Layering vaccination with antibiotic therapy results in protection and clearance of Burkholderia pseudomallei in Balb/c mice.

Melioidosis is a disease that is difficult to treat due to the causative organism, Burkholderia pseudomallei being inherently antibiotic resistant and it having the ability to invade, survive, and replicate in an intracellular environment. Combination therapy approaches are routinely being evaluated in animal models with the aim of improving the level of protection and clearance of colonizing bacteria detected. In this study, a subunit vaccine layered with the antibiotic finafloxacin was evaluated in vivo against an inhalational infection with B. pseudomallei in Balb/c mice. Groups of mice vaccinated, infected, and euthanized at antibiotic initiation had a reduced bacterial load compared to those that had not been immunized. In addition, the subunit vaccine provided a synergistic effect when it was delivered with a CpG ODN and finafloxacin was initiated at 48 h post-challenge. Vaccination was also shown to improve the outcome, in a composite measure of survival and clearance. In summary, layering a subunit vaccine with the antibiotic finafloxacin is a promising therapeutic alternative for use in the treatment of B. pseudomallei infections.

c-Src controls stability of sprouting blood vessels in the developing retina independently of cell-cell adhesion through focal adhesion assembly.

Endothelial cell adhesion is implicated in blood vessel sprout formation, yet how adhesion controls angiogenesis, and whether it occurs via rapid remodeling of adherens junctions or focal adhesion assembly, or both, remains poorly understood. Furthermore, how endothelial cell adhesion is controlled in particular tissues and under different conditions remains unexplored. Here, we have identified an unexpected role for spatiotemporal c-Src activity in sprouting angiogenesis in the retina, which is in contrast to the dominant focus on the role of c-Src in the maintenance of vascular integrity. Thus, mice specifically deficient in endothelial c-Src displayed significantly reduced blood vessel sprouting and loss in actin-rich filopodial protrusions at the vascular front of the developing retina. In contrast to what has been observed during vascular leakage, endothelial cell-cell adhesion was unaffected by loss of c-Src. Instead, decreased angiogenic sprouting was due to loss of focal adhesion assembly and cell-matrix adhesion, resulting in loss of sprout stability. These results demonstrate that c-Src signaling at specified endothelial cell membrane compartments (adherens junctions or focal adhesions) control vascular processes in a tissue- and context-dependent manner.

Phase-separated foci of EML4-ALK facilitate signalling and depend upon an active kinase conformation.

Variants of the oncogenic EML4-ALK fusion protein contain a similar region of ALK encompassing the kinase domain, but different portions of EML4. Here, we show that EML4-ALK V1 and V3 proteins form cytoplasmic foci that contain components of the MAPK, PLCγ and PI3K signalling pathways. The ALK inhibitors ceritinib and lorlatinib dissolve these foci and EML4-ALK V3 but not V1 protein re-localises to microtubules, an effect recapitulated in a catalytically inactive EML4-ALK mutant. Mutations that promote a constitutively active ALK stabilise the cytoplasmic foci even in the presence of these inhibitors. In contrast, the inhibitor alectinib increases foci formation of both wild-type and catalytically inactive EML4-ALK V3 proteins, but not a Lys-Glu salt bridge mutant. We propose that EML4-ALK foci formation occurs as a result of transient association of stable EML4-ALK trimers mediated through an active conformation of the ALK kinase domain. Our results demonstrate the formation of EML4-ALK cytoplasmic foci that orchestrate oncogenic signalling and reveal that their assembly depends upon the conformational state of the catalytic domain and can be differentially modulated by structurally divergent ALK inhibitors.

Initial heart rate score predicts new-onset atrial tachyarrhythmias in pacemaker patients.

