Atomic-Scale Mechanisms of MoS2 Oxidation for Kinetic Control of MoS2/MoO3 Interfaces.

Oxidation of transition metal dichalcogenides (TMDs) occurs readily under a variety of conditions. Therefore, understanding the oxidation processes is necessary for successful TMD handling and device fabrication. Here, we investigate atomic-scale oxidation mechanisms of the most widely studied TMD, MoS2. We find that thermal oxidation results in α-phase crystalline MoO3 with sharp interfaces, voids, and crystallographic alignment with the underlying MoS2. Experiments with remote substrates prove that thermal oxidation proceeds via vapor-phase mass transport and redeposition, a challenge to forming thin, conformal films. Oxygen plasma accelerates the kinetics of oxidation relative to the kinetics of mass transport, forming smooth and conformal oxides. The resulting amorphous MoO3 can be grown with subnanometer to several-nanometer thickness, and we calibrate the oxidation rate for different instruments and process parameters. Our results provide quantitative guidance for managing both the atomic scale structure and thin-film morphology of oxides in the design and processing of TMD devices.

Interplay between nuclear factor-κB, p38 MAPK, and glucocorticoid receptor signaling synergistically induces functional TLR2 in lung epithelial cells.

While glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to reduce the expression of many inflammatory genes, repression is not an invariable outcome. Here, we explore synergy occurring between synthetic glucocorticoids (dexamethasone and budesonide) and proinflammatory cytokines (IL1B and TNF) on the expression of the toll-like receptor 2 (TLR2). This effect is observed in epithelial cell lines and both undifferentiated and differentiated primary human bronchial epithelial cells (pHBECs). In A549 cells, IL1B-plus-glucocorticoid-induced TLR2 expression required nuclear factor (NF)-κB and GR. Likewise, in A549 cells, BEAS-2B cells, and pHBECs, chromatin immunoprecipitation identified GR- and NF-κB/p65-binding regions ∼32 kb (R1) and ∼7.3 kb (R2) upstream of the TLR2 gene. Treatment of BEAS-2B cells with TNF or/and dexamethasone followed by global run-on sequencing confirmed transcriptional activity at these regions. Furthermore, cloning R1 or R2 into luciferase reporters revealed transcriptional activation by budesonide or IL1B, respectively, while R1+R2 juxtaposition enabled synergistic activation by IL1B and budesonide. In addition, small-molecule inhibitors and siRNA knockdown showed p38α MAPK to negatively regulate both IL1B-induced TLR2 expression and R1+R2 reporter activity. Finally, agonism of IL1B-plus-dexamethasone-induced TLR2 in A549 cells and pHBECs stimulated NF-κB- and interferon regulatory factor-dependent reporter activity and chemokine release. We conclude that glucocorticoid-plus-cytokine-driven synergy at TLR2 involves GR and NF-κB acting via specific enhancer regions, which combined with the inhibition of p38α MAPK promotes TLR2 expression. Subsequent inflammatory effects that occur following TLR2 agonism may be pertinent in severe neutrophilic asthma or chronic obstructive pulmonary disease, where glucocorticoid-based therapies are less efficacious.

Genomic determinants implicated in the glucocorticoid-mediated induction of KLF9 in pulmonary epithelial cells.

Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However, some genes, including Krüppel-like factor 9 (KLF9), a putative transcriptional repressor, demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells, glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5'-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO) sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confirmed in A549 cells, plus submersion, and ALI culture of HBE cells. Cloning each GBS into luciferase reporters revealed glucocorticoid-induced activity requiring a glucocorticoid response element (GRE) within each distal GBS. While the proximal GBSs drove modest reporter induction by glucocorticoids, this region exhibited basal eRNA production, RNA polymerase II enrichment, and looping to the TSS, plausibly underlying constitutive KLF9 expression. Post glucocorticoid treatment, interactions between distal and proximal GBSs and the TSS correlated with KLF9 induction. CBP/P300 silencing reduced proximal GBS activity, but negligibly affected KLF9 expression. Overall, a model for glucocorticoid-mediated regulation of KLF9 involving multiple GBSs is depicted. This work unequivocally demonstrates that mechanistic insights gained from cell lines can translate to physiologically relevant systems.

