Classification of oligonucleotide fingerprints: application for microbial community and gene expression analyses

MOTIVATION: Oligonucleotide fingerprinting of ribosomal RNA genes (OFRG) is a procedure that sorts rRNA gene (rDNA) clones into taxonomic groups through a series of hybridization experiments. The hybridization signals are classified into three discrete values 0, 1 and N, where 0 and 1, respectively, specify negative and positive hybridization events and N designates an uncertain assignment. This study examined various approaches for classifying the values including Bayesian classification with normally distributed signal data, Bayesian classification with the exponentially distributed data, and with gamma distributed data, along with tree-based classification. All classification data were clustered using the unweighted pair group method with arithmetic mean. RESULTS: The performance of each classification/clustering procedure was compared with results from known reference data. Comparisons indicated that the approach using the Bayesian classification with normal densities followed by tree clustering out-performed all others. The paper includes a discussion of how this Bayesian approach may be useful for the analysis of gene expression data.     

Physiological and ecological significance of sunflecks for dipterocarp seedlings

Irradiance is highly dynamic in many plant canopies. Photosynthesis during sunflecks provides 10-90% of daily carbon gain. The survivorship of tree seedlings in the deeply shaded understorey of tropical rain forests is limited by their ability to maintain a positive carbon balance. Dipterocarp seedlings from the SE Asian rain forest were used as a model system to test novel aspects of the physiological and ecological significance of sunflecks. First, understorey seedlings experienced leaf temperatures up to 38 degrees C in association with sunflecks. Under controlled environment conditions, the inhibition of carbon gain at 38 degrees C, compared with 28 degrees C, was significantly greater during a sequence of sunflecks (-59%), than under uniform irradiance (-40%), providing the same total photosynthetic photon flux density (PPFD). Second, the relative enhancement effects of elevated [CO2] were greater under sunflecks (growth +60%, carbon gain +89%), compared with uniform irradiance (growth +25%, carbon gain +59%), supplying the same daily PPFD. Third, seedling growth rates in the forest understorey were 4-fold greater under a dynamic irradiance treatment characterized by long flecks, compared with a regime of short flecks. Therefore, stresses associated with dynamic irradiance may constrain photosynthetic carbon gain. Additionally, seedling photosynthesis and growth may be more responsive to interactions with abiotic factors, including future changes in climate, than previously estimated. The sensitivity of seedling growth to varying patterns of dynamic irradiance, and the increased likelihood of species-specific responses through interactions with environmental factors, indicates the potential for sunflecks to influence regeneration processes, and hence forest structure and composition.     

The influence of nitrogen on rain forest dipterocarp seedlings exposed to a large increase in irradiance

Dipterocarps dominate the canopy of lowland tropical rain forest in South‐east Asia. Seedlings of these species form diverse assemblages on the forest floor where low irradiance severely limits their growth. Further growth depends largely upon the increased irradiance that can occur with the creation of canopy gaps. However, the response of dipterocarp seedlings to increased irradiance and their subsequent establishment in the canopy may be influenced by the availability of other resources, such as nutrient availability. We investigated the influence of nitrogen supply on aspects of the photosynthetic physiology and growth of seedlings of four dipterocarp species (Shorea leprosula, Shorea johorensis, Shorea oleosa and Dryobalanops lanceolata) growing under low irradiance, during transfer from low to high irradiance, and during subsequent growth at high irradiance. All four species increased growth and photosynthetic capacity in response to N‐supply at high irradiances but not at low irradiance approximating that which can be expected to occur in the forest understorey. When seedlings grown at low irradiances and varying N‐supply were exposed to a large increase in irradiance, all species showed some degree of initial photodamage (measured through chlorophyll fluorescence), the extent of which was similar between species but differed markedly depending on the pre‐exposure growth irradiance and N‐supply. Greater photodamage occurred in seedlings grown at lower compared with higher N‐supply and irradiance. Despite these initial difference in the extent of this photodamage, all seedlings demonstrated a similar capacity to recover from damage. However, the alterations in the photosynthetic physiology of leaves during this recovery differed between species and depended on N‐supply. Under high N‐supply all species apart from S. oleosa increased photosynthetic capacity per unit chlorophyll following exposure to high irradiance by increasing photosynthetic capacity per unit leaf area while, under low N‐supply, an increase in photosynthetic capacity per unit leaf only occurred in D. lanceolata. Our results suggest that variations in N‐availability may have a much greater impact on the relative competitiveness of dipterocarp seedlings during the regenerative phase following canopy gap formation than physiological differences between seedlings. Our results demonstrate a potentially significant role for N‐availability in the regeneration dynamics and distribution of canopy‐dominating dipterocarp species.     

