Single-Molecule Sequencing Reveals Complex Genome Variation of Hepatitis B Virus during 15 Years of Chronic Infection following Liver Transplantation.

UNLABELLED: Chronic hepatitis B (CHB) is prevalent worldwide. The infectious agent, hepatitis B virus (HBV), replicates via an RNA intermediate and is error prone, leading to the rapid generation of closely related but not identical viral variants, including those that can escape host immune responses and antiviral treatments. The complexity of CHB can be further enhanced by the presence of HBV variants with large deletions in the genome generated via splicing (spHBV variants). Although spHBV variants are incapable of autonomous replication, their replication is rescued by wild-type HBV. spHBV variants have been shown to enhance wild-type virus replication, and their prevalence increases with liver disease progression. Single-molecule deep sequencing was performed on whole HBV genomes extracted from samples, including the liver explant, longitudinally collected from a subject with CHB over a 15-year period after liver transplantation. By employing novel bioinformatics methods, this analysis showed that the dynamics of the viral population across a period of changing treatment regimens was complex. The spHBV variants detected in the liver explant remained present posttransplantation, and a highly diverse novel spHBV population as well as variants with multiple deletions in the pre-S genes emerged. The identification of novel mutations outside the HBV reverse transcriptase gene that co-occurred with known drug resistance-associated mutations highlights the relevance of using full-genome deep sequencing and supports the hypothesis that drug resistance involves interactions across the full length of the HBV genome. IMPORTANCE: Single-molecule sequencing allowed the characterization, in unprecedented detail, of the evolution of HBV populations and offered unique insights into the dynamics of defective and spHBV variants following liver transplantation and complex treatment regimens. This analysis also showed the rapid adaptation of HBV populations to treatment regimens with evolving drug resistance phenotypes and evidence of purifying selection across the whole genome. Finally, the new open-source bioinformatics tools with the capacity to easily identify potential spliced variants from deep sequencing data are freely available.

[Ileus provoked by hairfollicles in form of a foreign body granuloma in the small intestine of a cat. A case report]. / Ileus hervorgerufen durch Granulationsgewebe mit eingeschlossenen Haarschäften im Dünndarm einer Katze. Ein Fallbericht.

Vomitus and anorexia is a frequent reason for presentation of cats at any age at the veterinarian. When an underlying ileus is suspected the risk of a neoplasia is considered to be high, especially in older patients. Due to high cost for diagnostics and treatment and a guarded prognosis owners frequently opt for euthanasia of those animals without verification of the tentative diagnosis. In this cat the ileus was caused by hairfollicles in form of a foreign body granuloma in the intestinal mucosa, which could be removed without difficulty by an enterectomy. So far no similar case report has been documented. This case demonstrates that in spite of a justified suspicion of a neoplasia in an elder animal this will not necessarily be verified by histopathological examination. Further diagnostic measures should always be performed to establish a prognosis for the patient.

[Diagnosis of malignant bone and soft tissue tumors]. / Diagnostik von malignen Knochen- und Weichteiltumoren.

Primary sarcoma of bone is a rare entity but nevertheless a significant cause of mortality in children and adolescents. The focus of the preoperative evaluation is to set up a histological diagnosis, define local tumor extent and develop a therapy regimen. In addition to patient history and clinical findings a radiograph in two orthogonal planes is still of great importance. MRI plays a major role in the further clarification of the diagnosis, while CT is valuable in the diagnosis of tumors of the axial skeleton as well as in systemic staging. A PET-CT can be performed to obtain an overview of further tumor sites. Open bone biopsy is the final diagnostic step and should be carried out at the institution where the definitive treatment will be performed. Complications such as fracture, neural lesions and spread of tumor cells are relatively rare if the biopsy is performed appropriately; however, patients should be instructed to strictly avoid weight-bearing on the affected extremity.

[The modular MML revision system in knee revision and tumor arthroplasty]. / Einsatz eines modularen Knierevisionssystems MML im Rahmen des Knieprothesenwechsels und der Tumorendoprothetik.

