Metagenomics harvested genus-specific single-stranded DNA-annealing proteins improve and expand recombineering in Pseudomonas species.

The widespread Pseudomonas genus comprises a collection of related species with remarkable abilities to degrade plastics and polluted wastes and to produce a broad set of valuable compounds, ranging from bulk chemicals to pharmaceuticals. Pseudomonas possess characteristics of tolerance and stress resistance making them valuable hosts for industrial and environmental biotechnology. However, efficient and high-throughput genetic engineering tools have limited metabolic engineering efforts and applications. To improve their genome editing capabilities, we first employed a computational biology workflow to generate a genus-specific library of potential single-stranded DNA-annealing proteins (SSAPs). Assessment of the library was performed in different Pseudomonas using a high-throughput pooled recombinase screen followed by Oxford Nanopore NGS analysis. Among different active variants with variable levels of allelic replacement frequency (ARF), efficient SSAPs were found and characterized for mediating recombineering in the four tested species. New variants yielded higher ARFs than existing ones in Pseudomonas putida and Pseudomonas aeruginosa, and expanded the field of recombineering in Pseudomonas taiwanensisand Pseudomonas fluorescens. These findings will enhance the mutagenesis capabilities of these members of the Pseudomonas genus, increasing the possibilities for biotransformation and enhancing their potential for synthetic biology applications. .

Structure-Guided Mutations in the Terminal Organelle Protein MG491 Cause Major Motility and Morphologic Alterations on Mycoplasma genitalium.

The emergent human pathogen Mycoplasma genitalium, with one of the smallest genomes among cells capable of growing in axenic cultures, presents a flask-shaped morphology due to a protrusion of the cell membrane, known as the terminal organelle, that is involved in cell adhesion and motility and is an important virulence factor of this microorganism. The terminal organelle is supported by a cytoskeleton complex of about 300 nm in length that includes three substructures: the terminal button, the rod and the wheel complex. The crystal structure of the MG491 protein, a proposed component of the wheel complex, has been determined at ~3 Å resolution. MG491 subunits are composed of a 60-residue N-terminus, a central three-helix-bundle spanning about 150 residues and a C-terminal region that appears to be quite flexible and contains the region that interacts with MG200, another key protein of the terminal organelle. The MG491 molecule is a tetramer presenting a unique organization as a dimer of asymmetric pairs of subunits. The asymmetric arrangement results in two very different intersubunit interfaces between the central three-helix-bundle domains, which correlates with the formation of only ~50% of the intersubunit disulfide bridges of the single cysteine residue found in MG491 (Cys87). Moreover, M. genitalium cells with a point mutation in the MG491 gene causing the change of Cys87 to Ser present a drastic reduction in motility (as determined by microcinematography) and important alterations in morphology (as determined by electron microscopy), while preserving normal levels of the terminal organelle proteins. Other variants of MG491, designed also according to the structural information, altered significantly the motility and/or the cell morphology. Together, these results indicate that MG491 plays a key role in the functioning, organization and stabilization of the terminal organelle.

A minimized motile machinery for Mycoplasma genitalium.

The cell wall-less bacterium Mycoplasma genitalium uses specialized adhesins located at the terminal organelle to adhere to host cells and surfaces. The terminal organelle is a polar structure protruding from the cell body that is internally supported by a cytoskeleton and also has an important role in cell motility. We have engineered a M. genitalium null mutant for MG491 protein showing a massive downstream destabilization of proteins involved in the terminal organelle organization. This mutant strain exhibited striking similarities with the previously isolated MG_218 null mutant strain. Upon introduction of an extra copy of MG_318 gene in both strains, the amount of main adhesins P140 and P110 dramatically increased. These strains were characterized by microcinematography, epifluorescence microscopy and cryo-electron microcopy, revealing the presence of motile cells and filaments in the absence of many proteins considered essential for cell adhesion and motility. These results indicate that adhesin complexes play a major role in the motile machinery of M. genitalium and demonstrate that the rod element of the cytoskeleton core is not the molecular motor propelling mycoplasma cells. These strains containing a minimized motile machinery also provide a valuable cell model to investigate the adhesion and gliding properties of this human pathogen.

