Analysis of the Airway Microbiota of Healthy Individuals and Patients with Chronic Obstructive Pulmonary Disease by T-RFLP and Clone Sequencing

Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease that affects a large number of patients and has significant impact. One hallmark of the disease is the presence of bacteria in the lower airways. Objective: The aim of this study was to analyze the detailed structure of microbial communities found in the lungs of healthy individuals and patients with COPD. Nine COPD patients as compared and 9 healthy individuals underwent flexible bronchoscopy and BAL was performed. Bacterial nucleic acids were subjected to terminal restriction fragment (TRF) length polymorphism and clone library analysis. Overall, we identified 326 T-RFLP band, 159 in patients and 167 in healthy controls. The results of the TRF analysis correlated partly with the data obtained from clone sequencing. Although the results of the sequencing showed high diversity, the genera Prevotella, Sphingomonas, Pseudomonas, Acinetobacter, Fusobacterium, Megasphaera, Veillonella, Staphylococcus, and Streptococcus constituted the major part of the core microbiome found in both groups. A TRF band possibly representing Pseudomonas sp. monoinfection was associated with a reduction of the microbial diversity. Non-cultural methods reveal the complexity of the pulmonary microbiome in healthy individuals and in patients with COPD. Alterations of the microbiome in pulmonary diseases are correlated with disease.     

CD62L (L-Selectin) Shedding for Assessment of Perioperative Immune Sensitivity in Patients Undergoing Cardiac Surgery with Cardiopulmonary Bypass

Objective

To investigate the suitability of blood granulocyte and monocyte sensitivity, as measured by the quantity of different agonists required to induce CD62L shedding, for assessment of perioperative immune changes in patients undergoing cardiac surgery with cardiopulmonary bypass.

Methods

Patients scheduled for aortocoronary bypass grafting or for valve surgery were included in this prospective observational study. Blood samples were drawn before anesthesia induction, directly after surgery and 48 hours after anesthesia induction. We determined the concentration of two different inflammatory stimuli – lipoteichoic acid (LTA) and tumor necrosis factor alpha (TNF) - required to induce shedding of 50% of surface CD62L from blood granulocytes and monocytes. In parallel monocyte surface human leukocyte antigen (HLA)-DR, and plasma interleukin (IL)-8, soluble (s)CD62L, soluble (s)Toll-like receptor (TLR)-2 and ADAM17 quantification were used to illustrate perioperative immunomodulation.

Results

25 patients were enrolled. Blood granulocytes and monocytes showed decreased sensitivity to the TLR 2/6 agonist Staphylococcus aureus LTA immediately after surgery (p = 0.001 and p = 0.004 respectively). In contrast, granulocytes (p = 0.01), but not monocytes (p = 0.057) displayed a decreased postoperative sensitivity to TNF. We confirmed the presence of a systemic inflammatory response and a decreased immune sensitivity in the post-surgical period by measuring significant increases in the perioperative plasma concentration of IL-8 (p≤0.001) and sTLR (p = 0.004), and decreases in monocyte HLA-DR (p<0.001), plasma sCD62L (p≤0.001). In contrast, ADAM17 plasma levels did not show significant differences over the observation period (p = 0.401).

Conclusions

Monitoring granulocyte and monocyte sensitivity using the “CD62L shedding assay” in the perioperative period in cardiac surgical patients treated with the use of cardiopulmonary bypass reveals common changes in sensitivity to TLR2/6 ligands and to TNF stimulus. Further long-term follow-up studies will address the predictive value of these observations for clinical purposes.     

Unusual N-Prenylation in Diazepinomicin Biosynthesis: The Farnesylation of a Benzodiazepine Substrate Is Catalyzed by a New Member of the ABBA Prenyltransferase Superfamily

The bacterium Micromonospora sp. RV115, isolated from a marine sponge, produces the unusual metabolite diazepinomicin, a prenylated benzodiazepine derivative. We have cloned the prenyltransferase gene dzmP from this organism, expressed it in Escherichia coli, and the resulting His₈-tagged protein was purified and investigated biochemically. It was found to catalyze the farnesylation of the amide nitrogen of dibenzodiazepinone. DzmP belongs to the ABBA prenyltransferases and is the first member of this superfamily which utilizes farnesyl diphosphate as genuine substrate. All previously discovered members utilize either dimethylallyl diphosphate (C₅) or geranyl diphosphate (C₁₀). Another putative diazepinomicin biosynthetic gene cluster was identified in the genome of Streptomyces griseoflavus Tü4000, suggesting that the formation of diazepinomicin is not restricted to the genus Micromonospora. The gene cluster contains a gene ssrg_00986 with 61.4% identity (amino acid level) to dzmP. The gene was expressed in E. coli, and the purified protein showed similar catalytic properties as DzmP. Both enzymes also accepted other phenolic or phenazine substrates. ABBA prenyltransferases are useful tools for chemoenzymatic synthesis, due to their nature as soluble, stable biocatalysts. The discovery of DzmP and Ssrg_00986 extends the isoprenoid substrate range of this superfamily. The observed prenylation of an amide nitrogen is an unusual biochemical reaction.     

