Discovery of cyanophycin dipeptide hydrolase enzymes suggests widespread utility of the natural biopolymer cyanophycin.

Cyanophycin is a bacterial polymer mainly used for nitrogen storage. It is composed of a peptide backbone of L-aspartate residues with L-arginines attached to their side chains through isopeptide bonds. Cyanophycin is degraded in two steps: Cyanophycinase cleaves the polymer into ß-Asp-Arg dipeptides, which are hydrolyzed into free Asp and Arg by enzymes possessing isoaspartyl dipeptide hydrolase activity. Two unrelated enzymes with this activity, isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA) have been shown to degrade ß-Asp-Arg dipeptides, but bacteria which encode cyanophycin-metabolizing genes can lack iaaA and iadA genes. In this study, we investigate a previously uncharacterized enzyme whose gene can cluster with cyanophycin-metabolizing genes. This enzyme, which we name cyanophycin dipeptide hydrolase (CphZ), is specific for dipeptides derived from cyanophycin degradation. Accordingly, a co-complex structure of CphZ and ß-Asp-Arg shows that CphZ, unlike IadA or IaaA, recognizes all portions of its ß-Asp-Arg substrate. Bioinformatic analyses showed that CphZ is found in very many proteobacteria and is homologous to an uncharacterized protein encoded in the "arginine/ornithine transport" (aot) operon of many pseudomonas species, including Pseudomonas aeruginosa. In vitro assays show that AotO is indeed a CphZ, and in cellulo growth experiments show that this enzyme and the aot operon allow P. aeruginosa to take up and use ß-Asp-Arg as a sole carbon and nitrogen source. Together the results establish the novel, highly specific enzyme subfamily of CphZs, suggesting that cyanophycin is potentially used by a much wider range of bacteria than previously appreciated.

Intracellular Pseudomonas aeruginosa within the Airway Epithelium of Cystic Fibrosis Lung Tissues.

Rationale: Pseudomonas aeruginosa is the major bacterial pathogen colonizing the airways of adult patients with cystic fibrosis (CF) and causes chronic infections that persist despite antibiotic therapy. Intracellular bacteria may represent an unrecognized reservoir of bacteria that evade the immune system and antibiotic therapy. Although the ability of P. aeruginosa to invade and survive within epithelial cells has been described in vitro in different epithelial cell models, evidence of this intracellular lifestyle in human lung tissues is currently lacking. Objectives: To detect and characterize intracellular P. aeruginosa in CF airway epithelium from human lung explant tissues. Methods: We sampled lung explant tissues from patients with CF undergoing lung transplantation and non-CF lung donor control tissue. We analyzed lung tissue sections for the presence of intracellular P. aeruginosa using quantitative culture and microscopy, in parallel to histopathology and airway morphometry. Measurements and Main Results: P. aeruginosa was isolated from the lungs of seven patients with CF undergoing lung transplantation. Microscopic assessment revealed the presence of intracellular P. aeruginosa within airway epithelial cells in three of the seven patients analyzed at a varying but low frequency. We observed those events occurring in lung regions with high bacterial burden. Conclusions: This is the first study describing the presence of intracellular P. aeruginosa in CF lung tissues. Although intracellular P. aeruginosa in airway epithelial cells is likely relatively rare, our findings highlight the plausible occurrence of this intracellular bacterial reservoir in chronic CF infections.

MARK2 phosphorylates eIF2α in response to proteotoxic stress.

The regulation of protein synthesis is essential for maintaining cellular homeostasis, especially during stress responses, and its dysregulation could underlie the development of human diseases. The critical step during translation regulation is the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α). Here we report the identification of a direct kinase of eIF2α, microtubule affinity-regulating kinase 2 (MARK2), which phosphorylates eIF2α in response to proteotoxic stress. The activity of MARK2 was confirmed in the cells lacking the 4 previously known eIF2α kinases. MARK2 itself was found to be a substrate of protein kinase C delta (PKCδ), which serves as a sensor for protein misfolding stress through a dynamic interaction with heat shock protein 90 (HSP90). Both MARK2 and PKCδ are activated via phosphorylation in proteotoxicity-associated neurodegenerative mouse models and in human patients with amyotrophic lateral sclerosis (ALS). These results reveal a PKCδ-MARK2-eIF2α cascade that may play a critical role in cellular proteotoxic stress responses and human diseases.