Altered Hypoxic-Adenosine Axis and Metabolism in Group III Pulmonary Hypertension.

Group III pulmonary hypertension (PH) is a highly prevalent and deadly lung disorder with limited treatment options other than transplantation. Group III PH affects patients with ongoing chronic lung injury, such as idiopathic pulmonary fibrosis (IPF). Between 30 and 40% of patients with IPF are diagnosed with PH. The diagnosis of PH has devastating consequences to these patients, leading to increased morbidity and mortality, yet the molecular mechanisms involved in the development of PH in patients with chronic lung disease remain elusive. Our hypothesis was that the hypoxic-adenosinergic system is enhanced in patients with group III PH compared with patients with IPF with no PH. Explanted lung tissue was analyzed for markers of the hypoxic-adenosine axis, including expression levels of hypoxia-inducible factor (HIF)-1A, adenosine A2B receptor, CD73, and equilibrative nucleotide transporter-1. In addition, we assessed whether altered mitochondrial metabolism was present in these samples. Increased expression of HIF-1A was observed in tissues from patients with group III PH. These changes were consistent with increased evidence of adenosine accumulation in group III PH. A novel observation of our study was of evidence suggesting altered mitochondrial metabolism in lung tissue from group III PH leading to increased succinate levels that are able to further stabilize HIF-1A. Our data demonstrate that the hypoxic-adenosine axis is up-regulated in group III PH and that subsequent succinate accumulation may play a part in the development of group III PH.

A major determinant for gliding motility in Mycoplasma genitalium: the interaction between the terminal organelle proteins MG200 and MG491.

Several mycoplasmas, such as the emergent human pathogen Mycoplasma genitalium, developed a complex polar structure, known as the terminal organelle (TO), responsible for a new type of cellular motility, which is involved in a variety of cell functions: cell division, adherence to host cells, and virulence. The TO cytoskeleton is organized as a multisubunit dynamic motor, including three main ultrastructures: the terminal button, the electrodense core, and the wheel complex. Here, we describe the interaction between MG200 and MG491, two of the main components of the TO wheel complex that connects the TO with the cell body and the cell membrane. The interaction between MG200 and MG491 has a KD in the 80 nm range, as determined by surface plasmon resonance. The interface between the two partners was confined to the "enriched in aromatic and glycine residues" (EAGR) box of MG200, previously described as a protein-protein interaction domain, and to a 25-residue-long peptide from the C-terminal region of MG491 by surface plasmon resonance and NMR spectroscopy studies. An atomic description of the MG200 EAGR box binding surface was also provided by solution NMR. An M. genitalium mutant lacking the MG491 segment corresponding to the peptide reveals specific alterations in cell motility and cell morphology indicating that the MG200-MG491 interaction plays a key role in the stability and functioning of the TO.

Macrophage bone morphogenic protein receptor 2 depletion in idiopathic pulmonary fibrosis and Group III pulmonary hypertension.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology. The development of pulmonary hypertension (PH) is considered the single most significant predictor of mortality in patients with chronic lung diseases. The processes that govern the progression and development of fibroproliferative and vascular lesions in IPF are not fully understood. Using human lung explant samples from patients with IPF with or without a diagnosis of PH as well as normal control tissue, we report reduced BMPR2 expression in patients with IPF or IPF+PH. These changes were consistent with dampened P-SMAD 1/5/8 and elevated P-SMAD 2/3, demonstrating reduced BMPR2 signaling and elevated TGF-ß activity in IPF. In the bleomycin (BLM) model of lung fibrosis and PH, we also report decreased BMPR2 expression compared with control animals that correlated with vascular remodeling and PH. We show that genetic abrogation or pharmacological inhibition of interleukin-6 leads to diminished markers of fibrosis and PH consistent with elevated levels of BMPR2 and reduced levels of a collection of microRNAs (miRs) that are able to degrade BMPR2. We also demonstrate that isolated bone marrow-derived macrophages from BLM-exposed mice show reduced BMPR2 levels upon exposure with IL6 or the IL6+IL6R complex that are consistent with immunohistochemistry showing reduced BMPR2 in CD206 expressing macrophages from lung sections from IPF and IPF+PH patients. In conclusion, our data suggest that depletion of BMPR2 mediated by a collection of miRs induced by IL6 and subsequent STAT3 phosphorylation as a novel mechanism participating to fibroproliferative and vascular injuries in IPF.

