Highly conserved and cis-acting lncRNAs produced from paralogous regions in the center of HOXA and HOXB clusters in the endoderm lineage

Long noncoding RNAs (lncRNAs) have been shown to play important roles in gene regulatory networks acting in early development. There has been rapid turnover of lncRNA loci during vertebrate evolution, with few human lncRNAs conserved beyond mammals. The sequences of these rare deeply conserved lncRNAs are typically not similar to each other. Here, we characterize HOXA-AS3 and HOXB-AS3, lncRNAs produced from the central regions of the HOXA and HOXB clusters. Sequence-similar orthologs of both lncRNAs are found in multiple vertebrate species and there is evident sequence similarity between their promoters, suggesting that the production of these lncRNAs predates the duplication of the HOX clusters at the root of the vertebrate lineage. This conservation extends to similar expression patterns of the two lncRNAs, in particular in cells transiently arising during early development or in the adult colon. Functionally, the RNA products of HOXA-AS3 and HOXB-AS3 regulate the expression of their overlapping HOX5–7 genes both in HT-29 cells and during differentiation of human embryonic stem cells. Beyond production of paralogous protein-coding and microRNA genes, the regulatory program in the HOX clusters therefore also relies on paralogous lncRNAs acting in restricted spatial and temporal windows of embryonic development and cell differentiation.     

Advances in plant growth-promoting bacterial inoculant technology: formulations and practical perspectives (1998–2013)

Background

Inoculation of plants to enhance yield of crops and performance of other plants is a century old, proven technology for rhizobia and a newer venue for plant growth-promoting bacteria and other plant symbionts. The two main aspects dominating the success of inoculation are the effectiveness of the bacterial isolate and the proper application technology.

Scope

An assessment of practical aspects of bacterial inoculants for contemporary agriculture and environmental restoration is critically evaluated from the point of view of their current technological status, current applications, and future use. This is done because there are windows of opportunity for new developments in applied research using renewable, non-contaminated natural resources and new venues for research. Special emphasis is given to formulations and polymeric carriers. This review concentrates on practical aspect of inoculation technology dating from 1998 to 2013. Earlier publications are mentioned only for clarification of a specific point.

Conclusions

This review discusses characteristics of a carrier for inoculants, formulations of inoculants including liquid, organic, inorganic, polymeric, and encapsulated formulations. Technical aspects include inoculation techniques (soil and seed application), mass culture production, bulk sterilization, seed coating, shelf-life, and effect of moisture. Future research venues needed are noted.     

Converting the highly amyloidogenic human calcitonin into a powerful fibril inhibitor by three-dimensional structure homology with a non-amyloidogenic analogue

Irreversible aggregation limits bioavailability and therapeutic activity of protein-based drugs. Here we show that an aggregation-resistant mutant can be engineered by structural homology with a non-amyloidogenic analogue and that the aggregation-resistant variant may act as an inhibitor. This strategy has successfully been applied to the amyloidogenic human calcitonin (hCT). Including only five residues from the non-amyloidogenic salmon calcitonin (sCT), we obtained a variant, polar human calcitonin (phCT), whose solution structure was shown by CD, NMR, and calculations to be practically identical to that of sCT. phCT was also observed to be a potent amyloidogenesis inhibitor of hCT when mixed with it in a 1:1 ratio. Fibrillation studies of phCT and the phCT-hCT mixture mimicked the sCT behavior in the kinetics and shapes of the fibrils with a dramatic reduction with respect to hCT. Finally, the effect of phCT alone and of the mixture on the intracellular cAMP level in T47D cells confirmed for the mutant and the mixture their calcitonin-like activity, exhibiting stimulation effects identical to those of sCT, the current therapeutic form. The strategy followed appears to be suitable to develop new forms of hCT with a striking reduction of aggregation and improved activity. Finally, the inhibitory properties of the aggregation-resistant analogue, if confirmed for other amyloidogenic peptides, may favor a new strategy for controlling fibril formation in a variety of human diseases.     