AIMS: Heart rate score (HRSc), the per cent of atrial paced and sensed event in the largest 10 b.p.m. rate histogram bin of a pacemaker, predicts survival in patients with cardiac devices. No correlation between HRSc and development of atrial fibrillation (AF) has been reported. In this study, we evaluated the relationship between pacemaker post-implantation HRSc and the incidence of newly developed atrial tachyarrhythmias (ATAs). METHODS AND RESULTS: Patients with dual-chamber pacemakers, implanted 2013-17, with the LATITUDE remote monitoring data with ≥600 000 beats of histogram data collected at baseline were included (N = 34 543). Heart rate score was determined from the initial 3-month post-implantation histogram data. Patients were excluded if they had ATAs, defined as atrial high-rate episodes >5 min or >1% of right atrial beats >170 b.p.m. during the initial 3 months post-implantation. New ATAs, after the baseline period, were defined by each of the following: >1, >10, or >25% of atrial beats >170 b.p.m. or atrial tachycardia response (ATR) events >24 h. Patients were followed a median of 2.8 (1.0-4.0) years. The incidence of ATAs increased in proportion to HRSc (log-rank P-value <0.001), and the initial HRSc ≥70% was associated with increased ATAs by all definitions. Patients with initial HRSc ≥70% were older, had a higher percentage of right atrium pacing (%RA pacing), had a lower percentage of right ventricular pacing (%RV pacing), and were more likely programmed with rate-response vs. subjects with HRSc <70%. Initial HRSc (hazard ratio: 1.07, 95% confidence interval: 1.05-1.09; P < 0.0001) independently predicted ATAs after adjusting for age, gender, %RV pacing, and rate-response programming. The %RA pacing and initial HRSc were correlated. CONCLUSION: Heart rate score independently predicts any subsequent duration of ATAs in pacemaker patients.

Avoiding unnecessary ventricular pacing is associated with reduced incidence of heart failure hospitalizations and persistent atrial fibrillation in pacemaker patients.

AIMS: In bradycardia patients treated with dual-chamber pacing, we aimed to evaluate whether pacing with atrioventricular (AV) delay management [AV hysteresis (AVH)], compared with standard pacing with fixed AV delays, reduces unnecessary ventricular pacing percentage (VPP) and is associated with better clinical outcomes. Main study endpoints were the incidence of heart failure hospitalizations (HFH), persistent atrial fibrillation (AF), and cardiac death. METHODS AND RESULTS: Data from two identical prospective observational studies, BRADYCARE I in the USA and BRADYCARE II in Europe, Africa, and Asia, were pooled. Overall, 2592 patients (75 ± 10 years, 45.1% female, 50% with AVH) had complete clinical and device data at 1-year follow-up and were analysed. Primary pacing indication was sinus node disease (SND) in 1177 (45.4%), AV block (AVB) in 974 (37.6%), and other indications in 441 (17.0%) patients. Pacing with AVH, compared with standard pacing, was associated with a lower 1-year incidence of HFH [1.3% vs. 3.1%, relative risk reduction (RRR) 57.5%, P = 0.002] and of persistent AF (5.3% vs. 7.7%, RRR = 31.1%, P = 0.028). Cardiac mortality was not different between groups (1.0% vs. 1.4%, RRR = 27.8%, P = 0.366). Pacing with AVH, compared with standard pacing, was associated with a lower (P < 0.001) median VPP in all patients (7% vs. 75%), in SND (3% vs. 44%), in AVB (25% vs. 98%), and in patients with other pacing indications (3% vs. 47%). CONCLUSION: Cardiac pacing with AV delay management via AVH is associated with reduced 1-year incidence of HFH and persistent AF, most likely due to a reduction in VPP compared to standard pacing.

Mitotic spindle association of TACC3 requires Aurora-A-dependent stabilization of a cryptic α-helix.

Aurora-A regulates the recruitment of TACC3 to the mitotic spindle through a phospho-dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora-A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora-A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical-repeat region of CHC, not a recognized phospho-reader domain. This potentially widespread mechanism of phospho-recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.

EML4-ALK V3 oncogenic fusion proteins promote microtubule stabilization and accelerated migration through NEK9 and NEK7.