Characterization of the Recombinant Exopeptidases PepX and PepN from Lactobacillus helveticus ATCC 12046 Important for Food Protein Hydrolysis

The proline-specific X-prolyl dipeptidyl aminopeptidase (PepX; EC 3.4.14.11) and the general aminopeptidase N (PepN; EC 3.4.11.2) from Lactobacillus helveticus ATCC 12046 were produced recombinantly in E. coli BL21(DE3) via bioreactor cultivation. The maximum enzymatic activity obtained for PepX was 800 µkat_{H-Ala-Pro-pNA} L⁻¹, which is approx. 195-fold higher than values published previously. To the best of our knowledge, PepN was expressed in E. coli at high levels for the first time. The PepN activity reached 1,000 µkat_H-Ala-pNA L⁻¹. After an automated chromatographic purification, both peptidases were biochemically and kinetically characterized in detail. Substrate inhibition of PepN and product inhibition of both PepX and PepN were discovered for the first time. An apo-enzyme of the Zn²⁺-dependent PepN was generated, which could be reactivated by several metal ions in the order of Co²⁺>Zn²⁺>Mn²⁺>Ca²⁺>Mg²⁺. PepX and PepN exhibited a clear synergistic effect in casein hydrolysis studies. Here, the relative degree of hydrolysis (rDH) was increased by approx. 132%. Due to the remarkable temperature stability at 50°C and the complementary substrate specificities of both peptidases, a future application in food protein hydrolysis might be possible.     

Echolocation versus echo suppression in humans

Several studies have shown that blind humans can gather spatial information through echolocation. However, when localizing sound sources, the precedence effect suppresses spatial information of echoes, and thereby conflicts with effective echolocation. This study investigates the interaction of echolocation and echo suppression in terms of discrimination suppression in virtual acoustic space. In the ‘Listening’ experiment, sighted subjects discriminated between positions of a single sound source, the leading or the lagging of two sources, respectively. In the ‘Echolocation’ experiment, the sources were replaced by reflectors. Here, the same subjects evaluated echoes generated in real time from self-produced vocalizations and thereby discriminated between positions of a single reflector, the leading or the lagging of two reflectors, respectively. Two key results were observed. First, sighted subjects can learn to discriminate positions of reflective surfaces echo-acoustically with accuracy comparable to sound source discrimination. Second, in the Listening experiment, the presence of the leading source affected discrimination of lagging sources much more than vice versa. In the Echolocation experiment, however, the presence of both the lead and the lag strongly affected discrimination. These data show that the classically described asymmetry in the perception of leading and lagging sounds is strongly diminished in an echolocation task. Additional control experiments showed that the effect is owing to both the direct sound of the vocalization that precedes the echoes and owing to the fact that the subjects actively vocalize in the echolocation task.     

Heterotrimeric Gαi proteins are regulated by lipopolysaccharide and are anti-inflammatory in endotoxemia and polymicrobial sepsis

Previous studies have implicated a role of heterotrimeric Gα_i proteins in lipopolysaccharide (LPS)-induced inflammatory responses. We hypothesized that Toll-like receptor (TLR) signaling regulates Gα_i proteins, which are anti-inflammatory in endotoxemia and polymicrobial sepsis. RAW 264.7 cells were stimulated with LPS and the Gα_i-GTP protein complex was immunoprecipitated with a Gα_i protein activation assay. In subsequent in vivo studies, the Gα_i protein inhibitor pertussis toxin (PTx) or G_i protein agonist mastoparan (MP-7) were administrated prior to endotoxemia. LPS-induced pro-inflammatory cytokines and mortality were determined. To examine the role of Gα_i2 in sepsis, Gα_i2 (−/−) and wildtype (WT) mice were subjected to cecal ligation and puncture (CLP) and monitored every 24 h for 120 h. Other mice were sacrificed 24 h after CLP. Peritoneal fluid, blood, and tissue samples were collected. Plasma pro-inflammatory cytokine production, bacterial load in peritoneal fluid, blood and lung tissue, myeloperoxidase (MPO) activity in lung and liver and different immune cell populations in spleen were studied. We found that Gα_i proteins are rapidly activated by LPS followed by rapid inactivation. These studies provide the first direct evidence that Gα_i proteins are modulated by TLR signaling. In following studies, PTx augmented LPS-induced plasma TNFα, IL-6, whereas MP-7 suppressed LPS-induced TNFα and decreased LPS-induced mortality. In sepsis studies, the survival rate post-CLP was significantly decreased in the Gα_i2 (−/−) mice compared to WT mice. CLP-induced plasma TNFα, IL-6, bacterial load in peritoneal fluid, blood and lung tissue and lung and liver MPO activity were significantly increased in Gα_i2 (−/−) compared to WT mice. Gα_i2 (−/−) mice also exhibited increased Th1 and Th2 responses compared to WT mice. Taken together, Gα_i proteins are activated by LPS and negatively regulate endotoxemia and sepsis. Understanding the role of Gα_i2 protein in regulation of the inflammatory response in sepsis may provide novel targets for treatment of sepsis.     

