Research Note: Validation of a new differentiation approach using the commercial ASAPTM media to detect the Salmonella 441/014 vaccine strain.

Vaccination is one of the most important control tools to reduce Salmonella in poultry production. In order for a live vaccine to be licensed for field use it should be provided with the detection methods to differentiate it from field strains. This paper aims to describe the validation of an alternative method for the differentiation of the Salmonella 441/014 vaccine strain from field strains, using a chromogenic Media, ASAP from bioMérieux. The ASAP-based differentiation method was compared with already authorized methods, namely the Anicon SE Kylt PCR DIVA 1 assay and Ceva S-Check Salmonella differentiation kit, following the ISO 16140-6:2019 validation method guidelines. A Generalised Linear Model was fitted to the data to determine the inclusivity and exclusivity of differentiation methods (PCR Kylt vs. S-Check vs. ASAPTM). Statistical differences were based on a P-value level of < 0.05 (SPSS Inc., Chicago, IL). In this study, we show that the ASAP media was able to differentiate Salmonella Enteritidis vaccine strains from field strains, obtaining 100% agreement between the three differentiation assays. This differentiation approach is quicker, easier to deploy and cheaper as compared to alternative methods.

Polyester microfiber impacts on coastal sediment organic matter consumption.

As plastic pollution continues to accumulate at the seafloor, concerns around benthic ecosystem functionality heightens. This research demonstrates the systematic effects of polyester microfibers on seafloor organic matter consumption rates, an important benthic ecosystem function connected to multiple reactions and processes. We used a field-based assay to measure the loss of organic matter, both with and without polyester microfiber contamination. We identified sediment organic matter content, mud content, and mean grain size as the main drivers of organic matter consumption, however, polyester microfiber contamination decoupled ecosystem relationships and altered observed organic matter cycling dynamics. Organic matter consumption rates varied across horizontal and vertical spaces, highlighting that consumption and associated plastic effects are dependent on environmental heterogeneity at both small (within sites) and larger (between sites) scales. Our results emphasize the important role habitat heterogeneity plays in seafloor organic matter consumption and the associated effects of plastic pollution on ecosystem function.

Tumorigenesis driven by the BRAFV600E oncoprotein requires secondary mutations that overcome its feedback inhibition of migration and invasion.

BRAFV600E mutation occurs in 46% of melanomas and drives high levels of ERK activity and ERK-dependent proliferation. However, BRAFV600E is insufficient to drive melanoma in GEMM models, and 82% of human benign nevi harbor BRAFV600E mutations. We show here that BRAFV600E inhibits mesenchymal migration by causing feedback inhibition of RAC1 activity. ERK pathway inhibition induces RAC1 activation and restores migration and invasion. In cells with BRAFV600E, mutant RAC1, overexpression of PREX1, PREX2, or PTEN inactivation restore RAC1 activity and cell motility. Together, these lesions occur in 48% of BRAFV600E melanomas. Thus, although BRAFV600E activation of ERK deregulates cell proliferation, it prevents full malignant transformation by causing feedback inhibition of cell migration. Secondary mutations are, therefore, required for tumorigenesis. One mechanism underlying tumor evolution may be the selection of lesions that rescue the deleterious effects of oncogenic drivers.

Thermodynamically Stable Functionalization of Microporous Aromatic Frameworks with Sulfonic Acid Groups by Inserting Methylene Spacers.

Porous aromatic frameworks (PAFs) are an auspicious class of materials that allow for the introduction of sulfonic acid groups at the aromatic core units by post-synthetic modification. This makes PAFs promising for proton-exchange materials. However, the limited thermal stability of sulfonic acid groups attached to aromatic cores prevents high-temperature applications. Here, we present a framework based on PAF-303 where the acid groups were added as methylene sulfonic acid side chains in a two-step post-synthetic route (SMPAF-303) via the intermediate chloromethylene PAF (ClMPAF-303). Elemental analysis, NMR spectroscopy, electrochemical impedance spectroscopy and X-ray photoelectron spectroscopy were used to characterize both frameworks and corroborate the successful attachment of the side chains. The resulting framework SMPAF-303 features high thermal stability and an ion-exchange capacity of about 1.7 mequiv g-1. The proton conductivity depends strongly on the adsorbed water level. It reaches from about 10-7 S cm-1 for 33% RH to about 10-1 S cm-1 for 100% RH. We attribute the strong change to a locally alternating polarity of the inner surfaces. The latter introduces bottleneck effects for the water molecule and oxonium ion diffusion at lower relative humidities, due to electrolyte clustering. When the pores are completely filled with water, these bottlenecks vanish, leading to an unhindered electrolyte diffusion through the framework, explaining the conductivity rise.

