Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

Background

In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate.

Results

U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.

Conclusion

The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.     

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.

     

Models for forest ecosystem management: a European perspective

BACKGROUND: Forest management in Europe is committed to sustainability. In the face of climate change and accompanying risks, however, planning in order to achieve this aim becomes increasingly challenging, underlining the need for new and innovative methods. Models potentially integrate a wide range of system knowledge and present scenarios of variables important for any management decision. In the past, however, model development has mainly focused on specific purposes whereas today we are increasingly aware of the need for the whole range of information that can be provided by models. It is therefore assumed helpful to review the various approaches that are available for specific tasks and to discuss how they can be used for future management strategies. SCOPE: Here we develop a concept for the role of models in forest ecosystem management based on historical analyses. Five paradigms of forest management are identified: (1) multiple uses, (2) dominant use, (3) environmentally sensitive multiple uses, (4) full ecosystem approach and (5) eco-regional perspective. An overview of model approaches is given that is dedicated to this purpose and to developments of different kinds of approaches. It is discussed how these models can contribute to goal setting, decision support and development of guidelines for forestry operations. Furthermore, it is shown how scenario analysis, including stand and landscape visualization, can be used to depict alternatives, make long-term consequences of different options transparent, and ease participation of different stakeholder groups and education. CONCLUSIONS: In our opinion, the current challenge of forest ecosystem management in Europe is to integrate system knowledge from different temporal and spatial scales and from various disciplines. For this purpose, using a set of models with different focus that can be selected from a kind of toolbox according to particular needs is more promising than developing one overarching model, covering ecological, production and landscape issues equally well.     

An in situ evidence for autocrine function of NO in the vasculature

The concept of endothelium derived relaxing factor (EDRF) implies that nitric oxide (NO) generated by NO synthase in the endothelium diffuses to the underlying vascular smooth muscle cells (VSMC) modulating thereby vascular tone. VSMC were regarded as passive recipients of NO from endothelial cells. However, this paradigm of a paracrine function of NO became currently subject to considerable debate. To address this issue, we examined the localization of enzymes engaged in l-arginine-NO-cGMP signaling in the rat blood vessels. Employing multiple immunocytochemical labeling complemented with signal amplification, electron microscopy, Western blotting, and RT-PCR, we found that NO synthase was differentially expressed in blood vessels depending on the blood vessel type. Moreover, the expression pattern of NO synthase in VSMC showed striking parallels with arginase and soluble guanylyl cyclase. Our findings challenge the commonly accepted view that the expression of NO synthase is restricted to vascular endothelial cells and lends further support to an alternative mechanism, by which constitutive local NOS expression in VSMC may modulate vascular functions in an endothelium-independent manner. Moreover, the co-expression of enzymes engaged in l-arginine-NO-cGMP signaling (NO synthase, arginase, and soluble guanylyl cyclase) in VSMC is indicative of an autocrine fashion of NO signaling in the vasculature in addition to the paracrine role of NO generated in the endothelium.     

DNA extraction from bronchial aspirates for molecular cytology: which method to take?

OBJECTIVE: To date, there are only few systematic reports on the quality of DNA extracted from routine diagnostic cytologic specimens. It was the aim of the present study to evaluate the ability of 50% ethanol/2% carbowax (Saccomanno fixative) to preserve bronchial secretions with high quality genomic DNA as well as to compare different DNA extraction methods. METHODS: DNA was extracted from 45 bronchial aspirates by four different extraction protocols. Beside DNA yield, DNA quality with regard to purity, integrity, and PCR success rate were investigated. RESULTS: No fragmentation of sample DNA due to the fixative was detected. It was preserved as high molecular weight DNA. DNA yield, purity, and integrity were dependent on the DNA extraction method to some extend. Irrespective of the DNA extraction method the PCR success rate for amplification of beta-globin gene fragments (268, 536, and 989 bp) was 100%. CONCLUSION: A fixative containing 50% ethanol/2% carbowax preserves high quality DNA which is well suited for PCR-based assays regardless of the extraction protocol used. The selection of the DNA extraction protocol has to be adjusted to the circumstances of application.     