Increasing age and a higher level of mobility lead to an increasing incidence in revision arthroplasty after total knee replacement and tumor surgery. So far, the reconstruction of large defects in bony and soft tissue environments can be accomplished by the modern modular components of revision implants. The consecutive reconstruction of the extensor mechanism in extended revision has its own drawbacks and is often associated with significant functional limitations for the patient. Specially designed implants and methods are required to generate good functional results. The modular knee revision system MML provides specific modifications of the tibial component for reconstruction of the extensor mechanism. Combined with artificial strips, an excellent functional outcome could be achieved. In this study, 70 patients were operated with the MML endoprosthesis in knee revision or tumor surgery. An excellent functional outcome could be determined. At 7 years after surgery, an average of 32+/-13 points was achieved on the Oxford Knee Score. The outcome measurement using the functional scoring system of the American Knee Society (AKS score) showed similarly good results with 71+/-25 points out of 100. A minor deficit of only 2 degrees in active extension could be observed after reconstruction of the extensor mechanism. In conclusion, we have demonstrated that the MML modular revision system is appropriate for reconstruction of segmental bone defects.

Determination of ochratoxin A in wine by liquid chromatography tandem mass spectrometry after combined anion-exchange/reversed-phase clean-up.

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus ochraceus and Penicillium verrucosum. It has been found and analysed in several foods and feeds. Owing to its toxicity and occurrence in food and feed, the European Community has issued directives and some countries have their own regulations for OTA contents in food, feed and beverages. This work describes a method for the determination of OTA in mulled and red wine. It is based on combined anion exchange/reversed-phase clean-up and was analysed by liquid chromatography coupled with tandem mass spectrometry (multiple reaction monitoring). The method was validated with natural contaminated and spiked wine samples with OTA contents from 1.34 to 3.48 microg kg(-1). Owing to its accuracy, good reproducibility and repeatability, this easy method is a good alternative to liquid chromatography-fluorescence detection methods.

Effect of positive end expiratory pressure on functional residual capacity and compliance in surfactant-treated preterm infants.

UNLABELLED: Positive end expiratory pressure is routinely used when ventilating preterm infants. Elevation of PEEP increases lung volume, as does surfactant treatment. The purpose of this study was to investigate the effect of various levels of PEEP within the range of 0.2 to 0.4 kPa on lung volume, compliance and gas exchange. We measured functional residual capacity, compliance of the respiratory system and arterial blood gases in 20 infants (median birth weight 1240 g, range 660-1690 g; median gestational age 28 weeks, range 24-32 weeks; postnatal age 3-4 days). The infants were studied at 72 hours after their last dose of natural surfactant. At this time the patients were routinely nursed at 0.3 kPa of PEEP, the PEEP level was lowered to 0.2 kPa or raised to 0.4 kPa in random order. The PEEP level was then changed to the third level 0.4 kPa or 0.2 kPa. Each new setting was maintained for 20 min before FRC, compliance and blood gases were measured. FRC was assessed using SF6 washout technique. Increasing PEEP from 0.2 to 0.3 to 0.4 kPa resulted in increases in FRC (p < 0.01) and oxygenation (ns) in all infants. In 16 infants compliance decreased and paCO2 increased with elevation of PEEP. Only in 4 infants compliance increased and CO2 fell. CONCLUSION: In the majority of our infants reduction of PEEP from 0.4 to 0.2 kPa resulted in increases in compliance and CO2 reduction. Our results might suggest that relatively low levels of PEEP < 0.3 kPa may be appropriate at 72 hours after surfactant replacement. Furthermore, these results underline the importance of PEEP test in clinical practice.

Effects of in vivo nitroglycerin treatment on activity and expression of the guanylyl cyclase and cGMP-dependent protein kinase and their downstream target vasodilator-stimulated phosphoprotein in aorta.

BACKGROUND: Chronic in vivo treatment with nitroglycerin (NTG) induces tolerance to nitrates and cross-tolerance to nitrovasodilators and endothelium-derived nitric oxide (NO). We previously identified increased vascular superoxide formation and reduced NO bioavailability as one causal mechanism. It is still controversial whether intracellular downstream signaling to nitrovasodilator-derived NO is affected as well. METHODS AND RESULTS: We therefore studied the effects of 3-day NTG treatment of rats and rabbits on activity and expression of the immediate NO target soluble guanylyl cyclase (sGC) and on the cGMP-activated protein kinase I (cGK-I). Tolerance was induced either by chronic NTG infusion via osmotic minipumps (rats) or by NTG patches (rabbits). Western blot analysis, semiquantitative reverse transcription-polymerase chain reaction, and Northern blot analysis revealed significant and comparable increases in the expression of sGC alpha(1) and beta(1) subunit protein and mRNA. Studies with the oxidative fluorescent dye hydroethidine revealed an increase in superoxide in the endothelium and smooth muscle. Stimulation with NADH increased superoxide signals in both layers. Although cGK-I expression in response to low-dose NTG was not changed, a strong reduction in vasodilator-stimulated phosphoprotein (VASP) serine239 phosphorylation (specific substrate of cGK-I) was observed in tolerant tissue from rats and rabbits. Concomitant in vivo and in vitro treatment with vitamin C improved tolerance, reduced oxidative stress, and improved P-VASP. CONCLUSIONS: We therefore conclude that increased expression of sGC in the setting of tolerance reflects a chronic inhibition rather than an induction of the sGC-cGK-I pathway and may be mediated at least in part by increased vascular superoxide.