"Candidatus Siderophilus nitratireducens": a putative nap-dependent nitrate-reducing iron oxidizer within the new order Siderophiliales.

Nitrate leaching from agricultural soils is increasingly found in groundwater, a primary source of drinking water worldwide. This nitrate influx can potentially stimulate the biological oxidation of iron in anoxic groundwater reservoirs. Nitrate-dependent iron-oxidizing (NDFO) bacteria have been extensively studied in laboratory settings, yet their ecophysiology in natural environments remains largely unknown. To this end, we established a pilot-scale filter on nitrate-rich groundwater to elucidate the structure and metabolism of nitrate-reducing iron-oxidizing microbiomes under oligotrophic conditions mimicking natural groundwaters. The enriched community stoichiometrically removed iron and nitrate consistently with the NDFO metabolism. Genome-resolved metagenomics revealed the underlying metabolic network between the dominant iron-dependent denitrifying autotrophs and the less abundant organoheterotrophs. The most abundant genome belonged to a new Candidate order, named Siderophiliales. This new species, "Candidatus Siderophilus nitratireducens," carries genes central genes to iron oxidation (cytochrome c cyc2), carbon fixation (rbc), and for the sole periplasmic nitrate reductase (nap). Using thermodynamics, we demonstrate that iron oxidation coupled to nap based dissimilatory reduction of nitrate to nitrite is energetically favorable under realistic Fe3+/Fe2+ and NO3-/NO2- concentration ratios. Ultimately, by bridging the gap between laboratory investigations and nitrate real-world conditions, this study provides insights into the intricate interplay between nitrate and iron in groundwater ecosystems, and expands our understanding of NDFOs taxonomic diversity and ecological role.

Equine neuroaxonal dystrophy/degenerative myeloencephalopathy in Gypsy Vanner horses.

BACKGROUND: Equine neuroaxonal dystrophy/degenerative myeloencephalopathy (eNAD/EDM) is a neurodegenerative disease that primarily affects young, genetically predisposed horses that are deficient in vitamin E. Equine NAD/EDM has not previously been documented in Gypsy Vanner horses (GVs). OBJECTIVES: To evaluate: (1) the clinical phenotype, blood vitamin E concentrations before and after supplementation and pedigree in a cohort of GV horses with a high prevalence of neurologic disease suspicious for eNAD/EDM and (2) to confirm eNAD/EDM in GVs through postmortem evaluation. ANIMALS: Twenty-six GVs from 1 farm in California and 2 cases from the Midwestern U.S. METHODS: Prospective observational study on Californian horses; all 26 GVs underwent neurologic examination. Pre-supplementation blood vitamin E concentration was assessed in 17- GVs. Twenty-three were supplemented orally with 10 IU/kg of liquid RRR-alpha-tocopherol once daily for 28 days. Vitamin E concentration was measured in 23 GVs after supplementation, of which 15 (65%) had pre-supplementation measurements. Two clinically affected GVs from California and the 2 Midwestern cases had necropsy confirmation of eNAD/EDM. RESULTS: Pre-supplementation blood vitamin E concentration was ≤2.0 µg/mL in 16/17 (94%) of GVs from California. Post-supplementation concentration varied, with a median of 3.39 µg/mL (range, 1.23-13.87 µg/mL), but only 12/23 (52%) were normal (≥3.0 µg/mL). Normalization of vitamin E was significantly associated with increasing age (P = .02). Euthanized horses (n = 4) had eNAD/EDM confirmed at necropsy. CONCLUSIONS AND CLINICAL IMPORTANCE: GVs could have a genetic predisposition to eNAD/EDM. Vitamin E supplementation should be considered and monitored in young GVs.

Diagnosis of thrombotic thrombocytopenic purpura: easy-to-use fiber optic surface plasmon resonance immunoassays for automated ADAMTS-13 antigen and conformation evaluation.