LasR-deficient Pseudomonas aeruginosa variants increase airway epithelial mICAM-1 expression and enhance neutrophilic lung inflammation.

Pseudomonas aeruginosa causes chronic airway infections, a major determinant of lung inflammation and damage in cystic fibrosis (CF). Loss-of-function lasR mutants commonly arise during chronic CF infections, are associated with accelerated lung function decline in CF patients and induce exaggerated neutrophilic inflammation in model systems. In this study, we investigated how lasR mutants modulate airway epithelial membrane bound ICAM-1 (mICAM-1), a surface adhesion molecule, and determined its impact on neutrophilic inflammation in vitro and in vivo. We demonstrated that LasR-deficient strains induce increased mICAM-1 levels in airway epithelial cells compared to wild-type strains, an effect attributable to the loss of mICAM-1 degradation by LasR-regulated proteases and associated with enhanced neutrophil adhesion. In a subacute airway infection model, we also observed that lasR mutant-infected mice displayed greater airway epithelial ICAM-1 expression and increased neutrophilic pulmonary inflammation. Our findings provide new insights into the intricate interplay between lasR mutants, LasR-regulated proteases and airway epithelial ICAM-1 expression, and reveal a new mechanism involved in the exaggerated inflammatory response induced by lasR mutants.

Failed Eradication Therapy of New-Onset Pseudomonas aeruginosa Infections in Children With Cystic Fibrosis Is Associated With Bacterial Resistance to Neutrophil Functions.

BACKGROUND: Antibiotics, such as inhaled tobramycin, are used to eradicate new-onset Pseudomonas aeruginosa (PA) infections in patients with cystic fibrosis (CF) but frequently fail due to reasons poorly understood. We hypothesized that PA isolates' resistance to neutrophil antibacterial functions was associated with failed eradication in patients harboring those strains. METHODS: We analyzed all PA isolates from a cohort of 39 CF children with new-onset PA infections undergoing tobramycin eradication therapy, where 30 patients had eradicated and 9 patients had persistent infection. We characterized several bacterial phenotypes and measured the isolates' susceptibility to neutrophil antibacterial functions using in vitro assays of phagocytosis and intracellular bacterial killing. RESULTS: PA isolates from persistent infections were more resistant to neutrophil functions, with lower phagocytosis and intracellular bacterial killing compared to those from eradicated infections. In multivariable analyses, in vitro neutrophil responses were positively associated with twitching motility, and negatively with mucoidy. In vitro neutrophil phagocytosis was a predictor of persistent infection following tobramycin even after adjustment for clinical risk factors. CONCLUSIONS: PA isolates from new-onset CF infection show strain-specific susceptibility to neutrophil antibacterial functions, and infection with PA isolates resistant to neutrophil phagocytosis is an independent risk factor for failed tobramycin eradication.

Cranberry-Derived Proanthocyanidins Potentiate ß-Lactam Antibiotics against Resistant Bacteria.