EML4-ALK is an oncogenic fusion present in ∼5% of non-small cell lung cancers. However, alternative breakpoints in the EML4 gene lead to distinct variants of EML4-ALK with different patient outcomes. Here, we show that, in cell models, EML4-ALK variant 3 (V3), which is linked to accelerated metastatic spread, causes microtubule stabilization, formation of extended cytoplasmic protrusions and increased cell migration. EML4-ALK V3 also recruits the NEK9 and NEK7 kinases to microtubules via the N-terminal EML4 microtubule-binding region. Overexpression of wild-type EML4, as well as constitutive activation of NEK9, also perturbs cell morphology and accelerates migration in a microtubule-dependent manner that requires the downstream kinase NEK7 but does not require ALK activity. Strikingly, elevated NEK9 expression is associated with reduced progression-free survival in EML4-ALK patients. Hence, we propose that EML4-ALK V3 promotes microtubule stabilization through NEK9 and NEK7, leading to increased cell migration. This represents a novel actionable pathway that could drive metastatic disease progression in EML4-ALK lung cancer.

Model systems for studying lipid oxidation associated with muscle foods: Methods, challenges, and prospects.

Lipid oxidation is a complex process in muscle-based foods (red meat, poultry and fish) causing severe quality deterioration, e.g., off-odors, discoloration, texture defects and nutritional loss. The complexity of muscle tissue -both composition and structure- poses as a formidable challenge in directly clarifying the mechanisms of lipid oxidation in muscle-based foods. Therefore, different in vitro model systems simulating different aspects of muscle have been used to study the pathways of lipid oxidation. In this review, we discuss the principle, preparation, implementation as well as advantages and disadvantages of seven commonly-studied model systems that mimic either compositional or structural aspects of actual meat: emulsions, fatty acid micelles, liposomes, microsomes, erythrocytes, washed muscle mince, and muscle homogenates. Furthermore, we evaluate the prospects of stem cells, tissue cultures and three-dimensional printing for future model system development. Based on this reviewing of oxidation models, tailoring correct model to different study aims could be facilitated, and readers are becoming acquainted with advantages and shortcomings. In addition, insight into recent technology developments, e.g., stem cell- and tissue-cultures as well as three-dimensional printing could provide new opportunities to overcome the current bottlenecks of lipid oxidation studies in muscle.

A seismically induced onshore surge deposit at the KPg boundary, North Dakota.

The most immediate effects of the terminal-Cretaceous Chicxulub impact, essential to understanding the global-scale environmental and biotic collapses that mark the Cretaceous-Paleogene extinction, are poorly resolved despite extensive previous work. Here, we help to resolve this by describing a rapidly emplaced, high-energy onshore surge deposit from the terrestrial Hell Creek Formation in Montana. Associated ejecta and a cap of iridium-rich impactite reveal that its emplacement coincided with the Chicxulub event. Acipenseriform fish, densely packed in the deposit, contain ejecta spherules in their gills and were buried by an inland-directed surge that inundated a deeply incised river channel before accretion of the fine-grained impactite. Although this deposit displays all of the physical characteristics of a tsunami runup, the timing (<1 hour postimpact) is instead consistent with the arrival of strong seismic waves from the magnitude Mw ∼10 to 11 earthquake generated by the Chicxulub impact, identifying a seismically coupled seiche inundation as the likely cause. Our findings present high-resolution chronology of the immediate aftereffects of the Chicxulub impact event in the Western Interior, and report an impact-triggered onshore mix of marine and terrestrial sedimentation-potentially a significant advancement for eventually resolving both the complex dynamics of debris ejection and the full nature and extent of biotic disruptions that took place in the first moments postimpact.

Modelling the effects of cold temperature during the reproductive stage on the yield of chickpea (Cicer arietinum L.).