Tricyclic quinoxalines as potent kinase inhibitors of PDGFR kinase,Flt3 and Kit

Here we report on novel quinoxalines as highly potent and selective inhibitors of the type III receptor tyrosine kinases PDGFR, FLT3, and KIT. These compounds, tricyclic quinoxalines, were generated in order to improve bioavailability over the highly hydrophobic bicyclic quinoxalines. Four of the highly active compounds were characterized in detail and are shown to inhibit PDGFR kinase activity of the isolated receptor as well as in intact cells in the sub-micromolar concentration range. We show that the most active inhibitor (compound 13, AGL 2043) is approximately 15-20 times more potent than its isomer (compound 14, AGL 2044). We therefore compared the three dimensional structures of the two compounds by X-ray crystallography. These compounds are also highly effective in blocking the kinase activity of FLT3, KIT, and the oncogenic protein Tel-PDGFR in intact cells. These compounds are potent inhibitors of the proliferation of pig heart smooth muscle cells. They fully arrest the growth of these cells and the effect is fully reversible. The chemical, biochemical and cellular properties of these compounds as well as the solubility properties make them suitable for development as anti-restenosis and anti-cancer agents.     

Adaptive Resistance Mutations at Suprainhibitory Concentrations Independent of SOS Mutagenesis

Emergence of resistant bacteria during antimicrobial treatment is one of the most critical and universal health threats. It is known that several stress-induced mutagenesis and heteroresistance mechanisms can enhance microbial adaptation to antibiotics. Here, we demonstrate that the pathogen Bartonella can undergo stress-induced mutagenesis despite the fact it lacks error-prone polymerases, the rpoS gene and functional UV-induced mutagenesis. We demonstrate that Bartonella acquire de novo single mutations during rifampicin exposure at suprainhibitory concentrations at a much higher rate than expected from spontaneous fluctuations. This is while exhibiting a minimal heteroresistance capacity. The emerged resistant mutants acquired a single rpoB mutation, whereas no other mutations were found in their whole genome. Interestingly, the emergence of resistance in Bartonella occurred only during gradual exposure to the antibiotic, indicating that Bartonella sense and react to the changing environment. Using a mathematical model, we demonstrated that, to reproduce the experimental results, mutation rates should be transiently increased over 1,000-folds, and a larger population size or greater heteroresistance capacity is required. RNA expression analysis suggests that the increased mutation rate is due to downregulation of key DNA repair genes (mutS, mutY, and recA), associated with DNA breaks caused by massive prophage inductions. These results provide new evidence of the hazard of antibiotic overuse in medicine and agriculture.     

Increased Growth of the Microalga Chlorella vulgaris when Coimmobilized and Cocultured in Alginate Beads with the Plant-Growth-Promoting Bacterium Azospirillum brasilense

Coimmobilization of the freshwater microalga Chlorella vulgaris and the plant-growth-promoting bacterium Azospirillum brasilense in small alginate beads resulted in a significantly increased growth of the microalga. Dry and fresh weight, total number of cells, size of the microalgal clusters (colonies) within the bead, number of microalgal cells per cluster, and the levels of microalgal pigments significantly increased. Light microscopy revealed that both microorganisms colonized the same cavities inside the beads, though the microalgae tended to concentrate in the more aerated periphery while the bacteria colonized the entire bead. The effect of indole-3-acetic acid addition to microalgal culture prior to immobilization of microorganisms in alginate beads partially imitated the effect of A. brasilense. We propose that coimmobilization of microalgae and plant-growth-promoting bacteria is an effective means of increasing microalgal populations within confined environments.     

Transmission of Alternaria macrospora in Cotton Seeds

Alternaria macrospora was isolated from seeds only after the natural opening of the bolls and exposure of seeds to an environment in which the fungus was present. The fungus lacks the ability to penetrate the boll wall and reach the seed site. Attempts to isolate the pathogen from seeds of immature bolls at different developmental stages failed. Internal infection by slow injection resulted in seed infection and partial shedding of the injected plant parts which was high in buds and decreased with the ripening to mature bolls. Severity of seed infection was not dependent on either inoculum level, boll physiological age or even if the boll itself was not diseased. Infection of flowers under field conditions caused flower shedding. Naturally infected seeds or inoculated seeds with inoculum levels of 100 spores/ml and above resulted in diseased cotyledons, the incidence of which was, for inoculated seeds, positively correlated with inoculum level. A small difference was observed between cultivars in susceptibility to artificial inoculation at the cotyledon stage. A. macrospora survived on commercial cotton seeds and on post-season plants left growing at the field edges. Survival in plant debris under field conditions was minimal and may only have a minor effect on field reinfestation.     

