DNase I and Proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses

Molecular techniques, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), are very sensitive, but may detect total DNA present in a sample, including extracellular DNA (eDNA) and DNA coming from live and dead cells. DNase I is an endonuclease that non-specifically cleaves single- and double-stranded DNA. This enzyme was tested in this study to analyze its capacity of digesting DNA coming from dead cells with damaged cell membranes, leaving DNA from living cells with intact cell membranes available for DNA-based methods. For this purpose, an optimized DNase I/Proteinase K (DNase/PK) protocol was developed. Intact Staphylococcus aureus cells, heat-killed Pseudomonas aeruginosa cells, free genomic DNA of Salmonella enterica, and a mixture of these targets were treated according to the developed DNase/PK protocol. In parallel, these samples were treated with propidium monoazide (PMA) as an already described assay for live-dead discrimination. Quantitative PCR and PCR-DGGE of the eubacterial 16S rDNA fragment were used to test the ability of the DNase/PK and PMA treatments to distinguish DNA coming from cells with intact cell membranes in the presence of DNA from dead cells and free genomic DNA. The methods were applied to three months old autochthonous drinking water biofilms from a pilot facility built at a German waterworks. Shifts in the DNA patterns observed after DGGE analysis demonstrated the applicability of DNase/PK as well as of the PMA treatment for natural biofilm investigation. However, the DNase/PK treatment demonstrated some practical advantages in comparison with the PMA treatment for live/dead discrimination of bacterial targets in drinking water systems.     

Does the magnetic field of a magnetic stirrer in an optical aggregometer affect concurrent platelet aggregation?

Platelets are subjected to extremely low frequency electromagnetic fields during standard aggregometry measurements owing to the use of a magnetic stir bar in the instrument. This study evaluates the effects of this magnetic field exposure on platelet aggregation by comparing the results obtained in a modified aggregometer. Blood samples from healthy volunteers were anticoagulated using citrate or heparin. Platelet‐rich plasma (PRP) samples were prepared. A mechanical stirring device was attached to the aggregometer instead of the magnetic stir bar system. The PRP samples were stirred using a stirring rod tip that did not produce any magnetic fields in one channel of the aggregometer; in the other channel, a stirring rod carrying a small magnet at its tip was used. As a result, a magnetic field in the extremely low frequency range and in the amplitude range of 1.9–65 mT was applied to the platelets assigned to the channel where the magnetic stirring rod tip was used. Aggregation was induced using adenosine diphosphate (ADP), collagen, or epinephrine. The slopes, maximum aggregation values, and areas under the aggregation curves were compared between the magnetic and neutral stirring rod tip groups. For samples stirred with the magnetic stirring rod tip, a significant decrease was observed in 12 of the 14 parameters evaluated for aggregations induced with ADP or collagen compared to the neutral stirring rod tip, regardless of the method used for anticoagulation. This observation indicates that the magnetic stir bars used in standard aggregometry may significantly alter aggregation parameters and platelets may be possible targets of electromagnetic fields. Bioelectromagnetics 34:349–357, 2013. © 2012 Wiley Periodicals, Inc.     

Acetate supplementation increases brain phosphocreatine and reduces AMP levels with no effect on mitochondrial biogenesis

Acetate supplementation in rats increases plasma acetate and brain acetyl-CoA levels. Although acetate is used as a marker to study glial energy metabolism, the effect that acetate supplementation has on normal brain energy stores has not been quantified. To determine the effect(s) that an increase in acetyl-CoA levels has on brain energy metabolism, we measured brain nucleotide, phosphagen and glycogen levels, and quantified cardiolipin content and mitochondrial number in rats subjected to acetate supplementation. Acetate supplementation was induced with glyceryl triacetate (GTA) by oral gavage (6 g/kg body weight). Rats used for biochemical analysis were euthanized using head-focused microwave irradiation at 2, and 4 h following treatment to immediately stop metabolism. We found that acetate did not alter brain ATP, ADP, NAD, GTP levels, or the energy charge ratio [ECR, (ATP + ½ ADP)/(ATP + ADP + AMP)] when compared to controls. However, after 4 h of treatment brain phosphocreatine levels were significantly elevated with a concomitant reduction in AMP levels with no change in glycogen levels. In parallel studies where rats were treated with GTA for 28 days, we found that acetate did not alter brain glycogen and mitochondrial biogenesis as determined by measuring brain cardiolipin content, the fatty acid composition of cardiolipin and using quantitative ultra-structural analysis to determine mitochondrial density/unit area of cytoplasm in hippocampal CA3 neurons. Collectively, these data suggest that an increase in brain acetyl-CoA levels by acetate supplementation does increase brain energy stores however it has no effect on brain glycogen and neuronal mitochondrial biogenesis.     