During the reproductive stage, chilling temperatures and frost reduce the yield of chickpea and limit its adaptation. The adverse effects of chilling temperature and frost in terms of the threshold temperatures, impact of cold duration, and genotype-by-environment-by-management interactions are not well quantified. Crop growth models that predict flowering time and yield under diverse climates can identify combinations of cultivars and sowing time to reduce frost risk in target environments. The Agricultural Production Systems Simulator (APSIM-chickpea) model uses daily temperatures to model basic crop growth but does not include penalties for either frost damage or cold temperatures during flowering and podding stages. Regression analysis overcame this limitation of the model for chickpea crops grown at 95 locations in Australia using 70 years of historic data incorporating three cultivars and three sowing times (early, mid, and late). We modified model parameters to include the effect of soil water on thermal time calculations, which significantly improved the prediction of flowering time. Simulated data, and data from field experiments grown in Australia (2013 to 2019), showed robust predictions for flowering time (n = 29; R2 = 0.97), and grain yield (n = 22; R2 = 0.63-0.70). In addition, we identified threshold cold temperatures that significantly affected predicted yield, and combinations of locations, variety, and sowing time where the overlap between peak cold temperatures and peak flowering was minimal. Our results showed that frost and/or cold temperature-induced yield losses are a major limitation in some unexpected Australian locations, e.g., inland, subtropical latitudes in Queensland. Intermediate sowing maximise yield, as it avoids cold temperature, late heat, and drought stresses potentially limiting yield in early and late sowing respectively.

Lipid oxidation and antioxidant delivery systems in muscle food.

Lipid oxidation accelerates quality deterioration in muscle-based foods (fish, red meat, and poultry), resulting in off-odors/flavors, color problems, texture defects, and safety concerns. Adding antioxidants is one approach to control lipid oxidation, and several delivery strategies have been applied, such as supplementing antioxidants to the feed, direct mixing into minces, or, for whole muscle pieces; spraying, glazing, and injection. However, some issues linked to these technologies hinder their wide utilization, such as low effectiveness, noncompatibility with clean label, and off-flavor. These shortcomings have promoted the development of new antioxidant delivery technologies. In this review, the main focus is on the principles, characteristics, and implementation of five novel antioxidant delivery methods in different types of muscle food products. Their advantages and drawbacks are also summarized, plus comments about future trends in this area. Among novel routes to deliver antioxidants to muscle foods are, for whole tissues, recyclable dipping solutions; for minces, encapsulation; and, for both minces and whole tissues, cross-processing with nonmuscle antioxidant-containing raw materials as well as applications of edible films/coatings and active packaging. Advantages of these technologies comprise, for example, low price, the possibility to control the antioxidant release rate, overcoming strong aromas from natural antioxidants, and allowing antioxidant-containing raw materials from the food industry to be valorized, providing an opportunity for more circular food production.

Finafloxacin Is an Effective Treatment for Inhalational Tularemia and Plague in Mouse Models of Infection.

Infection with aerosolized Francisella tularensis or Yersinia pestis can lead to lethal disease in humans if treatment is not initiated promptly. Finafloxacin is a novel fluoroquinolone which has demonstrated broad-spectrum activity against a range of bacterial species in vitro, in vivo, and in humans, activity which is superior in acidic, infection-relevant conditions. Human-equivalent doses of finafloxacin or ciprofloxacin were delivered at 24 h (representing prophylaxis) or at 72 or 38 h (representing treatment) postchallenge with F. tularensis or Y. pestis, respectively, in BALB/c mouse models. In addition, a short course of therapy (3 days) was compared to a longer course (7 days). Both therapies provided a high level of protection against both infections when administered at 24 h postchallenge, irrespective of the length of the dosing regimen; however, differences were observed when therapy was delayed. A benefit was demonstrated with finafloxacin compared to ciprofloxacin in both models when therapy was delivered later in the infection. These studies suggest that finafloxacin is an effective alternative therapeutic for the prophylaxis and treatment of inhalational infections with F. tularensis or Y. pestis.

Investigative study into whether an insect repellent has virucidal activity against SARS-CoV-2.

A small-scale study with Mosi-guard Natural spray, an insect repellent containing Citriodiol, was performed to determine if it has virucidal activity against SARS-CoV-2. A liquid test examined the activity of the insect repellent and the individual components for virucidal activity. A surface contact test looked at the activity of the insect repellent when impregnated on a latex surface as a synthetic skin for potential topical prophylactic application. Both Mosi-guard Natural spray and Citriodiol, as well as other components of the repellent, had virucidal activity in the liquid contact test. On a latex surface used to simulate treated skin, the titre of SARS-CoV-2 was less over time on the Mosi-guard Natural-treated surface but virus was still recovered.

