NADPH oxidase-derived H2O2 subverts pathogen signaling by oxidative phosphotyrosine conversion to PB-DOPA.

Strengthening the host immune system to fully exploit its potential as antimicrobial defense is vital in countering antibiotic resistance. Chemical compounds released during bidirectional host-pathogen cross-talk, which follows a sensing-response paradigm, can serve as protective mediators. A potent, diffusible messenger is hydrogen peroxide (H2O2), but its consequences on extracellular pathogens are unknown. Here we show that H2O2, released by the host on pathogen contact, subverts the tyrosine signaling network of a number of bacteria accustomed to low-oxygen environments. This defense mechanism uses heme-containing bacterial enzymes with peroxidase-like activity to facilitate phosphotyrosine (p-Tyr) oxidation. An intrabacterial reaction converts p-Tyr to protein-bound dopa (PB-DOPA) via a tyrosinyl radical intermediate, thereby altering antioxidant defense and inactivating enzymes involved in polysaccharide biosynthesis and metabolism. Disruption of bacterial signaling by DOPA modification reveals an infection containment strategy that weakens bacterial fitness and could be a blueprint for antivirulence approaches.

nandb-number and brightness in R with a novel automatic detrending algorithm.

SUMMARY: An R package for performing number and brightness image analysis, with the implementation of a novel automatic detrending algorithm. AVAILABILITY AND IMPLEMENTATION: Available at https://github.com/rorynolan/nandb for all platforms. CONTACT: rnolan@well.ox.ac.uk or spadilla@well.ox.ac.uk. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Serum surfactant protein D (SP-D) is a prognostic marker of poor outcome in patients with A/H1N1 virus infection.

INTRODUCTION: Surfactant protein D (SP-D) plays an important role in the innate responses against pathogens and its production is altered in lung disorders. METHODS: We studied the circulating levels of SP-D in 37 patients with acute respiratory distress syndrome due to the A/H1N1 virus infection and in 40 healthy controls. Cox logistic regression models were constructed to explore the association of SP-D levels and risk of death. RESULTS: Mortality rate after a 28-day was 32.42 %. Significant higher levels of SP-D were detected in A/H1N1 patients with fatal outcome (p < 0.05). After adjusting for confounding variables, levels of SP-D ≥250 ng/mL were associated with increased the risk of death (HR = 8.27, 95 % CI 1.1-64.1, p = 0.043). CONCLUSIONS: Our results revealed that higher circulating levels of SP-D are associated with higher mortality risk in critically ill A/H1N1 patients. SP-D might be a predictive factor of poor outcomes in viral pneumonia.

Microbacterium oxydans and Microbacterium liquefaciens: a biological alternative for the treatment of Ni-V-containing wastes.

The present study evaluated 15 isolates obtained of environmental samples capable of tolerating high Ni and V concentrations. Those coded as MNSH2-PHGII-1, MNSH2-PHGII-2 and MV-PHGII-2 showed a minimum inhibitory concentration higher than 200 ppm for Ni and V and showed removal percentages corresponding to 84, 75 and 26% for Ni and 60, 55 and 20.3% for V, respectively, in liquid medium. When spent catalyst was added at 16% (w/v) pulp density, the highest Ni and V removal corresponded to MNSH2-PHGII-1 and MNSH2-PHGII-2, which were identified as Microbacterium oxydans and Microbacterium liquefaciens respectively, Microbacterium oxydans was able to remove Ni at the extent of 45.4% and V at 30.4% while Microbacterium liquefaciens removed Ni at 51% and V at 41.4% from the spent catalyst. The isolate MV-PHGII-2 identified also as Microbacterium oxydans showed the lowest removal for Ni and V corresponding to 16% and 9.5%, respectively. This is the first report where strains of Microbacterium were tested for their abilities to remove Ni and V from spent catalyst, suggesting its potential use in the treatment of this solid industrial waste.

Hiatal Hernia of Stomach and Lesser Omentum in a Cadaver: Is It a Type III or IV?

