Intervertebral disc temperature mapping during disc biacuplasty in the human cadaver.

BACKGROUND: Intradiscal biacuplasty (IDB) is a novel heating therapy using cooled radiofrequency (RF), which may offer relief for discogenic pain. Effective neuroablation may be achieved intradiscally at higher lesion temperatures. The safety of intradiscal heating at elevated temperatures using cooled RF has never been reported. OBJECTIVE: The purpose of this study is to map the intradiscal and peridiscal temperatures when IDB is performed at increased temperature using a modified lesion approach. The resulting temperature profiles are used to assess the safety and theoretical efficacy of this approach to ablate nociceptors in the posterior annulus. STUDY DESIGN: Research article. METHODS: Eleven lumbar discs in a non-perfused human cadaver were treated by IDB. Temperature profiles in the disc during bipolar lesion at 50°C followed by 2 monopolar lesions at 60°C were mapped using custom thermocouples. Temperatures inside the disc, at the nerve roots, and in the midline ventral epidural space were monitored in real-time using a data-collection system with custom RF filters. SETTING: Human research laboratory. RESULTS: Higher maximum temperature was reached intradiscally, and a larger volume of tissue was exposed to neuroablative temperature (> 45°C). Temperature at the nerve roots and in the epidural space increased by 2.4°C ± 2.6°C and 4.9°C ± 1.9°C (mean ± SD), respectively, during bipolar lesion. Similarly, temperature increased by 2.2°C ± 1.9°C and 0.8°C ± 1.3°C at the nerve roots and in the epidural space, respectively, during monopolar lesion. LIMITATIONS: Limitations include the ex vivo setting which lacks perfusion and may not reproduce in vivo conditions such as cerebrospinal fluid dynamics. CONCLUSIONS: The modified treatment paradigm showed intradiscal heating is achieved and is concentrated in the posterior annulus, suggesting minimal risk of thermal damage to the neighboring neural structures. Clinical benefits should be evaluated.

Over-expression of AtPAP2 in Camelina sativa leads to faster plant growth and higher seed yield.

BACKGROUND: Lipids extracted from seeds of Camelina sativa have been successfully used as a reliable source of aviation biofuels. This biofuel is environmentally friendly because the drought resistance, frost tolerance and low fertilizer requirement of Camelina sativa allow it to grow on marginal lands. Improving the species growth and seed yield by genetic engineering is therefore a target for the biofuels industry. In Arabidopsis, overexpression of purple acid phosphatase 2 encoded by Arabidopsis (AtPAP2) promotes plant growth by modulating carbon metabolism. Overexpression lines bolt earlier and produce 50% more seeds per plant than wild type. In this study, we explored the effects of overexpressing AtPAP2 in Camelina sativa. RESULTS: Under controlled environmental conditions, overexpression of AtPAP2 in Camelina sativa resulted in longer hypocotyls, earlier flowering, faster growth rate, higher photosynthetic rate and stomatal conductance, increased seed yield and seed size in comparison with the wild-type line and null-lines. Similar to transgenic Arabidopsis, activity of sucrose phosphate synthase in leaves of transgenic Camelina was also significantly up-regulated. Sucrose produced in photosynthetic tissues supplies the building blocks for cellulose, starch and lipids for growth and fuel for anabolic metabolism. Changes in carbon flow and sink/source activities in transgenic lines may affect floral, architectural, and reproductive traits of plants. CONCLUSIONS: Lipids extracted from the seeds of Camelina sativa have been used as a major constituent of aviation biofuels. The improved growth rate and seed yield of transgenic Camelina under controlled environmental conditions have the potential to boost oil yield on an area basis in field conditions and thus make Camelina-based biofuels more environmentally friendly and economically attractive.

The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.

