Oxidants act as chemorepellents in Paramecium by stimulating an electrogenic plasma membrane reductase activity

Paramecium is a valuable eukaryotic model system for studying chemosensory transduction, adaptation and cellular sensory integration. While millimolar amounts of many attractants hyperpolarize and cause faster forward swimming, oxidants are repellents that depolarize and cause backward swimming at micromolar concentrations. The non-permeant oxidants cytochrome c, nitro blue tetrazolium and ferricyanide are repellents with half maximal concentrations of 0.4 M, 2.2 M and 100 M respectively. In vivo reductase activities follow the same order of potencies. The concentration dependence of the cytochrome c reductase activity is well correlated with cytochrome c-induced depolarizations. This suggests that plasma membrane reduction of external cytochrome c is electrogenic, causing membrane depolarization and chemorepulsion. The reductase activity also appears to be voltage dependent. Depolarization by either K+, Na+, Ca+ or Mg+ correlates with inhibition of both in vivo reductase activities and cytochrome c-induced membrane potential changes. These responses were also seen in deciliated cells, showing that the body plasma membrane is sufficient for the response. Both chloroquine and diphenyleneiodonium inhibited reductase activities but only at unusually high concentrations. This activity showed no pH dependence in the physiological range. We propose that a plasma membrane bound NADPH-dependent reductase controls oxidant-induced depolarizations and consequent chemorepulsion.Abbreviations bmv Body plasma membrane vesicles - BPS Bathophenanthroline disulfonate - cAMP Cyclic adenosine monophosphate - cmv Ciliary membrane vesicles - cyt c Cytochrome c - DPI Diphenyleneiodonium - EC ₅₀ Concentration for 50% effectiveness - FeCN Ferricyanide [Fe(CN)₆–3] - FeEDTA Ethylenediaminetetracetic acid (ferric-sodium salt) - GTP Guanosine 5-triphosphate - KCN Potassium cyanide - mM Millimolar - MOPS 3-(N-morpholino) propanesulfonic acid - mV Millivolts - NADH Nicotinamide adenine dinucleotide (reduced form) - NADPH Nicotinamide adenine dinucleotide phosphate (reduced form) - NBT Nitro blue tetrazolium - nm Nanometer - pCMB p-Chloromercuribenzoate - PMA Phorbol 12-myristate 13-acetate - s.d. Standard deviation - SOD Superoxide dismutase - Tris Tris(hydroxymethyl)aminomethane - M Micromolar     

Fatty acid efficiency profile in uncoupling of <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> mitochondria

A profile of free fatty acid (FFA) specificity in Acanthamoeba castellanii mitochondrial uncoupling is described. The FFA uncoupling specificity was observed as different abilities to stimulate resting respiration, to decrease resting membrane potential, and to decrease oxidative phosphorylation efficiency. Tested unsaturated FFA (C18–20) were more effective as uncouplers and protonophores when compared to tested saturated FFA (C8–18), with palmitic acid (C16:0) as the most active. As FFA efficiency in mitochondrial uncoupling is related to physiological changes of fatty acid composition (and thereby FFA availability) during growth of amoeba cells, it could be a way to regulate the activity of an uncoupling protein and thereby the efficiency of oxidative phosphorylation during a cell life of this unicellular organism. Aleksandra Swida and Małgorzata Czarna contributed equally to this work.     

Bacterial Distribution in the Lungs of Children with Protracted Bacterial Bronchitis

Objectives

Flexible bronchoscopy with bronchoalveolar lavage (FB-BAL) is increasingly used for the microbiological confirmation of protracted bacterial bronchitis (PBB) in children with a chronic wet cough. At our centre, when performing FB-BAL for microbiological diagnosis we sample 6 lobes (including lingula) as this is known to increase the rate of culture positive procedures in children with cystic fibrosis. We investigated if this is also the case in children with PBB.

Methods

We undertook a retrospective case note review of 50 children investigated for suspected PBB between May 2011 and November 2013.

Results

The median (IQR) age at bronchoscopy was 2.9 (1.7–4.4) years and the median (IQR) duration of cough was 11 (8.0–14) months. Positive cultures were obtained from 41/50 (82%) and 16 (39%) of these patients isolated ≥2 organisms. The commonest organisms isolated were Haemophilus influenzae (25 patients), Moraxella catarrhalis (14 patients), Staphylococcus aureus (11 patients) and Streptococcus pneumoniae (8 patients). If only one lobe had been sampled (as per the European Respiratory Society guidance) 17 different organisms would have been missed in 15 patients, 8 of whom would have had no organism cultured at all. The FB-BAL culture results led to an antibiotic other than co-amoxiclav being prescribed in 17/41 (41%) patients.