Adcitmer® , a new CD56-targeting monomethyl auristatin E-conjugated antibody, is a potential therapeutic approach in Merkel cell carcinoma.

BACKGROUND: Merkel cell carcinoma (MCC) is an aggressive skin cancer, whose tumour cells often express CD56. While immune checkpoint inhibitors constitute a major advance for treating patients with MCC with advanced disease, new therapeutic options are still urgently required. OBJECTIVES: To produce and evaluate the therapeutic performance of a new antibody-drug conjugate (Adcitmer® ) targeting CD56 in preclinical models of MCC. METHODS: CD56 expression was evaluated in a MCC cohort (immunohistochemistry on a tissue microarray of 90 tumour samples) and MCC cell lines. Interaction of an unconjugated CD56-targeting antibody with CD56+ MCC cell lines was investigated by immunohistochemistry and imaging flow cytometry. Adcitmer® product was generated by the bioconjugation of CD56-targeting antibody to a cytotoxic drug (monomethyl auristatin E) using the McSAF Inside® bioconjugation process. The chemical properties and homogeneity of Adcitmer® were characterized by hydrophobic interaction chromatography. Adcitmer® cytotoxicity was evaluated in vitro and in an MCC xenograft mice model. RESULTS: Similar to previous reports, CD56 was expressed by 66% of MCC tumours in our cohort, confirming its relevance as a therapeutic target. Specific binding and internalization of the unconjugated CD56-targeting antibody was validated in MCC cell lines. The high homogeneity of the newly generated Adcitmer® was confirmed by hydrophobic interaction chromatography. The CD56-mediated cytotoxicity of Adcitmer® was demonstrated in vitro in MCC cell lines. Moreover, Adcitmer® significantly reduced tumour growth in a MCC mouse model. CONCLUSIONS: Our study suggests that Adcitmer® should be further assessed as a therapeutic option in patients with MCC, as an alternative therapy or combined with immune checkpoint inhibitors.

Digitized collections elucidate invasion history and patterns of awn polymorphism in Microstegium vimineum.

PREMISE: Digitized collections can help illuminate the mechanisms behind the establishment and spread of invasive plants. These databases provide a record of traits in space and time that allows for investigation of abiotic and biotic factors that influence invasive species. METHODS: Over 1100 digitized herbarium records were examined to investigate the invasion history and trait variation of Microstegium vimineum. Presence-absence of awns was investigated to quantify geographic patterns of this polymorphic trait, which serves several functions in grasses, including diaspore burial and dispersal to germination sites. Floret traits were further quantified, and genomic analyses of contemporary samples were conducted to investigate the history of M. vimineum's introduction and spread into North America. RESULTS: Herbarium records revealed similar patterns of awn polymorphism in native and invaded ranges of M. vimineum, with awned forms predominating at higher latitudes and awnless forms at lower latitudes. Herbarium records and genomic data suggested initial introduction and spread of the awnless form in the southeastern United States, followed by a putative secondary invasion and spread of the awned form from eastern Pennsylvania. Awned forms have longer florets, and floret size varies significantly with latitude. There is evidence of a transition zone with short-awned specimens at mid-latitudes. Genomic analyses revealed two distinct clusters corresponding to awnless and awned forms, with evidence of admixture. CONCLUSIONS: Our results demonstrate the power of herbarium data to elucidate the invasion history of a problematic weed in North America and, together with genomic data, reveal a possible key trait in introduction success: presence or absence of an awn.

Compendium of sodium reduction strategies in foods: A scoping review.