Circular permutation: a different way to engineer enzyme structure and function

Protein engineers traditionally rely on amino acid substitutions to alter the functional properties of biomacromolecules, yet have largely overlooked the potential benefits of reorganizing the polypeptide chain of a protein by circular permutation (CP). By connecting the native protein termini via a covalent linker and introducing new ends through the cleavage of an existing peptide bond, CP can perturb local tertiary structure and protein dynamics, as well as introduce possible quaternary structure changes. In several recent studies, these effects have successfully been exploited to manipulate protein scaffolds, resulting in improved catalytic activity and altered substrate or ligand binding affinity, as well as enabling the design of novel biocatalysts and biosensors.     

Ethylene-regulated (methylsulfanyl)alkanoate ester biosynthesis is likely to be modulated by precursor availability in Actinidia chinensis genotypes

The limiting steps of ethylene-dependent (methylsulfanyl)alkanoate ester biosynthesis have been investigated in this study, using closely related Actinidia chinensis genotypes and the commercial cultivar ‘Hort16A’. Quantification of methylsulfanyl-compounds from the headspace of ethylene-producing kiwifruits revealed little variation in their volatile composition but remarkable differences in the magnitude of the fruit volatile levels. To test whether the variations in fruit volatile levels can be correlated with the genotype-specific apparent catalytic efficiency, the initial slope of the substrate response curve (V′_MaxK_M⁻¹ where V′_Max is the apparent V_Max in a crude extract) was evaluated for total alcohol acyltransferase (EC 2.3.1.84) activity. The V′_MaxK_M⁻¹ values of different (methylsulfanyl)alkyl-CoAs were in a similar range for most genotypes, which suggests substrate availability as the limiting factor for (methylsulfanyl)alkanoate ester synthesis in these kiwifruit. Furthermore, gene expression analysis of acyltransferase expressed sequence tags points towards the action of multiple isozymes for (methylsulfanyl)alkanoate ester synthesis, emphasizing the central role of substrate levels on final ester concentrations. Volatile levels of the potential precursor methional were increased in ethylene-producing A. chinensis kiwifruit and a close connection between (methylsulfanyl)alkanoate ester formation and ethylene synthesis in plants is proposed. Finally, a possible biosynthetic pathway is presented.     

Pacing Strategy and Change in Body Composition during a Deca Iron Triathlon

We investigated the timeline of performances in the three races of the 'World Challenge Deca Iron Triathlon', held in 2006, 2007 and 2009, where the athletes completed one Ironman triathlon daily on 10 consecutive days. The association of anthropometric characteristics such as body fat estimated using bioelectrical impedance analysis and previous experience in ultra-triathlon with race time was investigated using multiple linear regression analysis. Forty-nine athletes participated in these three races; 23 (47%) participants completed the race within 8,817 (1,322) min. Day 1 was the fastest with 762 (86) min; the slowest was Day 10 with 943 (167) min (P<0.05). The time per Ironman increased during the race (P<0.05). Body mass and fat mass decreased whereas lean body mass increased (P<0.05). Race time was related to both the number of finished Triple Iron triathlons (P=0.028) and the personal best time in a Triple Iron triathlon (P<0.0001). We concluded that performance in a Deca Iron triathlon decreased throughout the competition, with the fastest race on Day 1 and the slowest on Day 10. The number of finished Triple Iron triathlons and the personal best time in a Triple Iron triathlon, but not anthropometry, were related to race time. To conclude, athletes need to have a high number of previously completed Triple Iron triathlons, as well as a fast personal best time in a Triple Iron triathlon, in order to finish a Deca Iron triathlon successfully.     

Neural coding of echo-envelope disparities in echolocating bats

The effective use of echolocation requires not only measuring the delay between the emitted call and returning echo to estimate the distance of an ensonified object. To locate an object in azimuth and elevation, the bat’s auditory system must analyze the returning echoes in terms of their binaural properties, i.e., the echoes’ interaural intensity and time differences (IIDs and ITDs). The effectiveness of IIDs for echolocation is undisputed, but when bats ensonify complex objects, the temporal structure of echoes may facilitate the analysis of the echo envelope in terms of envelope ITDs. Using extracellular recordings from the auditory midbrain of the bat, Phyllostomus discolor, we found a population of neurons that are sensitive to envelope ITDs of echoes of their sonar calls. Moreover, the envelope-ITD sensitivity improved with increasing temporal fluctuations in the echo envelopes, a sonar parameter related to the spatial statistics of complex natural reflectors like vegetation. The data show that in bats envelope ITDs may be used not only to locate external, prey-generated rustling sounds but also in the context of echolocation. Specifically, the temporal fluctuations in the echo envelope, which are created when the sonar emission is reflected from a complex natural target, support ITD-mediated echolocation.