Differential proteomic analysis of a polymicrobial biofilm

Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia exist in a polymicrobial biofilm associated with chronic periodontitis. The aim of this study was to culture these three species as a polymicrobial biofilm and to determine proteins important for bacterial interactions. In a flow cell all three species attached and grew as a biofilm; however, after 90 h of culture P. gingivalis and T. denticola were closely associated and dominated the polymicrobial biofilm. For comparison, planktonic cultures of P. gingivalis and T. denticola were grown separately in continuous culture. Whole cell lysates were subjected to SDS-PAGE, followed by in-gel proteolytic H(2)(16)O/H(2)(18)O labeling. From two replicates, 135 and 174 P. gingivalis proteins and 134 and 194 T. denticola proteins were quantified by LC-MALDI TOF/TOF MS. The results suggest a change of strategy in iron acquisition by P. gingivalis due to large increases in the abundance of HusA and HusB in the polymicrobial biofilm while HmuY and other iron/haem transport systems decreased. Significant changes in the abundance of peptidases and enzymes involved in glutamate and glycine catabolism suggest syntrophy. These data indicate an intimate association between P. gingivalis and T. denticola in a biofilm that may play a role in disease pathogenesis.     

Microengineered systems with iPSC-derived cardiac and hepatic cells to evaluate drug adverse effects

Hepatic and cardiac drug adverse effects are among the leading causes of attrition in drug development programs, in part due to predictive failures of current animal or in vitro models. Hepatocytes and cardiomyocytes differentiated from human induced pluripotent stem cells (iPSCs) hold promise for predicting clinical drug effects, given their human-specific properties and their ability to harbor genetically determined characteristics that underlie inter-individual variations in drug response. Currently, the fetal-like properties and heterogeneity of hepatocytes and cardiomyocytes differentiated from iPSCs make them physiologically different from their counterparts isolated from primary tissues and limit their use for predicting clinical drug effects. To address this hurdle, there have been ongoing advances in differentiation and maturation protocols to improve the quality and use of iPSC-differentiated lineages. Among these are in vitro hepatic and cardiac cellular microsystems that can further enhance the physiology of cultured cells, can be used to better predict drug adverse effects, and investigate drug metabolism, pharmacokinetics, and pharmacodynamics to facilitate successful drug development. In this article, we discuss how cellular microsystems can establish microenvironments for these applications and propose how they could be used for potentially controlling the differentiation of hepatocytes or cardiomyocytes. The physiological relevance of cells is enhanced in cellular microsystems by simulating properties of tissue microenvironments, such as structural dimensionality, media flow, microfluidic control of media composition, and co-cultures with interacting cell types. Recent studies demonstrated that these properties also affect iPSC differentiations and we further elaborate on how they could control differentiation efficiency in microengineered devices. In summary, we describe recent advances in the field of cellular microsystems that can control the differentiation and maturation of hepatocytes and cardiomyocytes for drug evaluation. We also propose how future research with iPSCs within engineered microenvironments could enable their differentiation for scalable evaluations of drug effects.     

Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d- galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 &mgr;m recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.      

Fungal and bacterial contributions to nitrogen cycling in cheatgrass-invaded and uninvaded native sagebrush soils of the western USA

Aim

There is interest in determining how cheatgrass (Bromus tectorum L.) modifies N cycling in sagebrush (Artemisia tridentata Nutt.) soils of the western USA.

Methods

To gain insight into the roles of fungi and bacteria in N cycling of cheatgrass-invaded and uninvaded sagebrush soils, the fungal protein synthesis inhibitor, cycloheximide (CHX), and the bacteriocidal compound, bronopol (BRO) were combined with a ¹⁵NH₄ ⁺ isotope pool dilution approach.

Results

CHX reduced gross N mineralization to the same rate in both sagebrush and cheatgrass soils indicating a role for fungi in N mineralization in both soil types. In cheatgrass soils BRO completely inhibited gross N mineralization, whereas, in sagebrush soils a BRO-resistant gross N mineralization rate was detected that was slower than CHX sensitive gross N mineralization, suggesting that the microbial drivers of gross N mineralization were different in sagebrush and cheatgrass soils. Net N mineralization was stimulated to a higher rate in sagebrush than in cheatgrass soils by CHX, implying that a CHX inhibited N sink was larger in the former than the latter soils. Initial gross NH₄ ⁺ consumption rates were reduced significantly by both CHX and BRO in both soil types, yet, consumption rates recovered significantly between 24 and 48 h in CHX-treated sagebrush soils. The recovery of NH₄ ⁺ consumption in sagebrush soils corresponded with an increase in the rate of net nitrification.

Conclusions

These results suggest that cheatgrass invasion of sagebrush soils of the northern Great Basin reduces the capacity of the fungal N consumption sink, enhances the capacity of a CHX resistant N sink and alters the contributions of bacteria and fungi to gross N mineralization.

     

Quantifying the Hydroregime of a Temporary Pool Habitat: A Modelling Approach for Ephemeral Rock Pools in SE Botswana

Ecological and evolutionary processes in temporary rock pools operate within constraints imposed by their hydrologic regimes. These shallow pools flood when seasonal rains accumulate on impermeable substrates. Despite the ecological importance of hydrologic conditions for these ecosystems, we typically lack tools and empirical data required to understand the implications of hydrologic variability and climate change for biotic populations and communities in these habitats. In this study, we developed a hydrologic model to simulate rock pool hydrologic regimes based on rainfall, evapotranspiration, and basin geometry. The model was used to investigate long-term patterns of seasonal and inter-annual variation in hydroregime. In addition, hydrologic conditions associated with potential climate change scenarios were simulated and evaluated with respect to the biological requirements of the anostracan Branchipodopsis wolfi. The model’s output for daily inundation matched with field observations with an overall accuracy of 85% and correctly estimated complete hydroperiods with an overall accuracy of 70%. Simulations indicate large variation in individual hydroperiods (76–115%) as well as in the number of hydroperiods per year (19–23%). Furthermore, this study suggests that climate change may significantly alter the rock pool hydroregime. These findings confirm the hydrologic sensitivity of these ephemeral habitats to precipitation patterns, and their potential sensitivity to future climate change. Modelling indicates that the suitability of average inundation conditions for B. wolfi deteriorates significantly under future climate predictions. High levels of spatial and temporal variation in hydrologic conditions are dominant features of these habitats and an essential consideration for understanding population and community-level ecological processes.     

Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.