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Rhipicephalus microplus is one of the most important ectoparasites of cattle in tropical and subtropical regions. In ticks collected from cattle the pathogens Babesia bovis, Anaplasma spp. and Ehrlichia spp. can be detected. Here, we report the first detection of the pathogen Anaplasma phagocytophilum in Madagascar from ticks infesting cattle. Furthermore, we report for the first time Anaplasma ovis, Ehrlichia canis, Ehrlichia ewingii and Ehrlichia muris in both R. microplus and in Madagascar. We show no correlation between the detection of B. bovis, Ehrlichia spp. and Anaplasma spp. DNA within the same tick. Previous reports have demonstrated strong interactions between A. marginale, A. centrale and A. ovis in the same tick, as well between these pathogens and A. phagocytophilum. A strong correlation also existed between the occurrences of Ehrlichia species within the same tick. Our findings suggest that R. microplus ticks are potential vectors and reservoirs of many tick-borne diseases of cattle.

In the blast-furnace production, raw materials may account for approx. 50 % of the pig-iron manufacture costs. Therefore, any, even small, saving in the sphere of raw material purchasing will translate into the reduction in the cost of the pig-iron manufacture. The selection of appropriate supply sources and the associated raw material quality influencing the economic viability of the charge blend constitutes a multi-faceted optimization task. The paper presents a modified model for production cost estimation at the moment of making raw material purchasing, which is possible to be used in the logistics pillar of the WCM concept.

Introduction: Composite restorations undergo degradation in the oral cavity, which may compromise their aesthetic and functional properties, cause failure of marginal integrity, increased abrasion or even partial fracture of the filling. Instead of complete removal of such restorations, they can be repaired. Aim: To study the bond strength of a new composite resin to an aged composite substrate after a 4-month storage in distilled water or sodium laurylsulphate solution (SLS). Two various surface treatments and two different adhesive systems were tested. Materials and methods: Light cured specimens of a micro-hybrid composite (Filtek Z250, shade A2) were aged in distilled water (37 °C) for 5 months. Composite surface was prepared either by grinding (SiC paper P320, mean grain size 46 µm, equivalent to red diamond bur) or by air abrasion (Rondoflex; Al2O3 50 µm; 3.2 bar; 60 s). Composite build-ups were made from the same composite material using two adhesive systems (Optibond FL, Gluma Comfort Bond). The microtensile bond strength (n = 13-16) was measured after a 4-month storage of specimens at 37 °C in distilled water or in a solution of sodium lauryl sulfate (1.5 wt. %). Control specimens were stored in distilled water for 24 hours (37 °C). Results: A decrease of the bond strength compared to the control group on the ground surface after the 4-month storage in distilled water or SLS was found to be adhesive system- dependant. The bond strength for Optibond FL decreased by 52% in SLS only (p < 0.001), but for Gluma Comfort Bond it decreased by 45% in distilled water (p < 0.001) and by 61% in SLS (p < 0,001). On the air-abraded surface the bond strength decreased only for Optibond FL in SLS (p < 0.01), by 31%. Conclusion: Long-term durability of composite repair can be maintained by preparing the original composite surface using air abrasion. The effect of the adhesive system on the bond strength was not significant.

Peroxide bleaching systems may affect resin composite restorations through their strong oxidative effect. The objective was to investigate the influence of bleaching systems on long-term stability of marginal integrity of composite restorations and composite surface resistance. Class V cavities were prepared at the enamel-dentine junction and filled using a total-etch adhesive system Gluma Comfort Bond (n=36) and self-etching adhesive iBond (n=36) in combination with the microhybrid resin composite material Charisma. The peroxide bleaching gel Opalescence PF 20% (20 wt. % carbamide peroxide) was applied 25 times on the restorations. The control groups were exposed to distilled water for 24 hours and two months before a microleakage test was performed. Composite hardness was characterized after 0, 2, 7, 14 and 25 applications of the gel and compared with the control group (each n=5) exposed to distilled water for two months. Surface morphology was evaluated using scanning electron microscopy. After the gel application no significant changes in marginal integrity were found for both groups of samples. With Gluma Comfort Bond due to its high bonding performance and with iBond due to its decreased performance on enamel causing marginal integrity failure at the beginning of the experiment. On the other hand, pronounced surface porosity and decreased hardness of the composite material indicated its significant degradation in the presence of the bleaching gel.