A quantitative model for dermal infection and oedema in BALB/c mice pinna.

BACKGROUND: Pharmaceutical industry demands innovation for developing new molecules to improve effectiveness and safety of therapeutic medicines. Preclinical assays are the first tests performed to evaluate new therapeutic molecules using animal models. Currently, there are several models for evaluation of treatments, for dermal oedema or infection. However, the most common or usual way is to induce the inflammation with chemical substances instead of infectious agents. On the other hand, this kind of models require the implementation of histological techniques and the interpretation of pathologies to verify the effectiveness of the therapy under assessment. This work was focused on developing a quantitative model of infection and oedema in mouse pinna. The infection was achieved with a strain of Streptococcus pyogenes that was inoculated in an injury induced at the auricle of BALB/c mice, the induced oedema was recorded by measuring the ear thickness with a digital micrometer and histopathological analysis was performed to verify the damage. The presence of S. pyogenes at the infection site was determined every day by culture. RESULTS: Our results showed that S. pyogenes can infect the mouse pinna and that it can be recovered at least for up to 4 days from the infected site; we also found that S. pyogenes can induce a bigger oedema than the PBS-treated control for at least 7 days; our results were validated with an antibacterial and anti-inflammatory formulation made with ciprofloxacin and hydrocortisone. CONCLUSIONS: The model we developed led us to emulate a dermal infection and allowed us to objectively evaluate the increase or decrease of the oedema by measuring the thickness of the ear pinna, and to determine the presence of the pathogen in the infection site. We consider that the model could be useful for assessment of new anti-inflammatory or antibacterial therapies for dermal infections.

Blockade of IL-6 Trans signaling attenuates pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease with progressive fibrosis and death within 2-3 y of diagnosis. IPF incidence and prevalence rates are increasing annually with few effective treatments available. Inhibition of IL-6 results in the attenuation of pulmonary fibrosis in mice. It is unclear whether this is due to blockade of classical signaling, mediated by membrane-bound IL-6Rα, or trans signaling, mediated by soluble IL-6Rα (sIL-6Rα). Our study assessed the role of sIL-6Rα in IPF. We demonstrated elevations of sIL-6Rα in IPF patients and in mice during the onset and progression of fibrosis. We demonstrated that protease-mediated cleavage from lung macrophages was important in production of sIL-6Rα. In vivo neutralization of sIL-6Rα attenuated pulmonary fibrosis in mice as seen by reductions in myofibroblasts, fibronectin, and collagen in the lung. In vitro activation of IL-6 trans signaling enhanced fibroblast proliferation and extracellular matrix protein production, effects relevant in the progression of pulmonary fibrosis. Taken together, these findings demonstrate that the production of sIL-6Rα from macrophages in the diseased lung contributes to IL-6 trans signaling that in turn influences events crucial in pulmonary fibrosis.

Hypoxia-induced deoxycytidine kinase contributes to epithelial proliferation in pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a deadly lung disease with few therapeutic options. Apoptosis of alveolar epithelial cells, followed by abnormal tissue repair characterized by hyperplastic epithelial cell formation, is a pathogenic process that contributes to the progression of pulmonary fibrosis. However, the signaling pathways responsible for increased proliferation of epithelial cells remain poorly understood. OBJECTIVES: To investigate the role of deoxycytidine kinase (DCK), an important enzyme for the salvage of deoxynucleotides, in the progression of pulmonary fibrosis. METHODS: DCK expression was examined in the lungs of patients with IPF and mice exposed to bleomycin. The regulation of DCK expression by hypoxia was studied in vitro and the importance of DCK in experimental pulmonary fibrosis was examined using a DCK inhibitor and alveolar epithelial cell-specific knockout mice. MEASUREMENTS AND MAIN RESULTS: DCK was elevated in hyperplastic alveolar epithelial cells of patients with IPF and in mice exposed to bleomycin. Increased DCK was localized to cells associated with hypoxia, and hypoxia directly induced DCK in alveolar epithelial cells in vitro. Hypoxia-induced DCK expression was abolished by silencing hypoxia-inducible factor 1α and treatment of bleomycin-exposed mice with a DCK inhibitor attenuated pulmonary fibrosis in association with decreased epithelial cell proliferation. Furthermore, DCK expression, and proliferation of epithelial cells and pulmonary fibrosis was attenuated in mice with conditional deletion of hypoxia-inducible factor 1α in the alveolar epithelium. CONCLUSIONS: Our findings suggest that the induction of DCK after hypoxia plays a role in the progression of pulmonary fibrosis by contributing to alveolar epithelial cell proliferation.