A novel adaptive insertable cardiac monitor algorithm improves the detection of atrial fibrillation and atrial tachycardia in silico.

INTRODUCTION: Insertable cardiac monitors (ICMs) provide a minimally invasive method of continuous monitoring for abnormal heart rhythms. While the benefits of ICMs are clear, current algorithm performance can be improved. The objective of this study is to assess the performance of a novel adaptive atrial fibrillation (AF) detection algorithm and separately programmable atrial tachycardia (AT) algorithm. METHODS: A dual-stage detect-and-verify AF algorithm and separately programmable AT algorithm were developed. Sensitivity and PPV across a range of settings were determined in silico by comparison with an adjudicated Holter data set (n = 1966 with 229 patient days). Finally, the ability to improve performance through simulated remote programming was assessed. RESULTS: The dual-stage algorithm detected AF in all true AF patients (76/76) resulting in a patient-level sensitivity of 100%. Episode-level sensitivity and PPV ranged from 97.6% to 100% and 79.1% to 98.5%, respectively. Thirty-six false-positive episodes were observed and 32 (88.9%) of these were corrected with programming changes. Decoupling of AF and AT durations improved PPV from a range of 10%-22% to a range of 95%-100%. CONCLUSIONS: AF and AT algorithms were designed with novel features including an adaptive morphology assessment for AF detection and separately programmable durations for AT detection. In silico performance yielded improved PPVs while maintaining high sensitivity across a range of settings. Importantly, programming changes that may be made remotely with this system reduced false positives. These algorithms allow clinicians to individualize arrhythmia detection settings thereby improving data management and reducing clinic burden.

The Influence of High Flow Nasal Cannulae on the Outcomes of Severe Respiratory Disease in Children Admitted to a Regional Hospital in South Africa.

In settings where access to paediatric intensive care unit (PICU) facilities is constrained and transfer capacity is limited, High Flow Nasal Cannulae (HFNC) might fill an important service gap. The aim of this study was to document the effect of HFNC on the outcomes of children admitted with severe respiratory disease at a regional hospital without a PICU in Cape Town, South Africa. It is a 4-year retrospective analysis documenting two periods of 2 years each, one before (2013-15) and one after (2016-18) the initiation of HFNC use. Patients were between the ages of 2 months and 13 years and had been admitted to a paediatric ward. Outcomes were defined by the need for transfer to a tertiary hospital, the need for invasive ventilation and death. There were 90 instances of HFNC use with a significant reduction in the number of children who were transferred (59 vs. 31), invasively ventilated (20 vs. 6, p ≤ 0.01) and who died (3 vs. 0, p = 0.02). Before HFNC implementation, there was also a significantly greater proportion of transferred children who remained on low flow nasal cannulae (15 vs. 2, p ≤ 0.001) at the tertiary hospital. Children who failed HFNC use tended to do this within a day of initiation (Median 11 vs. 60 h for success, p ≤ 0.001). There were no complications related to its use. We believe that in our setting the utilization of HFNC has helped to timeously and accurately identify children needing to be transferred and may mitigate against severe respiratory disease progression.

A high-throughput ratiometric method for imaging hypertrophic growth in cultured primary cardiac myocytes.

Maladaptive hypertrophy of cardiac myocytes increases the risk of heart failure. The underlying signaling can be triggered and interrogated in cultured neonatal ventricular myocytes (NRVMs) using sophisticated pharmacological and genetic techniques. However, the methods for quantifying cell growth are, by comparison, inadequate. The lack of quantitative, calibratable and computationally-inexpensive high-throughput technology has limited the scope for using cultured myocytes in large-scale analyses. We present a ratiometric method for quantifying the hypertrophic growth of cultured myocytes, compatible with high-throughput imaging platforms. Protein biomass was assayed from sulforhodamine B (SRB) fluorescence, and image analysis calculated the quotient of signal from extra-nuclear and nuclear regions. The former readout relates to hypertrophic growth, whereas the latter is a reference for correcting protein-independent (e.g. equipment-related) variables. This ratiometric measure, when normalized to the number of cells, provides a robust quantification of cellular hypertrophy. The method was tested by comparing the efficacy of various chemical agonists to evoke hypertrophy, and verified using independent assays (myocyte area, transcripts of markers). The method's high resolving power and wide dynamic range were confirmed by the ability to generate concentration-response curves, track the time-course of hypertrophic responses with fine temporal resolution, describe drug/agonist interactions, and screen for novel anti-hypertrophic agents. The method can be implemented as an end-point in protocols investigating hypertrophy, and is compatible with automated plate-reader platforms for generating high-throughput data, thereby reducing investigator-bias. Finally, the computationally-minimal workflow required for obtaining measurements makes the method simple to implement in most laboratories.