Hiatal hernias, protrusions of abdominal viscera through the esophageal hiatus, are classified into four types. Types I and II involve ascent of the stomach without affecting the gastroesophageal junction. Types III and IV involve the gastroesophageal junction. Type IV specifically may have stomach as well as other abdominal organ involvement, such as pancreas or omentum. Among these types, type IV is the most complex and rare form, accounting for only 0.1% of all cases of hiatal hernias. This report presents a case of a type IV hiatal hernia involving the lesser omentum and a significant portion of the stomach in an 86-year-old male cadaver with a history of mediastinal surgery. To our knowledge, this presentation in a cadaver has not previously been reported in the literature.  This case highlights classification inconsistencies in the literature, particularly regarding type IV hiatal hernias. It is unclear given the current classification system, whether this presentation would be considered a type III or type IV hiatal hernia as it fits both criteria and there are several interpretations of the criteria of a type IV hiatal hernia. Inconsistencies in the classification system may impede standardization of care. This report highlights the need for a more precise classification system that better accounts for anatomical changes and clinical presentation.

An analytical workflow for the molecular dissection of irreversibly modified fluorescent proteins.

Owing to their ability to be genetically expressed in live cells, fluorescent proteins have become indispensable markers in cellular and biochemical studies. These proteins can undergo a number of covalent chemical modifications that may affect their photophysical properties. Among other mechanisms, such covalent modifications may be induced by reactive oxygen species (ROS), as generated along a variety of biological pathways or through the action of ionizing radiations. In a previous report [1], we showed that the exposure of cyan fluorescent protein (ECFP) to amounts of (•)OH that mimic the conditions of intracellular oxidative bursts (associated with intense ROS production) leads to observable changes in its photophysical properties in the absence of any direct oxidation of the ECFP chromophore. In the present work, we analyzed the associated structural modifications of the protein in depth. Following the quantified production of (•)OH, we devised a complete analytical workflow based on chromatography and mass spectrometry that allowed us to fully characterize the oxidation events. While methionine, tyrosine, and phenylalanine were the only amino acids that were found to be oxidized, semi-quantitative assessment of their oxidation levels showed that the protein is preferentially oxidized at eight residue positions. To account for the preferred oxidation of a few, poorly accessible methionine residues, we propose a multi-step reaction pathway supported by data from pulsed radiolysis experiments. The described experimental workflow is widely generalizable to other fluorescent proteins, and opens the door to the identification of crucial covalent modifications that affect their photophysics.

Disruption of the bone morphogenetic protein receptor 2 pathway in nitrofen-induced congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Congenital diaphragmatic hernia (CDH) remains a major therapeutic challenge despite advances in neonatal resuscitation and intensive care. The high mortality and morbidity in CDH has been attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PH). Bone morphogenetic protein receptor 2 (BMPR2) plays a key role in pulmonary vasculogenesis during the late stages of fetal lung development. BMPR2 is essential for control of endothelial and smooth muscle cell proliferation. Dysfunction of BMPR2 and downstream signaling have been shown to disturb the crucial balance of proliferation of smooth muscle cells contributing to the pathogenesis of human and experimental PH. We designed this study to investigate the hypothesis that BMPR2 signaling is disrupted in nitrofen-induced CDH. METHODS: Pregnant rats were treated with nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D21 and divided into CDH and control. Quantitative real-time polymerase chain reaction, Western blotting, and confocal-immunofluorescence were performed to determine pulmonary gene expression levels and protein expression of BMPR2 and related proteins. RESULTS: Pulmonary Bmpr2 gene expression levels were significantly decreased in nitrofen-induced CDH compared to controls. Western blotting and confocal microscopy revealed decreased pulmonary BMPR2 protein expression and increased activation of p38(MAPK) in CDH compared to controls. CONCLUSION: The observed disruption of the BMPR2 signaling pathway may lead to extensive vascular remodeling and contribute to PH in the nitrofen-induced CDH model. BMPR2 may therefore represent a potential target for the treatment of PH in CDH.

