Substratum preference of the caddisfly Helicopsyche borealis (Hagen) (Trichoptera: Helicopsychidae)

The position of Helicopsyche borealis (Hagen) (Trichoptera: Helicopsychidae) larvae on the substratum surface is dependent on the current regime but varies with larval size. All size classes of larvae chose significantly different positions on the substratum under high versus low current velocities. All size classes preferred exposed surfaces under low current velocities. Small larvae preferred the upper surfaces of substrata under low current velocities and were physically displaced under high current velocities. Larger larvae also occurred on upper surfaces, but were more evenly dispersed over all surfaces than smaller larvae, and tended to aggregate on down-stream faces of rocks during high flow.     

Population Dynamics of Soil Pseudomonads in the Rhizosphere of Potato (Solanum tuberosum L.)

Rhizosphere population dynamics of seven Pseudomonas fluorescens and Pseudomonas putida strains isolated from rhizospheres of various agricultural plants were studied on potato (Solanum tuberosum L.) in field soil under controlled environmental conditions. Rhizosphere populations of two strains (B10 and B4) were quantitatively related to initial seed piece inoculum levels when plants were grown at −0.3 bar matric potential. At a given inoculum level, rhizosphere populations of strain B4 were consistently greater than those of strain B10. In vivo growth curves on 4-cm root tip-proximal segments indicated that both strains grew at similar rates in the potato rhizosphere, but large populations of strain B10 were not maintained at 24°C after 7 h, whereas those of strain B4 were maintained for at least 40 h. Although both strains grew more rapidly in the rhizosphere at 24°C than at 12°C, their rhizosphere populations after seed piece inoculation were generally greater at 12 or 18°C, indicating that in vivo growth did not solely determine rhizosphere populations in these studies. In vitro osmotolerance of seven Pseudomonas strains (including strains B4 and B10) was correlated with their abilities to establish stable populations in the rhizosphere of potato. Stability of rhizosphere populations of the Pseudomonas strains studied here was maximized at low (i.e., 12°C) soil temperatures. These results indicate that Pseudomonas strains differ in their capacity to maintain stable rhizosphere populations in association with potato. This capacity, distinct from the ability to grow in the rhizosphere, may limit the establishment of rhizosphere populations under some environmental conditions.     

Inorganic and Metal-Organic Growth Requirements of the Genus Bacteroides

The inorganic and metal-organic growth requirements of ruminal and nonruminal Bacteroides species were compared. The heme requirement of many nonruminal Bacteroides species was similar to that of Bacteroides ruminicola subsp. ruminicola and was a general tetrapyrrole requirement. Some nonruminal Bacteroides species utilized succinate or alpha-ketoglutarate, as well as tetrapyrrole-containing compounds, in place of heme. Fe(+) as well as heme was required for maximal yields of some Bacteroides species. The divalent cation requirements of Bacteroides species are complex. Mg(2+) deletion from a medium containing Mg(2+), Ca(2+), Co(2+), and Mn(2+) reduced the yields of all isolates. Ca(2+) deletion from the same medium reduced the growth yields of Bacteroides fragilis, B. fundiliformis, and one strain of B. oralis. The effects of Mg(2+) and Ca(2+) on the growth of Bacteroides isolates was influenced by other divalent cations. Relatively large quantities of Na(+) were obligately required by all of the currently recognized predominant rumen Bacteroides species. Nonruminal Bacteroides species either did not require Na(+) or required only small amounts. The Na(+) requirement of some nonruminal Bacteroides species could be partially replaced by Li(+) or Cs(+). The Na(+) requirement of rumen Bacteroides species was absolute. The inorganic and metal-organic growth requirements of Bacteroides species appear useful as aids in species differentiation.     

Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle.

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.     

Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.     