A shear bond strength and fractography analysis was used to evaluate reliability of nine total- and self-etching adhesives OptiBond FL, Gluma Comfort Bond, Prime&Bond NT, AdheSE, Adper Prompt, Adper Prompt L-Pop, Clearfil SE Bond, Xeno III and iBond. A critical bond strength at which 10 % samples failed was used as a measure of the adhesive reliability. Significant differences in reliability and also in mode of their fracture were found between total- and self-etching systems and also in adhesives in these groups. The highest reliability and mixed mode of fracture were found with group of classical total-etch 3- and 2-step adhesives OptiBond FL a Gluma Comfort Bond and also with 2-step self-etching AdheSE and Clearfil SE Bond. With these adhesives both high bond strength and highly predictable properties can be expected. Compromised reliability as manifested in decreased bond strength and predominantly less energetically demanding cohesive fractures of the adhesive layer or less frequently mixed fractures occurred for Adper Promt, iBond a Xeno III. On the other hand, Prime & Bond NT and especially Adper Prompt L-Pop in blisters were characterized as systems with a low reliability. Adhesive properties of self-etching systems might decrease as a result of aging processes during their shelf-life.

Researchers have examined the importance of school administrative support for teacher safety, victimization, anxiety, and retention; however, studies to date have rarely focused on school administrators’ perceptions of support by their district leaders, and its relation to administrators’ anxiety/stress, safety, and their intentions to transfer or quit their jobs. In the current study of 457 PreK-12th grade school administrators in the United States, structural equation modeling was used to examine relations between administrators’ perceptions of support from their district leaders and their anxiety/stress, safety, and intentions to transfer or quit their jobs. Administrator experiences of violence by student offenders served as a moderator. Results indicated that administrators’ perceptions of district leaders’ support were associated with lower intentions to transfer or quit their positions both directly and indirectly as a function of decreased anxiety/stress. District support was positively related to administrator safety, particularly for administrators who reported experiencing more student violence. Findings highlight the importance of district support of administrators for reducing mental health concerns and transfer/quit intentions in the context of student violence against school administrators. Implications of findings for research and practice are presented.

Abstract Background Acute lung injury and the acute respiratory distress syndrome are characterized by pulmonary inflammation, reduced endothelial barrier integrity and filling of the alveolar space with protein rich edema fluid and infiltrating leukocytes. Animal models are critical to uncovering the pathologic mechanisms of this devastating syndrome. Intravital imaging of the intact lung via two-photon intravital microscopy has proven a valuable method to investigate lung injury in small rodent models through characterization of inflammatory cells and vascular changes in real time. However, respiratory motion complicates the analysis of these time series images and requires selective data extraction to stabilize the image. Consequently, analysis of individual alveoli may not provide a complete picture of the integrated mechanical, vascular and inflammatory processes occurring simultaneously in the intact lung. To address these challenges, we developed a web browser-based visualization application named Alveolus Analysis to process, analyze and graphically display intravital lung microscopy data. Results The designed tool takes raw temporal image data as input, performs image preprocessing and feature extraction offline, and visualizes the extracted information in a web browser-based interface. The interface allows users to explore multiple experiments in three panels corresponding to different levels of detail: summary statistics of alveolar/neutrophil behavior, characterization of alveolar dynamics including lung edema and inflammatory cells at specific time points, and cross-experiment analysis. We performed a case study on the utility of the visualization with two members or our research team and they found the tool useful because of its ability to preprocess data consistently and visualize information in a digestible and informative format. Conclusions Application of our software tool, Alveolus Analysis, to intravital lung microscopy data has the potential to enhance the information gained from these experiments and provide new insights into the pathologic mechanisms of inflammatory lung injury.

