Temporal and stoichiometric patterns of algal stimulation of litter-associated heterotrophic microbial activity

1. Periphyton communities associated with submerged plant detritus contain interacting autotrophic and heterotrophic microbes, and are sites of extracellular enzymatic activity. The strength and nature of these interactions might be expected to change over time as microbial communities develop on plant litter. Microbial interactions and enzymatic activity can be altered by nutrient availability, suggesting that litter stoichiometry could also affect these phenomena.
2. We grew wetland plants under ambient and nutrient-enriched conditions to generate plant litter of differing nutrient content. In two experiments, we investigated: (1) the influence of algal photosynthesis on fungal and bacterial production and the activities of four extracellular enzymes throughout a 54-day period of microbial colonisation and growth; and (2) the influence of litter stoichiometry on these relationships.
3. Ambient and nutrient-enriched standing-dead plant litter was collected and then submerged in wetland pools to allow for natural microbial colonisation and growth. Litter samples were periodically retrieved and transported to the laboratory for experiments manipulating photosynthesis using the photosystem II inhibitor DCMU (which effectively prevents algal photosynthetic activity). Algal (¹⁴C-bicarbonate), bacterial (³H-leucine), and fungal (¹⁴C-acetate) production, and β-glucosidase, β-xylosidase, leucine aminopeptidase, and phosphatase activities (MUF- or AMC-labelled fluorogenic substrates) were measured under conditions of active and inhibited algal photosynthesis.
4. Photosynthesis stimulated overall fungal and bacterial production in both experiments, although the strength of stimulation varied amongst sampling dates. Phosphatase activity was stimulated by photosynthesis during the first, but not the second, experiment. No other enzymatic responses to short-term photosynthesis manipulations were observed.
5. Microbial communities on high-nutrient litter occasionally showed increased extracellular enzyme activity, fungal growth rates, and bacterial production compared to communities on non-enriched litter, but algal and fungal production were not affected. Litter stoichiometry had no effects on fungal, bacterial, or enzymatic responses to photosynthesis, but the mean enzyme vector analysis angle (a measure of P- versus N-acquiring enzyme activity) was positively correlated to litter N:P, suggesting that elevated litter N:P led to an increase in the relative activity of P-acquiring enzymes.
6. These results supported the hypothesis that algal photosynthesis strongly influences heterotrophic microbial activity on macrophyte leaf litter, especially that of fungi, throughout microbial community development. However, the strength of this photosynthetic stimulation does not generally depend on small differences in litter nutrient content.
7. Stimulation of microbial heterotrophs by algal photosynthesis could drive diurnal shifts in periphyton community and aquatic ecosystem function, as well as linking green (photoautotroph-based) and brown (detrital-based) food webs.

     

Analysis of cell-type-specific chromatin modifications and gene expression in Drosophila neurons that direct reproductive behavior

Examining the role of chromatin modifications and gene expression in neurons is critical for understanding how the potential for behaviors are established and maintained. We investigate this question by examining Drosophila melanogaster fru P1 neurons that underlie reproductive behaviors in both sexes. We developed a method to purify cell-type-specific chromatin (Chromatag), using a tagged histone H2B variant that is expressed using the versatile Gal4/UAS gene expression system. Here, we use Chromatag to evaluate five chromatin modifications, at three life stages in both sexes. We find substantial changes in chromatin modification profiles across development and fewer differences between males and females. Additionally, we find chromatin modifications that persist in different sets of genes from pupal to adult stages, which may point to genes important for cell fate determination in fru P1 neurons. We generated cell-type-specific RNA-seq data sets, using translating ribosome affinity purification (TRAP). We identify actively translated genes in fru P1 neurons, revealing novel stage- and sex-differences in gene expression. We also find chromatin modification enrichment patterns that are associated with gene expression. Next, we use the chromatin modification data to identify cell-type-specific super-enhancer-containing genes. We show that genes with super-enhancers in fru P1 neurons differ across development and between the sexes. We validated that a set of genes are expressed in fru P1 neurons, which were chosen based on having a super-enhancer and TRAP-enriched expression in fru P1 neurons.     

Development of synthetic peptide substrates for the poliovirus 3C proteinase.

Picornaviruses, such as polio, translate their entire genome as a single polyprotein which must be proteolytically processed to produce the mature viral proteins. A majority of these cleavages are catalyzed by the virus-encoded cysteine proteinase, 3C. We report here the design and synthesis of a series of oligopeptide substrates, based upon native 3C cleavage sites, for an HPLC assay of poliovirus 3C proteinase activity. A similar series of peptides based upon human rhinovirus 3C cleavage sites was also examined. The enzyme shows a marked preference for those peptides with a proline in the P'2 position. A quenched fluorescent substrate suitable for continuous assay of 3C proteinase activity was also synthesized. Both the HPLC assay and the fluorescence assay were used to evaluate a number of potential 3C proteinase inhibitors.     

