A new African species of parasitic Dero (Annelida, Clitellata, Naididae) in the urinary tract of reed frogs.

A new species of naidid oligochaete, Dero rwandae, detected in the bladder and the Wolffian ducts of reed frogs Hyperolius kivuensis from Rwanda, is described. Until now, D. bauchiensis was the only endoparasitic Dero known to infect African frogs infesting the eyes and Harderian glands. To the best of our knowledge, the finding of D. rwandae is the first record of an African Dero species infecting the urinary tract of anurans. In general morphology, the two African Dero parasites resemble each other, but differences in the features of ventral setae morphology exist. Parts of the mitochondrial 16S rRNA locus and the nuclear 18S and 28S rRNA loci were sequenced to assess the phylogenetic relationships to other Dero spp. Among those few species, that are barcoded so far, the closest relative of the new taxon is D. superterrenus, a free-living South American species. The species groups formerly termed subgenera Allodero, Aulophorus and Dero within the genus Dero do not represent distinct evolutionary lineages and the genus is paraphyletic including Branchiodrilus.

Helminth endoparasites of the smooth newt Lissotriton vulgaris: linking morphological identification and molecular data.

The helminth endoparasites of many European amphibian species are often known exclusively from morphological descriptions. A molecular library of DNA sequence data linked to morphological identifications is still in its infancy. In this paper, we aim to contribute to such a library on the smooth newt Lissotriton vulgaris, the intermediate and definitive host of 31 helminth parasites, according to evidence published so far. Newts (n = 69) were collected at two study sites in western Germany and examined for the presence of helminths. A total of five helminth species were detected in 56 (81%) of the newts, but only one or two species infected a single host. Four out of five helminth species were identified morphologically and based on DNA sequences as Parastrigea robusta (metacercariae), Oswaldocruzia filiformis, Megalobatrachonema terdentatum (adults and larvae) and Cosmocerca longicauda, and the corresponding sequences were provided subsequently. Oswaldocruzia molgeta was confirmed to be a junior synonym of O. filiformis. Molecular data on a fifth species (a cosmocercid nematode) that could not be identified at species level were added to GenBank. These findings increased the molecular library on morphologically identified smooth newt parasites significantly, from 12 to 15 entries.

Coupled atomistic spin-lattice simulations of ultrafast demagnetization in 3d ferromagnets.

Despite decades of research, the role of the lattice and its coupling to the magnetisation during ultrafast demagnetisation processes is still not fully understood. Here we report on studies of both explicit and implicit lattice effects on laser induced ultrafast demagnetisation of bcc Fe and fcc Co. We do this using atomistic spin- and lattice dynamics simulations following a heat-conserving three-temperature model. We show that this type of Langevin-based simulation is able to reproduce observed trends of the ultrafast magnetization dynamics of fcc Co and bcc Fe. The parameters used in our models are all obtained from electronic structure theory, with the exception of the lattice dynamics damping term, where a range of parameters were investigated. It was found that while the explicit spin-lattice coupling in the studied systems does not impact the demagnetisation process notably, the lattice damping has a large influence on the details of the magnetization dynamics. The dynamics of Fe and Co following the absorption of a femtosecond laser pulse are compared with previous results for Ni and similarities and differences in the materials' behavior are analysed. For all elements investigated so far with this model, we obtain a linear relationship between the value of the maximally demagnetized state and the fluence of the laser pulse , which is in agreement with experiments. Moreover, we demonstrate that the demagnetization amplitude is largest for Ni and smallest for Co. This holds over a wide range of the reported electron-phonon couplings, and this demagnetization trend is in agreement with recent experiments.

Efficient gene transfer to airway epithelium using recombinant Sendai virus.

Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer.

A strange endocytobiont revealed as largest virus.

A lot of endocytobionts (or endosymbionts) have been discovered within free-living amoebae in recent years. In this article the results of a long lasting effort to derive valuable data about an extraordinary spore-like infectious microorganism (endocytobiont, endosymbiont) within host amoebae (Acanthamoeba sp.) recently isolated from the contact lens case of a patient with keratitis, are presented. It took some time until this endocytobiont could be attributed to the genus Pandoravirus following a publication of two other pandoraviruses isolated from aquatic environments. Consequently the molecular biological investigation led to the taxonomic affiliation of the endocytobiont with the genus Pandoravirus and to the description of a new Pandoravirus species, Pandoravirus inopinatum after whole-genome sequencing in 2015. The fact that it was isolated from a contact lens container of a keratitis patient gives another dimension to these findings showing paradigmatically, how readily these 'new' giant viruses get to humans.