Abstract Elevated ACE expression in tissues (reflected by blood ACE levels) is associated with increased risk of cardiovascular diseases and is also a marker for granulomatous diseases. We developed a new approach for characterization of ACE status in the blood—ACE phenotyping and established normal values of ACE levels 50–150% of control pooled plasma. ACE phenotyping was performed in citrated plasma of 120 patients with known interstitial lung diseases. In the 1st set of 100 patients we found 22 patients with ACE levels > 150%; ACE phenotyping also objectively identified the presence of ACE inhibitors in the plasma of 15 patients. After excluding these patients and patient with ACE mutation that increases ACE shedding, 17 patients were identified as a suspicious for systemic sarcoidosis based on elevation of blood ACE (> 150% of mean). A new parameter that we have established–ACE immunoreactivity (with mAb 9B9)—allowed us to detect 22 patients with decreased values (

Abstract Despite encouraging preclinical data, therapies to reduce ARDS mortality remains a globally unmet need, including during the COVID-19 pandemic. We previously identified extracellular nicotinamide phosphoribosyltransferase (eNAMPT) as a novel damage-associated molecular pattern protein (DAMP) via TLR4 ligation which regulates inflammatory cascade activation. eNAMPT is tightly linked to human ARDS by biomarker and genotyping studies in ARDS subjects. We now hypothesize that an eNAMPT-neutralizing mAb will significantly reduce the severity of ARDS lung inflammatory lung injury in diverse preclinical rat and porcine models. Sprague Dawley rats received eNAMPT mAb intravenously following exposure to intratracheal lipopolysaccharide (LPS) or to a traumatic blast (125 kPa) but prior to initiation of ventilator-induced lung injury (VILI) (4 h). Yucatan minipigs received intravenous eNAMPT mAb 2 h after initiation of septic shock and VILI (12 h). Each rat/porcine ARDS/VILI model was strongly associated with evidence of severe inflammatory lung injury with NFkB pathway activation and marked dysregulation of the Akt/mTORC2 signaling pathway. eNAMPT neutralization dramatically reduced inflammatory indices and the severity of lung injury in each rat/porcine ARDS/VILI model (~ 50% reduction) including reduction in serum lactate, and plasma levels of eNAMPT, IL-6, TNFα and Ang-2. The eNAMPT mAb further rectified NFkB pathway activation and preserved the Akt/mTORC2 signaling pathway. These results strongly support targeting the eNAMPT/TLR4 inflammatory pathway as a potential ARDS strategy to reduce inflammatory lung injury and ARDS mortality.

Increases in lung vascular permeability is a cardinal feature of inflammatory disease and represents an imbalance in vascular contractile forces and barrier‐restorative forces, with both forces highly dependent upon the actin cytoskeleton. The current study investigates the role of Ena‐VASP‐like (EVL), a member of the Ena‐VASP family known to regulate the actin cytoskeleton, in regulating vascular permeability responses and lung endothelial cell barrier integrity. Utilizing changes in transendothelial electricial resistance (TEER) to measure endothelial cell barrier responses, we demonstrate that EVL expression regulates endothelial cell responses to both sphingosine‐1‐phospate (S1P), a vascular barrier‐enhancing agonist, and to thrombin, a barrier‐disrupting stimulus. Total internal reflection fluorescence demonstrates that EVL is present in endothelial cell focal adhesions and impacts focal adhesion size, distribution, and the number of focal adhesions generated in response to S1P and thrombin challenge, with the focal adhesion kinase (FAK) a key contributor in S1P‐stimulated EVL‐transduced endothelial cell but a limited role in thrombin‐induced focal adhesion rearrangements. In summary, these data indicate that EVL is a focal adhesion protein intimately involved in regulation of cytoskeletal responses to endothelial cell barrier‐altering stimuli. Keywords: cytoskeleton, vascular barrier, sphingosine‐1‐phosphate, thrombin, focal adhesion kinase (FAK), Ena‐VASP like protein (EVL), cytoskeletal regulatory protein