The emergence and spread of extended-spectrum ß-lactamases (ESBLs), metallo-ß-lactamases (MBLs), or variant low-affinity penicillin-binding proteins (PBPs) pose a major threat to our ability to treat bacterial infection using ß-lactam antibiotics. Although combinations of ß-lactamase inhibitors with ß-lactam agents have been clinically successful, there are no MBL inhibitors in current therapeutic use. Furthermore, recent clinical use of new-generation cephalosporins targeting PBP2a, an altered PBP, has led to the emergence of resistance to these antimicrobial agents. Previous work shows that natural polyphenols such as cranberry-extracted proanthocyanidins (cPAC) can potentiate non-ß-lactam antibiotics against Gram-negative bacteria. This study extends beyond previous work by investigating the in vitro effect of cPAC in overcoming ESBL-, MBL-, and PBP2a-mediated ß-lactam resistance. The results show that cPAC exhibit variable potentiation of different ß-lactams against ß-lactam-resistant Enterobacteriaceae clinical isolates as well as ESBL- and MBL-producing E. coli We also discovered that cPAC have broad-spectrum inhibitory properties in vitro on the activity of different classes of ß-lactamases, including CTX-M3 ESBL and IMP-1 MBL. Furthermore, we observe that cPAC selectively potentiate oxacillin and carbenicillin against methicillin-resistant but not methicillin-sensitive staphylococci, suggesting that cPAC also interfere with PBP2a-mediated resistance. This study motivates the need for future work to identify the most bioactive compounds in cPAC and to evaluate their antibiotic-potentiating efficacy in vivoIMPORTANCE The emergence of ß-lactam-resistant Enterobacteriaceae and staphylococci compromises the effectiveness of ß-lactam-based therapy. By acquisition of ESBLs, MBLs, or PBPs, it is highly likely that bacteria may become completely resistant to the most effective ß-lactam agents in the near future. In this study, we described a natural extract rich in proanthocyanidins which exerts adjuvant properties by interfering with two different resistance mechanisms. By their broad-spectrum inhibitory ability, cranberry-extracted proanthocyanidins could have the potential to enhance the effectiveness of existing ß-lactam agents.

Liposomal Bupivacaine Versus Bupivacaine/Epinephrine Intercostal Nerve Block as Part of an Enhanced Recovery After Thoracic Surgery (ERATS) Care Pathway for Robotic Thoracic Surgery.

OBJECTIVES: To examine how postoperative pain control after robotic thoracoscopic surgery varies with liposomal bupivacaine (LipoB) versus 0.5% bupivacaine/1:200,000 epinephrine (Bupi/Epi) intercostal nerve blocks within the context of an enhanced recovery after thoracic surgery (ERATS) protocol. DESIGN: A retrospective analysis of a prospectively maintained database of patients undergoing robotic thoracoscopic procedures between September 1, 2018 and October 31, 2019 was conducted. SETTING: University of Miami, single-institutional. PARTICIPANTS: Patients. INTERVENTIONS: Two hundred fifty-two patients had either LipoB intercostal nerve blocks (n = 129) or Bupi/Epi intercostal nerve blocks (n = 123) when undergoing robotic thoracic surgery. MEASUREMENTS AND MAIN RESULTS: Comparative analysis of patient-reported pain levels, in-hospital and post-discharge opioid requirements, 90-day operative complications, length of hospital stay, and hospital costs was performed. Data were stratified to either anatomic lung resection or pulmonary wedge resection/mediastinal-pleural procedures. Bupi/Epi patients reported significantly more acute postoperative pain than LipoB patients, which correlated with higher in-hospital and post-discharge opioid requirements. There were no differences in postoperative complications, length of hospital stay, or hospital costs between the two groups. CONCLUSIONS: As part of an ERATS protocol, infiltration of intercostal spaces and surgical wounds with LipoB for robotic thoracoscopic procedures afforded better postoperative subjective pain control and decreased opioid requirements without an increase in hospital costs as compared with use of Bupi/Epi.

Superoxide dismutase activity confers (p)ppGpp-mediated antibiotic tolerance to stationary-phase Pseudomonas aeruginosa.

Metabolically quiescent bacteria represent a large proportion of those in natural and host environments, and they are often refractory to antibiotic treatment. Such drug tolerance is also observed in the laboratory during stationary phase, when bacteria face stress and starvation-induced growth arrest. Tolerance requires (p)ppGpp signaling, which mediates the stress and starvation stringent response (SR), but the downstream effectors that confer tolerance are unclear. We previously demonstrated that the SR is linked to increased antioxidant defenses in Pseudomonas aeruginosa We now demonstrate that superoxide dismutase (SOD) activity is a key factor in SR-mediated multidrug tolerance in stationary-phase P. aeruginosa Inactivation of the SR leads to loss of SOD activity and decreased multidrug tolerance during stationary phase. Genetic or chemical complementation of SOD activity of the ΔrelA spoT mutant (ΔSR) is sufficient to restore antibiotic tolerance to WT levels. Remarkably, we observe high membrane permeability and increased drug internalization upon ablation of SOD activity. Combined, our results highlight an unprecedented mode of SR-mediated multidrug tolerance in stationary-phase P. aeruginosa and suggest that inhibition of SOD activity may potentiate current antibiotics.