In response to health concerns generated by increased sodium intake, many new approaches have been studied to reduce the sodium content in processed food. It has been suggested that reducing sodium in the food supply may be the most appropriate solution. The aim of this scoping review was to establish what sodium reduction strategies are effective in maintaining acceptable sensory qualities for various food industry applications. Studies that evaluate and report on the effectiveness of a sodium reduction strategy relevant to food and included outcomes detailing how the strategies were received by human subjects using sensory data are included, as well as book chapters, literature reviews, and patents focusing on sodium reduction strategies. Only those published in English and since 1970 were included. Literature was obtained through Scopus, PubMed, EBSCOhost, and ScienceDirect databases, whereas patents were obtained through US Patent Trademark Office, Google Patents, and PATENTSCOPE databases. Two-hundred and seventy-seven primary studies, 27 literature reviews, 10 book chapters, and 143 patents were selected for inclusion. Data extracted included details such as analytical methods, broad and specific treatment categories, significant outcomes, and limitations among other material. Sodium reduction methods were categorized as either salt removal, salt replacement, flavor modification, functional modification, or physical modification. Although salt removal and salt replacement were the majority of included studies, future research would benefit from combining methods from other categories while investigating the impact on sensory characteristics, technological aspects, and consumer perception of the strategy.

ß 2-Adrenoceptor Agonists Promote Extracellular Signal-Regulated Kinase 1/2 Dephosphorylation in Human Airway Epithelial Cells by Canonical, cAMP-Driven Signaling Independently of ß-Arrestin 2.

Chronic use of ß 2-adrenoceptor agonists as a monotherapy in asthma is associated with a loss of disease control and an increased risk of mortality. Herein, we tested the hypothesis that ß 2-adrenoceptor agonists, including formoterol, promote biased, ß-arrestin (Arr) 2-dependent activation of the mitogen-activated protein kinases, ERK1/2, in human airway epithelial cells and, thereby, effect changes in gene expression that could contribute to their adverse clinical outcomes. Three airway epithelial cell models were used: the BEAS-2B cell line, human primary bronchial epithelial cells (HBEC) grown in submersion culture, and HBEC that were highly differentiated at an air-liquid interface. Unexpectedly, treatment of all epithelial cell models with formoterol decreased basal ERK1/2 phosphorylation. This was mediated by cAMP-dependent protein kinase and involved the inactivation of C-rapidly-activated fibrosarcoma, which attenuated downstream ERK1/2 activity, and the induction of dual-specificity phosphatase 1. Formoterol also inhibited the basal expression of early growth response-1, an ERK1/2-regulated gene that controls cell growth and repair in the airways. Neither carvedilol, a ß 2-adrenoceptor agonist biased toward ßArr2, nor formoterol promoted ERK1/2 phosphorylation in BEAS-2B cells, although ß 2-adrenoceptor desensitization was compromised in ARRB2-deficient cells. Collectively, these results contest the hypothesis that formoterol activates ERK1/2 in airway epithelia by nucleating a ßArr2 signaling complex; instead, they indicate that ß 2-adrenoceptor agonists inhibit constitutive ERK1/2 activity in a cAMP-dependent manner. These findings are the antithesis of results obtained using acutely challenged native and engineered HEK293 cells, which have been used extensively to study mechanisms of ERK1/2 activation, and highlight the cell type dependence of ß 2-adrenoceptor-mediated signaling. SIGNIFICANCE STATEMENT: It has been proposed that the adverse effects of ß 2-adrenoceptor agonist monotherapy in asthma are mediated by genomic mechanisms that occur principally in airway epithelial cells and are the result of ß-arrestin 2-dependent activation of ERK1/2. This study shows that ß 2-adrenoceptor agonists, paradoxically, reduced ERK1/2 phosphorylation in airway epithelia by disrupting upstream rat sarcoma-C-rapidly accelerated fibrosarcoma complex formation and inducing dual-specificity phosphatase 1. Moreover, these effects were cAMP-dependent protein kinase-dependent, suggesting that ß 2-adrenoceptor agonists were not biased toward ß-arrestin 2 and acted via canonical, cAMP-dependent signaling.

Astrocytes are necessary for blood-brain barrier maintenance in the adult mouse brain.