Abstract Purpose In acute respiratory distress syndrome (ARDS), dead space fraction has been independently associated with mortality. We hypothesized that early measurement of the difference between arterial and end-tidal CO2 (arterial-ET difference), a surrogate for dead space fraction, would predict mortality in mechanically ventilated patients with ARDS. Methods We performed two separate exploratory analyses. We first used publicly available databases from the ALTA, EDEN, and OMEGA ARDS Network trials (N = 124) as a derivation cohort to test our hypothesis. We then performed a separate retrospective analysis of patients with ARDS using University of Chicago patients (N = 302) as a validation cohort. Results The ARDS Network derivation cohort demonstrated arterial-ET difference, vasopressor requirement, age, and APACHE III to be associated with mortality by univariable analysis. By multivariable analysis, only the arterial-ET difference remained significant (P = 0.047). In a separate analysis, the modified Enghoff equation ((PaCO2–PETCO2)/PaCO2) was used in place of the arterial-ET difference and did not alter the results. The University of Chicago cohort found arterial-ET difference, age, ventilator mode, vasopressor requirement, and APACHE II to be associated with mortality in a univariate analysis. By multivariable analysis, the arterial-ET difference continued to be predictive of mortality (P = 0.031). In the validation cohort, substitution of the arterial-ET difference for the modified Enghoff equation showed similar results. Conclusion Arterial to end-tidal CO2 (ETCO2) difference is an independent predictor of mortality in patients with ARDS.

Background: Numerous potential ARDS therapeutics, based upon preclinical successful rodent studies that utilized LPS challenge without mechanical ventilation, have failed in Phase 2/3 clinical trials. Recently, ALT-100 mAb, a novel biologic that neutralizes the TLR4 ligand and DAMP, eNAMPT (extracellular nicotinamide phosphoribosyltransferase), was shown to reduce septic shock/VILI-induced porcine lung injury when delivered 2 h after injury onset. We now examine the ALT-100 mAb efficacy on acute kidney injury (AKI) and lung fluid balance in a porcine ARDS/VILI model when delivered 6 h post injury.Methods/Results: Compared to control PBS-treated pigs, exposure of ALT-100 mAb-treated pigs (0.4 mg/kg, 2 h or 6 h after injury initiation) to LPS-induced pneumonia/septic shock and VILI (12 h), demonstrated significantly diminished lung injury severity (histology, BAL PMNs, plasma cytokines), biochemical/genomic evidence of NF-kB/MAP kinase/cytokine receptor signaling, and AKI (histology, plasma lipocalin). ALT-100 mAb treatment effectively preserved lung fluid balance reflected by reduced BAL protein/tissue albumin levels, lung wet/dry tissue ratios, ultrasound-derived B lines, and chest radiograph opacities. Delayed ALT-100 mAb at 2 h was significantly more protective than 6 h delivery only for plasma eNAMPT while trending toward greater protection for remaining inflammatory indices. Delayed ALT-100 treatment also decreased lung/renal injury indices in LPS/VILI-exposed rats when delivered up to 12 h after LPS.Conclusions: These studies indicate the delayed delivery of the eNAMPT-neutralizing ALT-100 mAb reduces inflammatory lung injury, preserves lung fluid balance, and reduces multi-organ dysfunction, and may potentially address the unmet need for novel therapeutics that reduce ARDS/VILI mortality.

Abstract Global knockout of the nonmuscle isoform of myosin light‐chain kinase (nmMLCK), a primary cellular regulator of cytoskeletal machinery, is strongly protective in preclinical murine models of inflammatory lung injury. The current study was designed to assess the specific contribution of endothelial cell (EC) nmMLCK to the severity of murine inflammatory lung injury produced by lipopolysaccharide (LPS) and mechanical ventilation ventilator‐induced lung injury or ventilation (VILI). Responses to combined LPS/VILI exposure were assessed in: (i) wild‐type (WT) C57BL/6J mice; (ii) transgenic mice with global deletion of nmMLCK (nmMylk−/−); (iii) transgenic nmMylk−/− mice with overexpression of nmMLCK restricted to the endothelium (nmMylk−/−/ec‐tg+). Lung inflammation indices included lung histology, bronchoalveolar lavage (BAL) polymorphonuclear leukocytes (PMNs), lung protein biochemistry, tissue albumin levels, Evans blue dye (EBD) lung extravasation, and plasma cytokines (interleukin‐6 [IL‐6], keratinocyte chemoattractant [KC]/IL‐8, IL‐1bβ, extracellular nicotinamide phosphoribosyltransferase, tumor necrosis factor‐α). Compared to WT C57BL/6J mice, the severity of LPS/VILI‐induced lung injury was markedly reduced in mice with global nmMLCK deletion reflected by reductions in histologic inflammatory lung injury, BAL PMN counts, mitogen‐activated protein kinase, and NF‐kB pathway activation in lung homogenates, plasma cytokine levels, and parameters of lung permeability (increased BAL protein, tissue albumin levels, EBD lung extravasation). In contrast, mice with restricted overexpression of nmMLCK in EC (nmMylk−/−/ec‐tg+) showed significant persistence of LPS/VILI‐induced lung injury severity compared to WT mice. In conclusion, these studies strongly endorse the role of EC nmMLCK in driving the severity of preclinical inflammatory lung injury. Precise targeting of EC nmMLCK may represent an attractive therapeutic strategy to reduce lung inflammation and both lung and systemic vascular permeability.

