Molecular analysis of Dehalococcoides 16S ribosomal DNA from chloroethene-contaminated sites throughout North America and Europe

The environmental distribution of Dehalococcoides group organisms and their association with chloroethene-contaminated sites were examined. Samples from 24 chloroethene-dechlorinating sites scattered throughout North America and Europe were tested for the presence of members of the Dehalococcoides group by using a PCR assay developed to detect Dehalococcoides 16S rRNA gene (rDNA) sequences. Sequences identified by sequence analysis as sequences of members of the Dehalococcoides group were detected at 21 sites. Full dechlorination of chloroethenes to ethene occurred at these sites. Dehalococcoides sequences were not detected in samples from three sites at which partial dechlorination of chloroethenes occurred, where dechlorination appeared to stop at 1,2-cis-dichloroethene. Phylogenetic analysis of the 16S rDNA amplicons confirmed that Dehalococcoides sequences formed a unique 16S rDNA group. These 16S rDNA sequences were divided into three subgroups based on specific base substitution patterns in variable regions 2 and 6 of the Dehalococcoides 16S rDNA sequence. Analyses also demonstrated that specific base substitution patterns were signature patterns. The specific base substitutions distinguished the three sequence subgroups phylogenetically. These results demonstrated that members of the Dehalococcoides group are widely distributed in nature and can be found in a variety of geological formations and in different climatic zones. Furthermore, the association of these organisms with full dechlorination of chloroethenes suggests that they are promising candidates for engineered bioremediation and may be important contributors to natural attenuation of chloroethenes.     

Mutational analysis of the serotonin receptor 5HT2c in severe early-onset human obesity

Deletion of the serotonin receptor 5HT2c in mice results in increased food intake and obesity. We screened 95 individuals with severe early-onset obesity for mutations in the coding sequence of this gene. We found a novel missense variant c.1255A > G (Thr419Ala) in a single Caucasian subject that was not found in 192 Caucasian control subjects. In transiently-transfected COS cells, the Thr419Ala variant was indistinguishable from the wild-type receptor in its ability to generate inositol phosphate, although differences in coupling to other pathways were not excluded. Three previously unreported silent variants: IVS3 + 30G > A, IVS3 + 80C > G and IVS4 - 31A > G were found with prevalences of 11.5%, 0.5% and 17.9%, respectively. In conclusion, mutations in 5HT2c are unlikely to be a common cause of severe early-onset human obesity. The identification of several novel polymorphisms at this locus may aid future genetic epidemiological studies.     

Stable gene expression from a mammalian artificial chromosome

We have investigated the potential of PAC-based vectors as a route to the incorporation of a gene in a mammalian artificial chromosome (MAC). Previously we demonstrated that a PAC (PAC7c5) containing α-satellite DNA generated mitotically stable MACs in human cells. To determine whether a functional HPRT gene could be assembled in a MAC, PAC7c5 was co-transfected with a second PAC containing a 140 kb human HPRT gene into HPRT-deficient HT1080 cells. Lines were isolated containing a MAC hybridizing with both α-satellite and HPRT probes. The MACs segregated efficiently, associated with kinetochore proteins and stably expressed HPRT message after 60 days without selection. Complementation of the parental HPRT deficiency was confirmed phenotypically by growth on HAT selection. These results suggest that MACs could be further developed for delivering a range of genomic copies of genes into cells and that stable transgene expression can be achieved.     

Oral microbial biofilm stimulation of epithelial cell responses

Oral bacterial biofilms trigger chronic inflammatory responses in the host that can result in the tissue destructive events of periodontitis. However, the characteristics of the capacity of specific host cell types to respond to these biofilms remain ill-defined. This report describes the use of a novel model of bacterial biofilms to stimulate oral epithelial cells and profile select cytokines and chemokines that contribute to the local inflammatory environment in the periodontium. Monoinfection biofilms were developed with Streptococcus sanguinis, Streptococcus oralis, Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis on rigid gas-permeable contact lenses. Biofilms, as well as planktonic cultures of these same bacterial species, were incubated under anaerobic conditions with a human oral epithelial cell line, OKF4, for up to 24h. Gro-1α, IL1α, IL-6, IL-8, TGFα, Fractalkine, MIP-1α, and IP-10 were shown to be produced in response to a range of the planktonic or biofilm forms of these species. P. gingivalis biofilms significantly inhibited the production of all of these cytokines and chemokines, except MIP-1α. Generally, the biofilms of all species inhibited Gro-1α, TGFα, and Fractalkine production, while F. nucleatum biofilms stimulated significant increases in IL-1α, IL-6, IL-8, and IP-10. A. naeslundii biofilms induced elevated levels of IL-6, IL-8 and IP-10. The oral streptococcal species in biofilms or planktonic forms were poor stimulants for any of these mediators from the epithelial cells. The results of these studies demonstrate that oral bacteria in biofilms elicit a substantially different profile of responses compared to planktonic bacteria of the same species. Moreover, certain oral species are highly stimulatory when in biofilms and interact with host cell receptors to trigger pathways of responses that appear quite divergent from individual bacteria.     