BACKGROUND: Laboratory diagnosis of immune-mediated thrombotic thrombocytopenic purpura (iTTP) remains challenging when ADAMTS-13 activity ranges between 10% and 20%. To prevent misdiagnosis, open ADAMTS-13 conformation gained clinical attention as a novel biomarker, especially to diagnose acute iTTP in patients with diagnostic undecisive ADAMTS-13 activity. Plasma ADAMTS-13 conformation analysis corrects for ADAMTS-13 antigen, with both parameters being characterized in enzyme-linked immunosorbent assay (ELISA)-based reference assays requiring expert technicians. OBJECTIVES: To design ADAMTS-13 antigen and conformation assays on automated, easy-to-use fiber optic surface plasmon resonance (FO-SPR) technology to promote assay accessibility and diagnose challenging iTTP patients. METHODS: ADAMTS-13 antigen and conformation assays were designed on FO-SPR technology. Plasma of 20 healthy donors and 20 acute iTTP patients were quantified, and data from FO-SPR and ELISA reference assays were compared. RESULTS: Following assay design, both antigen and conformation FO-SPR assays were optimized and characterized, presenting strong analytical sensitivity (detection limit of 0.001 µg/mL) and repeatability (interassay variation of 14.4%). Comparative analysis suggested positive correlation (Spearman r of 0.92) and good agreement between FO-SPR and ELISA assays. As expected, FO-SPR assays showed a closed or open ADAMTS-13 conformation in healthy donors and acute iTTP patients, respectively. CONCLUSION: Both ADAMTS-13 antigen and conformation assays were transferred onto automated, easy-to-use FO-SPR technology, displaying potent analytical sensitivity and reproducibility. ADAMTS-13 antigen and conformation were determined for healthy donors and acute iTTP patients showing strong correlation with ELISA reference. Introducing FO-SPR technology in clinical context could support routine diagnosis of acute iTTP patients, notably when ADAMTS-13 activity fluctuates between 10% and 20%.

R-tPA Resistance Is Specific for Platelet-Rich Stroke Thrombi and Can Be Overcome by Targeting Nonfibrin Components.

BACKGROUND: Resistance to r-tPA (recombinant tissue-type plasminogen activator) is a well-known but poorly understood phenomenon that hampers successful recanalization in patients with acute ischemic stroke. Using clinically relevant thrombi from patients with acute ischemic stroke, we investigated if and how thrombus composition impacts r-tPA-mediated lysis. In addition, we explored strategies to overcome r-tPA resistance. METHODS: Thrombi were split into 2 parts, 1 of which was used for thrombolysis and the other for detailed histological analysis. Thrombolysis was performed in normal human plasma using r-tPA alone, using r-tPA in combination with DNase-1 or using r-tPA in combination with N,N'-diacetyl-l-cystine. Thrombus lysis was calculated as the percentage of residual thrombus weight compared with its initial weight and the degree of lysis was linked to thrombus composition determined via histology. RESULTS: Interestingly, we found that the efficacy of r-tPA-mediated thrombolysis was strongly correlated with the composition of the thrombi. Thrombi containing high amounts of red blood cells and low amounts of DNA and von Willebrand Factor were efficiently degraded by r-tPA, whereas thrombi containing low amounts of red blood cells and higher amounts of DNA and von Willebrand Factor were resistant to r-tPA. Importantly, combination of r-tPA with DNase-1 or N,N'-diacetyl-l-cystine significantly and specifically improved the lysis of these r-tPA-resistant thrombi. CONCLUSIONS: Using patient thrombus material, our results for the first time show that the composition of stroke thrombi largely determines their susceptibility to r-tPA-mediated thrombolysis. Red blood cell-poor thrombi have a specific resistance to r-tPA, which can be overcome by targeting nonfibrin components using DNase-1 or N,N'-diacetyl-l-cystine.

The association between psoriasis and chronic obstructive pulmonary disease: a systematic review and meta-analysis.