Abstract Electrical cell‐substrate impedance sensing (ECIS) is an in vitro methodology for measuring the barrier integrity of a variety of cell types, including pulmonary endothelial cells. These experiments are frequently used for in vitro assessment of lung injury. The data derived from ECIS experiments consists of repeated measures of resistance across an endothelial monolayer. As such, these data reflect the dynamic changes in electrical resistance that occur over time. Currently methodologies for assessing ECIS data rely on single point assessments of barrier function, such as the maximal drop in trans‐endothelial electrical resistance (TERMax). However, this approach ignores the myriad of changes in resistance that occur before and after the TERMax data point. Herein, we utilize polynomial curve fitting on experimentally generated ECIS data, thus allowing for comparing ECIS experiments by examining the mean polynomial coefficients between groups. We show that polynomial curves accurately fit a variety of ECIS data, and that concordance between TERMax and coefficient analysis varies by type of stimulus, suggesting that TERMax differences may not always correlate with a significant difference in the overall shape of the ECIS profile. Lastly, we identify factors that impact coefficient values obtained in our analyses, including the length of time devoted to baseline measurements before addition of stimuli. Polynomial coefficient analysis is another tool that can be used for more comprehensive interrogation of ECIS data to better understand the biological underpinnings that lead to changes in barrier dysfunction in vitro.

Castration-resistant prostate cancer (CRPC) is defined by resistance of the tumor to androgen deprivation therapy (ADT). Several molecular changes, particularly in the AR signaling cascade, have been described that may explain ADT resistance. The variety of changes may also explain why the response to novel therapies varies between patients. Testing the specific molecular changes may be a major step towards personalized treatment of CRPC patients. The aim of our study was to evaluate the molecular changes in the AR signaling cascade in CRPC patients. We have developed and validated several methods which are easy to use, and require little tissue material, for exploring AR signaling pathway changes simultaneously. We found that the AR signaling pathway is still active in the majority of our CRPC patients, due to molecular changes in AR signaling components. There was heterogeneity in the molecular changes observed, but we could classify the patients into 4 major subgroups which are: AR mutation, AR amplification, active intratumoral steroidogenesis, and combination of AR amplification and active intratumoral steroidogenesis. We suggest characterizing the AR signaling pathway in CRPC patients before beginning any new treatment, and a recent fresh tissue sample from the prostate or a metastatic site should be obtained for the purpose of this characterization.

A 52-year-old man presented to our hospital complaining of general malaise, cough, and fever. Total body computed tomography revealed scattered pneumonia and urethral foreign bodies that had been inserted during adolescence. Candida glabrata was detected in blood and urine cultures. Based on these findings, the patient was diagnosed with candidemia that developed due to Candida urinary tract infection, complicated by septic pulmonary embolism and severe diabetes mellitus. Candidemia likely persisted despite the initiation of intravenous antifungal therapy and control of blood sugar level. Therefore, surgical removal of the urethral foreign bodies was performed, which resulted in resolution of the patient’s symptoms. Herein, we report a rare case of candidemia complicated by Candida urinary tract infection that developed due to the long-term presence of urethral foreign bodies. A multidisciplinary therapeutic approach, including surgical removal of the infected foreign bodies, is effective in such cases. This case indicates that long-term presence of foreign bodies and acquired immune dysfunction can be risk factors for candidemia. Therefore, detailed history should be obtained and systemic examination should be performed to identify the complicating risk factors on diagnosis of candidemia.