A Case Report of Hyperostosis Frontalis Interna.

A routine dissection of an 89-year-old female cadaver who had died of cardiopulmonary arrest revealed a unique case of hyperostosis frontalis interna (HFI). Multiple layers of spongy bone growth deep to the internal table were coupled with asymmetrical nodular growths. Slight superior sagittal sinus growth was also noted, which is atypical of this condition. Additionally, this cadaver represents one of the rarer and more severe forms of HFI, class C. A clear consensus on whether HFI presents a clinical risk has not been reached. We hope that this report on a unique manifestation of HFI will help clinicians in evaluating patients with this condition.

Cellular uptake mediated off/on responsive near-infrared fluorescent nanoparticles.

Fluorescence imaging, utilizing molecular fluorophores, often acts as a central tool for the investigation of fundamental biological processes and offers huge future potential for human imaging coupled to therapeutic procedures. An often encountered limitation with fluorescence imaging is the difficulty in discriminating nonspecific background fluorophore emission from a fluorophore localized at a specific region of interest. This limits imaging to individual time points at which background fluorescence has been minimized. It would be of significant advantage if the fluorescence output could be modulated from off to on in response to specific biological events as this would permit imaging of such events in real time without background interference. Here we report our approach to achieve this for the most fundamental of cellular processes, i.e. endocytosis. We describe a new near-infrared off to on fluorescence switchable nanoparticle construct that is capable of switching its fluorescence on following cellular uptake but remains switched off in extracellular environments. This permits continuous real-time imaging of the uptake process as extracellular particles are nonfluorescent. The principles behind the fluorescence off/on switch can be understood by encapsulation of particles in cellular organelles which effect a microenvironmental change establishing a fluorescence signal.

CXCL17 Is Dispensable during Hypervirulent Mycobacterium tuberculosis HN878 Infection in Mice.

CXCL17 is a novel mucosal chemokine that mediates myeloid cell recruitment and bactericidal activity and highly expressed in the respiratory tract. However, its role in tuberculosis (TB) immunopathogenesis or protection remains unknown. In this study, we evaluated the function of CXCL17 in a mouse model of aerosol infection with the clinical W-Beijing lineage Mycobacterium tuberculosis hypervirulent HN878 strain. Our results show that CXCL17 production increases in the lung of M. tuberculosis-infected mice during acute and chronic stages of infection. Moreover, in vitro M. tuberculosis infection of epithelial cells and myeloid cells induces production of CXCL17. In humans, lower serum CXCL17 levels are observed among active pulmonary TB patients when compared with subjects with latent TB infection and healthy controls, suggesting a protective role. However, mice treated with rCXCL17 show similar lung bacterial burden and inflammation compared with control animals, despite an increased lung myeloid cell accumulation. Finally, CXCL17-/- mice are not more susceptible to TB than wild-type animals. These findings suggest that CXCL17 is induced in both murine epithelial and myeloid cells upon M. tuberculosis infection and increased expression during human latent TB infection. However, CXCL17 may have a dispensable role during pulmonary TB.

Altered anoctamin-1 and tyrosine phosphorylation in congenital ureteropelvic junction obstruction.

PURPOSE: Ureteropelvic junction (UPJ) obstruction is the most common cause of congenital hydronephrosis in children. The pathophysiology of UPJ obstruction and the exact mechanism of pelviureteral peristalsis are poorly understood. Anoctamin-1 (ANO1), a Ca2+-activated chloride channel, has been shown to play a key role in muscle wall contractions in the gastrointestinal tract. We designed this study to investigate the hypothesis that ANO1 is expressed in smooth muscle cells (SMCs) of the human UPJ and that tyrosine phosphorylation is altered in UPJ obstruction. MATERIALS AND METHODS: Fresh frozen specimens of UPJ obstruction (n = 28) and control specimens from patients who underwent Wilms' tumor nephrectomy (n = 20) were prepared. Western blot (WB) was performed to evaluate levels of ANO1 protein expression and changes in tyrosine phosphorylation. In addition analysis of ANO1 and phalloidin using confocal-immunofluoresence-double staining and 3D reconstruction were carried out. RESULTS: Our WB results revealed increased tyrosine phosphorylation in UPJ obstruction samples compared to controls, and decreased ANO1 expression in UPJ obstruction. Confocal microscopy showed that ANO1 immunoreactivity was decreased in SMCs of UPJ obstruction compared to controls. CONCLUSIONS: We provide evidence, for the first time, of the presence of ANO1 expression in the human UPJ. We speculate that altered tyrosine phosphorylation, observed in UPJ obstruction, may lead to a failure of transmission of peristaltic waves in UPJ obstruction by inhibiting Ca2+-activated chloride channels in SMCs.