Mitochondrial mRNA editing in Trypanosoma brucei requires the specific interaction of a guide RNA with its cognate mRNA. Hundreds of gRNAs are involved in the editing process, each needing to target their specific editing domain within the target message. We hypothesized that the structure surrounding the mRNA target may be a limiting factor and involved in the regulation process. In this study, we selected four mRNAs with distinct target structures and investigated how sequence and structure affected efficient gRNA targeting. Two of the mRNAs, including the ATPase subunit 6 and ND7-550 (5' end of NADH dehydrogenase subunit 7) that have open, accessible anchor binding sites show very efficient gRNA targeting. Electrophoretic mobility shift assays indicate that the cognate gRNA for ND7-550 had 10-fold higher affinity for its mRNA than the A6 pair. Surface plasmon resonance studies indicate that the difference in affinity was due to a four-fold faster association rate. As expected, mRNAs with considerable structure surrounding the anchor binding sites were less accessible and had very low affinity for their cognate gRNAs. In vitro editing assays indicate that efficient pairing is crucial for gRNA directed cleavage. However, only the A6 substrate showed gRNA-directed cleavage at the correct editing site. This suggests that different gRNA/mRNA pairs may require different "sets" of accessory factors for efficient editing. By characterizing a number of different gRNA/mRNA interactions, we may be able to define a "bank" of RNA editing substrates with different putative chaperone and other co-factor requirements. This will allow the more efficient identification and characterization of transcript specific RNA editing accessory proteins.

The use of immobilized neurotrophins to support neuron survival and guide nerve fiber growth in compartmentalized chambers.

We answered two major questions: (1) does retrograde signaling involve retrograde transport of nerve growth factor (NGF); and (2) is a gradient of immobilized NGF sufficient to promote and guide local axonal growth? To answer these questions, we developed a technique that resulted in stably immobilized NGF and combined this with compartmented chambers. NGF was photochemically-immobilized on a chitosan surface either in the cell body (CB) compartment, distal axon (DA) compartment, or both. Neuron survival and axon outgrowth were found to be insignificantly different from positive controls where soluble NGF was present. When NGF was immobilized on chitosan surfaces in the DA compartment, and in the absence of soluble NGF, neuron survival was observed, likely due to the retrograde signal of the activated TrkA receptor and NGF-induced signals, but not the retrograde signal of NGF itself. Axons were guided towards the higher end of the step concentration gradient of NGF that was photoimmobilized on the chitosan surface in the DA compartment by laser confocal patterning, demonstrating axonal guidance. These studies provide better insight into NGF signaling mechanisms which are important to both understanding developmental disorders and degenerative diseases of the nervous system, as well as improving design strategies to promote nerve regeneration after injury.

Miniaturized system of neurotrophin patterning for guided regeneration.

Understanding the fundamentals of cell behaviour is imperative for designing and improving engineering strategies for regenerative medicine. By combining the precision of confocal microscopy with photochemistry, nerve growth factor (NGF) was chemically immobilized on chitosan films either in distinct areas or as concentration gradients. Using rhodamine as a proxy for NGF, a series of immobilized concentration gradients were created, using the number of rastering scans within a defined area and the distance between each area as a way to control the resulting gradient. The same photochemistry was applied to create NGF patterns on chitosan films which were visualized by immunostaining, and the immobilized NGF remained bioactive as demonstrated with a neuron survival assay. Neuron survival was 73.2+/-1.3% after 3 days of culture on chitosan films with 30 ng/cm(2) of homogenously immobilized NGF, which was comparable to 74.8+/-3.4% neuron survival on chitosan with 50 ng/ml of soluble NGF present. Interestingly, when neurons were plated on a chitosan film that had distinct immobilized NGF-patterned areas surrounded by unmodified chitosan, the neurons remained predominantly as single cells in the NGF-patterned regions, but formed aggregates outside of these patterns on the plain chitosan film. Thus, the immobilized NGF pattern influenced neuron behaviour and can be used to further probe mechanisms of other neuron behaviour such as axon guidance. Importantly, the versatility of the confocal laser patterning technique reported here can be extended to other factors to elucidate fundamental cell functions, and hence design strategies in regenerative medicine.