Summary: Lung alveolar epithelium is composed of alveolar type I (AT1) and type II (AT2) cells. AT1 cells mediate gas exchange, whereas AT2 cells act as progenitor cells to repair injured alveoli. Lung microvascular endothelial cells (LMVECs) play a crucial but still poorly understood role in regulating alveolar repair. Here, we studied the role of the LMVEC-derived bioactive lipid sphingosine-1-phosphate (S1P) in promoting alveolar repair using mice with endothelial-specific deletion of sphingosine kinase 1 (Sphk1), the key enzyme promoting S1P generation. These mutant lungs developed airspace-enlargement lesions and exhibited a reduced number of AT1 cells after Pseudomonas-aeruginosa-induced lung injury. We demonstrated that S1P released by LMVECs acted via its receptor, S1PR2, on AT2 cells and induced nuclear translocation of yes-associated protein (YAP), a regulator of AT2 to AT1 transition. Thus, angiocrine S1P released after injury acts via the S1PR2-YAP signaling axis on AT2 cells to promote AT2 to AT1 differentiation required for alveolar repair.

Abstract Approximately 20% of patients with bladder cancer are diagnosed with muscle‐invasive disease (MIBC). The treatment involves radical cystectomy, but almost 50% of patients with MIBC eventually relapse and develop metastasis. The use of platinum‐based chemotherapy in the neoadjuvant setting or for metastatic patients has been shown to improve the overall survival in a subset of patients. Unfortunately, no biomarkers are available to select patients with MIBC who will benefit from chemotherapy or to monitor the efficacy of the treatment. Recently, long noncoding RNAs (lncRNAs) were shown to regulate a variety of processes involved in the development and progression of cancer, including bladder cancer. Moreover, several lncRNAs have been shown to play a role in chemotherapy resistance. Here, we analyzed lncRNA expression associated with response to platinum‐based chemotherapy in metastatic MIBC using data from the MiTranscriptome lncRNA expression database. Expression of the lncRNA, LINC00857, was found to be upregulated in tumors from patients that did not respond to platinum‐based chemotherapy. Moreover, high expression of LINC00857 is correlated with shorter recurrence‐free and overall survival of patients with MIBC. Knockdown of LINC00857 significantly decreased cell viability of bladder cancer cell lines through the induction of apoptosis. Furthermore, LINC00857 knockdown sensitized UM‐UC‐3 and T24 bladder cancer cells to cisplatin, via the negative regulation of the LMAN1 gene. Our data indicate that LINC00857 plays an important role in the regulation of response to platinum‐based chemotherapy. LINC00857 potentially could serve as a novel prognostic and predictive biomarker and might be a therapeutic target to overcome cisplatin resistance in patients with MIBC.

Abstract Exploration of human pulmonary artery endothelial cell (EC) as a prototypical biomechanical system has important pathophysiologic relevance because this cell type plays a key role in the development of a wide variety of clinical conditions. The complex hierarchical organization ranging from the molecular scale up to the cellular level has an intimate and intricate relationship to the barrier function between lung tissue and blood. To understand the innate molecule-cell-tissue relationship across varied length-scales, the functional role of c-Abl kinase in the cytoskeletal nano-biomechanics of ECs in response to barrier-altering agonists was investigated using atomic force microscopy. Concurrently, the spatially specific arrangement of cytoskeleton structure and dynamic distribution of critical proteins were examined using scanning electron microscopy and immunofluorescence. Reduction in c-Abl expression by siRNA attenuates both thrombin- and sphingosine 1-phosphate (S1P)-mediated structural changes in ECs, specifically spatially-defined changes in elastic modulus and distribution of critical proteins. These results indicate that c-Abl kinase is an important determinant of cortical actin-based cytoskeletal rearrangement. Our findings directly bridge the gap between kinase activity, structural complexity, and functional connectivity across varied length-scales, and suggest that manipulation of c-Abl kinase activity may be a potential target for the treatment of pulmonary barrier disorders.