Conclusions

Bacterial distribution in the lungs of children with PBB is heterogeneous and organisms may therefore be missed if only one lobe is sampled at FB-BAL. Positive FB-BAL results are useful in children with PBB and can influence treatment.     

The Vaginal Microbiota: What Have We Learned after a Decade of Molecular Characterization?

We conducted a systematic review of the Medline database (U.S. National Library of Medicine, National Institutes of Health, Bethesda, MD, U.S.A) to determine if consistent molecular vaginal microbiota (VMB) composition patterns can be discerned after a decade of molecular testing, and to evaluate demographic, behavioral and clinical determinants of VMB compositions. Studies were eligible when published between 1 January 2008 and 15 November 2013, and if at least one molecular technique (sequencing, PCR, DNA fingerprinting, or DNA hybridization) was used to characterize the VMB. Sixty three eligible studies were identified. These studies have now conclusively shown that lactobacilli-dominated VMB are associated with a healthy vaginal micro-environment and that bacterial vaginosis (BV) is best described as a polybacterial dysbiosis. The extent of dysbiosis correlates well with Nugent score and vaginal pH but not with the other Amsel criteria. Lactobacillus crispatus is more beneficial than L. iners. Longitudinal studies have shown that a L. crispatus-dominated VMB is more likely to shift to a L. iners-dominated or mixed lactobacilli VMB than to full dysbiosis. Data on VMB determinants are scarce and inconsistent, but dysbiosis is consistently associated with HIV, human papillomavirus (HPV), and Trichomonas vaginalis infection. In contrast, vaginal colonization with Candida spp. is more common in women with a lactobacilli-dominated VMB than in women with dysbiosis. Cervicovaginal mucosal immune responses to molecular VMB compositions have not yet been properly characterized. Molecular techniques have now become more affordable, and we make a case for incorporating them into larger epidemiological studies to address knowledge gaps in etiology and pathogenesis of dysbiosis, associations of different dysbiotic states with clinical outcomes, and to evaluate interventions aimed at restoring and maintaining a lactobacilli-dominated VMB.     

Importance of flow division on transition to turbulence within an arteriovenous graft

Transitional blood flow in an arteriovenous graft under various conditions of flow division was examined through direct numerical simulation. This junction consists of an inlet vessel (prosthetic graft) connected to a host vessel (vein) at an acute angle (21.6 degrees ). Inlet Reynolds numbers, based on mean velocity and graft inlet diameter, ranged from 800 to 1400. Various flow divisions between the two ends of the host vessel (i.e., the proximal venous segment, PVS, and distal venous segment, DVS) were considered (PVS:DVS ratios of 100:0, 85:15, 70:30 and 115:(15)). The numerical technique employed the spectral element method which is a high-order discretization ideally suited to the simulation of transitional flows in complex domains. High velocity and pressure fluctuations were observed for the PVS:DVS=70:30 and 85:15 cases and absent from the 100:0 and 115:(15) cases; the results indicate the importance of flow division on the development of turbulence in this junction. Transition to turbulence was observed at Reynolds numbers as low as 1000 and 800 under flow divisions of 85:15 and 70:30, respectively, significantly lower than the critical value of 2100. The frequency spectra of velocity fluctuations indicated a significant intensity within the frequency range of approximately 300Hz downstream of the junction. An adverse pressure gradient developed in the PVS when graft inflow divided into opposite directions in the junction. This pressure gradient had a destabilizing effect on the flow and enhanced transition to turbulence in the PVS. These findings suggest that measurements of in vivo flow rates at the inlet and outlets are critical for the accurate prediction of arteriovenous hemodynamics. A potential clinical application of these results might be to close off the DVS during graft construction to ensure a 100:0 flow division.     