Comparative Efficacy of 177Lu and 90Y for Anti-CD20 Pretargeted Radioimmunotherapy in Murine Lymphoma Xenograft Models

Purpose

Pretargeted radioimmunotherapy (PRIT) is a multi-step method of selectively delivering high doses of radiotherapy to tumor cells while minimizing exposure to surrounding tissues. Yttrium-90 (⁹⁰Y) and lutetium-177 (¹⁷⁷Lu) are two of the most promising beta-particle emitting radionuclides used for radioimmunotherapy, which despite having similar chemistries differ distinctly in terms of radiophysical features. These differences may have important consequences for the absorbed dose to tumors and normal organs. Whereas ⁹⁰Y has been successfully applied in a number of preclinical and clinical radioimmunotherapy settings, there have been few published pretargeting studies with ¹⁷⁷Lu. We therefore compared the therapeutic potential of targeting either ⁹⁰Y or ¹⁷⁷Lu to human B-cell lymphoma xenografts in mice.

Methods

Parallel experiments evaluating the biodistribution, imaging, dosimetry, therapeutic efficacy, and toxicity were performed in female athymic nude mice bearing either Ramos (Burkitt lymphoma) or Granta (mantle cell lymphoma) xenografts, utilizing an anti-CD20 antibody-streptavidin conjugate (1F5-SA) and an ⁹⁰Y- or ¹⁷⁷Lu-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-biotin second step reagent.

Results

The two radionuclides displayed comparable biodistributions in tumors and normal organs; however, the absorbed radiation dose delivered to tumor was more than twice as high for ⁹⁰Y (1.3 Gy/MBq) as for ¹⁷⁷Lu (0.6 Gy/MBq). More importantly, therapy with ⁹⁰Y-DOTA-biotin was dramatically more effective than with ¹⁷⁷Lu-DOTA-biotin, with 100% of Ramos xenograft-bearing mice cured with 37 MBq ⁹⁰Y, whereas 0% were cured using identical amounts of ¹⁷⁷Lu-DOTA-biotin. Similar results were observed in mice bearing Granta xenografts, with 80% of the mice cured with ⁹⁰Y-PRIT and 0% cured with ¹⁷⁷Lu-PRIT. Toxicities were comparable with both isotopes.

Conclusion

⁹⁰Y was therapeutically superior to ¹⁷⁷Lu for streptavidin-biotin PRIT approaches in these human lymphoma xenograft models.     

Fasting Glucose Levels Correlate with Disease Severity of Guillain-Barré Syndrome

Objective

A potential association between diabetes and Guillain-Barré syndrome (GBS) has been indicated by a few case studies. We retrospectively analyzed the clinical features of a large cohort of GBS patients to explore the relationship between the level of fasting plasma glucose (FPG) obtained in the acute phase at admission and the severity of GBS.

Methods

Three hundred and four GBS patients were divided into two groups, one with normal FPG and the other with high FPG levels according to the international standards of FPG.

Results

The GBS disability scale score was positively, the Medical Research Council (MRC) sum score was negatively correlated to the level of FPG, but not to blood HBA1c or CSF glucose concentrations. A relatively higher FPG level was observed in older and younger GBS patients, and more often in those with cranial nerve involvement, autonomic deficit, dyspnea and ventilator dependence than in patients without these clinical characteristics. Importantly, higher levels of FPG at admission were associated with poorer short-term prognosis measured by the MRC sum score and the GBS disability scale at discharge.

Conclusions

Our data demonstrates that FPG in the acute phase of GBS correlates with the severity of GBS and may predict the short-term prognosis of GBS.     

Contribution of subsurface peat to CO2 and CH4 fluxes in a neotropical peatland

Tropical peatlands play an important role in the global carbon cycling but little is known about factors regulating carbon dioxide (CO₂) and methane (CH₄) fluxes from these ecosystems. Here, we test the hypotheses that (i) CO₂ and CH₄ are produced mainly from surface peat and (ii) that the contribution of subsurface peat to net C emissions is governed by substrate availability. To achieve this, in situ and ex situ CO₂ and CH₄ fluxes were determined throughout the peat profiles under three vegetation types along a nutrient gradient in a tropical ombrotrophic peatland in Panama. The peat was also characterized with respect to its organic composition using ¹³C solid state cross‐polarization magic‐angle spinning nuclear magnetic resonance spectroscopy. Deep peat contributed substantially to CO₂ effluxes both with respect to actual in situ and potential ex situ fluxes. CH₄ was produced throughout the peat profile with distinct subsurface peaks, but net emission was limited by oxidation in the surface layers. CO₂ and CH₄ production were strongly substrate‐limited and a large proportion of the variance in their production (30% and 63%, respectively) was related to the quantity of carbohydrates in the peat. Furthermore, CO₂ and CH₄ production differed between vegetation types, suggesting that the quality of plant‐derived carbon inputs is an important driver of trace gas production throughout the peat profile. We conclude that the production of both CO₂ and CH₄ from subsurface peat is a substantial component of the net efflux of these gases, but that gas production through the peat profile is regulated in part by the degree of decomposition of the peat.     

