Differences in Hyporheic-Zone Microbial Community Structure along a Heavy-Metal Contamination Gradient

The hyporheic zone of a river is nonphotic, has steep chemical and redox gradients, and has a heterotrophic food web based on the consumption of organic carbon entrained from downwelling surface water or from upwelling groundwater. The microbial communities in the hyporheic zone are an important component of these heterotrophic food webs and perform essential functions in lotic ecosystems. Using a suite of methods (denaturing gradient gel electrophoresis, 16S rRNA phylogeny, phospholipid fatty acid analysis, direct microscopic enumeration, and quantitative PCR), we compared the microbial communities inhabiting the hyporheic zone of six different river sites that encompass a wide range of sediment metal loads resulting from large base-metal mining activity in the region. There was no correlation between sediment metal content and the total hyporheic microbial biomass present within each site. However, microbial community structure showed a significant linear relationship with the sediment metal loads. The abundances of four phylogenetic groups (groups I, II, III, and IV) most closely related to α-, β-, and γ-proteobacteria and the cyanobacteria, respectively, were determined. The sediment metal content gradient was positively correlated with group III abundance and negatively correlated with group II abundance. No correlation was apparent with regard to group I or IV abundance. This is the first documentation of a relationship between fluvially deposited heavy-metal contamination and hyporheic microbial community structure. The information presented here may be useful in predicting long-term effects of heavy-metal contamination in streams and provides a basis for further studies of metal effects on hyporheic microbial communities.     

Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

Seasonal Dynamics of Shallow-Hyporheic-Zone Microbial Community Structure along a Heavy-Metal Contamination Gradient

Heavy metals contaminate numerous freshwater streams and rivers worldwide. Previous work by this group demonstrated a relationship between the structure of hyporheic microbial communities and the fluvial deposition of heavy metals along a contamination gradient during the fall season. Seasonal variation has been documented in microbial communities in numerous terrestrial and aquatic environments, including the hyporheic zone. The current study was designed to assess whether relationships between hyporheic microbial community structure and heavy-metal contamination vary seasonally by monitoring community structure along a heavy-metal contamination gradient for more than a year. No relationship between total bacterial abundance and heavy metals was observed (R² = 0.02, P = 0.83). However, denaturing gradient gel electrophoresis pattern analysis indicated a strong and consistent linear relationship between the difference in microbial community composition (populations present) and the difference in the heavy metal content of hyporheic sediments throughout the year (R² = 0.58, P < 0.001). Correlations between heavy-metal contamination and the abundance of four specific phylogenetic groups (most closely related to the α, β, and γ-proteobacteria and cyanobacteria) were apparent only during the fall and early winter, when the majority of organic matter is deposited into regional streams. These seasonal data suggest that the abundance of susceptible populations responds to heavy metals primarily during seasons when the potential for growth is highest.     

Seasonal dynamics of shallow-hyporheic-zone microbial community structure along a heavy-metal contamination gradient

Heavy metals contaminate numerous freshwater streams and rivers worldwide. Previous work by this group demonstrated a relationship between the structure of hyporheic microbial communities and the fluvial deposition of heavy metals along a contamination gradient during the fall season. Seasonal variation has been documented in microbial communities in numerous terrestrial and aquatic environments, including the hyporheic zone. The current study was designed to assess whether relationships between hyporheic microbial community structure and heavy-metal contamination vary seasonally by monitoring community structure along a heavy-metal contamination gradient for more than a year. No relationship between total bacterial abundance and heavy metals was observed (R(2) = 0.02, P = 0.83). However, denaturing gradient gel electrophoresis pattern analysis indicated a strong and consistent linear relationship between the difference in microbial community composition (populations present) and the difference in the heavy metal content of hyporheic sediments throughout the year (R(2) = 0.58, P < 0.001). Correlations between heavy-metal contamination and the abundance of four specific phylogenetic groups (most closely related to the alpha, beta, and gamma-proteobacteria and cyanobacteria) were apparent only during the fall and early winter, when the majority of organic matter is deposited into regional streams. These seasonal data suggest that the abundance of susceptible populations responds to heavy metals primarily during seasons when the potential for growth is highest.     

