding of the algae powder.     

A missense mutation,S349P,completely inactivates phenylalanine hydroxylase in North African Jews with phenylketonuria

The majority of hyperphenylalaninemias (HPAs) result from mutations at the gene for phenylalanine hydroxylase (PAH). The broad phenotypic variability of these conditions, ranging from phenylketonuria (PKU) to mild benign HPA, is underlain by a wide spectrum of mutations giving rise to various genotypic combinations. Mutant PAH alleles, labeled by specific polymorphic haplotypes and mutations, are becoming useful markers in human population genetics. We report here a mutant PAH allele found in Jews from Morocco and Tunisia, marked by haplotype 4 and a missense mutation, TCA^SerCCA^pro, at codon 349 in exon 10 of the gene. In vitro expression of the mutation showed normal levels of mRNA with virtually no enzymatic activity or protein immunoreactivity, pointing to a highly unstable protein. A homozygote for this mutation showed the most severe (classical) type of PKU, while compound heterozygotes showed two other types of HPA — atypical PKU and high benign HPA — illustrating the interplay between different mutations that gives rise to various HPAs.     

Expression of <Emphasis Type="Italic">MAX2</Emphasis> under <Emphasis Type="Italic">SCARECROW</Emphasis> promoter enhances the strigolactone/MAX2 dependent response of <Emphasis Type="Italic">Arabidopsis</Emphasis> roots to low-phosphate conditions

Main conclusion

MAX2/strigolactone signaling in the endodermis and/or quiescent center of the root is partiallysufficient to exert changes in F-actin density and cellular trafficking in the root epidermis, and alter gene expression during plant response to low Pi conditions.Strigolactones (SLs) are a new group of plant hormones that regulate different developmental processes in the plant via MAX2, an F-box protein that interacts with their receptor. SLs and MAX2 are necessary for the marked increase in root-hair (RH) density in seedlings under conditions of phosphate (Pi) deprivation. This marked elevation was associated with an active reduction in actin-filament density and endosomal movement in root epidermal cells. Also, expression of MAX2 under the SCARECROW (SCR) promoter was sufficient to confer SL sensitivity in roots, suggesting that SL signaling pathways act through a root-specific, yet non-cell-autonomous regulatory mode of action. Here we show evidence for a non-cell autonomous signaling of SL/MAX2, originating from the root endodermis, and necessary for seedling response to conditions of Pi deprivation. SCR-derived expression of MAX2 in max2-1 mutant background promoted the root low Pi response, whereas supplementation of the synthetic SL GR24 to these SCR:MAX2 expressing lines further enhanced this response. Moreover, the SCR:MAX2 expression led to changes in actin density and endosome movement in epidermal cells and in TIR1 and PHO2 gene expression. These results demonstrate that MAX2 signaling in the endodermis and/or quiescent center is partially sufficient to exert changes in F-actin density and cellular trafficking in the epidermis, and alter gene expression under low Pi conditions.