Excessive inflammation in the lung is a primary cause of acute respiratory distress syndrome (ARDS). CD26/dipeptidyl peptidase-4 (DPP4) is a transmembrane protein that is expressed in various cell types and exerts multiple pleiotropic effects. We recently reported that pharmacological CD26/DPP4 inhibition ameliorated lipopolysaccharide (LPS)-induced lung injury in mice and exerted anti-inflammatory effects on human lung microvascular endothelial cells (HLMVECs), in vitro. However, the mechanistic roles of CD26/DPP4 in lung injury and its effects on HLMVECs remain unclear. In this study, transcriptome analysis, followed by various confirmation experiments using siRNA in cultured HLMVECs, are performed to evaluate the role of CD26/DPP4 in response to the pro-inflammatory involved in inflammation, barrier function, and regenerative processes in HLMVECs after pro-inflammatory stimulation. These are all functions that are closely related to the pathophysiology and repair process of lung injury. Confirmatory experiments using flow cytometry; enzyme-linked immunosorbent assay; quantitative polymerase chain reaction; dextran permeability assay; WST-8 assay; wound healing assay; and tube formation assay, reveal that the reduction of CD26/DPP4 via siRNA is associated with altered parameters of inflammation, barrier function, and the regenerative processes in HLMVECs. Thus, CD26/DPP4 can play a pathological role in mediating injury in pulmonary endothelial cells. CD26/DPP4 inhibition can be a new therapeutic strategy for inflammatory lung diseases, involving pulmonary vascular damage.

Pneumolysin (PLY) is a pore-forming toxin of Streptococcus pneumoniae that contributes substantially to the inflammatory processes underlying pneumococcal pneumonia and lung injury. Host responses against S. pneumoniae are regulated in part by neutrophils and platelets, both individually and in cooperative interaction. Previous studies have shown that PLY can target both neutrophils and platelets, however, the mechanisms by which PLY directly affects these cells and alters their interactions are not completely understood. In this study, we characterize the effects of PLY on neutrophils and platelets and explore the mechanisms by which PLY may induce neutrophil–platelet interactions. In vitro studies demonstrated that PLY causes the formation of neutrophil extracellular traps (NETs) and the release of extracellular vesicles (EVs) from both human and murine neutrophils. In vivo, neutrophil EV (nEV) levels were increased in mice infected with S. pneumoniae. In platelets, treatment with PLY induced the cell surface expression of P-selectin (CD62P) and binding to annexin V and caused a significant release of platelet EVs (pl-EVs). Moreover, PLY-induced nEVs but not NETs promoted platelet activation. The pretreatment of nEVs with proteinase K inhibited platelet activation, indicating that the surface proteins of nEVs play a role in this process. Our findings demonstrate that PLY activates neutrophils and platelets to release EVs and support an important role for neutrophil EVs in modulating platelet functions in pneumococcal infections.

Summary: Although much progress has been made in understanding type II theta rhythm generation under urethane anesthesia, less is known about the mechanisms underlying type I theta generation during active exploration. To better understand the contributions of cholinergic and NMDA receptor activation to type I theta generation, we recorded hippocampal theta oscillations from freely moving mice with local infusion of cholinergic or NMDA receptor antagonists to either the hippocampus or the entorhinal cortex (EC). We found that cholinergic receptors in the hippocampus, but not the EC, and NMDA receptors in the EC, but not the hippocampus, are critical for open-field theta generation and Y-maze performance. We further found that muscarinic M1 receptors located on pyramidal neurons, but not interneurons, are critical for cholinergic modulation of hippocampal synapses, theta generation, and Y-maze performance. These results suggest that hippocampus and EC neurons recruit cholinergic-dependent and NMDA-receptor-dependent mechanisms, respectively, to generate theta oscillations to support behavioral performance. : Gu et al. find that the entorhinal cortex and hippocampus recruit NMDA-receptor-dependent and cholinergic-dependent mechanisms, respectively, to generate hippocampal theta oscillations in freely moving mice. Muscarinic M1 receptors on pyramidal neurons are important for theta generation, providing potential cellular mechanisms underlying theta generation. Keywords: theta, hippocampus, entorhinal cortex, NMDA receptor, cholinergic receptor, muscarinic, medial septum, Y-maze