Evaluation of two DNA template preparation methods for post-immunomagnetic separation detection of Cryptosporidium parvum in foods and beverages by PCR

Cryptosporidium parvum oocysts were recovered by immunomagnetic separation from six artificially contaminated foods. Two DNA isolation methods were subsequently evaluated by PCR. The FTA Concentrator-PS filter provided rapid and reproducible detection, although variability increased at lower inoculum levels (88% and 15% detection in high- and low-inoculum-level samples, respectively). Total DNA extraction generated consistent results at all oocyst levels but resulted in longer analysis time (100% and 59% detection in high- and low-inoculum-level samples, respectively). Also reflected in this study was that the matrix played an important role in the ability to recover oocysts, as sample turbidity, pH, and PCR inhibitors all influenced detection.     

Structure and seasonal dynamics of hyporheic zone microbial communities in free-stone rivers of the estern United States

The hyporheic zone of a river is characterized by being nonphotic, exhibiting chemical/redox gradients, and having a heterotrophic food web based on the consumption of organic carbon entrained from surface waters. Hyporheic microbial communities constitute the base of food webs in these environments and are important for maintaining a functioning lotic ecosystem. While microbial communities of rivers dominated by fine-grained sediments are relatively well studied, little is known about the structure and seasonal dynamics of microbial communities inhabiting the predominantly gravel and cobble hyporheic zones of rivers of the western United States. Here, we present the first molecular analysis of hyporheic microbial communities of three different stream types (based on mean base discharge, substratum type, and drainage area), in Montana. Utilizing 16S rDNA phylogeny, DGGE pattern analysis, and qPCR, we have analyzed the prokaryotic communities living on the 1.7 to 2.36 mm grain-size fraction of hyporheic sediments from three separate riffles in each stream. DGGE analysis showed clear seasonal community patterns, indicated similar community composition between different riffles within a stream (95.6–96.6% similarity), and allowed differentiation between communities in different streams. Each river supported a unique complement of species; however, several phylogenetic groups were conserved between all three streams including Pseudomonads and members of the genera Aquabacterium, Rhodoferax, Hyphomicrobium, and Pirellula. Each group showed pronounced seasonal trends in abundance, with peaks during the Fall. The Hyphomicrobium group was numerically dominant throughout the year in all three streams. This work provides a framework for investigating the effects of various environmental factors and anthropogenic effects on microbial communities inhabiting the hyporheic zone.     

A benefit-finding intervention for family caregivers of persons with Alzheimer disease: study protocol of a randomized controlled trial

Background

Patients with ST-elevation myocardial infarction (STEMI) not treated with primary or rescue percutaneous coronary intervention (PCI) are at risk for recurrent ischemia, especially when viability in the infarct-area is present. Therefore, an invasive strategy with PCI of the infarct-related coronary artery in patients with viability would reduce the occurrence of a composite end point of death, reinfarction, or unstable angina (UA).

Methods

Patients admitted with an (sub)acute myocardial infarction, who were not treated by primary or rescue PCI, and who were stable during the first 48 hours after the acute event, were screened for the study. Eventually, we randomly assigned 216 patients with viability (demonstrated with low-dose dobutamine echocardiography) to an invasive or a conservative strategy. In the invasive strategy stenting of the infarct-related coronary artery was intended with abciximab as adjunct treatment. Seventy-five (75) patients without viability served as registry group. The primary endpoint was the composite of death from any cause, recurrent myocardial infarction (MI) and unstable angina at one year. As secondary endpoint the need for (repeat) revascularization procedures and anginal status were recorded.