Automated lumen segmentation using multi-frame convolutional neural networks in intravascular ultrasound datasets.

Aims: Assessment of minimum lumen areas in intravascular ultrasound (IVUS) pullbacks is time-consuming and demands adequately trained personnel. In this work, we introduce a novel and fully automated pipeline to segment the lumen boundary in IVUS datasets. Methods and results: First, an automated gating is applied to select end-diastolic frames and bypass saw-tooth artefacts. Second, within a machine learning (ML) environment, we automatically segment the lumen boundary using a multi-frame (MF) convolutional neural network (MFCNN). Finally, we use the theory of Gaussian processes (GPs) to regress the final lumen boundary. The dataset consisted of 85 IVUS pullbacks (52 patients). The dataset was partitioned at the pullback-level using 73 pullbacks for training (20 586 frames), 6 pullbacks for validation (1692 frames), and 6 for testing (1692 frames). The degree of overlapping, between the ground truth and ML contours, median (interquartile range, IQR) systematically increased from 0.896 (0.874-0.933) for MF1 to 0.925 (0.911-0.948) for MF11. The median (IQR) of the distance error was also reduced from 3.83 (2.94-4.98)% for MF1 to 3.02 (2.25-3.95)% for MF11-GP. The corresponding median (IQR) in the lumen area error remained between 5.49 (2.50-10.50)% for MF1 and 5.12 (2.15-9.00)% for MF11-GP. The dispersion in the relative distance and area errors consistently decreased as we increased the number of frames, and also when the GP regressor was coupled to the MFCNN output. Conclusion: These results demonstrate that the proposed ML approach is suitable to effectively segment the lumen boundary in IVUS scans, reducing the burden of costly and time-consuming manual delineation.

Mass spectrometry-based structural dissection of fluorescent proteins.

Fluorescent proteins (FPs) are essential for live cell studies using fluorescence microscopy. To date, the molecular basis for FPs' irreversible photobleaching and the nature of the associated photoproducts are a matter of debate. Mass spectrometry, which should be an ideal technique for the structural dissection of FPs, cannot be harnessed efficiently due to their extreme resistance to trypsinolysis, due to the compactness of the barrel structure containing the chromopeptide. We devised a mild endoproteolysis procedure that affords a peptide mass fingerprint almost totally covering the sequence, thus allowing high-resolution mass spectrometric investigations of the protein structure.

The surgical option in the management of acute pulmonary embolism.