Hypoxia-induced deoxycytidine kinase expression contributes to apoptosis in chronic lung disease.

Chronic obstructive pulmonary disease (COPD) is characterized by persistent inflammation and tissue remodeling and is a leading cause of death in the United States. Increased apoptosis of pulmonary epithelial cells is thought to play a role in COPD development and progression. Identification of signaling pathways resulting in increased apoptosis in COPD can be used in the development of novel therapeutic interventions. Deoxyadenosine (dAdo) is a DNA breakdown product that amplifies lymphocyte apoptosis by being phosphorylated to deoxyadenosine triphosphate (dATP). dAdo is maintained at low levels by adenosine deaminase (ADA). This study demonstrated that mice lacking ADA developed COPD manifestations in association with elevated dAdo and dATP levels and increased apoptosis in the lung. Deoxycitidine kinase (DCK), a major enzyme for dAdo phosphorylation, was up-regulated in mouse and human airway epithelial cells in association with air-space enlargement. Hypoxia was identified as a novel regulator of DCK, and inhibition of DCK resulted in diminished dAdo-mediated apoptosis in the lungs. Our results suggest that activating the dAdo-DCK-dATP pathway directly results in increased apoptosis in the lungs of mice with air-space enlargement and suggests a novel therapeutic target for the treatment of COPD.

Adenosine A2B receptor and hyaluronan modulate pulmonary hypertension associated with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death worldwide. The development of pulmonary hypertension (PH) in patients with COPD is strongly associated with increased mortality. Chronic inflammation and changes to the lung extracellular matrix (ECM) have been implicated in the pathogenesis of COPD, yet the mechanisms that lead to PH secondary to COPD remain unknown. Our experiments using human lung tissue show increased expression levels of the adenosine A2B receptor (ADORA2B) and a heightened deposition of hyaluronan (HA; a component of the ECM) in remodeled vessels of patients with PH associated with COPD. We also demonstrate that the expression of HA synthase 2 correlates with mean pulmonary arterial pressures in patients with COPD, with and without a secondary diagnosis of PH. Using an animal model of airspace enlargement and PH, we show that the blockade of ADORA2B is able to attenuate the development of a PH phenotype that correlates with reduced levels of HA deposition in the vessels and the down-regulation of genes involved in the synthesis of HA.

ReScribe: An Unrestrained Tool Combining Multiplex Recombineering and Minimal-PAM ScCas9 for Genome Recoding Pseudomonas putida.

Genome recoding enables incorporating new functions into the DNA of microorganisms. By reassigning codons to noncanonical amino acids, the generation of new-to-nature proteins offers countless opportunities for bioproduction and biocontainment in industrial chassis. A key bottleneck in genome recoding efforts, however, is the low efficiency of recombineering, which hinders large-scale applications at acceptable speed and cost. To relieve this bottleneck, we developed ReScribe, a highly optimized recombineering tool enhanced by CRISPR-Cas9-mediated counterselection built upon the minimal PAM 5'-NNG-3' of the Streptococcus canis Cas9 (ScCas9). As a proof of concept, we used ReScribe to generate a minimally recoded strain of the industrial chassis Pseudomonas putida by replacing TAG stop codons (functioning as PAMs) of essential metabolic genes with the synonymous TAA. We showed that ReScribe enables nearly 100% engineering efficiency of multiple loci in P. putida, opening promising avenues for genome editing and applications thereof in this bacterium and beyond.

A navigation guide of synthetic biology tools for Pseudomonas putida.