Predictive survival markers in patients with surgically resected non-small cell lung carcinoma.

Among patients with resected non-small cell lung carcinoma, about 50% will present a tumor recurrence. Thus, it would be of major importance to be able to predict and try to prevent these relapses by an active chemotherapy and/or radiotherapy. In an attempt to answer this question, the tumors of 227 patients with a surgically resected non-small cell lung carcinoma were evaluated as follows: tumors were classified as squamous cell carcinoma (n = 132) or adenocarcinoma (n = 95), and tumor differentiation was evaluated for each type. Then, all tumors were classified in respect to their pathological TNM staging (WHO) and screened by immunohistochemistry for the detection of the expression of the following antigens: Bcl-2, A+B+H blood group antigens, c-erb-b2, p53, and Pan-Ras antigens. Furthermore, adenocarcinomas were screened for the presence of point mutations in Ki-Ras codons 1-31. Finally, the patient blood group was defined, and patient survival was analyzed using nonparametric tests and proportional hazard Cox models. Using Kaplan-Meier survival curves, disease pathological TNM staging was shown to be a strong predictive factor of survival for both squamous cell carcinoma and adenocarcinoma. Patients with squamous cell carcinoma experienced fewer relapses than those with adenocarcinoma (42% versus 63%; P = 0.0002) and had a significantly better survival. All evaluated antigens were more often present in squamous cell carcinoma than in adenocarcinoma except for Pan-Ras (three times more frequent in adenocarcinoma). In patients with squamous cell carcinoma, only tumor staging had a significant prognosis value (P = 0.01). In patients with lung adenocarcinoma, a well-differentiated tumor (P = 0.009) as well as a positive Bcl-2 staining (P = 0.009) and an A+B+H antigen tumor staining (P = 0.024) were associated with a better survival. In contrast, patients with a stage I or II disease and a p53-positive tumor staining and patients with the O blood group (P = 0.01) had a shorter survival. Interestingly, no relation with patient survival was related to c-erb-b2 and Pan-Ras staining. Finally, 12 point mutations were found out of 81 tumors (15%) evaluated for Ki-Ras codons 1-31; they involved codon 12 but also 8, 14, and 15 without any relationship to survival. In respect to lung adenocarcinoma, using Cox proportional hazard models stratified on tumor staging, the following markers were shown to be related to survival: (a) Independent markers of longer survival (ie., high histological degree of tumor differentiation and positive Bcl-2 and A+B+H blood group antigen expression by tumor cells); and (b) Independent markers of shorter survival (i.e., O blood group for all patients and p53 tumor staining in patients with stage I and II diseases). This study suggests that, in patients who undergo surgery for lung adenocarcinoma, the presence or absence of these criteria could be used to define a subset of patients who may benefit from a more specific follow-up.

Whole-genome sequencing of a pandoravirus isolated from keratitis-inducing acanthamoeba.

Following the recent discovery of two Pandoravirus species in 2013, a previously described endocytobiont isolated from the inflamed eye of a patient with keratitis was subjected to whole-genome sequencing (WGS). Here, we present the complete genome sequence of a new Pandoravirus isolate.

CFTR gene mutations in adults with disseminated bronchiectasis.

The severity and type of clinical manifestations are variable in patients with cystic fibrosis (CF). The respiratory syndromes in these patients consist of lung infections associated with disseminated bronchiectasis (DB), asthma, and chronic obstructive pulmonary disease. To investigate the possible involvement of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in chronic pulmonary disease in adults, we studied 32 DB patients with a clinically isolated respiratory syndrome. Careful analysis of all the CFTR gene exons and their flanking regions revealed a significantly increased frequency of CFTR gene mutations in these patients. Thirteen CFTR gene mutations were identified in sixteen different alleles. Six of these mutations, which have previously been reported as CF defects, were found on nine alleles. A further four, two of which had not previously been described (D192N and 406-2 AdeltaC), are potentially disease-causing mutations. We also identified three rare substitutions (R31C, L997F, T1220I), which could be involved in mild CFTR gene disease. Four patients were compound heterozygotes, one carried two CFTR gene mutations (possibly allelic) and six were heterozygous for a mutation. These results indicate that CFTR gene mutations may play a role in bronchiectatic lung disease, possibly in a multifactorial context. These findings have implications for genetic counselling of DB patients and their families.