Hemoptysis is frequently fatal if blood comes directly from the aorta commentary.

Physicians throughout the world, across various specialties, are faced with diagnostic challenges of appropriately identifying the source of hemotosysis, which could range from a simple treatable infection, to the more ominous massive hemorrhage from the aorta requiring emergency, life saving surgery. Aortobronchopulmonary fistula, which is an abnormal communication between the thoracic aorta and the pulmonary tree, is an uncommon but often lethal condition if not promptly surgically intervened. Over the decades, the underlying cause has shifted, from primarily due to an aortic infection, such as tuberculosis, to now secondarily as a result of endovascular repair of the intrathoracic aorta. The best treatment modality, whether open surgical repair, endovascular management, or hybrid approach continues to be debated given the high operative morbidity and mortality of open repair and need to address the pulmonary communication, with optimal management still undetermined.

Use of venovenous extracorporeal membrane oxygenation for resection of a large paratracheal mass causing critical tracheal stenosis: A case report.

Critical airway stenosis is challenging for surgeons and anesthesiologists to secure a reliable airway for ventilation. The use of venovenous (VV)-extracorporeal membrane oxygenation (ECMO) has been described as a strategy to provide adequate gas exchange in such instances. We present a case of a young female with a complex paratracheal mass significantly compressing the trachea; a planned intraoperative VV-ECMO was instituted to allow safe orotracheal intubation of a double-lumen endotracheal tube for lung isolation and tumor resection.

Prevalence, Serovar, and Antimicrobial Resistance of Nontyphoidal Salmonella in Vegetable, Fruit, and Water Samples in Ho Chi Minh City, Vietnam.

In this study, we investigated the prevalence, serovar distribution, and antimicrobial resistance pattern of Salmonella isolates from vegetable, fruit, and water samples in Ho Chi Minh City, Vietnam. Salmonella was detected in 75% (30/40), 57.1% (12/21), 17.5% (28/160), and 2.5% (1/40) of river water, irrigation water, vegetable, and ice water samples, respectively. However, no Salmonella was isolated from 160 fruit and 40 tap water samples examined. A total of 102 isolates obtained from 71 samples belonged to 34 different serovars, of which Salmonella Rissen was the most prevalent, followed by Salmonella London, Salmonella Hvittingfoss, and Salmonella Weltevreden. Certain Salmonella serovars such as Newport, Rissen, and Weltevreden were isolated from both vegetable and water samples. Antimicrobial resistance was most commonly observed against tetracycline (35.3%), followed by chloramphenicol (34.3%), ampicillin (31.4%), trimethoprim/sulfamethoxazole (23.5%), and nalidixic acid (10.8%). Of 102 isolates analyzed, 52 (51%) showed resistance to at least 1 antimicrobial class whereas 27 (26.5%) showed multidrug resistant (MDR) phenotype, being resistant to at least three different classes of antimicrobials. Determination of the presence and type of ß-lactamase genes showed the cooccurrence of blaTEM-1 and blaCMY-2 in one Salmonella Agona isolate from a river water sample. Taken together, these data indicated that both environmental water and vegetables were contaminated with Salmonella, including MDR strains, and that environmental water used in irrigation might have been the source of Salmonella contamination in the vegetables.

Hydrophilic Mechano-Bactericidal Nanopillars Require External Forces to Rapidly Kill Bacteria.

Nanopillars have been shown to mechanically damage bacteria, suggesting a promising strategy for future antibacterial surfaces. However, the mechanisms underlying this phenomena remain unclear, which ultimately limits translational potential toward real-world applications. Using real-time and end-point analysis techniques, we demonstrate that in contrast to initial expectations, bacteria on multiple hydrophilic "mechano-bactericidal" surfaces remained viable unless exposed to a moving air-liquid interface, which caused considerable cell death. Reasoning that normal forces arising from surface tension may underlie this mechano-bactericidal activity, we developed computational and experimental models to estimate, manipulate, and recreate the impact of these forces. Our experiments together demonstrate that a critical level of external force acting on cells attached to nanopillar surfaces can rapidly deform and rupture bacteria. These studies provide fundamental physical insight into how nanopillar surfaces can serve as effective antibacterial materials and suggest use-conditions under which such nanotechnology approaches may provide practical value.

