Multilayered VBC score predicts sgRNAs that efficiently generate loss-of-function alleles.

CRISPR-Cas9 screens have emerged as a transformative approach to systematically probe gene functions. The quality and success of these screens depends on the frequencies of loss-of-function alleles, particularly in negative-selection screens widely applied for probing essential genes. Using optimized screening workflows, we performed essentialome screens in cancer cell lines and embryonic stem cells and achieved dropout efficiencies that could not be explained by common frameshift frequencies. We find that these superior effect sizes are mainly determined by the impact of in-frame mutations on protein function, which can be predicted based on amino acid composition and conservation. We integrate protein features into a 'Bioscore' and fuse it with improved predictors of single-guide RNA activity and indel formation to establish a score that captures all relevant processes in CRISPR-Cas9 mutagenesis. This Vienna Bioactivity CRISPR score (www.vbc-score.org) outperforms previous prediction tools and enables the selection of sgRNAs that effectively produce loss-of-function alleles.

Cyclic PaO2 oscillations assessed in the renal microcirculation: correlation with tidal volume in a porcine model of lung lavage.

BACKGROUND: Oscillations of the arterial partial pressure of oxygen induced by varying shunt fractions occur during cyclic alveolar recruitment within the injured lung. Recently, these were proposed as a pathomechanism that may be relevant for remote organ injury following acute respiratory distress syndrome. This study examines the transmission of oxygen oscillations to the renal tissue and their tidal volume dependency. METHODS: Lung injury was induced by repetitive bronchoalveolar lavage in eight anaesthetized pigs. Cyclic alveolar recruitment was provoked by high tidal volume ventilation. Oscillations of the arterial partial pressure of oxygen were measured in real-time in the macrocirculation by multi-frequency phase fluorimetry and in the renal microcirculation by combined white-light spectrometry and laser-Doppler flowmetry during tidal volume down-titration. RESULTS: Significant respiratory-dependent oxygen oscillations were detected in the macrocirculation and transmitted to the renal microcirculation in a substantial extent. The amplitudes of these oscillations significantly correlate to the applied tidal volume and are minimized during down-titration. CONCLUSIONS: In a porcine model oscillations of the arterial partial pressure of oxygen are induced by cyclic alveolar recruitment and transmitted to the renal microcirculation in a tidal volume-dependent fashion. They might play a role in organ crosstalk and remote organ damage following lung injury.

Systemic PaO2 Oscillations Cause Mild Brain Injury in a Pig Model.

OBJECTIVE: Systemic PaO2 oscillations occur during cyclic recruitment and derecruitment of atelectasis in acute respiratory failure and might harm brain tissue integrity. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Adult anesthetized pigs. INTERVENTIONS: Pigs were randomized to a control group (anesthesia and extracorporeal circulation for 20 hr with constant PaO2, n = 10) or an oscillation group (anesthesia and extracorporeal circulation for 20 hr with artificial PaO2 oscillations [3 cycles min⁻¹], n = 10). Five additional animals served as native group (n = 5). MEASUREMENTS AND MAIN RESULTS: Outcome following exposure to artificial PaO2 oscillations compared with constant PaO2 levels was measured using 1) immunohistochemistry, 2) real-time polymerase chain reaction for inflammatory markers, 3) receptor autoradiography, and 4) transcriptome analysis in the hippocampus. Our study shows that PaO2 oscillations are transmitted to brain tissue as detected by novel ultrarapid oxygen sensing technology. PaO2 oscillations cause significant decrease in NISSL-stained neurons (p < 0.05) and induce inflammation (p < 0.05) in the hippocampus and a shift of the balance of hippocampal neurotransmitter receptor densities toward inhibition (p < 0.05). A pathway analysis suggests that cerebral immune and acute-phase response may play a role in mediating PaO2 oscillation-induced brain injury. CONCLUSIONS: Artificial PaO2 oscillations cause mild brain injury mediated by inflammatory pathways. Although artificial PaO2 oscillations and endogenous PaO2 oscillations in lung-diseased patients have different origins, it is likely that they share the same noxious effect on the brain. Therefore, PaO2 oscillations might represent a newly detected pathway potentially contributing to the crosstalk between acute lung and remote brain injury.