Thrombin stimulates Na+-H+ exchange across the human platelet plasma membrane.

We have investigated the release of protons from thrombin-stimulated platelets. Addition of thrombin to suspensions of washed platelets resulted in fast liberation of H+. In the presence of 0.1 mM amiloride, a potent inhibitor of the Na+/H+ transport system, the amount of protons liberated was decreased by about 50%, and was further reduced to about 15% by 1 mM amiloride. Similar inhibition of H+ release was observed after Na+ in the incubating medium had been replaced by choline. We conclude that one of the earliest events in thrombin-stimulated platelets consists of the activation of an Na+/H+ countertransport, which leads to an increase in intracellular pH.

Inhibition of Na+/H+ exchange reduces Ca2+ mobilization without affecting the initial cleavage of phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets.

Stimulation of human platelets increases cytoplasmic pH (pHi) via activation of Na+/H+ exchange. We have determined the effect of inhibiting Na+/H+ exchange on (i) thrombin-induced Ca2+ mobilization and (ii) turnover of 32P-labelled phospholipids. Blocking Na+/H+ exchange by removal of extracellular Na+ or by ethylisopropylamiloride (EIPA) inhibited Ca2+ mobilization induced by 0.2 U/ml thrombin, whereas increasing pHi by NH4Cl enhanced the thrombin-induced increase in cytosolic free Ca2+. The effect of EIPA was bypassed after increasing pHi by moneasin. The thrombin-induced cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) was unaffected by treatments that blocked Na+/H+ exchange or increased pHi. It is concluded that activation of Na+/H+ exchange is a prerequisite for Ca2+ mobilization in human platelets but not for the stimulus-induced hydrolysis of PIP2.

Effects of levodropropizine on vagal afferent C-fibres in the cat.

1. Levodropropizine (LVDP) is an effective antitussive drug. Its effects on single-unit discharge of vagal afferent C-fibres were tested in anaesthetized cats to assess whether an inhibition of vagal C-fibres is involved in its antitussive properties. Vagal C-fibres, identified by their response to phenylbiguanide (PBG), were recorded via suction electrodes from the distal part of the cut vagus. Based on their response to lung inflation, C-fibres were classified as pulmonary (19 fibres) or non-pulmonary (6 fibres). 2. PBG increased the discharge rate of both C-fibre types and activated a respiratory reflex causing apnoea. This reflex was abolished when the second vagus nerve was cut as well, while PBG-mediated stimulation of the C-fibres was not affected by vagotomy. 3. LVDP was administered intravenously and the C-fibre response to PBG was compared with that before administration of the drug. LVDP reduced both the duration of apnoea and the response of the C-fibre to PBG. 4. Comparison of the C-fibre responses to PBG and to a mixture of PBG and LVDP revealed that the period of apnoea was shortened and the discharge rate of the C-fibre reduced when LVDP was present. 5. The LVDP-induced inhibition of the C-fibre response to PBG was on average 50% in pulmonary and 25% in non-pulmonary fibres. 6. These results suggest that LVDP significantly reduces the response of vagal C-fibres to chemical stimuli. It is, thus, likely that the antitussive effect of LVDP is mediated through its inhibitory action on C-fibres.

Activity-related pH changes in respiratory neurones and glial cells of cats.

Intracellular pH (pHi) and membrane potential (Em) were measured in vivo in expiratory neurones and glial cells in the medulla of anaesthetized cats using double-barrelled H(+)-sensitive microelectrodes. In glial cells, stimulation of spinal pathways evoked a depolarization of up to 12 mV amplitude and an increase of pHi (7.25 +/- 0.15) by maximally 0.1 pH unit. IN expiratory neurones, pHi (7.15 +/- 0.18) fell by up to 0.2 pH unit during inspiratory inhibition. In axons of expiratory neurones, pHi remained unaffected during rhythmic action potential discharges. We suggest that the glial alkalinization is due to activation of Na+/HCO3- cotransport, whereas the neuronal acidification is caused by efflux of HCO3- via receptor-coupled anion channels.

Respiratory response to positive inspiratory pressure in the cat: effects of CO2 and vagal integrity.