Influence of single-level lumbar degenerative disc disease on the behavior of the adjacent segments—A finite element model study

The current study investigated mechanical predictors for the development of adjacent disc degeneration. A 3-D finite element model of a lumbar spine was modified to simulate two grades of degeneration at the L4–L5 disc. Degeneration was modeled by changes in geometry and material properties. All models were subjected to follower preloads of 800 N and moment loads in the three principal directions of motion using a hybrid protocol. Degeneration caused changes in the loading and motion patterns of the segments above and below the degenerated disc. At the level (L3–L4) above the degenerated disc, the motion increased due to moderate degeneration by 21% under lateral bending, 26% under axial rotation and 28% under flexion/extension. At the level (L5-S1) below the degenerated disc, motion increased only during lateral bending by 20% due to moderate degeneration. Both the L3–L4 and L5-S1 segment showed a monotonic increase in both the maximum von Mises stress and shear stress in the annulus as degeneration progressed for all loading directions, expect extension at L3–L4. The most significant increase in stress was observed at the L5-S1 level during axial rotation with nearly a ten-fold increase in the maximum shear stress and 103% increase in the maximum von Mises stress. The L5-S1 segment also showed a progressive increase in facet contact force for all loading directions with degeneration. Nucleus pressure did not increase significantly for any loading direction at either the caudal or cephalic adjacent segment. Results suggest that single-level degeneration can increase the risk for injury at the adjacent levels.     

Mechanomyographic and electromyographic responses to repeated concentric muscle actions of the quadriceps femoris.

In comparison to isometric muscle action models, little is known about the electromyographic (EMG) and mechanomyographic (MMG) amplitude and mean power frequency (MPF) responses to fatiguing dynamic muscle actions. Simultaneous examination of the EMG and MMG amplitude and MPF may provide additional insight with regard to the motor control strategies utilized by the superficial muscles of the quadriceps femoris during a concentric fatiguing task. Thus, the purpose of this study was to examine the EMG and MMG amplitude and MPF responses of the vastus lateralis (VL), rectus femoris (RF), and vastus medialis (VM) during repeated, concentric muscle actions of the dominant leg. Seventeen adults (21.8+/-1.7 yr) performed 50 consecutive, maximal concentric muscle actions of the dominant leg extensors on a Biodex System 3 Dynamometer at velocities of 60 degrees s(-1) and 300 degrees s(-1). Bipolar surface electrode arrangements were placed over the mid portion of the VL, RF, and VM muscles with a MMG contact sensor placed adjacent to the superior EMG electrode on each muscle. Torque, MMG and EMG amplitude and MPF values were calculated for each of the 50 repetitions. All values were normalized to the value recorded during the first repetition and then averaged across all subjects. The cubic decreases in torque at 60 degrees s(-1) (R2 = 0.972) and 300 degrees s(-1) (R2 = 0.931) was associated with a decline in torque of 59+/-24% and 53+/-11%, respectively. The muscle and velocity specific responses for the MMG amplitude and MPF demonstrated that each of the superficial muscles of the quadriceps femoris uniquely contributed to the control of force output across the 50 repetitions. These results suggested that the MMG responses for the VL, RF, VM during a fatiguing task may be influenced by a number of factors such as fiber type differences, alterations in activation strategy including motor unit recruitment and firing rate and possibly muscle wisdom.     

The addition of CO2 to traditional taste solutions alters taste quality

Previous studies of the effect of carbonation on taste perception have suggested that it may be negligible, manifesting primarily in increases in the perceived intensity of weak salt and sour stimuli. Assuming CO2 solutions in the mouth stimulate only trigeminal nerve endings, this result is not altogether surprising; however, there are neurophysiological data indicating that CO2 stimulates gustatory as well as trigeminal fibers. In that case, carbonation might alter the quality profile of a stimulus without producing substantial changes in overall taste intensity--much as occurs when qualitatively different taste stimuli are mixed. To address this possibility, subjects were asked to rate the total taste intensity of moderate concentrations of stimuli representing each of the basic tastes and their binary combinations, with an without added carbonation. They then subdivided total taste intensity into the proportions of sweetness, saltiness, sourness, bitterness and 'other taste qualities' they perceived. The addition of carbonation produced only small increases in ratings of total taste intensity. However, rather dramatic alterations in the quality profiles of stimuli were observed, particularly for sweet and salty tastes. The nature of the interaction is consistent with a direct effect of carbonation/CO2 on the gustatory system, although the possibility that at least some of the observed effects reflect trigeminal-gustatory interactions cannot be ruled out.