Results

The primary combined endpoint of death, recurrent MI and unstable angina was 7.5% (8/106) in the invasive group and 17.3% (19/110) in the conservative group (Hazard ratio 0.42; 95% confidence interval [CI] 0.18-0.96; p = 0.032). During follow up revascularization-procedures were performed in 6.6% (7/106) in the invasive group and 31.8% (35/110) in the conservative group (Hazard ratio 0.18; 95% CI 0.13-0.43; p < 0.0001). A low rate of recurrent ischemia was found in the non-viable group (5.4%) in comparison to the viable-conservative group (14.5%). (Hazard-ratio 0.35; 95% CI 0.17-1.00; p = 0.051).

Conclusion

We demonstrated that after acute MI (treated with thrombolysis or without reperfusion therapy) patients with viability in the infarct-area benefit from a strategy of early in-hospital stenting of the infarct-related coronary artery. This treatment results in a long-term uneventful clinical course. The study confirmed the low risk of recurrent ischemia in patients without viability.

Trial registration

ClinicalTrials.gov: NCT00149591.     

Diffraction-Enhanced Computed Tomographic Imaging of Growing Piglet Joints by Using a Synchrotron Light Source

The objective of this project was to develop and test a new technology for imaging growing joints by means of diffraction-enhanced imaging (DEI) combined with CT and using a synchrotron radiation source. DEI–CT images of an explanted 4-wk-old piglet stifle joint were acquired by using a 40-keV beam. The series of scanned slices was later ‘stitched’ together, forming a 3D dataset. High-resolution DEI-CT images demonstrated fine detail within all joint structures and tissues. Striking detail of vasculature traversing between bone and cartilage, a characteristic of growing but not mature joints, was demonstrated. This report documents for the first time that DEI combined with CT and a synchrotron radiation source can generate more detailed images of intact, growing joints than can currently available conventional imaging modalities.Abbreviations: DEI, diffraction-enhanced imagingDiffraction-enhanced imaging (DEI) is a biomedical imaging technique that, compared with conventional radiography, generates very detailed images with more edge contrast but deposits a lower radiation dose to the object. DEI generates enhanced contrast both from absorption, the process involved in conventional radiography, and from of X-ray refraction, a process that harnesses photons that otherwise typically are imperceptibly diffracted.⁴ The DEI technique collects information from X-rays that are refracted as they pass through tissues that have different refractive indices as it almost completely removes diffracted X-rays. In comparison, conventional radiography produces images from X-rays that are attenuated by the tissues through which they pass, but X-rays that are refracted within those same tissues confound, rather than clarify, image contrast. The creation of contrast from the refraction of X-rays, rather than exclusively from absorption, yields images that display more detail with clearer distinction between tissue interfaces. Refraction-based imaging can reveal tiny structures that are transparent to X-ray attenuation but have sufficient variation in density to produce refraction contrast. Furthermore, refraction-based imaging decreases the required radiation dose.²¹To obviate the superimposing effects in a 2-dimensional DEI refraction image, we considered that combining CT with DEI would yield images with even greater clarity. CT allows a 3D representation of the sample, such that contrast from features at different depths are no longer superimposed on one another but can be separated and viewed as independent structures. Although this advantage is valuable in traditional absorption imaging, the additional features that provide contrast in a refraction-based image enhance the value of CT. Combining DEI technology, which is capable of imaging soft-tissue detail, with CT, which allows segregation of the contrast images at different depths, overcomes limitations of conventional X-ray imaging, namely lack of distinction of soft tissues and 2-dimensionality. As we report here, DEI combined with CT and a synchrotron-generated X-ray source yields 3D images of growing joint tissues at a resolution on the order of micrometers, which is much higher than can be generated using conventional imaging techniques.A synchrotron radiation source was required for the development of DEI because a synchrotron currently is the only source capable of providing an intensely brilliant light (millions of times brighter than sunlight and conventional X-ray sources), is highly collimated (light rays in the beam remain parallel with negligible dispersion over distance), can be made to be monochromatic (having a single wavelength), and can be tuned precisely to an array of energy ranges. The Canadian Light Source (www.lightsource.ca), which began operations in 2005, is one of only 47 synchrotron facilities worldwide and the only such facility in Canada. Although nonsynchrotron sources of X-rays for DEI–CT are conceivable,^16,18 such technology requires considerable image-acquisition time. Regardless, the quality of images generated by using synchrotron technology likely would remain the standard with which any new nonsynchrotron DEI–CT technological innovations would be compared.