Effects of inspiratory pressure support (IPS) on respiration and activity of inspiratory muscles were tested in eight anesthetized cats by recording the diaphragmatic electromyogram (EMGdi) and respiratory variables at four levels of positive inspiratory airway pressure (5, 10, 15, and 20 cmH2O); onset of IPS was triggered by the inspiratory effort of the animal. When IPS was applied with room air (IPSAir) the respiratory frequency (f) was reduced compared with spontaneous breathing and the tidal volume (VT) was significantly increased, which resulted in a fall of arterial PCO2 (PaCO2) at IPS airway pressures (Paw) above 5 cmH2O. Despite this increase in VT, the amplitude of the integrated EMGdi (Adi) was reduced during IPS at all levels of Paw. When arterial hypocapnia is corrected by addition of CO2 to the inspirate, the values of VT at any given Paw were virtually identical with those during IPSAir, but IPS-mediated changes in f and Adi were smaller than those during IPSAir. IPS was also performed after bilateral vagotomy. Vagotomy itself caused VT and Adi to increase, and f to decrease, during spontaneous breathing. In comparison with the corresponding treatment before vagotomy, IPSAir led to a less severe reduction in Adi. As a result, VT was more enhanced and Paco2 was more reduced after vagotomy than before, both during spontaneous breathing and during IPSAir at all levels of Paw. When, however, isocapnia was restored with IPS with CO2 in the vagotomized animal, diaphragmatic activity and f became very similar to their values during spontaneous breathing, whereas VT remained elevated as a result of the high positive airway pressure. Our data suggest that in anesthetized cats IPS leads to a diminution of diaphragm activity and that this reduction can be entirely attributed to 1) the hypocapnia, resulting from increased VT, and 2) the stimulation of pulmonary vagal afferent fibers at positive airway pressure.

Effects of thromboxane on respiration and pulmonary circulation in the cat: role of vagus nerve.

Infusion of stoichiometrically equal quantities of acid and base (neutral acid-base infusion) in the cat elicits pulmonary hypertension and rapid shallow breathing (J. Appl. Physiol. 62: 2362-2370, 1987), and thromboxane A2 (TxA2), released from platelets, is responsible for these effects (Respir. Physiol. 71: 169-183, 1988). To investigate the involvement of vagal afferent fibers in these responses, we reversibly blocked signal conduction in the vagus of the cat by bilaterally cooling the vagus nerves to 1 degree C and measured the cardiorespiratory parameters in response to neutral acid-base infusion and infusion of the TxA2 mimetic U-46619. Vagal cooling before infusion caused tidal volume (VT) to increase and respiratory frequency (fresp) to decrease, whereby total ventilation (VE) was slightly enhanced, but did not affect right ventricular blood pressure (Prv). Infusion of neutral acid-base after vagal cooling prompted Prv to rise, on average from 35 Torr to a peak of 60 Torr, and a similar rise was elicited by infusion of U-46619. However, vagal cooling abolished any effect on VT or fresp of both acid-base and U-46619 infusion. After rewarming the vagus nerves, infusion of U-46619 caused fresp to increase and VT to decrease (rapid shallow breathing) with a concomitant rise in Prv, similar to what had been observed in the earlier studies. Our data suggest that the effects of TxA2 and of its mimetic U-46619 on respiration are mediated by the stimulation of vagal afferent fibers, whereas pulmonary hypertension is unrelated to vagal activity.

Respiratory response to inhaled CO2 during positive inspiratory pressure in humans.

To investigate ventilatory CO2 sensitivity during inspiratory pressure support (IPS), we administered inspiratory CO2 [fractional concn (FICO2) 0.01, 0.03, or 0.05] in eight normal subjects without (CTRL) or with (Pinsp) positive inspiratory airway pressure (5 or 10 cmH2O). At CTRL and low IPS, CO2 inhalation led to a significant increase in tidal volume (VT) with nearly identical slopes in the plot of VT vs. end-tidal PCO2. At the high IPS level, VT at FICO2 of 0 was significantly above the value at lower Pinsp and did not increase with CO2 unless FICO2 was elevated to > 0.03. There was very little effect of either Pinsp or FICO2 on respiratory frequency and respiratory timing. The data suggest that the CO2 sensitivity of ventilation is similar at low levels of IPS as during CTRL. However, at high levels of IPS, VT is determined largely by the passive inflation and, thus, independent of CO2. CO2 has to be elevated to increase the respiratory drive before VT becomes CO2 sensitive.