A Label-free Selected Reaction Monitoring Workflow Identifies a Subset of Pregnancy Specific Glycoproteins as Potential Predictive Markers of Early-onset Pre-eclampsia

Pre-eclampsia (PE) is a serious complication of pregnancy with potentially life threatening consequences for both mother and baby. Presently there is no test with the required performance to predict which healthy first-time mothers will go on to develop PE. The high specificity, sensitivity, and multiplexed nature of selected reaction monitoring holds great potential as a tool for the verification and validation of putative candidate biomarkersfor disease states. Realization of this potential involves establishing a high throughput, cost effective, reproducible sample preparation workflow. We have developed a semi-automated HPLC-based sample preparation workflow before a label-free selected reaction monitoring approach. This workflow has been applied to the search for novel predictive biomarkers for PE.To discover novel candidate biomarkers for PE, we used isobaric tagging to identify several potential biomarker proteins in plasma obtained at 15 weeks gestation from nulliparous women who later developed PE compared with pregnant women who remained healthy. Such a study generates a number of “candidate” biomarkers that require further testing in larger patient cohorts. As proof-of-principle, two of these proteins were taken forward for verification in a 100 women (58 PE, 42 controls) using label-free SRM. We obtained reproducible protein quantitation across the 100 samples and demonstrated significant changes in protein levels, even with as little as 20% change in protein concentration. The SRM data correlated with a commercial ELISA, suggesting that this is a robust workflow suitable for rapid, affordable, label-free verification of which candidate biomarkers should be taken forward for thorough investigation. A subset of pregnancy-specific glycoproteins (PSGs) had value as novel predictive markers for PE.The identification of clinically relevant plasma biomarkers with diagnostic and/or predictive value continues to challenge the proteomics field. Whereas once the biomarker pipeline was described as a two part discovery and validation process, there is increasing consensus that an intermediate step is required in which the proteins identified in the discovery phase are technically verified in 50 to 200 samples. This verification step identifies false positives from the discovery phase and allows prioritization of proteins to be taken into large-scale clinical validation studies (1). Although commercial ELISA kits may be used in this phase, these are unavailable for many proteins, are expensive, and may lack specificity. In addition, sample requirements may be too high to perform ELISA on all candidates, especially if many proteins are identified as potential markers by low powered, high penetration discovery workflows.Selected reaction monitoring (SRM)¹ mass spectrometry has great potential as an alternative verification method (2–6) as it can be multiplexed, customized, and is highly specific. This potential has not been exploited to date, largely because of technical issues developing a low-cost, reproducible workflow encompassing plasma and serum preparation and LC/MS analysis with the capability to measure protein levels reproducible in hundreds of samples. With traditional stable isotope dilution SRM (SID-SRM), the high cost of accurately quantified, purified stable isotope encoded peptides or proteins may be prohibitive for the verification of multiple peptides from many proteins. Label-free relatively quantitative methods are increasingly popular in discovery proteomics but to a much lesser extent in targeted SRM studies (7, 8).For any SRM method, sample preparation workflows must balance the extent of enrichment and fractionation to enable quantification of lower abundance proteins, against increased technical variability (which is influenced by the number of sample handling steps) and reduced multiplexed potential as a consequence of fractionating peptides from the protein of interest into several distinct fractions. It is also essential that the true technical variation in the workflow is quantitatively evaluated from freezer to MS analysis, rather than just the variation within the LC-SRM part of the experiment. As a paradigm for a label-free SRM assay, we developed our workflow and applied it to the verification of candidate biomarkers that indicate the risk of pre-eclampsia (PE).PE affects 2–8% of pregnancies, and is characterized by hypertension and proteinuria, which may progress to severe maternal complications or death (9). Because delivery of the infant is the only effective intervention, a third of babies are born premature and fetal or newborn mortality is increased three- to 10-fold (10). Its complex etiology involves abnormal placentation, an altered immune response and a sensitized maternal vascular endothelium (11). Prediction of the condition in early pregnancy would allow prevention strategies, such as low dose aspirin, to be targeted to high risk women. In first-time pregnant women, a group particularly at risk, biomarkers continue to fall short of a test that would be useful or cost effective in clinical practice (12–14). Better-performing novel biomarkers are required.The aim of this study was to identify candidate predictive biomarkers for PE and then develop a verification assay using mass spectrometry to determine whether these should be taken forward into more extensive and expensive validation studies. Initial discovery experiments were employed using a pooled sample iTRAQ approach using two different MS platforms to increase plasma proteome coverage. Among the set of proteins discovered, we then developed a label-free SRM assay for relative quantification of CXCL7 (Platelet basic protein; PBP) and members of the Pregnancy specific glycoprotein (PSG) family in a 100-sample set from the international SCreeningfOr Pregnancy Endpoints (SCOPE) study (www.scopestudy.net). Our workflow allowed the specificity and linearity of response for each peptide to be determined, along with true technical variability. Although absolute concentration and LOD/LOQ cannot be calculated using this approach, we aimed to test the hypothesis that a label-free SRM approach could provide a rapid, robust, and efficient screen of candidate plasma biomarkers.     