Peptide surface modification of methacrylamide chitosan for neural tissue engineering applications.

Nerve fibres are guided to their targets by the combined actions of chemotactic and haptotactic stimuli; however, translating these stimuli to a scaffold that will promote nerve regeneration is nontrivial. In pursuit of this goal, we synthesized and characterized cell-adhesive, biodegradable chitosan scaffolds. Chitosan amine groups were reacted with methacrylic anhydride resulting in a water soluble methacrylamide chitosan (MC) that was then crosslinked by radical polymerization resulting in a scaffold. Biodegradability by lysozyme and penetrability of the scaffold by rat superior cervical ganglion (SCG) neurons were studied. Maleimide-terminated cell adhesive peptides, mi-GDPGYIGSR and mi-GQASSIKVAV, were coupled to a thiolated form of MC to promote cell adhesion. The MC scaffold was found to be porous, biodegradable, and to allow neurite penetration. Interestingly, all of these properties were found to depend upon the amount of initiator used in crosslinking. Covalent modification of the MC scaffold with cell adhesive peptides significantly improved neuronal adhesion and neurite outgrowth. The MC can be crosslinked to form a novel scaffold, where our results demonstrate its suitability in neural tissue engineering and its potential for other engineered tissues, such as cartilage repair, where chitosan has already demonstrated some utility.

Identification of pentatricopeptide repeat proteins in Trypanosoma brucei.

A new class of organellar proteins, characterized by pentatricopeptide repeat (PPR) motifs, has been identified in plants. These proteins contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding a strand of RNA. All PPR proteins characterized to date appear to be involved in RNA processing pathways in organelles. Twenty-three PPR proteins have been identified in Trypanosoma brucei and database research indicates that most of these proteins are predicted to contain the traditional mitochondrial target sequence. Orthologues of each of the 23 proteins have also been identified in Leishmania major and Trypanosoma cruzi, indicating that these proteins represent a highly conserved class of proteins within the kinetoplastid family. Preliminary experiments using RNAi to specifically silence one identified PPR gene (TbPPRl- Tb927.2.3180), indicate that cells depleted of TbPPRl transcripts show a slow growth phenotype and altered mitochondrial maxicircle RNA profiles. This initial characterization suggests that PPR proteins will play important roles in the complex RNA processing required for mitochondrial gene expression in trypanosomes.

Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of a gRNA bound to its cognate mRNA.

Expression of mitochondrial genes in Trypanosoma brucei requires RNA editing of its mRNA transcripts. During editing, uridylates are precisely inserted and deleted as directed by the gRNA template to create the protein open reading frame. This process involves the bimolecular interaction of the gRNA with its cognate pre-edited mRNA and the assembly of a protein complex with the enzymatic machinery required. While a considerable amount of work has been done identifying the protein components of the editing complex, very little is known about how a functional editosome is assembled. In addition, the importance of RNA structure in establishing a functional editing complex is poorly understood. Work in our lab suggests that different mRNA/gRNA pairs can form similar secondary structures suggesting that a common core architecture may be important for editosome recognition and function. Using solution structure probing, we have investigated the structure of the initiating gRNA, gCYb-558, in the mRNA/gRNA complex with pre-edited apocytochrome b mRNA. Our data indicate that the stem-loop formed by the guiding region of the gRNA alone is maintained in its interaction with the pre-edited message. In addition, our data suggest that a gRNA stem-loop structure is maintained through the first few editing events by the use of alternative base-pairing with the U-tail.

Poly(ethylene glycol) (PEG) enhances dynamic surface activity of a bovine lipid extract surfactant (BLES).