Cardiorespiratory changes during HCl infusion unrelated to decreases in circulating blood pH.

To test the hypothesis that infusion of HCl changes blood pressure and respiration independent of decreases in circulating blood pH, an extracorporeal arteriovenous shunt (20 ml/min) between the femoral artery and vein was installed in anesthetized cats. Into this loop, acid (0.25 M HCl) and, approximately 10 cm downstream, base (0.25 M NaOH) could be infused simultaneously. Likewise, either acid or base could be infused individually. Right ventricular (Prv) and arterial (Pa) blood pressure, tidal volume (VT), and respiratory frequency (fresp) were recorded as well as blood gases and pH in arterial, right ventricular, and shunt loop blood at the reentrance into the animal. When HCl and NaOH were infused simultaneously and at equimolar rates (0.2 mmol/min for 10 min), there was a large increase in Prv, with little change or decrease in Pa. Respiratory frequency was increased, but total ventilation was not elevated because of a concomitant fall in VT. The rise in Prv and increase in fresp were transient in that they could only be evoked during the first HCl-NaOH infusion in a given animal. Repetitive infusions of HCl-NaOH into the same animal failed to elicit the response. Similar transient acid effects were evoked when HCl was infused without NaOH but not when NaOH was infused without HCl. During the second and third infusion of HCl, ventilatory responses were elicited that were explainable by stimulation of known chemoreceptors. The transient rise in Prv and fresp evoked by acid infusion might be explained by release of an agent from blood elements at the tip of the HCl infusion catheter, which in turn would constrict pulmonary vessels and influence breathing.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of O2 uptake and release by human erythrocytes studied by a stopped-flow technique.

The kinetics of O2 uptake into and release from human erythrocytes was investigated at 37 degrees C by a stopped-flow technique. From the time course of O2 saturation (SO2) change a specific transfer conductance of erythrocytes for O2 (GO2) was calculated. The following results were obtained: 1) GO2 decreased in the course of O2 uptake, but initial GO2 was nearly independent of SO2 at which uptake started; 2) addition of albumin to the medium reduced GO2; 3) increasing dithionite concentration in the medium in O2-release experiments progressively enhanced GO2, which became virtually constant for nearly the entire course of release; and 4) O2 uptake and O2 release (without dithoite) in the same SO2 range yielded very similar GO2. These results suggested that O2 uptake and release were importantly limited by diffusion through the external medium and that in the SO2 range between 0.3 and 0.8, chemical reaction exerted little limiting effect. Since O2 release at the highest dithionite concentration (40 mmol/l) appeared to be virtually unlimited by external diffusion, GO2 measured under these conditions, averaging 8.7 ml X min-1 X Torr-1 X ml erythrocytes-1, was considered to mainly reflect intracellular diffusion limitation. The corresponding specific transfer conductance for O2 transfer in whole blood (hematocrit, 0.45) is 3.9 ml X min-1 X Torr-1 X ml blood-1.

Micropipette system for sampling and injecting small volumes.

We have developed a micropipette system that allows sampling of preset volumes of liquids and injection of preset small amounts of liquids, using thermal expansion of an oil phase in a micropipette, achieved by increasing or decreasing the current applied to heat the shaft of the micropipette. The system has been tested to inject or collect volumes of about 20 nl, but smaller and larger volumes are possible. The amount injected or collected for a given temperature step of the micropipette is largely independent of the physical properties, e.g. of viscosity, into which the sample is injected or from which it is collected. The pipette tip is not measurably heated when heating the shaft for injection or collection, thus avoiding damage of the biological material into which the injection occurs or from which it is sampled.

Ion-sensitive microelectrode system with short response time.

The measurement of changes in ion activity (e.g., pH) in neurons requires fine tip-sized double-barreled microelectrodes: one channel being equipped with an ion-selective liquid membrane, the other used for measurement of the membrane potential. The limited bandwidth and the differing transfer functions for electrical and ionic signals necessitate frequency response linearization networks to ensure that the output signal of the electrode is a faithful image of the input signal. We have developed a linearization network to ensure a rapid response time for ion-sensitive microelectrodes. To test the response characteristic we have developed a test system that allows the pH at the electrode tip to be changed within 1 ms. Application of these techniques to electrodes of 1 micron tip diameter results in a 90% response time to a pH step of approximately 60 ms and of approximately 2 ms with electrodes with 20 micron tip.