Next-generation polymerized human hemoglobins in hepatic bioreactor simulations

Hepatic hollow fiber (HF) bioreactors can be used to provide temporary support to patients experiencing liver failure. Before being connected to the patient's circulation, cells in the bioreactor must be exposed to a range of physiological O₂ concentrations as observed in the liver sinusoid to ensure proper performance. This zonation in cellular oxygenation promotes differences in hepatocyte phenotype and may better approximate the performance of a real liver within the bioreactor. Polymerized human hemoglobin (PolyhHb) locked in the tense quaternary state (T-state) has the potential to both supply and regulate O₂ transport to cultured hepatocytes in the bioreactor due to its low O₂ affinity. In this study, T-state PolyhHb production and purification processes were optimized to minimize the concentration of low-molecular-weight PolyhHb species in solution. Deconvolution of size-exclusion chromatography spectra was performed to calculate the distribution of polymeric Hb species in the final product. Fluid flow and mass transport within a single fiber of a hepatic HF bioreactor was computationally modeled with finite element methods to simulate the effects of employing T-state PolyhHb to facilitate O₂ transport in a hepatic bioreactor system. Optimal bioreactor performance was defined as having a combined hypoxic and hyperoxic volume fraction in the extracapillary space of less than 0.05 where multiple zones were observed. The Damköhler number and Sherwood number had strong inverse relationships at each cell density and fiber thickness combination. These results suggest that targeting a specific Damköhler number may be beneficial for optimal hepatic HF bioreactor operation.     

A comparison of the effects of aspirin and histamine on fluid balance in the recruited lung

Salicylate administration has been reported to increase the flow of protein-rich lymph from lungs of animals, however, the mechanism of this response is unclear. In the present study we measured pulmonary hemodynamics and lung fluid and lung fluid and protein flux in anesthetized sheep, surgically prepared for the collection of lung lymph, in order to examine the possible effect of aspirin (ASP) on lung vascular permeability. ASP was given during recruitment of pulmonary microvascular surface area induced by sustained elevation of left atrial pressure (Pla) (Group 1) or continuous infusion of adenosine triphosphate (ATP) (Group 2). We compared the results of ASP administration to those found in similarly prepared animals given histamine (H) during like periods of increased Pla (Group 3) or ATP infusion (Group 4). ASP administration resulted in increased lymphatic protein clearance (Cp) in both Groups 1 and 2. In Group 1, following the characteristic increase in lung lymph flow (Q1) and fall in the ratio of lung lymph to plasma protein concentration (L/P) produced by Pla elevation, ASP administration resulted in a further increase in Q1 and a significant increase in L/P. The results found in ASP animals are qualitatively similar to those observed in Groups 3 and 4 after H. While we cannot specifically rule out a hemodynamic effect of the drug, our results suggest the increased protein flux observed following ASP administration was mediated at least in part through and increase in lung microvascular permeability.     

Comparison of afferent and efferent lung lymph in the sheep

Efferent lymph collected from the caudal mediastinal lymph node (CMN) in the sheep lung lymph fistula model has been reported to represent free pulmonary interstitial fluid. Studies that utilize this model assume that nodal transit does not alter the composition of lymph. We collected afferent lymph from the tracheobronchial node (TBN) while simultaneously collecting CMN efferent lymph in acutely prepared sheep. We compared afferent and efferent lymph protein concentrations (CA and CE) and changes in flow rates (QLA and QLE) during base line and periods of elevated left atrial pressure (Pla). As a result of elevated Pla, QLA and QLE increased and the afferent lymph-to-plasma protein concentration ratio (CA/Cp) and the efferent lymph-to-plasma protein concentration ratio (CE/Cp) fell. The CA/Cp was significantly lower than the CE/Cp during base line (0.67 vs. 0.80) and periods of elevated Pla (0.41 vs. 0.61). Although we cannot exclude regional permeability differences, the difference between CA/Cp and CE/Cp is most likely due to the concentration of lymph within the CMN. Our data suggest nodal modification of CA is correlated with the afferent lymph-to-plasma colloid osmotic pressure ratio (pi A/pi p) and further suggest that nodal alteration of lymph during elevated Pla is due to the influence of decreased pi A/pi p at the blood-to-lymph barrier. We conclude that afferent lymph is a more accurate representation of lung free interstitial fluid because collection of pulmonary afferent lymph obviates the complications introduced by the CMN. Studies utilizing efferent lymph may have overestimated lung microvascular permeability in the acute sheep preparation.