In the adult brain, multiple cell types are known to produce factors that regulate blood-brain barrier (BBB) properties, including astrocytes. Yet several recent studies disputed a role for mature astrocytes at the BBB. To determine if astrocytes contribute a nonredundant and necessary function in maintaining the adult BBB, we used a mouse model of tamoxifen-inducible astrocyte ablation. In adult mice, tamoxifen induction caused sparse apoptotic astrocyte cell death within 2 hr. Indicative of BBB damage, leakage of the small molecule Cadaverine, and the large plasma protein fibrinogen into the brain parenchyma indicative of BBB damage was detected as early as astrocyte ablation was present. Vessels within and close to regions of astrocyte loss had lower expression of the tight junction protein zonula occludens-1 while endothelial glucose transporter 1 expression was undisturbed. Cadaverine leakage persisted for several weeks suggesting a lack of barrier repair. This is consistent with the finding that ablated astrocytes were not replaced. Adjacent astrocytes responded with partial nonproliferative astrogliosis, characterized by morphological changes and delayed phosphorylation of STAT3, which restricted dye leakage to the brain and vessel surface areas lacking coverage by astrocytes 1 month after ablation. In conclusion, astrocytes are necessary to maintain BBB integrity in the adult brain. BBB-regulating factors secreted by other cell types, such as pericytes, are not sufficient to compensate for astrocyte loss.

A toolbox for studying respiratory viral infections using air-liquid interface cultures of human airway epithelial cells.

Submerged cultures of primary human airway epithelial cells or human airway epithelial cell lines have been a mainstay of airway epithelial biology research for decades due to their robust in vitro proliferative capacity, relatively low maintenance culture conditions, and clinically translatable results to nasal or bronchial brushings. With the development and improvement of air-liquid interface (ALI) cultures of human airway epithelial cells, such cultures have been considered superior to immortalized cell lines and primary cell monolayers, as such cultures effectively recapitulate in vivo epithelial architecture and cell types. Although ALI culture growth protocols are well-established and widely available, many researchers have avoided their use, as ALI cultures not only take longer to grow but also present technical challenges and limitations that make in vitro intracellular and structural assays taxing. Challenges arise relating to their complex structure, requirements for air exposure, the constraints of transwell growth apparatus, and interference in assays caused by mucus secretion. Although few publications briefly describe technical adaptations for some assays, there is still considerable trial and error required for researchers to establish consistent and reliable assay adaptations, often becoming a deterrent for pursuing mechanistic investigation. We have created a user-friendly toolbox detailing comprehensive protocols for numerous techniques and assay adaptations, particularly focusing on respiratory virus infections. By expanding the repertoire of ALI culture-adapted in vitro assays, we hope to facilitate the widespread adoption of this valuable culture system for mechanistic investigations of respiratory viral infections or other epithelial-pathogen models.

Prostanoid Receptors of the EP4-Subtype Mediate Gene Expression Changes in Human Airway Epithelial Cells with Potential Anti-Inflammatory Activity.

There is a clear, unmet clinical need to identify new drugs to treat individuals with asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF) in whom current medications are either inactive or suboptimal. In preclinical models, EP4-receptor agonists display efficacy, but their mechanism of action is unclear. In this study, using human bronchial epithelial cells as a therapeutically relevant drug target, we hypothesized that changes in gene expression may play an important role. Several prostanoid receptor mRNAs were detected in BEAS-2B cells, human primary bronchial epithelial cells (HBECs) grown in submersion culture and HBECs grown at an air-liquid interface with PTGER4 predominating. By using the activation of a cAMP response element reporter in BEAS-2B cells as a surrogate of gene expression, Schild analysis determined that PTGER4 mRNAs encoded functional EP4-receptors. Moreover, inhibitors of phosphodiesterase 4 (roflumilast N-oxide [RNO]) and cAMP-dependent protein kinase augmented and attenuated, respectively, reporter activation induced by 2-[3-[(1R,2S,3R)-3-hydroxy-2-[(E,3S)-3-hydroxy-5-[2-(methoxymethyl)phenyl]pent-1-enyl]-5-oxo-cyclopentyl]sulphanylpropylsulphanyl] acetic acid (ONO-AE1-329), a selective EP4-receptor agonist. ONO-AE1-329 also enhanced dexamethasone-induced activation of a glucocorticoid response element reporter in BEAS-2B cells, which was similarly potentiated by RNO. In each airway epithelial cell variant, numerous genes that may impart therapeutic benefit in asthma, COPD, and/or IPF were differentially expressed by ONO-AE1-329, and those changes were often augmented by RNO and/or dexamethasone. We submit that an EP4-receptor agonist, either alone or as a combination therapy, may be beneficial in individuals with chronic lung diseases in whom current treatment options are inadequate. SIGNIFICANCE STATEMENT: Using human bronchial epithelial cells as a therapeutically relevant drug target, we report that EP4-receptor activation promoted gene expression changes that could provide therapeutic benefit in individuals with asthma, COPD, and IPF in whom current treatment options are ineffective or suboptimal.