[Case report: 40-year-old patient with massive bleeding undergoing cesarean section]. / Kasuistik - 40-jährige Patientin mit Massivblutung im Rahmen einer Sectio caesarea.

We report a case of a 40-year-old patient suffering major bleeding when undergoing cesarean section for delivery with placenta previa. After surgery the patient suffered from severe bleeding several times and again underwent surgery with abdominal packing. After an abdominal compartment syndrome and an ARDS a post transfusion purpura was found to be the cause of the repeated bleeding and could successfully be treated with a cycle of plasmapheresis.

Inhalation therapy with the synthetic TIP-like peptide AP318 attenuates pulmonary inflammation in a porcine sepsis model.

BACKGROUND: The lectin-like domain of TNF-α can be mimicked by synthetic TIP peptides and represents an innovative pharmacologic option to treat edematous respiratory failure. TIP inhalation was shown to reduce pulmonary edema and improve gas exchange. In addition to its edema resolution effect, TIP peptides may exert some anti-inflammatory properties. The present study therefore investigates the influence of the inhaled TIP peptide AP318 on intrapulmonary inflammatory response in a porcine model of systemic sepsis. METHODS: In a randomized-blinded setting lung injury was induced in 18 pigs by lipopolysaccharide-infusion and a second hit with a short period of ventilator-induced lung stress, followed by a six-hour observation period. The animals received either two inhalations with the peptide (AP318, 2×1 mg kg(-1)) or vehicle. Post-mortem pulmonary expression of inflammatory and mechanotransduction markers were determined by real-time polymerase chain reaction (IL-1ß, IL-6, TNF-α, COX-2, iNOS, amphiregulin, and tenascin-c). Furthermore, regional histopathological lung injury, edema formation and systemic inflammation were quantified. RESULTS: Despite similar systemic response to lipopolysaccharide infusion in both groups, pulmonary inflammation (IL-6, TNF-α, COX-2, tenascin-c) was significantly mitigated by AP318. Furthermore, a Western blot analysis shows a significantly lower of COX-2 protein level. The present sepsis model caused minor lung edema formation and moderate gas exchange impairment. Six hours after onset pathologic scoring showed no improvement, while gas exchange parameters and pulmonary edema formation were similar in the two groups. CONCLUSION: In summary, AP318 significantly attenuated intrapulmonary inflammatory response even without the presence or resolution of severe pulmonary edema in a porcine model of systemic sepsis-associated lung injury. These findings suggest an anti-inflammatory mechanism of the lectin-like domain beyond mere edema reabsorption in endotoxemic lung injury in vivo.

Low tidal volume pressure support versus controlled ventilation in early experimental sepsis in pigs.

BACKGROUND: In moderate acute respiratory distress syndrome (ARDS) several studies support the usage of assisted spontaneous breathing modes. Only limited data, however, focus on the application in systemic sepsis and developing lung injury. The present study examines the effects of immediate initiation of pressure support ventilation (PSV) in a model of sepsis-induced ARDS. METHODS: 18 anesthetized pigs received a two-staged continuous lipopolysaccharide infusion to induce lung injury. The animals were randomly assigned to PSV or volume controlled (VCV) lung protective ventilation (tidal volume each 6 ml kg-1, n = 2x9) over six hours. Gas exchange parameters, hemodynamics, systemic inflammation, and ventilation distribution by multiple inert gas elimination and electrical impedance tomography were assessed. The post mortem analysis included histopathological scoring, wet to dry ratio, and alveolar protein content. RESULTS: Within six hours both groups developed a mild to moderate ARDS with comparable systemic inflammatory response and without signs of improving gas exchange parameters during PSV. The PSV group showed signs of more homogenous ventilation distribution by electrical impedance tomography, but only slightly less hyperinflated lung compartments by multiple inert gas elimination. Post mortem and histopathological assessment yielded no significant intergroup differences. CONCLUSIONS: In a porcine model of sepsis-induced mild ARDS immediate PSV was not superior to VCV. This contrasts with several experimental studies from non-septic mild to moderate ARDS. The present study therefore assumes that not only severity, but also etiology of lung injury considerably influences the response to early initiation of PSV.