OBJECTIVE: Massive or submassive pulmonary embolism (PE) carries a high mortality. Traditionally this condition has been treated with thrombolysis or anticoagulation and support measures. Surgical embolectomy is carried out in situations of hemodynamic instability or contraindication for thrombolysis. We present our results of surgical embolectomy in patients with massive and submassive PE. METHODS: Over a three-year period, we have carried out 20 surgical embolectomies for acute PE. The mean age was 66 years, and there were 11 males. In all cases, the diagnosis had been made by a computerized tomography (CT) pulmonary artery angiography. Nine patients (45%) arrived to the operating theater on inotropes, and two of them (10%) with ventilatory support. All patients underwent a median sternotomy, bicaval cannulation for institution of cardiopulmonary bypass (CPB), and main pulmonary arteriotomy for the removal of the thrombus. RESULTS: The mean bypass time was 45 minutes. Two patients (12%) died after being unable to wean off CPB due to right heart failure. Among the 15 survivors, the median ventilation time in the intensive care unit was 24 hours. Twelve patients (60%) required inotropic support postoperatively for right heart failure. All but one survivor (94%) underwent an insertion of a permanent inferior vena cava filter and were anticoagulated with coumarin. The mean follow-up is 9.8 months and is 100% complete, with a survival of 94.5%. All patients were in the World Health Organization (WHO) functional class I, with no re-admissions for respiratory failure. CONCLUSION: In patients with acute massive or submassive PE, surgical embolectomy offers a valid therapeutic strategy. A right-sided heart failure is the main complication of this condition.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Dynamin-2 Stabilizes the HIV-1 Fusion Pore with a Low Oligomeric State.

One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry, its exact role during the first steps of HIV-1 infection is not well characterized. Here, we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall, our data suggest that dynamin-2, as a tetramer, might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion.

Defects in NADPH Oxidase Genes NOX1 and DUOX2 in Very Early Onset Inflammatory Bowel Disease.

BACKGROUND & AIMS: Defects in intestinal innate defense systems predispose patients to inflammatory bowel disease (IBD). Reactive oxygen species (ROS) generated by nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases in the mucosal barrier maintain gut homeostasis and defend against pathogenic attack. We hypothesized that molecular genetic defects in intestinal NADPH oxidases might be present in children with IBD. METHODS: After targeted exome sequencing of epithelial NADPH oxidases NOX1 and DUOX2 on 209 children with very early onset inflammatory bowel disease (VEOIBD), the identified mutations were validated using Sanger Sequencing. A structural analysis of NOX1 and DUOX2 variants was performed by homology in silico modeling. The functional characterization included ROS generation in model cell lines and in in vivo transduced murine crypts, protein expression, intracellular localization, and cell-based infection studies with the enteric pathogens Campylobacter jejuni and enteropathogenic Escherichia coli. RESULTS: We identified missense mutations in NOX1 (c.988G>A, p.Pro330Ser; c.967G>A, p.Asp360Asn) and DUOX2 (c.4474G>A, p.Arg1211Cys; c.3631C>T, p.Arg1492Cys) in 5 of 209 VEOIBD patients. The NOX1 p.Asp360Asn variant was replicated in a male Ashkenazi Jewish ulcerative colitis cohort. All NOX1 and DUOX2 variants showed reduced ROS production compared with wild-type enzymes. Despite appropriate cellular localization and comparable pathogen-stimulated translocation of altered oxidases, cells harboring NOX1 or DUOX2 variants had defective host resistance to infection with C. jejuni. CONCLUSIONS: This study identifies the first inactivating missense variants in NOX1 and DUOX2 associated with VEOIBD. Defective ROS production from intestinal epithelial cells constitutes a risk factor for developing VEOIBD.

Prenatal retinoic acid increases lipofibroblast expression in hypoplastic rat lungs with experimental congenital diaphragmatic hernia.

BACKGROUND/PURPOSE: Prenatal administration of all-trans retinoic acid (ATRA) has been shown to stimulate alveolarization in nitrofen-induced pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial lipofibroblasts (LIFs), characterized by adipocyte differentiation-related protein (ADRP), play a critical role in alveolar development by coordinating lipid homeostasis. Previous studies have demonstrated that ATRA positively affects LIF expression in developing lungs. We hypothesized that pulmonary LIF expression is increased after prenatal ATRA treatment in the nitrofen model of CDH-associated PH. METHODS: Timed-pregnant rats were treated with nitrofen or vehicle on E9.5, followed by injection of ATRA or placebo on E18.5, E19.5, and E20.5. Fetal lungs were dissected on E21.5 and divided into Control+Placebo, Control+ATRA, Nitrofen+Placebo, and Nitrofen+ATRA. Pulmonary gene expression levels of ADRP were analyzed by quantitative real-time polymerase chain reaction, and LIF expression was investigated by ADRP immunohistochemistry, oil-red-O-, and immunofluorescence-double-staining. RESULTS: Relative mRNA expression of pulmonary ADRP was significantly increased in Nitrofen+ATRA compared to Nitrofen+Placebo (0.31±0.02 vs. 0.08±0.01; P<0.0001). ADRP immunoreactivity and oil-red-O-staining were markedly increased in alveolar interstitium of Nitrofen+ATRA compared to Nitrofen+Placebo. Immunofluorescence-double-staining confirmed markedly increased LIF expression in alveolar walls of Nitrofen+ATRA compared to Nitrofen+Placebo. CONCLUSIONS: Increased LIF expression after prenatal treatment with ATRA in nitrofen-induced PH suggests that ATRA may have a therapeutic potential in attenuating CDH-associated PH by stimulating alveolar development.