Importance of the large copepod Paraeuchaeta antarctica (Giesbrecht, 1902) in coastal waters and the diet of seabirds at Kerguelen, Southern Ocean

The importance of the euchaetid copepod Paraeuchaeta antarcticain the subantarctic pelagic ecosystem was quantified in thecoastal waters of the Golfe du Morbihan at Kerguelen Islandsby comparing food samples from two diving seabirds with concurrentnet samples taken within the predator foraging area. Paraeuchaetaantarctica occurred in very high densities (up to 30 individualsm^-3 and 96 mg dry weight m^-3) in the water column, being moreabundant in the deepest part of the gulf than in shallow watersor at the more offshore shelf stations. The common diving petrelfeeds almost exclusively on crustaceans, its diet being dominatedby the amphipod Themisto gaudichaudii (52% by number and 84%by reconstituted mass) and P. antarctica (33% and 16%, respectively).Rockhopper penguins preyed upon crustaceans and fish, with fourtaxa being important, namely T. gaudichaudii (37% and 23%, respectively),Euphausia vallentini (24% and 41%), postlarval fish (10% and24%), and P. antarctica (13% and 3%). Paraeuchaeta antarcticadominated numerically in 21% of the diving-petrel food samplesand in 12% of penguin samples. The two bird species segregatedby preying upon different developmental stages of P. antarctica,diving petrels fed equally on CV of both sexes and CVI, whilepenguins fed on CVI only. Comparison of P. antarctica foundin net and food samples indicated no prey selection by commondiving petrels that caught the different copepod stages in proportionto their availability in the water column. On the other hand,the diving performance of penguins, which is better than thatof the petrels (mean maximum dive depths 69 m for penguins versus32 m in petrels), allows them to catch CVI in deeper waters,probably near the bottom. Our study shows that P. antarcticais a major component of the coastal macrozooplankton communityand a significant prey for two species of diving seabirds inhabitingKerguelen. This is also the first record of a copepod speciesas a prey for penguin, and the first to highlight P. antarcticain the food of austral seabirds.     

Sleep and circadian phase in a ship's crew

Numerous factors influence the increased health risks of seamen. This study investigated sleep (by actigraphy) and the adaptation of the internal clock in watch-keeping crew compared to day workers, as possible contributory factors. Fourteen watch keepers, 4 h on, 8 h off (0800-1200/2000-2400 h, 1200-1600/2400-0400 h, 1600-2000/0400-0800 h) (fixed schedule, n = 6; rotating by delay weekly, n = 8), and 12 day workers participated during a voyage from the United Kingdom to Antarctica. They kept daily sleep diaries and wore wrist monitors for continuous recording of activity. Sleep parameters were derived from activity using the manufacturer's software and analyzed by repeated-measures ANOVA using SAS 8.2. Sequential urine samples were collected for 48 h weekly for 6-sulphatoxymelatonin measurement as an index of circadian rhythm timing. Individuals working watches of 1200-1600/2400-0400 h and 1600-2000/0400-0800 h had 2 sleeps daily, analyzed separately as main sleep (longest) and 2nd sleep. Main sleep duration was shorter in watch keepers than in day workers (p < 0.0001). Objective sleep quality was significantly compromised in rotaters compared to both day workers and fixed watch keepers, the most striking comparisons being sleep efficiency (percentage desired sleep time spent sleeping) main sleep (p < 0.0001) and sleep fragmentation (an index of restlessness) main sleep (p < 0.0001). The 2nd sleep was substantially less efficient than was the main sleep (p < 0.0001) for all watch keepers. There were few significant differences in sleep between the different watches in rotating watch keepers. Circadian timing remained constant in day workers. Timing of the 6-sulphatoxymelatonin rhythm was later for the watch of 1200-1600/2400-0400 h than for all others (1200-1600/2400-0400 h, 5.90 +/- 0.85 h; 1600-2000/0400-0800 h, 1.5 +/- 0.64 h; 0800-1200/ 2000-2400 h, 2.72 +/- 0.76 h; days, 2.09 +/- 0.68 h [decimal hours, mean +/- SEM]: ANOVA, p < 0.01). This study identifies weekly changes in watch time as a cause of poor sleep in watch keepers. The most likely mechanism is the inability of the internal clock to adapt rapidly to abrupt changes in schedule.     

Evolution and diversity of Rickettsia bacteria

Background  

Rickettsia are intracellular symbionts of eukaryotes that are best known for infecting and causing serious diseases in humans and other mammals. All known vertebrate-associated Rickettsia are vectored by arthropods as part of their life-cycle, and many other Rickettsia are found exclusively in arthropods with no known secondary host. However, little is known about the biology of these latter strains. Here, we have identified 20 new strains of Rickettsia from arthropods, and constructed a multi-gene phylogeny of the entire genus which includes these new strains.