Rhinovirus and Bacteria Synergistically Induce IL-17C Release from Human Airway Epithelial Cells To Promote Neutrophil Recruitment.

Virus-bacteria coinfections are associated with more severe exacerbations and increased risk of hospital readmission in patients with chronic obstructive pulmonary disease (COPD). The airway epithelium responds to such infections by releasing proinflammatory and antimicrobial cytokines, including IL-17C. However, the regulation and role of IL-17C is not well understood. In this study, we examine the mechanisms regulating IL-17C production and its potential role in COPD exacerbations. Human bronchial epithelial cells (HBE) obtained from normal, nontransplanted lungs or from brushings of nonsmokers, healthy smokers, or COPD patients were exposed to bacteria and/or human rhinovirus (HRV). RNA and protein were collected for analysis, and signaling pathways were assessed with pharmacological agonists, inhibitors, or small interfering RNAs. HBE were also stimulated with IL-17C to assess function. HRV-bacterial coinfections synergistically induced IL-17C expression. This induction was dependent on HRV replication and required NF-κB-mediated signaling. Synergy was lost in the presence of an inhibitor of the p38 MAP kinase pathway. HBE exposed to IL-17C show increased gene expression of CXCL1, CXCL2, NFKBIZ, and TFRC, and release CXCL1 protein, a neutrophil chemoattractant. Knockdown of IL-17C significantly reduced induction of CXCL1 in response to HRV-bacterial coinfection as well as neutrophil chemotaxis. HBE from healthy smokers release less IL-17C than cells from nonsmokers, but cells from COPD patients release significantly more IL-17C compared with either nonsmokers or healthy smokers. These data suggest that IL-17C may contribute to microbial-induced COPD exacerbations by promoting neutrophil recruitment.

Mastectomy in 25 small ruminants (2002-2019).

OBJECTIVE: To describe the outcome of small ruminants treated with unilateral and bilateral mastectomy by using three surgical techniques. STUDY DESIGN: Retrospective study. ANIMALS: Twenty-five small ruminants (24 goats and one sheep). METHODS: Medical records of animals that underwent mastectomy between November 1, 2002, and May 1, 2019, were reviewed. Follow-up information was obtained by telephone questionnaire with owners. Signalment, surgical data, intraoperative and postoperative complications, bacterial culture results, histopathologic diagnoses, short- and long-term outcomes, and other procedures performed were recorded. RESULT: Procedures consisted of six unilateral (with an elliptical incision) and 19 total (with inverted cloverleaf or elliptical skin incisions) mastectomies. All animals survived to hospital discharge. Intraoperative complications included contamination of the surgical site with mammary-gland fluid, hemorrhage, and difficulty dissecting skin from the mammary gland. Postoperative complications included seroma formation (7/25), surgical-site infection (5/25), and dehiscence of the skin incision (3/25). Mammary neoplasia was diagnosed in seven of 15 animals with histopathologic examination. No association was detected between surgical technique, diagnosis of neoplasia, and long-term outcome. Overall, client satisfaction was high. CONCLUSION: Mastectomy was effective at removing abnormally enlarged udders secondary to chronic mastitis, inappropriate lactation, idiopathic causes, or neoplasia and was associated with a low rate of complications in small ruminants. CLINICAL SIGNIFICANCE: Unilateral mastectomy with an elliptical skin incision or total mastectomy, preferably with inverted cloverleaf skin incision, may be indicated to remove diseased mammary tissue in small ruminants and can result in long-term survival with low morbidity and cosmetically pleasing results.

Caudal paramedian approach to cryptorchidectomy in 29 small ruminants (2011-2019).