Balancing redox cofactor generation and ATP synthesis: Key microaerobic responses in thermophilic fermentations

Geobacillus thermoglucosidasius is a Gram‐positive, thermophilic bacterium capable of ethanologenic fermentation of both C5 and C6 sugars and may have possible use for commercial bioethanol production [Tang et al., 2009; Taylor et al. (2009) Trends Biotechnol 27(7): 398–405]. Little is known about the physiological changes that accompany a switch from aerobic (high redox) to microaerobic/fermentative (low redox) conditions in thermophilic organisms. The changes in the central metabolic pathways in response to a switch in redox potential were analyzed using quantitative real‐time PCR and proteomics. During low redox (fermentative) states, results indicated that glycolysis was uniformly up‐regulated, the Krebs (tricarboxylic acid or TCA) cycle non‐uniformly down‐regulated and that there was little to no change in the pentose phosphate pathway. Acetate accumulation was accounted for by strong down‐regulation of the acetate CoA ligase gene (acs) in addition to up‐regulation of the pta and ackA genes (involved in acetate production), thus conserving ATP while reducing flux through the TCA cycle. Substitution of an NADH dehydrogenase (down‐regulated) by an up‐regulated NADH:FAD oxidoreductase and up‐regulation of an ATP synthase subunit, alongside the observed shifts in the TCA cycle, suggested that an oxygen‐scavenging electron transport chain likely remained active during low redox conditions. Together with the observed up‐regulation of a glyoxalase and down‐regulation of superoxide dismutase, thought to provide protection against the accumulation of toxic phosphorylated glycolytic intermediates and reactive oxygen species, respectively, the changes observed in G. thermoglucosidasius NCIMB 11955 under conditions of aerobic‐to‐microaerobic switching were consistent with responses to low pO₂ stress. Biotechnol. Bioeng. 2013; 110: 1057–1065. © 2012 Wiley Periodicals, Inc.     

Laminar shear stress elicit distinct endothelial cell e‐selectin expression pattern via TNFα and IL‐1β activation

The ability to discriminate cell adhesion molecule expression between healthy and inflamed endothelium is critical for therapeutic intervention in many diseases. This study explores the effect of laminar flow on TNFα‐induced E‐selectin surface expression levels in human umbilical vein endothelial cells (HUVECs) relative to IL‐1β‐induced expression via flow chamber assays. HUVECs grown in static culture were either directly (naïve) activated with cytokine in the presence of laminar shear or pre‐exposed to 12 h of laminar shear (shear‐conditioned) prior to simultaneous shear and cytokine activation. Naïve cells activated with cytokine in static served as control. Depending on the cell shear history, fluid shear is found to differently affect TNFα‐induced relative to IL‐1β‐induced HUVEC expression of E‐selectin. Specifically, E‐selectin surface expression by naïve HUVECs is enhanced in the 8–12 h activation time range with simultaneous exposure to shear and TNFα (shear‐TNFα) relative to TNFα static control whereas enhanced E‐selectin expression is observed in the 4–24 h range for shear‐IL‐1β treatment relative to IL‐1β static control. While exposure of HUVECs to shear preconditioning mutes shear‐TNFα‐induced E‐selectin expression, it enhances or down‐regulates shear‐IL‐1β‐induced expression dependent on the activation period. Under dual‐cytokine‐shear conditions, IL‐1β signaling dominates. Overall, a better understanding of E‐selectin expression pattern by human ECs relative to the combined interaction of cytokines, shear profile and history can help elucidate many disease pathologies. Biotechnol. Bioeng. 2013; 110: 999–1003. © 2012 Wiley Periodicals, Inc.     