INTRODUCTION: Psoriasis is a chronic T-cell-mediated inflammatory and proliferative skin disease. Chronic obstructive pulmonary disease (COPD) is an inflammatory disease of the airways. COPD has been studied as a comorbidity of psoriasis, but the association needs further study, hence the objective of this study. EVIDENCE ACQUISITION: A systematic review was performed using the database PubMed and 155 records were found including the ones found through references. Seven records were found eligible for this study including six observational studies and one experimental study with a total of 229,075 participants. The odds ratio of COPD in patients with psoriasis and healthy subjects was analysed using a random effects model. EVIDENCE SYNTHESIS: The pooled data showed a significant association (OR=1.77, 95% CI [1.32; 2.39]) between psoriasis and COPD with high inter-study heterogeneity (I2=96%). Sub-analyses of the different types of studies (cohort study: OR=2.53 [2.43; 2.63], case-control study: OR=1.6 [0.03; 100.96] and cross-sectional study: OR=1.57 [0.58; 4.22]) and smoking status (OR=1.7 [0.69; 4.14]) were also performed to further examine the association. CONCLUSIONS: There is a significant association between psoriasis and COPD, but the underlying mechanism and how smoking status affects the results remain unclear and need further study. Physicians should be aware of the risk and its seriousness to provide better and more targeted treatment.

Pretreatment with a dual antiplatelet and anticoagulant (APAC) reduces ischemia-reperfusion injury in a mouse model of temporary middle cerebral artery occlusion-implications for neurovascular procedures.

BACKGROUND: Several neurovascular procedures require temporary occlusion of cerebral arteries, leading to ischemia of unpredictable length, occasionally causing brain infarction. Experimental models of cerebral ischemia-reperfusion injury have established that platelet adhesion and coagulation play detrimental roles in reperfusion injury following transient cerebral ischemia. Therefore, in a model of cerebral ischemia-reperfusion injury (IRI), we investigated the therapeutic potential of a dual antiplatelet and anticoagulant (APAC) heparin proteoglycan mimetic which is able to bind to vascular injury sites. METHODS: Brain ischemia was induced in mice by transient occlusion of the right middle cerebral artery for 60 min. APAC, unfractionated heparin (UFH) (both at heparin equivalent doses of 0.5 mg/kg), or vehicle was intravenously administered 10 min before or 60 min after the start of ischemia. At 24 h later, mice were scored for their neurological and motor behavior, and brain damage was quantified. RESULTS: Both APAC and UFH administered before the onset of ischemia reduced brain injury. APAC and UFH pretreated mice had better neurological and motor functions (p < 0.05 and p < 0.01, respectively) and had significantly reduced cerebral infarct sizes (p < 0.01 and p < 0.001, respectively) at 24 h after transient occlusion compared with vehicle-treated mice. Importantly, no macroscopic bleeding complications were observed in either APAC- or UFH-treated animals. However, when APAC or UFH was administered 60 min after the start of ischemia, the therapeutic effect was lost, but without hemorrhaging either. CONCLUSIONS: Pretreatment with APAC or UFH was safe and effective in reducing brain injury in a model of cerebral ischemia induced by transient middle cerebral artery occlusion. Further studies on the use of APAC to limit ischemic injury during temporary occlusion in neurovascular procedures are indicated.

A Patient With Oral Discomfort and Reduced Oral Aperture.

A patient had burning and pain in the mouth, reduced oral aperture, white-tan plaques on the oral mucosa, and thickened buccal mucosae bilaterally; biopsy of the lower labial mucosa showed subepithelial fibrosis. She had no history of cigarette smoking or use of chewing tobacco but had current and past history of chewing areca nuts. What is the diagnosis and what would you do next?

Ulvans are not equal - Linkage and substitution patterns in ulvan polysaccharides differ with Ulva morphology.

Ulva are hardy green seaweeds that contain the sulfated polysaccharide ulvan and grow in two distinct morphologies: foliose and tubular. The authors hypothesise that ulvan from tubular species are more structurally complex than ulvans from foliose species. Herein, using standardised methods, the glycosyl linkage positions and sulfate ester substitutions of constituent monosaccharides of ulvan isolated from foliose (U. lacinulata and U. stenophylloides) and tubular (U. prolifera and U. ralfsii) species of Ulva were investigated. Comparison of native ulvans with 80 and 100 °C desulfated counterparts indicated that 4-linked rhamnose is predominantly 3-O-sulfated in all four ulvans. Ulvans from the foliose species predominantly contained →3,4)-Rhap-(1→, →4)-GlcAp-(1→ and →4)-IdoAp-(1→, collectively accounting for 67 to 81 mol% of the total linkages. In contrast, these same linkages in ulvans from the tubular species only collectively accounted for 29 to 36 mol%. Instead, ulvan from tubular species contained a combination of →2,3,4)-Rhap-(1→, terminal Rhap-(1→, →4)-GlcAp-(1→, →4)-Xylp-(1→, and/or →4)-Galp-(1→ in high proportions; some of the latter three residues were also likely O-2 sulfated. The results presented here suggest that ulvan from foliose species are predominantly unbranched polysaccharides composed of repeat disaccharides while ulvans from tubular species contain a greater diversity of branch and sulfate substitution locations.