OBJECTIVE: To describe a caudal paramedian approach to cryptorchidectomy in small ruminants. STUDY DESIGN: Retrospective case series. ANIMALS: Sheep (n = 20) and goats (n = 9) with cryptorchidism. METHODS: Medical records from January 2011 to July 2019 of small ruminants that underwent caudal paramedian cryptorchidectomy at our institution were retrospectively reviewed. Data from animal signalment, operative and postoperative complications, and client satisfaction were evaluated. RESULTS: Twenty sheep and nine goats underwent caudal paramedian cryptorchidectomy during the study period. The median age of rams was 2 months, and the median age of bucks was 3 months; median weights for rams and bucks were 20.5 kg (range, 14.5-41.3) and 28.1 kg (range, 12.9-82), respectively. Cryptorchidism was bilateral in 27.6% (8/29) of cases and unilateral in 72.4% (21/29). Among unilateral cases, 85.7% (18/21) were right sided and 14.3% (3/21) were left sided. Twenty-four of 29 (82.8%) cases were performed under sedation. One operative complication occurred in a 4-year-old 82 kg buck (rate, 3.4% [95% CI: 0.1%-17.2%]). Postoperatively, there were two minor and one major complications (rates, 6.9% [95% CI: 0.8%-22.1%] and 3.4% [95% CI: 0.1%-17.2%], respectively). Long-term follow-up (range, 8-117 months) reports described owner satisfaction and all animals doing well at the time of follow-up telephone call. CONCLUSION: Caudal paramedian approach to cryptorchidectomy was safely performed in small ruminants less than 4 months old. CLINICAL SIGNIFICANCE: Ease of surgical technique, minimal operative and postoperative complications, and owner satisfaction make this a suitable method for cryptorchidectomy.

Expanding the Dimensions of a Small, Two-Dimensional Diffraction Detector.

We report an approach to expand the effective number of pixels available to small, two-dimensional electron detectors. To do so, we acquire subsections of a diffraction pattern that are then accurately stitched together in post-processing. Using an electron microscopy pixel array detector (EMPAD) that has only 128 × 128 pixels, we show that the field of view can be expanded while achieving high reciprocal-space sampling. Further, we highlight the need to properly account for the detector position (rotation) and the non-orthonormal diffraction shift axes to achieve an accurate reconstruction. Applying the method, we provide examples of spot and convergent beam diffraction patterns acquired with a pixelated detector.

Exact analytic solution for a chaotic hybrid dynamical system and its electronic realization.

A novel hybrid dynamical system comprising a continuous and a discrete state is introduced and shown to exhibit chaotic dynamics. The system includes an unstable first-order filter subject to asynchronous switching of a set point according to a feedback rule. Regular samples of the continuous state yield a one-dimensional return map that is a tent function. An exact analytic solution is derived using a nonlinear transformation of a previously solved chaotic oscillator. Conjugacy to a symbolic dynamics is shown, and a practical realization of the system in an electronic circuit is demonstrated.

Generating and Detecting Solvable Chaos at Radio Frequencies with Consideration to Multi-User Ranging.

High entropy waveforms exhibit desirable correlation properties in radar and sonar applications when multiple systems are used in close proximity. Unfortunately, the information content of these signals can impose high sampling requirements for digital detection techniques. Solvable chaotic oscillators have been proposed to address such issues due to their simple, matched filters, where hardware has been demonstrated with a bandwidth of 10-20 kHz. To extend applications of these systems, we present theory, design, and experimental verification of solvable chaos at 1 MHz using simple off-the-shelf components. The waveforms produced by this system were propagated over a 2.45 GHz RF link and detected with an RLC-based, purely analog matched filter. Further, we show that properties of this special class of chaotic systems can be exploited to yield RF noise sources that are generally advantageous for multi-user ranging applications when compared to conventional techniques. The result is a simple, low-cost, and potentially low-power RF ranging system that requires very little digital signal processing.

Disseminated Rhodococcus equi in an Anglo-Nubian goat.

Disseminated Rhodococcus equi infection was diagnosed in an Anglo-Nubian goat presenting for non-weight bearing lameness of the right pelvic limb. Radiographs showed a moth-eaten osteolytic lesion in the proximal tibia suggestive of an aggressive bone lesion. Two pulmonary nodules were also present on thoracic radiographs. Initial antemortem cytology of the tibial lesion was suggestive of Rhodococcosis and the goat was sent to necropsy. Necropsy and bacterial culture confirmed the diagnosis of disseminated R. equi infection in the right tibia, lungs, and liver.