Altered Antibody Profiles against Common Infectious Agents in Chronic Disease

Despite the important diagnostic value of evaluating antibody responses to individual human pathogens, antibody profiles against multiple infectious agents have not been used to explore health and disease mainly for technical reasons.  We hypothesized that the interplay between infection and chronic disease might be revealed by profiling antibodies against multiple agents. Here, the levels of antibodies against a panel of 13 common infectious agents were evaluated with the quantitative Luciferase Immunoprecipitation Systems (LIPS) in patients from three disease cohorts including those with pathogenic anti-interferon-γ autoantibodies (IFN-γ AAB), HIV and Sjögren’s syndrome (SjS) to determine if their antibody profiles differed from control subjects.  The IFN-γ AAB patients compared to controls demonstrated statistically higher levels of antibodies against VZV (p=0.0003), EBV (p=0.002), CMV (p=0.003), and C. albicans (p=0.03), but lower antibody levels against poliovirus (p=0.04). Comparison of HIV patients with blood donor controls revealed that the patients had higher levels of antibodies against CMV (p=0.0008), HSV-2 (p=0.0008), EBV (p=0.001), and C. albicans (p=0.01), but showed decreased levels of antibodies against coxsackievirus B4 (p=0.0008), poliovirus (p=0.0005),   and HHV-6B (p=0.002). Lastly, SjS patients had higher levels of anti-EBV antibodies (p=0.03), but lower antibody levels against several enteroviruses including a newly identified picornavirus, HCoSV-A (p=0.004), coxsackievirus B4 (p=0.04), and poliovirus (p=0.02). For the IFN-γ AAB and HIV cohorts, principal component analysis revealed unique antibody clusters that showed the potential to discriminate patients from controls.  The results suggest that antibody profiles against these and likely other common infectious agents may yield insight into the interplay between exposure to infectious agents, dysbiosis, adaptive immunity and disease activity.     

Efficacy of a Food Safety Comic Book on Knowledge and Self-Reported Behavior for Persons Living with AIDS

Introduction

Persons living with AIDS are highly vulnerable to foodborne enteric infections with the potential for substantial morbidity and mortality. Educational materials about foodborne enteric infections intended for this immunocompromised population have not been assessed for their efficacy in improving knowledge or encouraging behavior change.

Methods/Results

AIDS patients in four healthcare facilities in Chicago, New Orleans, and Puerto Rico were recruited using fliers and word of mouth to healthcare providers. Those who contacted research staff were interviewed to determine food safety knowledge gaps and risky behaviors. A food safety educational comic book that targeted knowledge gaps was created, piloted, and provided to these patients who were instructed to read it and return at least 2 weeks later for a follow-up interview. The overall food safety score was determined by the number of the 26 knowledge/belief/behavior questions from the survey answered correctly. Among 150 patients who participated in both the baseline and follow-up questionnaire, the intervention resulted in a substantial increase in the food safety score (baseline 59%, post-intervention 81%, p<0.001). The intervention produced a significant increase in all the food safety knowledge, belief, and behavior items that comprised the food safety score. Many of these increases were from baseline knowledge below 80 percent to well above 90%. Most (85%) of the patients stated they made a change to their behavior since receiving the educational booklet.

Conclusion

This comic book format intervention to educate persons living with AIDS was highly effective. Future studies should examine to what extent long-term behavioral changes result.