TIP peptide inhalation in experimental acute lung injury: effect of repetitive dosage and different synthetic variants.

BACKGROUND: Inhalation of TIP peptides that mimic the lectin-like domain of TNF-α is a novel approach to attenuate pulmonary oedema on the threshold to clinical application. A placebo-controlled porcine model of acute respiratory distress syndrome (ARDS) demonstrated a reduced thermodilution-derived extravascular lung water index (EVLWI) and improved gas exchange through TIP peptide inhalation within three hours. Based on these findings, the present study compares a single versus a repetitive inhalation of a TIP peptide (TIP-A) and two alternate peptide versions (TIP-A, TIP-B). METHODS: Following animal care committee approval ARDS was induced by bronchoalveolar lavage followed by injurious ventilation in 21 anaesthetized pigs. A randomised-blinded three-group setting compared the single-dosed peptide variants TIP-A and TIP-B as well as single versus repetitive inhalation of TIP-A (n = 7 per group). Over two three-hour intervals parameters of gas exchange, transpulmonary thermodilution, calculated alveolar fluid clearance, and ventilation/perfusion-distribution were assessed. Post-mortem measurements included pulmonary wet/dry ratio and haemorrhage/congestion scoring. RESULTS: The repetitive TIP-A inhalation led to a significantly lower wet/dry ratio than a single dose and a small but significantly lower EVLWI. However, EVLWI changes over time and the derived alveolar fluid clearance did not differ significantly. The comparison of TIP-A and B showed no relevant differences. Gas exchange and ventilation/perfusion-distribution significantly improved in all groups without intergroup differences. No differences were found in haemorrhage/congestion scoring. CONCLUSIONS: In comparison to a single application the repetitive inhalation of a TIP peptide in three-hour intervals may lead to a small additional reduction the lung water content. Two alternate TIP peptide versions showed interchangeable characteristics.

Hyaluronic acid plasma levels during high versus low tidal volume ventilation in a porcine sepsis model.

BACKGROUND: Shedding of the endothelial glycocalyx can be observed regularly during sepsis. Moreover, sepsis may be associated with acute respiratory distress syndrome (ARDS), which requires lung protective ventilation with the two cornerstones of application of low tidal volume and positive end-expiratory pressure. This study investigated the effect of a lung protective ventilation on the integrity of the endothelial glycocalyx in comparison to a high tidal volume ventilation mode in a porcine model of sepsis-induced ARDS. METHODS: After approval by the State and Institutional Animal Care Committee, 20 male pigs were anesthetized and received a continuous infusion of lipopolysaccharide to induce septic shock. The animals were randomly assigned to either low tidal volume ventilation, high tidal volume ventilation, or no-LPS-group groups and observed for 6 h. In addition to the gas exchange parameters and hematologic analyses, the serum hyaluronic acid concentrations were determined from central venous blood and from pre- and postpulmonary and pre- and postcerebral circulation. Post-mortem analysis included histopathological evaluation and determination of the pulmonary and cerebral wet-to-dry ratios. RESULTS: Both sepsis groups developed ARDS within 6 h of the experiment and showed significantly increased serum levels of hyaluronic acid in comparison to the no-LPS-group. No significant differences in the hyaluronic acid concentrations were detected before and after pulmonary and cerebral circulation. There was also no significant difference in the serum hyaluronic acid concentrations between the two sepsis groups. Post-mortem analysis showed no significant difference between the two sepsis groups. CONCLUSION: In a porcine model of septic shock and ARDS, the serum hyaluronic acid levels were significantly elevated in both sepsis groups in comparison to the no-LPS-group. Intergroup comparison between lung protective ventilated and high tidal ventilated animals revealed no significant differences in the serum hyaluronic acid levels.