Enhancing the Effect of Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Signaling and Arginine Deprivation in Melanoma.

Melanoma as a very aggressive type of cancer is still in urgent need of improved treatment. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and arginine deiminase (ADI-PEG20) are two of many suggested drugs for treating melanoma. Both have shown anti-tumor activities without harming normal cells. However, resistance to both drugs has also been noted. Studies on the mechanism of action of and resistance to these drugs provide multiple targets that can be utilized to increase the efficacy and overcome the resistance. As a result, combination strategies have been proposed for these drug candidates with various other agents, and achieved enhanced or synergistic anti-tumor effect. The combination of TRAIL and ADI-PEG20 as one example can greatly enhance the cytotoxicity to melanoma cells including those resistant to the single component of this combination. It is found that combination treatment generally can alter the expression of the components of cell signaling in melanoma cells to favor cell death. In this paper, the signaling of TRAIL and ADI-PEG20-induced arginine deprivation including the main mechanism of resistance to these drugs and exemplary combination strategies is discussed. Finally, factors hampering the clinical application of both drugs, current and future development to overcome these hurdles are briefly discussed.

Enhancement in virtual learning cannot substitute for hands-on training in cardiothoracic surgery.

This program director survey attempts to determine how coronavirus 2019 pandemic is impacting current training in cardiothoracic surgery. A transition to virtual didactic sessions may prove beneficial with increasing attendance. On the other hand, decreasing live simulation and case volumes may jeopardize achieving competency in surgical skills.

Surgical management of mediastinal paraganglioma: All hands on deck!

Mediastinal paragangliomas are very uncommon neuroendocrine neoplasms. Due to their tissue of origin (sympathetic ganglia of the great vessels), they tend to arise deep within pericardial space and, more importantly, intimately attached to great vessels, which makes surgical resection, even with cardiopulmonary bypass, very challenging. This commentary accompanies the case report describing complex surgical management of a paraganglioma located in the anterior mediastinum that was initially thought to be a thymoma.

Ethanol Stimulates Trehalose Production through a SpoT-DksA-AlgU-Dependent Pathway in Pseudomonas aeruginosa.

Pseudomonas aeruginosa frequently resides among ethanol-producing microbes, making its response to the microbially produced concentrations of ethanol relevant to understanding its biology. Our transcriptome analysis found that genes involved in trehalose metabolism were induced by low concentrations of ethanol, and biochemical assays showed that levels of intracellular trehalose increased significantly upon growth with ethanol. The increase in trehalose was dependent on the TreYZ pathway but not other trehalose-metabolic enzymes (TreS or TreA). The sigma factor AlgU (AlgT), a homolog of RpoE in other species, was required for increased expression of the treZ gene and trehalose levels, but induction was not controlled by the well-characterized proteolysis of its anti-sigma factor, MucA. Growth with ethanol led to increased SpoT-dependent (p)ppGpp accumulation, which stimulates AlgU-dependent transcription of treZ and other AlgU-regulated genes through DksA, a (p)ppGpp and RNA polymerase binding protein. Ethanol stimulation of trehalose also required acylhomoserine lactone (AHL)-mediated quorum sensing (QS), as induction was not observed in a ΔlasR ΔrhlR strain. A network analysis using a model, eADAGE, built from publicly available P. aeruginosa transcriptome data sets (J. Tan, G. Doing, K. A. Lewis, C. E. Price, et al., Cell Syst 5:63-71, 2017, https://doi.org/10.1016/j.cels.2017.06.003) provided strong support for our model in which treZ and coregulated genes are controlled by both AlgU- and AHL-mediated QS. Consistent with (p)ppGpp- and AHL-mediated quorum-sensing regulation, ethanol, even when added at the time of culture inoculation, stimulated treZ transcript levels and trehalose production in cells from post-exponential-phase cultures but not in cells from exponential-phase cultures. These data highlight the integration of growth and cell density cues in the P. aeruginosa transcriptional response to ethanol.IMPORTANCEPseudomonas aeruginosa is often found with bacteria and fungi that produce fermentation products, including ethanol. At concentrations similar to those produced by environmental microbes, we found that ethanol stimulated expression of trehalose-biosynthetic genes and cellular levels of trehalose, a disaccharide that protects against environmental stresses. The induction of trehalose by ethanol required the alternative sigma factor AlgU through DksA- and SpoT-dependent (p)ppGpp. Trehalose accumulation also required AHL quorum sensing and occurred only in post-exponential-phase cultures. This work highlights how cells integrate cell density and growth cues in their responses to products made by other microbes and reveals a new role for (p)ppGpp in the regulation of AlgU activity.