The Fire Refuge Value of Patches of a Fire‐Sensitive Tree in Fire‐prone Savannas: Callitris intratropica in Northern Australia

Patches of fire‐sensitive vegetation often occur within fire‐prone tropical savannas, and are indicative of localized areas where fire regimes are less severe. These may act as important fire refugia for fire‐sensitive biota. The fire‐sensitive tree Callitris intratropica occurs in small patches throughout the fire‐prone northern Australian savannas, and is widely seen as an indicator of low‐severity fire regimes and of good ecosystem health. Here, we address the question: to what extent do Callitris patches act as refuges for other fire‐sensitive biota, and therefore play a broader conservation role? We contrast floral and faunal species composition between Callitris patches and surrounding eucalypt savanna, using three case studies. In the first case study, a floristic analysis of 47 Callitris patches across Western Australia's Kimberley region showed that woody species in these patches were overwhelmingly widespread, fire‐tolerant savanna taxa. No species of special conservation concern occurred disproportionately within Callitris patches. Similarly, there was no concentration of fire‐sensitive fauna or flora in five Callitris patches in the East Kimberley. Finally, there was no difference in ant species composition among 12 Callitris patches and surrounding eucalypt savannas in Kakadu National Park, Northern Territory, and there were no fire‐sensitive ant species in Callitris patches. Our three case studies from throughout the northwestern Australia provide no evidence that Callitris patches act as important refuges for fire‐sensitive flora or fauna within fire‐prone eucalypt savannas. This calls into question the notion that Callitris is a strong indicator of general ecosystem health.     

The Potential Role of Scattered Trees for Ant Conservation in an Agriculturally Dominated Neotropical Landscape

Although biological conservation has traditionally focused on the protection of pristine areas, it is becoming increasingly evident that efforts should also be made toward the maintenance of biological diversity in human‐managed ecosystems. We evaluated the potential role of scattered, remnant trees in enhancing ant diversity in agricultural lands within a biodiversity hotspot, namely the Cerrado savannas of central Brazil. Ant collections were performed in, beneath, and away from the crowns of a native tree species growing in planted pastures, soy fields, and in small (<400 ha) savanna reserves. Significantly more ant species were found beneath than 30 m away from scattered trees. The magnitude of this effect was similar in soy fields and in pastures at all scales of our analyses, except at the landscape scale where the effect was comparatively greater in soy fields. Most of the species that occurred more frequently beneath than away from trees nest on ground, indicating that the conservation value of scattered trees goes beyond the mere protection of arboreal ant species. Soy plantations presented a particular species‐poor ground‐dwelling fauna when compared with savannas, whereas differences in the number and composition of arboreal species were less marked. In contrast, the diversity of arboreal‐ and ground‐dwelling ants recorded in and beneath pasture trees was nearly as high as the one found in savannas. It is suggested that pasture trees can have an important value for ant conservation off reserves, particularly in regions where most of the native vegetation has already been cleared.     

Divergent requirements for fibroblast growth factor signaling in zebrafish maxillary barbel and caudal fin regeneration

The zebrafish maxillary barbel is an integumentary organ containing skin, glands, pigment cells, taste buds, nerves, and endothelial vessels. The maxillary barbel can regenerate (LeClair & Topczewski 2010); however, little is known about its molecular regulation. We have studied fibroblast growth factor (FGF) pathway molecules during barbel regeneration, comparing this system to a well‐known regenerating appendage, the zebrafish caudal fin. Multiple FGF ligands (fgf20a, fgf24), receptors (fgfr1‐4) and downstream targets (pea3, il17d) are expressed in normal and regenerating barbel tissue, confirming FGF activation. To test if specific FGF pathways were required for barbel regeneration, we performed simultaneous barbel and caudal fin amputations in two temperature‐dependent zebrafish lines. Zebrafish homozygous for a point mutation in fgf20a, a factor essential for caudal fin blastema formation, regrew maxillary barbels normally, indicating that the requirement for this ligand is appendage‐specific. Global overexpression of a dominant negative FGF receptor, Tg(hsp70l:dn‐fgfr1:EGFP)^pd1 completely blocked fin outgrowth but only partially inhibited barbel outgrowth, suggesting reduced requirements for FGFs in barbel tissue. Maxillary barbels expressing dn‐fgfr1 regenerated peripheral nerves, dermal connective tissue, endothelial tubes, and a glandular epithelium; in contrast to a recent report in which dn‐fgfr1 overexpression blocks pharyngeal taste bud formation in zebrafish larvae (Kapsimali et al. 2011), we observed robust formation of calretinin‐positive tastebuds. These are the first experiments to explore the molecular mechanisms of maxillary barbel regeneration. Our results suggest heterogeneous requirements for FGF signaling in the regeneration of different zebrafish appendages (caudal fin versus maxillary barbel) and taste buds of different embryonic origin (pharyngeal endoderm versus barbel ectoderm).