Rational synthesis of a heparan sulfate saccharide that promotes the activity of BMP2.

Heparan sulfate (HS) is a glycosaminoglycan (GAG) found throughout nature and is involved in a wide range of functions including modulation of cell signalling via sequestration of growth factors. Current consensus is that the specificity of HS motifs for protein binding are individual for each protein. Given the structural complexity of HS the synthesis of libraries of these compounds to probe this is not trivial. Herein we present the synthesis of an HS decamer, the design of which was undertaken rationally from previously published data for HS binding to the growth factor BMP-2. The biological activity of this HS decamer was assessed in vitro, showing that it had the ability to both bind BMP-2 and increase its thermal stability as well as enhancing the bioactivity of BMP-2 in vitro in C2C12 cells. At the same time no undesired anticoagulant effect was observed. This decamer was then analysed in vivo in a rabbit model where higher bone formation, bone mineral density (BMD) and trabecular thickness were observed over an empty defect or collagen implant alone. This indicated that the HS decamer was effective in promoting bone regeneration in vivo.

Effects of power training in older patients with multiple sclerosis on neurodegeneration, neuromuscular function, and physical function. A study protocol for the "power training in older multiple sclerosis patients (PoTOMS) randomized control trial.

Introduction: Approximately one-third of all persons with multiple sclerosis (pwMS) are older, i.e., having an age ≥60 years. Whilst ageing and MS separately elicit deteriorating effects on brain morphology, neuromuscular function, and physical function, the combination of ageing and MS may pose a particular challenge. To counteract such detrimental changes, power training (i.e., a type of resistance exercise focusing on moderate-to-high loading at maximal intended movement velocity) presents itself as a viable and highly effective solution. Power training is known to positively impact physical function, neuromuscular function, as well as brain morphology. Existing evidence is promising but limited to young and middle-aged pwMS, with the effects of power training remaining to be elucidated in older pwMS. Methods: The presented 'Power Training in Older MS patients (PoTOMS)' trial is a national, multi-center, parallel-group, randomized controlled trial. The trial compares 24 weeks of usual care(n = 30) to 24 weeks of usual care and power training (n = 30). The primary outcome is whole brain atrophy rate. The secondary outcomes include changes in brain micro and macro structures, neuromuscular function, physical function, cognitive function, bone health, and patient-reported outcomes. Ethics and dissemination: The presented study is approved by The Regional Ethics Committee (reference number 1-10-72-222-20) and registered at the Danish Data Protection Agency (reference number 2016-051-000001). All study findings will be published in scientific peer-reviewed journals and presented at relevant scientific conferences independent of the results. The www.clinicaltrials.gov identifier is NCT04762342.

Spatial heterogeneity in collective electrotaxis: continuum modelling and applications to optimal control.

Collective electrotaxis is a phenomenon that occurs when a cellular collective, for example an epithelial monolayer, is subjected to an electric field. Biologically, it is well known that the velocity of migration during the collective electrotaxis of large epithelia exhibits significant spatial heterogeneity. In this work, we demonstrate that the heterogeneity of velocities in the electrotaxing epithelium can be accounted for by a continuum model of cue competition in different tissue regions. Having established a working model of competing migratory cues in the migrating epithelium, we develop and validate a reaction-convection-diffusion model that describes the movement of an epithelial monolayer as it undergoes electrotaxis. We use the model to predict how tissue size and geometry affect the collective migration of MDCK monolayers, and to propose several ways in which electric fields can be designed such that they give rise to a desired spatial pattern of collective migration. We conclude with two examples that demonstrate practical applications of the method in designing bespoke stimulation protocols.