¹⁴Despite refinements in medical imaging, conventional radiography, CT scanning, and MRI still are insufficient to discern fine details, particularly in growing joints in which soft tissues (including cartilage) predominate and change with physiologic growth. The impetus for the current research was to develop an imaging technique that better demonstrated normal joint characteristics during growth and, in the future, could be applied to pathologic joints for experimental research and eventually clinical applications. In particular, we were motivated by a need to more effectively and reliably image growing joints affected by arthritis, a disease associated with alterations of bone and cartilage growth, tissue morphology and vascularity. Childhood arthritis research likely will benefit from having an improved imaging technique to aid in early diagnosis, monitor disease progression, and assess responses to therapies. The long-term outcomes of childhood arthritis are improved with early diagnosis and prompt and effective response to treatment interventions. Clinical and laboratory-based indicators of inflammation are not always adequate to detect and monitor subclinical intraarticular inflammation which, as with overt disease, can lead to progressive joint damage. Imaging can augment clinical and laboratory assessment of arthritis activity, but even the most sensitive currently available modalities are unable to detect all joint pathology.In juvenile arthritis, joint-imaging outcomes are difficult to evaluate because variations associated with normal growth cannot always be easily discerned from variations induced by the disease. Conventional radiography tends to detect advanced joint damage that has affected bone, but cartilage can be assessed only indirectly, and soft tissue abnormalities cannot be fully evaluated. Consequently, conventional radiography has insufficient sensitivity and specificity to be considered useful for diagnosing or monitoring children with inflammatory joint disease.^6,20 MRI, which evaluates both soft tissues and osteochondral structures, can be used to detect cartilage loss, bone erosions, and synovial hypertrophy in children and adolescents, and contrast-enhanced MRI detects active synovitis.^1,10 However, standardized approaches to acquire and interpret MRI data are not established for children in general and, in particular, for children with arthritis;^12,15 it is not always clear, for example, if observed thinning of cartilage is physiologic or pathologic. Furthermore, although MRI is more sensitive than conventional radiography, MRI too has limited precision in detecting fine structures and pathologic changes; a clinical MRI has less than 50% sensitivity in detecting cartilage damage that subsequently is seen arthroscopically.^8,13CT offers another option for joint visualization, given that it provides high-resolution, 3D images of bone from any angle. Despite its high spatial resolution, however, CT cannot match MRI''s soft-tissue contrast resolution, because CT provides negligible variability of attenuation coefficients of soft tissues so attenuation is nearly the same for cartilage, muscles, and ligaments. Furthermore, CT''s value is offset by the necessity for radiation exposure, a particular concern in the pediatric population. Therefore, for joint research and clinical applications, each of the conventional imaging techniques currently available has limitations. A safe, higher resolution imaging system that generates good contrast for all joint structures is required.Because the DEI technique initially was developed by using a synchrotron light source, we similarly used synchrotron technology in the current experiments. In contrast to conventional X-ray tubes, a synchrotron generates light by using radiofrequency waves and electromagnets to energize and accelerate electrons, thus producing brilliant, highly focused light from the entire wavelength spectrum, including X-rays. For the development and evaluation of DEI–CT imaging of joints, we chose to use healthy commercial piglet stifle joints because porcine stifle joints are anatomically similar to human knees.⁵ In addition, pigs grow quickly, reaching skeletal maturity at the distal femur and proximal tibia in 20 mo,¹⁹ thus allowing for the use of the pig as a model to study growth patterns in normal and disease states in a relatively short time period. The current study aimed to develop and test a new technology for imaging growing joints by using DEI combined with CT and a synchrotron radiation source. This report is the first to document the application of DEI–CT for imaging intact, growing joints.