Cigarette smoke (CS) is the primary cause of Chronic Obstructive Pulmonary Disease (COPD), and an important pathophysiologic event in COPD is CS-induced apoptosis in lung endothelial cells (EC). Cortactin (CTTN) is a cytoskeletal actin-binding regulatory protein with modulation by Src-mediated tyrosine phosphorylation. Based upon data demonstrating reduced CTTN mRNA levels in the lungs of smokers compared to non-smokers, we hypothesized a functional role for CTTN in CS-induced mitochondrial ROS generation and apoptosis in lung EC. Exposure of cultured human lung EC to CS condensate (CSC) led to the rearrangement of the actin cytoskeleton and increased CTTN tyrosine phosphorylation (within hours). Exposure to CS significantly increased EC mitochondrial ROS generation and EC apoptosis. The functional role of CTTN in these CSC-induced EC responses was explored using cortactin siRNA to reduce its expression, and by using a blocking peptide for the CTTN SH3 domain, which is critical to cytoskeletal interactions. CTTN siRNA or blockade of its SH3 domain resulted in significantly increased EC mitochondrial ROS and apoptosis and augmented CSC-induced effects. Exposure of lung EC to e-cigarette condensate demonstrated similar results, with CTTN siRNA or SH3 domain blocking peptide increasing lung EC apoptosis. These data demonstrate a novel role for CTTN in modulating lung EC apoptosis induced by CS or e-cigarettes potentially providing new insights into COPD pathogenesis.

Lung endothelial dysfunction is a key feature of acute lung injury (ALI) and clinical acute respiratory distress syndrome (ARDS). Previous studies have identified the lipid-generating enzyme, group V phospholipase A2 (gVPLA2), as a mediator of lung endothelial barrier disruption and inflammation. The current study aimed to determine the role of gVPLA2 in mediating lung endothelial responses to methicillin-resistant Staphylococcus aureus (MRSA, USA300 strain), a major cause of ALI/ARDS. In vitro studies assessed the effects of gVPLA2 inhibition on lung endothelial cell (EC) permeability after exposure to heat-killed (HK) MRSA. In vivo studies assessed the effects of intratracheal live or HK-MRSA on multiple indices of ALI in wild-type (WT) and gVPLA2-deficient (KO) mice. In vitro, HK-MRSA increased gVPLA2 expression and permeability in human lung EC. Inhibition of gVPLA2 with either the PLA2 inhibitor, LY311727, or with a specific monoclonal antibody, attenuated the barrier disruption caused by HK-MRSA. LY311727 also reduced HK-MRSA-induced permeability in mouse lung EC isolated from WT but not gVPLA2-KO mice. In vivo, live MRSA caused significantly less ALI in gVPLA2 KO mice compared to WT, findings confirmed by intravital microscopy assessment in HK-MRSA-treated mice. After targeted delivery of gVPLA2 plasmid to lung endothelium using ACE antibody-conjugated liposomes, MRSA-induced ALI was significantly increased in gVPLA2-KO mice, indicating that lung endothelial expression of gVPLA2 is critical in vivo. In summary, these results demonstrate an important role for gVPLA2 in mediating MRSA-induced lung EC permeability and ALI. Thus, gVPLA2 may represent a novel therapeutic target in ALI/ARDS caused by bacterial infection.

Centralized infrastructure to support analyses involving complexity beyond genome-wide association studies is broadly needed. Here, Ritchie and colleagues develop PLATO, a software tool to process and integrate various methods for this task.

Abstract Many neurotransmitters directly inhibit neurons by activating G protein-gated inwardly rectifying K+ (GIRK) channels, thereby moderating the influence of excitatory input on neuronal excitability. While most neuronal GIRK channels are formed by GIRK1 and GIRK2 subunits, distinct GIRK2 isoforms generated by alternative splicing have been identified. Here, we compared the trafficking and function of two isoforms (GIRK2a and GIRK2c) expressed individually in hippocampal pyramidal neurons lacking GIRK2. GIRK2a and GIRK2c supported comparable somato-dendritic GIRK currents in Girk2 −/− pyramidal neurons, although GIRK2c achieved a more uniform subcellular distribution in pyramidal neurons and supported inhibitory postsynaptic currents in distal dendrites better than GIRK2a. While over-expression of either isoform in dorsal CA1 pyramidal neurons restored contextual fear learning in a conditional Girk2 −/− mouse line, GIRK2a also enhanced cue fear learning. Collectively, these data indicate that GIRK2 isoform balance within a neuron can impact the processing of afferent inhibitory input and associated behavior.