Demonstration of the broad spectrum in vitro activity of finafloxacin against pathogens of biodefence interest.

This study investigated the in vitro activity of finafloxacin against panels of the biodefence pathogens. Broth microdilution assays were performed at neutral and acidic pH, to determine the effectiveness of the antibiotics in conditions typical of an intracellular environment. In all instances, finafloxacin demonstrated superior activity at low pH. These results highlight the importance of evaluating antimicrobial efficacy in conditions relevant to those encountered in vivo.

Subclinical atrial fibrillation detection with a floating atrial sensing dipole in single lead implantable cardioverter-defibrillator systems: Results of the SENSE trial.

INTRODUCTION: Subclinical atrial fibrillation (AF), in the form of cardiac implantable device-detected atrial high rate episodes (AHREs), has been associated with increased thromboembolism. An implantable cardioverter-defibrillator (ICD) lead with a floating atrial dipole may permit a single lead (DX) ICD system to detect AHREs. We sought to assess the utility of the DX ICD system for subclinical AF detection in patients, with a prospective multicenter, cohort-controlled trial. METHODS AND RESULTS: One hundred fifty patients without prior history of AF (age 59 ± 13 years; 108 [72%] male) were enrolled into the DX cohort and implanted with a Biotronik DX ICD system at eight centers. Age-, sex-, and left ventricular ejection fraction-matched single- and dual-chamber ICD cohorts were derived from a Cornell database and from the IMPACT trial, respectively. The primary endpoint were AHRE detection at 12 months. During median 12 months follow-up, AHREs were detected in 19 (13%) patients in the DX, 8 (5.3%) in the single-chamber, and 19 (13%) in the dual-chamber cohorts. The rate of AHRE detection was significantly higher in the DX cohort compared to the single-chamber cohort (P = .026), but not significantly different compared to the dual-chamber cohort. There were no inappropriate ICD therapies in the DX cohort. At 12 months, only 3.0% of patients in the DX cohort had sensed atrial amplitudes less than 1.0 mV. CONCLUSION: Use of a DX ICD lead allows subclinical AF detection with a single lead DX system that is superior to that of a conventional single-chamber ICD system.

Structural basis of N-Myc binding by Aurora-A and its destabilization by kinase inhibitors.

Myc family proteins promote cancer by inducing widespread changes in gene expression. Their rapid turnover by the ubiquitin-proteasome pathway is regulated through phosphorylation of Myc Box I and ubiquitination by the E3 ubiquitin ligase SCFFbxW7 However, N-Myc protein (the product of the MYCN oncogene) is stabilized in neuroblastoma by the protein kinase Aurora-A in a manner that is sensitive to certain Aurora-A-selective inhibitors. Here we identify a direct interaction between the catalytic domain of Aurora-A and a site flanking Myc Box I that also binds SCFFbxW7 We determined the crystal structure of the complex between Aurora-A and this region of N-Myc to 1.72-Å resolution. The structure indicates that the conformation of Aurora-A induced by compounds such as alisertib and CD532 is not compatible with the binding of N-Myc, explaining the activity of these compounds in neuroblastoma cells and providing a rational basis for the design of cancer therapeutics optimized for destabilization of the complex. We also propose a model for the stabilization mechanism in which binding to Aurora-A alters how N-Myc interacts with SCFFbxW7 to disfavor the generation of Lys48-linked polyubiquitin chains.