Pseudomonas putida is a microbial chassis of huge potential for industrial and environmental biotechnology, owing to its remarkable metabolic versatility and ability to sustain difficult redox reactions and operational stresses, among other attractive characteristics. A wealth of genetic and in silico tools have been developed to enable the unravelling of its physiology and improvement of its performance. However, the rise of this microbe as a promising platform for biotechnological applications has resulted in diversification of tools and methods rather than standardization and convergence. As a consequence, multiple tools for the same purpose have been generated, whilst most of them have not been embraced by the scientific community, which has led to compartmentalization and inefficient use of resources. Inspired by this and by the substantial increase in popularity of P. putida, we aim herein to bring together and assess all currently available (wet and dry) synthetic biology tools specific for this microbe, focusing on the last 5 years. We provide information on the principles, functionality, advantages and limitations, with special focus on their use in metabolic engineering. Additionally, we compare the tool portfolio for P. putida with those for other bacterial chassis and discuss potential future directions for tool development. Therefore, this review is intended as a reference guide for experts and new 'users' of this promising chassis.

A RAGE Based Strategy for the Genome Engineering of the Human Respiratory Pathogen Mycoplasma pneumoniae.

Genome engineering of microorganisms has become a standard in microbial biotechnologies. Several efficient tools are available for the genetic manipulation of model bacteria such as Escherichia coli and Bacillus subtilis, or the yeast Saccharomyces cerevisiae. Difficulties arise when transferring these tools to nonmodel organisms. Synthetic biology strategies relying on genome transplantation (GT) aim at using yeast cells for engineering bacterial genomes cloned as artificial chromosomes. However, these strategies remain unsuccessful for many bacteria, including Mycoplasma pneumoniae (MPN), a human pathogen infecting the respiratory tract that has been extensively studied as a model for systems biology of simple unicellular organisms. Here, we have designed a novel strategy for genome engineering based on the recombinase-assisted genomic engineering (RAGE) technology for editing the MPN genome. Using this strategy, we have introduced a 15 kbp fragment at a specific locus of the MPN genome and replaced 38 kbp from its genome by engineered versions modified either in yeast or in E. coli. A strain harboring a synthetic version of this fragment cleared of 13 nonessential genes could also be built and propagated in vitro. These strains were depleted of known virulence factors aiming at creating an avirulent chassis for SynBio applications. Such a chassis and technology are a step forward to build vaccines or deliver therapeutic compounds in the lungs to prevent or cure respiratory diseases in humans.

Model-driven design allows growth of Mycoplasma pneumoniae on serum-free media.

Mycoplasma pneumoniae is a slow-growing, human pathogen that causes atypical pneumonia. Because it lacks a cell wall, many antibiotics are ineffective. Due to its reduced genome and dearth of many biosynthetic pathways, this fastidious bacterium depends on rich, undefined medium for growth, which makes large-scale cultivation challenging and expensive. To understand factors limiting growth, we developed a genome-scale, constraint-based model of M. pneumoniae called iEG158_mpn to describe the metabolic potential of this bacterium. We have put special emphasis on cell membrane formation to identify key lipid components to maximize bacterial growth. We have used this knowledge to predict essential components validated with in vitro serum-free media able to sustain growth. Our findings also show that glycolysis and lipid metabolism are much less efficient under hypoxia; these findings suggest that factors other than metabolism and membrane formation alone affect the growth of M. pneumoniae. Altogether, our modelling approach allowed us to optimize medium composition, enabled growth in defined media and streamlined operational requirements, thereby providing the basis for stable, reproducible and less expensive production.

Quantitative assessment of Mycoplasma hemadsorption activity by flow cytometry.

A number of adherent mycoplasmas have developed highly complex polar structures that are involved in diverse aspects of the biology of these microorganisms and play a key role as virulence factors by promoting adhesion to host cells in the first stages of infection. Attachment activity of mycoplasma cells has been traditionally investigated by determining their hemadsorption ability to red blood cells and it is a distinctive trait widely examined when characterizing the different mycoplasma species. Despite the fact that protocols to qualitatively determine the hemadsorption or hemagglutination of mycoplasmas are straightforward, current methods when investigating hemadsorption at the quantitative level are expensive and poorly reproducible. By using flow cytometry, we have developed a procedure to quantify rapidly and accurately the hemadsorption activity of mycoplasmas in the presence of SYBR Green I, a vital fluorochrome that stains nucleic acids, allowing to resolve erythrocyte and mycoplasma cells by their different size and fluorescence. This method is very reproducible and permits the kinetic analysis of the obtained data and a precise hemadsorption quantification based on standard binding parameters such as the dissociation constant K d. The procedure we developed could be easily implemented in a standardized assay to test the hemadsorption activity of the growing number of clinical isolates and mutant strains of different mycoplasma species, providing valuable data about the virulence of these microorganisms.