Ethanol causes the redistribution of L1 cell adhesion molecule in lipid rafts

Fetal alcohol spectrum disorder is estimated to affect 1% of live births. The similarities between children with fetal alcohol syndrome and those with mutations in the gene encoding L1 cell adhesion molecule (L1) implicates L1 as a target of ethanol developmental neurotoxicity. Ethanol specifically inhibits the neurite outgrowth promoting function of L1 at pharmacologic concentrations. Emerging evidence shows that localized disruption of the lipid rafts reduces L1-mediated neurite outgrowth. We hypothesize that ethanol impairment of the association of L1 with lipid rafts is a mechanism underlying ethanol's inhibition of L1-mediated neurite outgrowth. In this study, we examine the effects of ethanol on the association of L1 and lipid rafts. We show that, in vitro, L1 but not N-cadherin shifts into lipid rafts following treatment with 25 mM ethanol. The ethanol concentrations causing this effect are similar to those inhibiting L1-mediated neurite outgrowth. Increasing chain length of the alcohol demonstrates the same cutoff as that previously shown for inhibition of L1-L1 binding. In addition, in cerebellar granule neurons in which lipid rafts are disrupted with methyl-beta-cyclodextrin, the rate of L1-mediated neurite outgrowth on L1-Fc is reduced to background rate and that this background rate is not ethanol sensitive. These data indicate that ethanol may inhibit L1-mediated neurite outgrowth by retarding L1 trafficking through a lipid raft compartment.     

Matching fusion protein systems for affinity analysis of two interacting families of proteins: the cohesin-dockerin interaction

Cellulosomes are multi-enzyme complexes that orchestrate the efficient degradation of cellulose and related plant cell wall polysaccharides. The complex is maintained by the high-affinity protein-protein interaction between two complementary modules: the cohesin and the dockerin. In order to characterize the interaction between different cohesins and dockerins, we have developed matching fusion-protein systems, which harbor either the cohesin or the dockerin component. For this purpose, corresponding plasmid cassettes were designed, which encoded for the following carrier proteins: (i) a thermostable xylanase with an appended His-tag; and (ii) a highly stable cellulose-binding module (CBM). The resultant xylanase-dockerin and CBM-cohesin fusion products exhibited high expression levels of soluble protein. The expressed, affinity-purified proteins were extremely stable, and the functionality of the cohesin or dockerin component was retained. The fusion protein system was used to establish a sensitive and reliable, semi-quantitative enzyme-linked affinity assay for determining multiple samples of cohesin-dockerin interactions in microtiter plates. A variety of cohesin-dockerin systems, which had been examined previously using other methodologies, were revisited applying the affinity-based enzyme assay, the results of which served to verify the validity of the approach.     

It's time to swim! Zebrafish and the circadian clock

The zebrafish represents a fascinating model for studying key aspects of the vertebrate circadian timing system. Easy access to early embryonic development has made this species ideal for investigating how the clock is first established during embryogenesis. In particular, the molecular basis for the functional development of the zebrafish pineal gland has received much attention. In addition to this dedicated clock and photoreceptor organ, and unlike the situation in mammals, the clocks in zebrafish peripheral tissues and even cell lines are entrainable by direct exposure to light thus providing unique insight into the function and evolution of the light input pathway. Finally, the small size, low maintenance costs and high fecundity of this fish together with the availability of genetic tools make this an attractive model for forward genetic analysis of the circadian clock. Here, we review the work that has established the zebrafish as a valuable clock model organism and highlight the key questions that will shape the future direction of research.