Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.     

Inferring differences in the distribution of reaction rates across conditions

Elucidating changes in the distribution of reaction rates in metabolic pathways under different conditions is a central challenge in systems biology. Here we present a method for inferring regulation mechanisms responsible for changes in the distribution of reaction rates across conditions from correlations in time-resolved data. A reversal of correlations between conditions reveals information about regulation mechanisms. With the use of a small in silico hypothetical network, based on only the topology and directionality of a known pathway, several regulation scenarios can be formulated. Confronting these scenarios with experimental data results in a short list of possible pathway regulation mechanisms associated with the reversal of correlations between conditions. This procedure allows for the formulation of regulation scenarios without detailed prior knowledge of kinetics and for the inference of reaction rate changes without rate information. The method was applied to experimental time-resolved metabolomics data from multiple short-term perturbation-response experiments in S. cerevisiae across aerobic and anaerobic conditions. The method's output was validated against a detailed kinetic model of glycolysis in S. cerevisiae, which showed that the method can indeed infer the correct regulation scenario.     

Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.

     

The apoptotic pathway contributing to the deletion of naive CD8 T cells during the induction of peripheral tolerance to a cross-presented self-antigen

The maintenance of T cell tolerance in the periphery proceeds through several mechanisms, including anergy, immuno-regulation, and deletion via apoptosis. We examined the mechanism underlying the induction of CD8 T cell peripheral tolerance to a self-Ag expressed on pancreatic islet beta-cells. Following adoptive transfer, Ag-specific clone 4 T cells underwent deletion independently of extrinsic death receptors, including Fas, TNFR1, or TNFR2. Additional experiments revealed that the induction of clone 4 T cell apoptosis during peripheral tolerance occurred via an intrinsic death pathway that could be inhibited by overexpression of Bcl-2 or targeted deletion of the proapoptotic molecule, Bim, thereby resulting in accumulation of activated clone 4 T cells. Over-expression of Bcl-2 in clone 4 T cells promoted the development of effector function and insulitis whereas Bim-/- clone 4 cells were not autoaggressive. Examination of the upstream molecular mechanisms contributing to clone 4 T cell apoptosis revealed that it proceeded in a p53, E2F1, and E2F2-independent manner. Taken together, these data reveal that initiation of clone 4 T cell apoptosis during the induction of peripheral tolerance to a cross-presented self-Ag occurs through a Bcl-2-sensitive and at least partially Bim-dependent mechanism.     

Statistical data processing in clinical proteomics

This review discusses data analysis strategies for the discovery of biomarkers in clinical proteomics. Proteomics studies produce large amounts of data, characterized by few samples of which many variables are measured. A wealth of classification methods exists for extracting information from the data. Feature selection plays an important role in reducing the dimensionality of the data prior to classification and in discovering biomarker leads. The question which classification strategy works best is yet unanswered. Validation is a crucial step for biomarker leads towards clinical use. Here we only discuss statistical validation, recognizing that biological and clinical validation is of utmost importance. First, there is the need for validated model selection to develop a generalized classifier that predicts new samples correctly. A cross-validation loop that is wrapped around the model development procedure assesses the performance using unseen data. The significance of the model should be tested; we use permutations of the data for comparison with uninformative data. This procedure also tests the correctness of the performance validation. Preferably, a new set of samples is measured to test the classifier and rule out results specific for a machine, analyst, laboratory or the first set of samples. This is not yet standard practice. We present a modular framework that combines feature selection, classification, biomarker discovery and statistical validation; these data analysis aspects are all discussed in this review. The feature selection, classification and biomarker discovery modules can be incorporated or omitted to the preference of the researcher. The validation modules, however, should not be optional. In each module, the researcher can select from a wide range of methods, since there is not one unique way that leads to the correct model and proper validation. We discuss many possibilities for feature selection, classification and biomarker discovery. For validation we advice a combination of cross-validation and permutation testing, a validation strategy supported in the literature.     

Molecular characterization of the brassinosteroid-deficient lkb mutant in pea

The brassinosteriod-deficient lkb mutant of garden pea (Pisum sativum L.) is characterized by an erectoides phenotype (reduced internode length, thickened stems, epinastic leaves), which is rescued by application of exogenous brassinolide. We show that the LKB gene is the Arabidopsis DIMINUTO/DWARF-1 (DIM/DWF1) homologue of pea. The DIM/DWF1 homologue from lkb plants contains a mutation that may result in reduced enzyme function, thus resulting in the previously shown accumulation of 24-methylenecholesterol and a deficiency of its hydrogenated product, campesterol. This ultimately leads to a deficiency of the biologically active brassionolide. The mutation in the lkb sequence cosegregates with the lkb phenotype. Northern analyis of the LKB gene revealed that the gene is ubiquitously expressed around the plant and that there is no evidence for negative feedback regulation of the gene.     

Anaerobic Ammonia Oxidation in the Presence of Nitrogen Oxides (NOx) by Two Different Lithotrophs

The anaerobic ammonia-oxidizing activity of the planctomycete Candidatus “Brocadia anammoxidans” was not inhibited by NO concentrations up to 600 ppm and NO₂ concentrations up to 100 ppm. B. anammoxidans was able to convert (detoxify) NO, which might explain the high NO tolerance of this organism. In the presence of NO₂, the specific ammonia oxidation activity of B. anammoxidans increased, and Nitrosomonas-like microorganisms recovered an NO₂-dependent anaerobic ammonia oxidation activity. Addition of NO₂ to a mixed population of B. anammoxidans and Nitrosomonas induced simultaneous specific anaerobic ammonia oxidation activities of up to 5.5 mmol of NH₄⁺ g of protein⁻¹ h⁻¹ by B. anammoxidans and up to 1.5 mmol of NH₄⁺ g of protein⁻¹ h⁻¹ by Nitrosomonas. The stoichiometry of the converted N compounds (NO₂⁻/NH₃ ratio) and the microbial community structure were strongly influenced by NO₂. The combined activity of B. anammoxidans and Nitrosomonas-like ammonia oxidizers might be of relevance in natural environments and for technical applications.     

Improved nitrogen removal by application of new nitrogen-cycle bacteria

In order to meet increasingly stringentEuropean discharge standards, new applicationsand control strategies for the sustainableremoval of ammonia from wastewater have to beimplemented. In this paper we discuss anitrogen removal system based on the processesof partial nitrification and anoxic ammoniaoxidation (anammox). The anammox process offersgreat opportunities to remove ammonia in fullyautotrophic systems with biomass retention. Noorganic carbon is needed in such nitrogenremoval system, since ammonia is used aselectron donor for nitrite reduction. Thenitrite can be produced from ammonia inoxygen-limited biofilm systems or in continuousprocesses without biomass retention. Forsuccessful implementation of the combinedprocesses, accurate biosensors for measuringammonia and nitrite concentrations, insight inthe complex microbial communities involved, andnew control strategies have to be developed andevaluated.