Intraoperative surgical sealant application during cardiac defect repair.

Bleeding can occur as a sequela to cardiac surgery. Surgical products-such as conventional sutures and clips, and somewhat less conventional sealants-have been developed to prevent this event. Among these, CoSeal is a sealant used at our institution; here we report the cases of 2 patients in whom CoSeal was used successfully as either a supplement or an alternative to suture repair. This sealant was found to be useful in attaining hemostasis both in high-pressure ventricular repair and in the rupture of a friable coronary sinus adjacent to vital structures (in this instance, a left circumflex coronary artery).

Improved short-term outcomes with off-pump reoperative coronary artery bypass grafting.

OBJECTIVE: Reoperative coronary surgery patients are usually sicker and older, and the procedure is more technically demanding. Comparisons between coronary surgery with (coronary artery bypass [CAB] surgery on cardiopulmonary bypass [CPB]) and without (off-pump CAB [OPCAB]) the pump have been conducted; however, few studies showed results in reoperative cases. We investigate the potential superiority of one technique over the other in redo coronary surgeries. METHODS: Our institutional Society of Thoracic Surgery database was used to gather the data for 266 isolated reoperative coronary artery surgeries from January 2004 to July 2011. These were divided into the CAB surgery in CPB group (n = 204) and the OPCAB group (n = 62). RESULTS: Baseline characteristics of the two groups were similar, except for a significantly higher prevalence of cerebrovascular disease among the off-pump group (P = 0.01). There was also a trend toward fewer vessels bypassed among the same group (P = 0.07). Risk adjustment was done using multivariable analyses for detection of independent effects. The use of CPB was an independent predictor of increased rates of postoperative events (odds ratio, 3.9; P = 0.004) and atrial fibrillation (odds ratio, 5.9; P < 0.005) and longer intensive care unit (0.006) and hospital stay (0.004). CONCLUSIONS: Redo OPCAB seems to offer favorable short-term outcomes compared with redo CAB. Our results suggest a reduced rate of overall postoperative events, decreased new postoperative atrial fibrillation, reduced hours stayed in the intensive care unit, and fewer days stayed from surgery to discharge. This was not associated with an increase in morbidity and mortality. A randomized study with a larger number of patients and with a longer follow-up is needed.

Contemporary surgical management of cardiac paragangliomas.

BACKGROUND: Cardiac paragangliomas are an extremely rare subset of chromaffin cell tumors that develop from neural crest cells. METHODS: Between March 2004 and October 2010, 7 male patients from our two institutions who underwent surgical resection of cardiac paraganglioma were retrospectively reviewed. RESULTS: In 5 patients, paragangliomas originated from the roof of the left atrium, and in 2 patients, they originated from the aortic root. Hospital mortality was 14%. CONCLUSIONS: Complete surgical resection remains the mainstay of therapy and can be curative, but carries a significant risk of intraoperative bleeding and usually requires cardiopulmonary bypass and often complex resection techniques, including cardiac autotransplantation.

Advanced in aortic root surgery.

The aortic root is often affected by aneurysmal degeneration of the ascending aorta or dissection. It is important for the clinician to be familiar with current guidelines and recommendations for detection, monitoring, and intervention of the aneurysmal aortic root. Timely surgical referral to an experienced aortic center allows for close monitoring and possible intervention that may preserve the aortic valve in appropriate cases and avoid disastrous complications such as aortic dissection, rupture, or death. Patients with bicuspid aortic valve syndrome or connective tissue disorders (e.g. Marfan syndrome) are particularly at risk and should be followed aggressively. Whenever possible, attempts should be made to preserve or repair the aortic valve using valve-sparing aortic root replacement techniques. This article provides an overview of recent advances in management of the aortic root, including guidelines for surgical intervention, technical procedures, and outcomes.