Exosome-producing follicle associated epithelium is not involved in uptake of PrPd from the gut of sheep (Ovis aries): an ultrastructural study

In natural or experimental oral scrapie infection of sheep, disease associated prion protein (PrP(d)) often first accumulates in Peyer's patch (PP) follicles. The route by which infectivity reaches the follicles is unknown, however, intestinal epithelial cells may participate in intestinal antigenic presentation by delivering exosomes as vehicles of luminal antigens. In a previous study using an intestinal loop model, following inoculation of scrapie brain homogenate, inoculum associated PrP(d) was detected by light microscopy shortly (15 minutes to 3.5 hours) after inoculation in the villous lacteals and sub-mucosal lymphatics. No PrP(d) was located within the follicle-associated epithelium (FAE), sub-FAE domes or the PP follicles. To evaluate this gut loop model and the transportation routes in more detail, we used electron microscopy (EM) to study intestinal tissues exposed to scrapie or control homogenates for 15 minutes to 10 days. In addition, immuno-EM was used to investigate whether exosomes produced in the FAE may possess small amounts of PrP(d) that were not detectable by light microscopy. This study showed that the integrity of the intestinal epithelium was sustained in the intestinal loop model. Despite prominent transcytotic activity and exosome release from the FAE of the ileal PP in sheep, these structures were not associated with transportation of PrP(d) across the mucosa. The study did not determine how infectivity reaches the follicles of PPs. The possibility that the infectious agent is transported across the FAE remains a possibility if it occurs in a form that is undetectable by the methods used in this study. Infectivity may also be transported via lymph to the blood and further to all other lymphoid tissues including the PP follicles, but the early presence of PrP(d) in the PP follicles during scrapie infection argues against such a mechanism.     

Evaluation of approaches for measuring taxonomic completeness of lake profundal macroinvertebrate assemblages

1. Benthic macroinvertebrates (MI) are commonly used to assess freshwater ecosystems with the reference condition approach. Such assessments necessitate control for natural community variation, either by categorical typologies or by predictive models that have been widely and successfully developed for running water biota but not previously for lake profundal invertebrates. 2. We evaluated four modelling techniques [multivariate regression tree (MRT), limiting environmental differences, nonparametric multiplicative regression (NPMR) and River Invertebrate Prediction And Classification System (RIVPACS) and the operative Finnish lake typology for assessing taxonomic completeness (observed‐to‐expected number of taxa, O/E) of profundal MI assemblages. We used data from 74 and 33 minimally disturbed reference lake basins for calibration and validation of the approaches, respectively, and 72 test basins subject to various anthropogenic pressures to evaluate sensitivity to detect impact. Either all predicted taxa (threshold probability of capture P_t = 0+) or only those predicted to be captured with ≥0.25 probability were used to calculate O/E. 3. With P_t = 0.25, all four modelling approaches were accurate (mean O/E = 0.966–1.053) but imprecise (SD of O/E = 0.279–0.304) in predicting the fauna actually observed in validation sites. All models were subtly more precise than a null model (mean 1.038, SD 0.343) or the typology (1.046, 0.327). The taxon‐specific NPMR model was slightly more precise than the other three models based on site groupings. 4. The O/E values correlated relatively weakly (r = 0.55–0.86) among the approaches, which thus produced contrasting lake‐specific assessments, despite their seemingly comparable performances. Indeed, typology, suggesting that MI assemblages were impaired in 56% of test sites, was more sensitive than the other approaches (26–46%) as an indicator of human‐induced deterioration. However, this greater ostensible sensitivity seemed to be biased, as lake morphometry, a main driver of natural community variation, remained uncontrolled by the typology. 5. Generally, our exercise illustrates the inconclusiveness of the common validation criteria for the assessment methods. The apparent poor predictability of the profundal fauna, irrespective of the method, may partly stem from large observation error, which could be alleviated by more intensive sampling. However, instead of an O/E‐taxa index, some other metric encompassing quantitative aspects might be preferable for assessing these species‐poor communities.