Differential transcriptional modulation of biological processes in adipocyte triglyceride lipase and hormone-sensitive lipase-deficient mice.

Adipocyte triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are intracellular lipases that mobilize triglycerides, the main energy source in mammals. Deletion of genes encoding ATGL (Pnpla2) or HSL (Lipe) in mice results in striking phenotypic differences, suggesting distinct roles for these lipases. The goal of the present study was to identify the biological processes that are modulated in the metabolic tissues of ATGL- and HSL-deficient mice. DNA microarrays were employed to provide full genome coverage concerning the types of genes that are differentially expressed in wild-type and mutant mice. For both mouse models, transcript signatures were identified in white adipose tissue, brown adipose tissue (BAT), skeletal muscle (SM), cardiac muscle (CM), and liver. Genetic ablation of ATGL and HSL alters the transcript levels of a large number of genes in metabolic tissues. The genes affected in the two models are, however, largely different ones. Indeed, only one biological process was modulated in the same way in both mouse models, namely the down-regulation of fatty acid metabolism in BAT. The most pronounced modulation of biological processes was observed in ATGL-/- CM, in which a concerted down-regulation of transcripts associated with oxidative pathways was observed. In HSL-/- mice, in contrast, the most marked changes were seen in SM, namely, alterations in transcript levels reflecting a change of energy source from lipid to carbohydrate. The transcript signatures also provided novel insights into the metabolic derangements that are characteristic of ATGL-/- mice. Our findings suggest that ATGL and HSL differentially modulate biological processes in metabolic tissues. We hypothesize that the intermediary metabolites of the lipolytic pathways are signaling molecules and activators of a wide range of biochemical and cellular processes in mammals.

Random allogeneic blood transfusion in pigs: characterisation of a novel experimental model.

BACKGROUND: Organ cross-talk describes interactions between a primary affected organ and a secondarily injured remote organ, particularly in lung-brain interactions. A common theory is the systemic distribution of inflammatory mediators that are released by the affected organ and transferred through the bloodstream. The present study characterises the baseline immunogenic effects of a novel experimental model of random allogeneic blood transfusion in pigs designed to analyse the role of the bloodstream in organ cross-talk. METHODS: After approval of the State and Institutional Animal Care Committee, 20 anesthetized pig were randomized in a donor and an acceptor (each n = 8): the acceptor animals each received high-volume whole blood transfusion from the donor (35-40 ml kg-1). Four animals received balanced electrolyte solution instead of blood transfusion (control group; n = 4). Afterwards the animals underwent extended cardiorespiratory monitoring for eight hours. Post mortem assessment included pulmonary, cerebral and systemic mediators of early inflammatory response (IL-6, TNF-alpha, iNOS), wet to dry ratio, and lung histology. RESULTS: No adverse events or incompatibilities occurred during the blood transfusion procedures. Systemic cytokine levels and pulmonary function were unaffected. Lung histopathology scoring did not display relevant intergroup differences. Neither within the lung nor within the brain an up-regulation of inflammatory mediators was detected. High volume random allogeneic blood transfusion in pigs neither impaired pulmonary integrity nor induced systemic, lung, or brain inflammatory response. CONCLUSION: This approach can represent a novel experimental model to characterize the blood-bound transmission in remote organ injury.

Effect of gelatin-polysuccinat on cerebral oxygenation and microcirculation in a porcine haemorrhagic shock model.