Spatiotemporal alterations in Sprouty-2 expression and tyrosine phosphorylation in nitrofen-induced pulmonary hypoplasia.

BACKGROUND/PURPOSE: Pulmonary hypoplasia (PH) is a life-threatening condition of newborns presenting with congenital diaphragmatic hernia (CDH). Sprouty-2 functions as a key regulator of fibroblast growth factor receptor (FGFR) signalling in developing foetal lungs. It has been reported that FGFR-mediated alveolarization is disrupted in nitrofen-induced PH. Sprouty-2 knockouts show severe defects in lung morphogenesis similar to nitrofen-induced PH. Upon FGFR stimulation, Sprouty-2 is tyrosine-phosphorylated, which is essential for its physiological function during foetal lung development. We hypothesized that Sprouty-2 expression and tyrosine phosphorylation are altered in nitrofen-induced PH. METHODS: Time-pregnant rats received either nitrofen or vehicle on gestation day 9 (D9). Foetal lungs were dissected on D18 and D21. Pulmonary Sprouty-2 gene and protein expression levels were analyzed by qRT-PCR, Western blotting and immunohistochemical staining. RESULTS: Relative mRNA expression of Sprouty-2 was significantly decreased in hypoplastic lungs without CDH (0.1050±0.01 vs. 0.3125±0.01; P<.0001) and with CDH (0.1671±0.01 vs. 0.3125±0.01; P<.0001) compared to controls on D18. Protein levels of Sprouty-2 were markedly decreased in hypoplastic lungs on D18 with decreased tyrosine phosphorylation levels on D18 and D21 detected at the molecular weight of Sprouty-2 consistent with Sprouty-2 tyrosine phosphorylation. Sprouty-2 immunoreactivity was markedly decreased in hypoplastic lungs on D18 and D21. CONCLUSION: Spatiotemporal alterations in pulmonary Sprouty-2 expression and tyrosine phosphorylation during the late stages of foetal lung development may interfere with FGFR-mediated alveolarization in nitrofen-induced PH.

Cj1411c encodes for a cytochrome P450 involved in Campylobacter jejuni 81-176 pathogenicity.

Cytochrome P450s are b-heme-containing enzymes that are able to introduce oxygen atoms into a wide variety of organic substrates. They are extremely widespread in nature having diverse functions at both biochemical and physiological level. The genome of C. jejuni 81-176 encodes a single cytochrome P450 (Cj1411c) that has no close homologues. Cj1411c is unusual in its genomic location within a cluster involved in the biosynthesis of outer surface structures. Here we show that E. coli expressed and affinity-purified C. jejuni cytochrome P450 is lipophilic, containing one equivalent Cys-ligated heme. Immunoblotting confirmed the association of cytochrome P450 with membrane fractions. A Cj1411c deletion mutant had significantly reduced ability to infect human cells and was less able to survive following exposure to human serum when compared to the wild type strain. Phenotypically following staining with Alcian blue, we show that a Cj1411c deletion mutant produces significantly less capsular polysaccharide. This study describes the first known membrane-bound bacterial cytochrome P450 and its involvement in Campylobacter virulence.