Rhinovirus replication and innate immunity in highly differentiated human airway epithelial cells.

BACKGROUND: Human rhinovirus (HRV) infections are the primary cause of the common cold and are a major trigger for exacerbations of lower airway diseases, such as asthma and chronic obstructive pulmonary diseases. Although human bronchial epithelial cells (HBE) are the natural host for HRV infections, much of our understanding of how HRV replicates and induces host antiviral responses is based on studies using non-airway cell lines (e.g. HeLa cells). The current study examines the replication cycle of HRV, and host cell responses, in highly differentiated cultures of HBE. METHODS: Highly differentiated cultures of HBE were exposed to initial infectious doses ranging from 104 to 101 50% tissue culture-infective dose (TCID50) of purified HRV-16, and responses were monitored up to 144 h after infection. Viral genomic RNA and negative strand RNA template levels were monitored, along with levels of type I and II interferons and selected antivirals. RESULTS: Regardless of initial infectious dose, relatively constant levels of both genomic and negative strand RNA are generated during replication, with negative strand copy numbers being10,000-fold lower than those of genomic strands. Infections were limited to a small percentage of ciliated cells and did not result in any overt signs of epithelial death. Importantly, regardless of infectious dose, HRV-16 infections were cleared by HBE in the absence of immune cells. Levels of type I and type III interferons (IFNs) varied with initial infectious dose, implying that factors other than levels of double-stranded RNA regulate IFN induction, but the time-course of HRV-16 clearance HBE was the same regardless of levels of IFNs produced. Patterns of antiviral viperin and ISG15 expression suggest they may be generated in an IFN-independent manner during HRV-16 infections. CONCLUSIONS: These data challenge a number of aspects of dogma generated from studies in HeLa cells and emphasize the importance of appropriate cell context when studying HRV infections.

A digital matched filter for reverse time chaos.

The use of reverse time chaos allows the realization of hardware chaotic systems that can operate at speeds equivalent to existing state of the art while requiring significantly less complex circuitry. Matched filter decoding is possible for the reverse time system since it exhibits a closed form solution formed partially by a linear basis pulse. Coefficients have been calculated and are used to realize the matched filter digitally as a finite impulse response filter. Numerical simulations confirm that this correctly implements a matched filter that can be used for detection of the chaotic signal. In addition, the direct form of the filter has been implemented in hardware description language and demonstrates performance in agreement with numerical results.

Acyl-ACP substrate recognition in Burkholderia mallei BmaI1 acyl-homoserine lactone synthase.

The acyl-homoserine lactone (AHL) autoinducer mediated quorum sensing regulates virulence in several pathogenic bacteria. The hallmark of an efficient quorum sensing system relies on the tight specificity in the signal generated by each bacterium. Since AHL signal specificity is derived from the acyl-chain of the acyl-ACP (ACP = acyl carrier protein) substrate, AHL synthase enzymes must recognize and react with the native acyl-ACP with high catalytic efficiency while keeping reaction rates with non-native acyl-ACPs low. The mechanism of acyl-ACP substrate recognition in these enzymes, however, remains elusive. In this study, we investigated differences in catalytic efficiencies for shorter and longer chain acyl-ACP substrates reacting with an octanoyl-homoserine lactone synthase Burkholderia mallei BmaI1. With the exception of two-carbon shorter hexanoyl-ACP, the catalytic efficiencies of butyryl-ACP, decanoyl-ACP, and octanoyl-CoA reacting with BmaI1 decreased by greater than 20-fold compared to the native octanoyl-ACP substrate. Furthermore, we also noticed kinetic cooperativity when BmaI1 reacted with non-native acyl-donor substrates. Our kinetic data suggest that non-native acyl-ACP substrates are unable to form a stable and productive BmaI1·acyl-ACP·SAM ternary complex and are thus effectively discriminated by the enzyme. These results offer insights into the molecular basis of substrate recognition for the BmaI1 enzyme.