Prognostic significance of DNA cytometry in carcinoma of the uterine cervix FIGO stage IB and II.

OBJECTIVE: To assess the prognostic value of DNA-image cytometry in cervical carcinoma of the uterus and its relation to other established prognostic factors. STUDY DESIGN: The study included 116 cases of cervical carcinoma FIGO stages IB and II which were treated with radical abdominal hysterectomy. The median follow-up was 55 months (range 1-162 months). DNA image cytometry was performed on cytologic specimens prepared by enzymatic cell separation from formalin-fixed, paraffin-embedded tissues. DNA stemline ploidy, DNA stemline aneuploidy, 5c exceeding rate, 9c exceeding rate, 2c deviation index, and DNA malignancy grade were computed. DNA-variables as well as various clinical and histological variables were related to survival rates. RESULTS: In multivariate statistical analysis DNA stemline ploidy using 2.2c as a cut-off value and FIGO stage showed to be statistically significant available presurgery predictors of survival, whereas the postsurgical parameters lymphonodal status, tumor size and parametrial involvement were significantly correlated with survival. The synopsis of all parameters in a multivariate Cox model indicated that - with declining relevance - the number of positive pelvic lymph nodes, DNA stemline ploidy using a cut-off level at a modal value of 2.2c, largest pelvic lymph node, 5c exceeding rate, and ratio of carcinoma area to cervix area, were of predictive value for survival. CONCLUSIONS: Our results suggest that prognostic information deducted from classical staging parameters is successfully complemented by DNA image cytometry which can be applied pretherapeutically.     

Electron and X-ray microscopic analyses of reaggregated materials obtained after fractionation of dissolved sporopollenin

Summary Exines fromTypha angustifolia L. pollen were dissolved in hot 2-aminoethanol. The solubilisate was successively fractionated and reaggregated via a dialysis cascade with dialysis tubings of different exclusion volumina. Four fractions of reaggregated material with different molecular mass were obtained. Fraction 1 with a molecular mass above 25,000 Da, fraction 2 with a molecular mass between 10,000–25,000 Da, fraction 3 with a molecular mass between 5,000–10,000 Da, and fraction 4 of a molecular mass lower than 5,000 Da. The fractions were comparatively analysed by scanning and transmission electron microscopy and X-ray microscopy. The material of the fractions with a molecular mass above 10,000 Da exhibit high congruence to the initial material. Analysis of the reaggregated material with the lowest molecular mass revealed special distinct substructures which in form and size showed high similarities to substructures of exines described in literature. In detail, spherical substructures consisting of an electron-dense core surrounded by an electron-transparent corona and in addition elongated substructures with a distinctive surface sculpture were detected.Abbreviations SEM scanning electron microscopy - TEM transmission electron microscopy     

Immunogold electron microscopy of soluble proteins: localization of Bet v I major allergen in ultra-thin sections of birch pollen after anhydrous fixation techniques

To localize the highly water-soluble major allergen Bet v I in ultra-thin sections of birch pollen, pollen grains were cracked, air-dried, and processed for electron microscopy using one of the following preparation techniques: fixation in aqueous p-formaldehyde + cetylpyridinium chloride; fixation in p-formaldehyde vapor; fixation in benzoquinone vapor; inert dehydration; or no fixation. Afterwards the pollen grains were embedded in Lowicryl K4M resin at low temperature. Ultra-thin sections were cut and incubated with a monoclonal antibody against Bet v I, followed by a gold-labeled secondary antibody. In some experiments, commercial rabbit IgG antibodies against birth pollen allergens were also used, followed by incubation with the protein A-gold complex. Bet v I could be localized only after vapor fixation and in the inert dehydrated specimens. Best preservation of ultrastructure and antigenicity was obtained after p-formaldehyde vapor fixation. Bet v I antibody binding sites were detected only in the cytoplasmic matrix of the pollen grain, never in the pollen wall. Commercial rabbit antibodies bound to cytoplasm and wall of all prepared specimens, even after aqueous fixation. This might be explained by the assumption that these antibodies recognize a variety of antigenic and allergenic structures, not all of which are so highly soluble as Bet v I.     

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