Comparison of lung volume measurements by antero-posterior chest X-ray and the SF6 washout technique in mechanically ventilated infants.

While anterior/posterior chest x-rays (CXR) are routinely performed to estimate lung volume (LV) and adjust ventilator settings, the precise measurement of LV requires additional sophistication. In 31 infants ventilated because of surfactant deficiency (n=23), bronchopulmonary dysplasia (n=7), or pulmonary hypoplasia (n=1) with either intermittent positive pressure (n=18) or high frequency oscillation (n=13) (gestational age 23-39 weeks (median 26 weeks); birthweight 550-2780 g (median 840 g); age at measurement 1-91 days (median 6 days); weight at study time (WST) 675-3000 g (median 938 g)), we investigated whether LV, as measured by the sulfur hexafluoride (SF6) washout technique, could by estimated from CXR by: 1) the sum (A+B) of the right (A) and left (B) lung fields areas; 2) the product (LxW) of the distances from the right apex to the right costophrenic angle (L) and between both costophrenic angles (W); 3) the diaphragm position relative to the posterior parts of the ribs (DP); and 4) the lung radiolucency (RL, grades 0-4). Correlations between A+B (r=0.44) or LxW (r=0.37) and LV were poor, but improved when A+B, LxW, and LV were normalized to WST: (A+B)/WST vs. LV/WST (r=0.74), and LxW/WST vs. LV/WST (r=0.67). DP (r=0.13) and RL (Spearman's rho=0.17) did not correlate with LV/WST. A multiple linear regression analysis led to the following best-fit equation: LV/WST=2.58 (A+B)/WST - 5.47 DP + 42.2 (r=0.83). We concluded that an estimate of LV from CXR lacked sufficient accuracy. DP and RL did not correlate with LV measured by SF6 washout.

The effect of positive endexpiratory pressure, peak inspiratory pressure, and inspiratory time on functional residual capacity in mechanically ventilated preterm infants.

UNLABELLED: In mechanical ventilation of preterm infants, positive endexpiratory pressure (PEEP) is widely used to prevent alveolar collapse, maintain functional residual capacity (FRC) and improve oxygenation. Prolongation of inspiratory time (ti) and increase of peak inspiratory pressure (PIP) are also used for this purpose. We investigated the effect of variations of PEEP, PIP and ti on FRC in ten infants with hyaline membrane disease and onset of bronchopulmonary dysplasia (BPD, n = 7), pulmonary hypertension (n = 1), pulmonary hypoplasia (n = 1) or severe BPD (n = 1) (gestational age 24-39 weeks, median 26 weeks; birth weight 590-2960 g, 785 g; chronological age 7 84 days, 19 days; weight 689-4650 g, 1185 g). FRC, measured using the sulphur hexafluoride washout technique, was between 6.2 and 48.3 ml/kg (median 21.5 ml/kg). PEEP was changed stepwise 2-5 times in each patient (median 3) and mean airway pressure (MAP) was modified independently of PEEP by changing PIP 0 2 times (median 1) and ti 0(2 times (median 2). Changes of FRC correlated well with modifications of PEEP in each patient (r = 0.90, range 0.71 0.99). The slope factors of linear correlations had a median value of 2.94 ml/cm H20 per kg, which was significantly different from zero (P < 0.01) and significantly higher than the slope factors of linear correlations between FRC and MAP after modifications of PIP or ti (P < 0.01). The latter two were statistically not different from zero. The quotients deltaFRC/deltaMAP were significantly higher after adjustments of PEEP than after adjustments of PIP or ti (P < 0.01). The time lag between the change of PEEP and the stabilization of FRC on a new level ranged from 2 to 14 min (median 5). CONCLUSION: FRC is mainly determined by PEEP but not by PIP or ti. Stabilization of FRC after a change of PEEP can last up to 14 min. Its duration is unpredictable and has to be waited for when testing pulmonary function in ventilated preterm infants.

Effects of mean airway pressure on lung volume during high-frequency oscillatory ventilation of preterm infants.