Shortage or malfunction of pulmonary surfactant in alveolar space leads to a critical condition termed respiratory distress syndrome (RDS). Surfactant replacement therapy, the major method to treat RDS, is an expensive treatment. In this paper, the effect of poly(ethylene glycol) (PEG) to improve dynamic surface activity of a bovine lipid extract surfactant (BLES) was studied by axisymmetric drop shape analysis (ADSA) and a captive bubble method. The activity of BLES+PEG mixtures was compared to that of a natural surfactant containing surfactant proteins A and D. When PEG was added into BLES mixtures, the surface tension hysteresis of BLES films was minimized when the films were compressed by more than 50%. PEG also helps to quickly restore surfactant films after film collapse. Thus, as far as surface tension effects go, the findings suggest that PEG might be used as a substitute for surfactant-associated protein SP-A in therapeutic surfactant products, and might also be used to reduce the amount of BLES required in clinical applications.

Poly(ethylene glycol) enhances the surface activity of a pulmonary surfactant.

The primary role of lung surfactant is to reduce surface tension at the air-liquid interface of alveoli during respiration. Axisymmetric drop shape analysis (ADSA) was used to study the effect of poly(ethylene glycol) (PEG) on the rate of surface film formation of a bovine lipid extract surfactant (BLES), a therapeutic lung surfactant preparation. PEG of molecular weights 3,350; 8,000; 10,000; 35,000; and 300,000 in combination with a BLES mixture of 0.5 mg/mL was studied. The adsorption rate of BLES alone at 0.5 mg/mL was much slower than that of a natural lung surfactant at the same concentration; more than 200 s are required to reach the equilibrium surface tension of 25 mJ/m(2). PEG, while not surface active itself, enhances the adsorption of BLES to an extent depending on its concentration and molecular weight. These findings suggest that depletion attraction induced by higher molecular weight PEG (in the range of 8,000 to 35,000) may be responsible for increasing the adsorption rate of BLES at low concentration. The results provide a basis for using PEG as an additive to BLES to reduce its required concentration in clinical treatment, thus reducing the cost for surfactant replacement therapy.

Constrained sessile drop as a new configuration to measure low surface tension in lung surfactant systems.

Existing methodology for surface tension measurements based on drop shapes suffers from the shortcoming that it is not capable to function at very low surface tension if the liquid dispersion is opaque, such as therapeutic lung surfactants at clinically relevant concentrations. The novel configuration proposed here removes the two big restrictions, i.e., the film leakage problem that is encountered with such methods as the pulsating bubble surfactometer as well as the pendant drop arrangement, and the problem of the opaqueness of the liquid, as in the original captive bubble arrangement. A sharp knife edge is the key design feature in the constrained sessile drop that avoids film leakage at low surface tension. The use of the constrained sessile drop configuration in conjunction with axisymmetric drop shape analysis to measure surface tension allows complete automation of the setup. Dynamic studies with lung surfactant can be performed readily by changing the volume of a sessile drop, and thus the surface area, by means of a motor-driven syringe. To illustrate the validity of using this configuration, experiments were performed using an exogenous lung surfactant preparation, bovine lipid extract surfactant (BLES) at 5.0 mg/ml. A comparison of results obtained for BLES at low concentration between the constrained sessile drop and captive bubble arrangement shows excellent agreement between the two approaches. When the surface area of the BLES film (0.5 mg/ml) was compressed by about the same amount in both systems, the minimum surface tensions attained were identical within the 95% confidence limits.

Evidence for U-tail stabilization of gRNA/mRNA interactions in kinetoplastid RNA editing.