Rare metastatic patterns after malignant transformation of serous borderline tumor of the ovary.

Serous borderline tumors are rare, benign ovarian neoplasms that may recur and undergo malignant transformation to low-grade serous carcinomas (LGSCs). In this report, a 50-year-old female with a remote history of a serous borderline ovarian tumor experienced a recurrence of LGSC, presenting as a large solitary subcutaneous mass anterior to the sternum after a 33-year disease-free interval. The described case highlights the unpredictable nature of this disease and the importance of implementing lifelong surveillance strategies.

Hypercoagulability After Resection of Thoracic Malignancy: A Prospective Evaluation.

BACKGROUND: Rates of venous thromboembolism are increased in thoracic malignancy; however, coagulation patterns are not established. We hypothesize that patients with esophageal and lung malignancy have similar hypercoagulable pre- and postoperative profiles as defined by rotational thromboelastometry (ROTEM). METHODS: Prospective study was conducted in 47 patients with esophageal and lung cancer undergoing surgical resection. ROTEM evaluated pre/postoperative coagulation status. RESULTS: Patients with thoracic malignancy were hypercoagulable by ROTEM, but not by conventional coagulation tests. Preoperative hypercoagulability was higher in lung versus esophageal cancer (64 vs. 16%, p = 0.001). Lung cancer patients that were hypercoagulable preoperatively demonstrated decreased maximum clot firmness (MCF) (p = 0.044) and increased clot time (p = 0.049) after surgical resection, suggesting reversal of hypercoagulability. Resection of esophageal cancer increased hypercoagulability (16 vs. 56%, p = 0.002) via elevated MCF (reflecting platelet activity). Hypercoagulability remained at follow-up clinic for both lung and esophageal cancer patients. CONCLUSIONS: Hypercoagulability in patients with lung malignancies reversed following complete surgical resection, whereas hypercoagulability occurred only postoperatively in those with esophageal malignancies. In both, hypercoagulability was associated with fibrin and platelet function.

Nimbandiolactone-21 and nimbandioloxyfuran, two new 28-norlimonoids from the leaves of Azadirachta indica (Meliaceae).

From an EtOAc-soluble fraction of the leaves of Azadirachta indica, two new 28-norlimonoids named nimbandiolactone-21 (1) and nimbandioloxyfuran (2), together with nimbandiolactone-23 (3), were isolated. Their relative structures were elucidated based on NMR spectroscopic interpretation and biosynthetic consideration. Nimbandioloxyfuran (2) and nimbandiolactone-23 (3) showed potent α-glucosidase inhibitory activity, with the IC50 values of 46.2 and 38.7 µM, respectively.

Inference for dynamic and latent variable models via iterated, perturbed Bayes maps.

Iterated filtering algorithms are stochastic optimization procedures for latent variable models that recursively combine parameter perturbations with latent variable reconstruction. Previously, theoretical support for these algorithms has been based on the use of conditional moments of perturbed parameters to approximate derivatives of the log likelihood function. Here, a theoretical approach is introduced based on the convergence of an iterated Bayes map. An algorithm supported by this theory displays substantial numerical improvement on the computational challenge of inferring parameters of a partially observed Markov process.