BACKGROUND: During early treatment of haemorrhagic shock maintenance of cerebral and end-organ oxygen supply by fluid resuscitation is mandatory. Gelatin-polysuccinat (GP) recently regained attention despite a still unclear risk profile and widely unknown effects on cerebral and peripheral microcirculation. This study investigates the effects of GP versus balanced electrolyte solution (BEL) with focus on cerebral regional oxygen saturation and peripheral microcirculation in a porcine haemorrhagic shock model. METHODS: After Animal Care Committee approval haemorrhagic shock was induced by arterial blood withdrawal in 27 anaesthetized pigs. Consequently, the animals received rapid fluid resuscitation by either GP or BEL to replace the removed amount of blood, or remained untreated (n = 3 × 9). Over two hours cerebral regional oxygen saturation by near-infrared spectroscopy and peripheral buccal microcirculation by combined white-light spectrometry and laser-Doppler flowmetry were recorded. Secondary parameters included extended haemodynamics, spirometry, haematological and blood gas parameters. RESULTS: Both fluid resuscitation regimes sufficiently stabilized the macro- and microcirculation in haemorrhagic shock with a more pronounced effect following GP infusion. GP administration led to a persisting, critical impairment of cerebral regional oxygen saturation through considerable haemodilution. Survival rates were 100% in both fluid resuscitation groups, but only 33% in the untreated control. CONCLUSION: Equal amounts of GP and BEL sufficiently stabilize systemic circulation and microcirculatory perfusion. Forced fluid resuscitation by GP should be applied with caution to prevent haemodilution-induced impairment of cerebral oxygen delivery.

Lung injury does not aggravate mechanical ventilation-induced early cerebral inflammation or apoptosis in an animal model.

INTRODUCTION: The acute respiratory distress syndrome is not only associated with a high mortality, but also goes along with cognitive impairment in survivors. The cause for this cognitive impairment is still not clear. One possible mechanism could be cerebral inflammation as result of a "lung-brain-crosstalk". Even mechanical ventilation itself can induce cerebral inflammation. We hypothesized, that an acute lung injury aggravates the cerebral inflammation induced by mechanical ventilation itself and leads to neuronal damage. METHODS: After approval of the institutional and state animal care committee 20 pigs were randomized to one of three groups: lung injury by central venous injection of oleic acid (n = 8), lung injury by bronchoalveolar lavage in combination with one hour of injurious ventilation (n = 8) or control (n = 6). Brain tissue of four native animals from a different study served as native group. For six hours all animals were ventilated with a tidal volume of 7 ml kg-1 and a scheme for positive end-expiratory pressure and inspired oxygen fraction, which was adapted from the ARDS network tables. Afterwards the animals were killed and the brains were harvested for histological (number of neurons and microglia) and molecular biologic (TNFalpha, IL-1beta, and IL-6) examinations. RESULTS: There was no difference in the number of neurons or microglia cells between the groups. TNFalpha was significantly higher in all groups compared to native (p < 0.05), IL-6 was only increased in the lavage group compared to native (p < 0.05), IL-1beta showed no difference between the groups. DISCUSSION: With our data we can confirm earlier results, that mechanical ventilation itself seems to trigger cerebral inflammation. This is not aggravated by acute lung injury, at least not within the first 6 hours after onset. Nevertheless, it seems too early to dismiss the idea of lung-injury induced cerebral inflammation, as 6 hours might be just not enough time to see any profound effect.

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome.

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

Immiscible hydrous Fe-Ca-P melt and the origin of iron oxide-apatite ore deposits.

The origin of iron oxide-apatite deposits is controversial. Silicate liquid immiscibility and separation of an iron-rich melt has been invoked, but Fe-Ca-P-rich and Si-poor melts similar in composition to the ore have never been observed in natural or synthetic magmatic systems. Here we report experiments on intermediate magmas that develop liquid immiscibility at 100 MPa, 1000-1040 °C, and oxygen fugacity conditions (fO2) of ∆FMQ = 0.5-3.3 (FMQ = fayalite-magnetite-quartz equilibrium). Some of the immiscible melts are highly enriched in iron and phosphorous ± calcium, and strongly depleted in silicon (<5 wt.% SiO2). These Si-poor melts are in equilibrium with a rhyolitic conjugate and are produced under oxidized conditions (~FMQ + 3.3), high water activity (aH2O ≥ 0.7), and in fluorine-bearing systems (1 wt.%). Our results show that increasing aH2O and fO2 enlarges the two-liquid field thus allowing the Fe-Ca-P melt to separate easily from host silicic magma and produce iron oxide-apatite ores.