The most dramatic example of RNA editing is found in the mitochondria of trypanosomes. In these organisms, U-insertions/deletions can create mRNAs that are twice as large as the gene that encodes them. Guide RNAs (gRNAs) that are complementary to short stretches of the mature message direct the precise placements of the U residues. The binding of gRNA to mRNA is a fundamental step in RNA editing and understanding the relative importance of the elements that confer affinity and specificity on this interaction is critical to our understanding of the editing process. In this study, we have analyzed the relative binding affinities of two different gRNA/mRNA pairs. The affinity of gA6-14 for its message (ATPase 6) is high, with an apparent K(D) in the 5-10 nM range. In contrast, gCYb-558 has a low affinity for its cognate mRNA. Deletion of the gRNA U-tail caused a significant reduction in the binding affinity for only the gCYb-558 pair, and was observed only under physiological magnesium conditions. These results indicate that the U-tail contribution can differ substantially between the different gRNA/mRNA pairs. In addition, our results suggest that the efficiency of gRNA/mRNA interaction is highly dependent on thermodynamic parameters determined by the local sequences and their adopted structures surrounding the anchor-binding site.

Diverse microbial communities inhabiting ferromanganese deposits in Lechuguilla and Spider Caves.

Lechuguilla Cave is an ancient, deep, oligotrophic subterranean environment that contains an abundance of low-density ferromanganese deposits, the origin of which is uncertain. To assess the possibility that biotic factors may be involved in the production of these deposits and to investigate the nature of the microbial community in these materials, we carried out culture-independent, small subunit ribosomal RNA (SSU rRNA) sequence-based studies from two sites and from manganese and iron enrichment cultures inoculated with ferromanganese deposits from Lechuguilla and Spider Caves. Sequence analysis showed the presence of some organisms whose closest relatives are known iron- and manganese-oxidizing/reducing bacteria, including Hyphomicrobium, Pedomicrobium, Leptospirillum, Stenotrophomonas and Pantoea. The dominant clone types in one site grouped with mesophilic Archaea in both the Crenarchaeota and Euryarchaeota. The second site was dominated almost entirely by lactobacilli. Other clone sequences were most closely related to those of nitrite-oxidizing bacteria, nitrogen-fixing bacteria, actinomycetes and beta- and gamma-Proteobacteria. Geochemical analyses showed a fourfold enrichment of oxidized iron and manganese from bedrock to darkest ferromanganese deposits. These data support our hypothesis that microorganisms may contribute to the formation of manganese and iron oxide-rich deposits and a diverse microbial community is present in these unusual secondary mineral formations.

Connexin 26 35delG does not represent a mutational hotspot.

Non-syndromic hearing impairment (NSHI) is the most common form of deafness and presents with no other symptoms or sensory defects. Mutations in the gap junction gene GJB2 account for a high proportion of recessive NSHI. The GJB2 gene encodes connexin 26, which forms plasma membrane channels between cochlear cells. In Caucasian populations a single mutation, 35delG, accounts for most cases of NSHI. This mutation appears to be most prevalent in individuals of Mediterranean European descent, with carrier frequencies estimated as being as high as one in thirty. The 35delG region may be a mutational hotspot. The mutation arises from the deletion of a guanine from a six-guanine stretch and nearby microsatellite markers show little evidence for linkage disequilibrium. We believe that 35delG is an old mutation in a chromosomal region of high recombination. The genetic context of the 35delG mutation was examined to distinguish between an old or a recurring mutation. We identified two single-nucleotide polymorphisms (SNPs) immediately upstream of the first exon of GJB2. Polymerase chain reaction/restriction fragment length polymorphism analysis determined the SNP genotype of 35delG containing chromosomes from various populations, including Italy, Brazil, and North America. We found the same, relatively rare, polymorphism associated with the 35delG mutation in all populations studied. We have also examined microsatellite markers D13S175, which is 80 kb telomeric to GJB2, and D13S1316, which is 80 kb centromeric to GJB2. D13S175 appears to be in weak linkage disequilibrium with 35delG, while D13S1316 is less so. SNPs located between the 35delG mutation and the microsatellite markers show strong evidence of linkage disequilibrium. Taken together, these results indicate there has been substantial recombination near the 35delG mutation; however, we present evidence that the 35delG mutation arose in European and Middle Eastern populations from a single mutational event on a founder chromosome.