Measurement of mean lung volume (MLV) in high-frequency oscillatory ventilation (HFO) may be useful for optimizing the high lung volume strategy, but has not been available until now. We have measured MLV by means of the sulfur hexafluoride (SF6) washout method in 13 premature infants ventilated with HFO because of respiratory distress syndrome (gestational age, 23 to 31 wk [median, 25 + 6/7 wk]; birthweight, 630 to 1,140 g [790 g]; age at measurement, 2 to 10 d [4 d]; weight, 675 to 1,250 g [850 g]). To evaluate the relationship between MLV and mean airway pressure (MAP), the latter was systematically varied between the measurements. With clinically selected MAP, MLV was between 23.3 and 41.9 ml/kg (median, 33.5 ml/kg) and was strongly dependent on MAP in each patient; linear regression analyses resulted in slope factors between 1.0 and 6.9 ml/cm H2O/kg (median, 2.83 ml/cm H2O/kg), with correlation coefficients between 0.77 and 0.99 (median, 0.94). Stabilization of MLV after modification of MAP took 2 to 25 min (median, 9 min). We conclude that the selection of MAP on a clinical basis leads to a wide range of different MLVs. Despite the strong dependence of MLV on MAP, the prediction of MLV solely based on MAP was impossible because of large patient to patient variability of linear regressions. Alveolar recruitment and derecruitment may take up to 25 min after MAP changes.

Measurement of functional residual capacity by sulfur hexafluoride in small-volume lungs during spontaneous breathing and mechanical ventilation.

We modified a sulfur hexafluoride (SF6) washout technique to allow functional residual capacity (FRC) determinations in small-volume lungs both during spontaneous breathing and controlled mechanical ventilation. This method facilitates measurements in subjects who attempt spontaneous breaths between ventilator-generated breaths. We wished to confirm the accuracy and precision of the measurements and the method's sensitivity to change. The method uses a pneumotach together with a fast, mainstream infrared SF6 sensor mounted between the endotracheal tube and the ventilator circuit. A low flow of pure SF6 is delivered into the constant gas flow of the ventilator circuit to wash in tracer gas at a concentration of less than 2%. The flow signal and the instantaneous SF6 concentration is processed on-line by a computer. The calibration of the SF6 sensor's nonlinear signal and the ability of the flow sensor to reflect flow values precisely near zero flow had a major impact on the accuracy of the FRC estimate. This accuracy was tested by comparing measured FRC values with a dummy lung's true FRC that was varied from 7 to 70 mL. The comparison differed by 0.7 +/- 3.2% (mean +/- SD; range, -5.1 to 7.8%). As a measure of reproducibility (precision) across 20 FRC determinations in five adult rabbits, the average coefficient of variation was 1.7% (range, 0.57 to 4.33%) during continuous positive airway pressure and 1.98% (range, 0.35 to 3.81%) during controlled mechanical ventilation. The method proved sensitive to changes in FRC related to changes in airway pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Resistive and elastic unloading to assist spontaneous breathing does not change functional residual capacity.

Resistive and/or elastic unloading (a negative ventilator impedance, otherwise termed proportional assist ventilation) may be a useful means to assist spontaneous breathing. This only applies if the ventilator accurately provides pressure changes at the airway opening (P(ao)) proportional to the instantaneous flow and/or volume signal of spontaneous breathing and no significant phase lag. We designed such an infant ventilator, which controls the P(ao) by a negative feedback loop, and superimposes a second positive feedback circuit to generate unloading. To test this mode and the ventilator's accuracy in performing the synchronized pressure changes, we examined the functional residual capacity (FRC). We hypothesized that unloading by itself would not alter FRC because P(ao) should return to a preset baseline at end-expiration, and furthermore, that FRC could be actively altered by changing the baseline. Five anesthetized, tracheotomized, spontaneously breathing rabbits [respiratory system compliance 22.4 +/- 4.6 mL/kPa (mean +/- SEM)] were exposed to end-expiratory P(ao) levels of 0, 0.2, 0.4, and 0.6 kPa. At each of these levels a period of regular continuous positive airway pressure (CPAP) was alternated with a period of unloading (-40 mL/kPa ventilator compliance combined with -3 kPa/L/s ventilator resistance). FRC measured by a sulfur hexafluoride washout technique was virtually identical on CPAP and during unloading at equal end-expiratory P(ao) (difference, 1.41% +/- 0.95%), but FRC increased upon elevation of the end-expiratory P(ao) by 29.4 +/- 3.6 mL/kPa on CPAP and 30.2 +/- 3.2 at unloading (difference NS). We conclude that FRC is not destabilized by unloading, but that during unloading, as during CPAP, it depends on the end-expiratory P(ao).