Structure and post-translational modifications of the web silk protein spidroin-1 from Nephila spiders.

Spidroin-1 is one of the major ampullate silk proteins produced by spiders for use in the construction of the frame and radii of orb webs, and as a dragline to escape from predators. Only partial sequences of spidroin-1 produced by Nephila clavipes have been reported up to now, and there is no information on post-translational modifications (PTMs). A gel-based mass spectrometry strategy with ETD and CID fragmentation methods were used to sequence and determine the presence/location of any PTMs on the spidroin-1. Sequence coverage of 98.06%, 95.05%, and 98.37% were obtained for N. clavipes, Nephila edulis and for Nephila madagascariensis, respectively. Phosphorylation was the major PTM observed with 8 phosphorylation sites considered reliable on spidroin-1 produced by N. clavipes, 4 in N. madagascariensis and 2 for N. edulis. Dityrosine and 3,4-dihydroxyphenylalanine (formed by oxidation of the spidroin-1) were observed, although the mechanism by which they are formed (i.e. exposure to UV radiation or to peroxidases in the major ampullate silk gland) is uncertain. Herein we present structural information on the spidroin-1 produced by three different Nephila species; these findings may be valuable for understanding the physicochemical properties of the silk proteins and moreover, future designs of recombinantly produced spider silk proteins. Biotechnological significance The present investigation shows for the first time spidroin structure and post-translational modifications observed on the major ampullate silk spidroin-1. The many site specific phosphorylations (localized within the structural motifs) along with the probably photoinduction of hydroxylations may be relevant for scientists in material science, biology, biochemistry and environmental scientists. Up to now all the mechanical properties of the spidroin have been characterized without any consideration about the existence of PTMs in the sequence of spidroins. Thus, these findings for major ampullate silk spidroin-1 from Nephila spiders provide the basis for mechanical-elastic property studies of silk for biotechnological and biomedical potential applications. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Molecular processes during fat cell development revealed by gene expression profiling and functional annotation.

BACKGROUND: Large-scale transcription profiling of cell models and model organisms can identify novel molecular components involved in fat cell development. Detailed characterization of the sequences of identified gene products has not been done and global mechanisms have not been investigated. We evaluated the extent to which molecular processes can be revealed by expression profiling and functional annotation of genes that are differentially expressed during fat cell development. RESULTS: Mouse microarrays with more than 27,000 elements were developed, and transcriptional profiles of 3T3-L1 cells (pre-adipocyte cells) were monitored during differentiation. In total, 780 differentially expressed expressed sequence tags (ESTs) were subjected to in-depth bioinformatics analyses. The analysis of 3'-untranslated region sequences from 395 ESTs showed that 71% of the differentially expressed genes could be regulated by microRNAs. A molecular atlas of fat cell development was then constructed by de novo functional annotation on a sequence segment/domain-wise basis of 659 protein sequences, and subsequent mapping onto known pathways, possible cellular roles, and subcellular localizations. Key enzymes in 27 out of 36 investigated metabolic pathways were regulated at the transcriptional level, typically at the rate-limiting steps in these pathways. Also, coexpressed genes rarely shared consensus transcription-factor binding sites, and were typically not clustered in adjacent chromosomal regions, but were instead widely dispersed throughout the genome. CONCLUSIONS: Large-scale transcription profiling in conjunction with sophisticated bioinformatics analyses can provide not only a list of novel players in a particular setting but also a global view on biological processes and molecular networks.