Amoebae as training grounds for microbial pathogens.

Grazing of amoebae on microorganisms represents one of the oldest predator-prey dynamic relationships in nature. It represents a genetic "melting pot" for an ancient and continuous multi-directional inter- and intra-kingdom horizontal gene transfer between amoebae and its preys, intracellular microbial residents, endosymbionts, and giant viruses, which has shaped the evolution, selection, and adaptation of microbes that evade degradation by predatory amoeba. Unicellular phagocytic amoebae are thought to be the ancient ancestors of macrophages with highly conserved eukaryotic processes. Selection and evolution of microbes within amoeba through their evolution to target highly conserved eukaryotic processes have facilitated the expansion of their host range to mammals, causing various infectious diseases. Legionella and environmental Chlamydia harbor an immense number of eukaryotic-like proteins that are involved in ubiquitin-related processes or are tandem repeats-containing proteins involved in protein-protein and protein-chromatin interactions. Some of these eukaryotic-like proteins exhibit novel domain architecture and novel enzymatic functions absent in mammalian cells, such as ubiquitin ligases, likely acquired from amoebae. Mammalian cells and amoebae may respond similarly to microbial factors that target highly conserved eukaryotic processes, but mammalian cells may undergo an accidental response to amoeba-adapted microbial factors. We discuss specific examples of microbes that have evolved to evade amoeba predation, including the bacterial pathogens- Legionella, Chlamydia, Coxiella, Rickettssia, Francisella, Mycobacteria, Salmonella, Bartonella, Rhodococcus, Pseudomonas, Vibrio, Helicobacter, Campylobacter, and Aliarcobacter. We also discuss the fungi Cryptococcus, and Asperigillus, as well as amoebae mimiviruses/giant viruses. We propose that amoeba-microbe interactions will continue to be a major "training ground" for the evolution, selection, adaptation, and emergence of microbial pathogens equipped with unique pathogenic tools to infect mammalian hosts. However, our progress will continue to be highly dependent on additional genomic, biochemical, and cellular data of unicellular eukaryotes.

Feeder-free culture of human pluripotent stem cells drives MDM4-mediated gain of chromosome 1q.

Culture-acquired variants in human pluripotent stem cells (hPSCs) hinder their applications in research and clinic. However, the mechanisms that underpin selection of variants remain unclear. Here, through analysis of comprehensive karyotyping datasets from over 23,000 hPSC cultures of more than 1,500 lines, we explored how culture conditions shape variant selection. Strikingly, we identified an association of chromosome 1q gains with feeder-free cultures and noted a rise in its prevalence in recent years, coinciding with increased usage of feeder-free regimens. Competition experiments of multiple isogenic lines with and without a chromosome 1q gain confirmed that 1q variants have an advantage in feeder-free (E8/vitronectin), but not feeder-based, culture. Mechanistically, we show that overexpression of MDM4, located on chromosome 1q, drives variants' advantage in E8/vitronectin by alleviating genome damage-induced apoptosis, which is lower in feeder-based conditions. Our study explains condition-dependent patterns of hPSC aberrations and offers insights into the mechanisms of variant selection.

Immune Checkpoint Inhibitors in Hepatocellular Carcinoma and Their Hepatic-Related Side Effects: A Review.

Primary liver cancer is one of the leading causes of cancer mortality worldwide, with hepatocellular carcinoma (HCC) being the most prevalent type of liver cancer. The prognosis of patients with advanced, unresectable HCC has historically been poor. However, with the emergence of immunotherapy, specifically immune checkpoint inhibitors (ICIs), there is reason for optimism. Nevertheless, ICIs do not come without risk, especially when administered in patients with HCC, given their potential underlying poor hepatic reserve. Given their novelty in the management of HCC, there are few studies to date specifically investigating ICI-related side effects on the liver in patients with underlying HCC. This review will serve as a guide for clinicians on ICIs' role in the management of HCC and their potential side effect profile. There will be a discussion on ICI-related hepatotoxicity, the potential for hepatitis B and C reactivation with ICI use, the potential for the development of autoimmune hepatitis with ICI use, and the risk of gastrointestinal bleeding with ICI use. As ICIs become more commonplace as a treatment option in patients with advanced HCC, it is imperative that clinicians not only understand the mechanism of action of such agents but also understand and are able to identify hepatic-related side effects.

Long-term colchicine for the prevention of vascular recurrent events in non-cardioembolic stroke (CONVINCE): a randomised controlled trial.

BACKGROUND: Anti-inflammatory therapy with long-term colchicine prevented vascular recurrence in coronary disease. Unlike coronary disease, which is typically caused by atherosclerosis, ischaemic stroke is caused by diverse mechanisms including atherosclerosis and small vessel disease or is frequently due to an unknown cause. We aimed to investigate the hypothesis that long-term colchicine would reduce recurrent events after ischaemic stroke. METHODS: We did a randomised, parallel-group, open-label, blinded endpoint assessed trial comparing long-term colchicine (0·5 mg orally per day) plus guideline-based usual care with usual care only. Hospital-based patients with non-severe, non-cardioembolic ischaemic stroke or high-risk transient ischaemic attack were eligible. The primary endpoint was a composite of first fatal or non-fatal recurrent ischaemic stroke, myocardial infarction, cardiac arrest, or hospitalisation (defined as an admission to an inpatient unit or a visit to an emergency department that resulted in at least a 24 h stay [or a change in calendar date if the hospital admission or discharge times were not available]) for unstable angina. The p value for significance was 0·048 to adjust for two prespecified interim analyses conducted by the data monitoring committee, for which the steering committee and trial investigators remained blinded. The trial was registered at ClinicalTrials.gov (NCT02898610) and is completed. FINDINGS: 3154 patients were randomly assigned between Dec 19, 2016, and Nov 21, 2022, with the last follow-up on Jan 31, 2024. The trial finished before the anticipated number of outcomes was accrued (367 outcomes planned) due to budget constraints attributable to the COVID-19 pandemic. Ten patients withdrew consent for analysis of their data, leaving 3144 patients in the intention-to-treat analysis: 1569 (colchicine and usual care) and 1575 (usual care alone). A primary endpoint occurred in 338 patients, 153 (9·8%) of 1569 patients allocated to colchicine and usual care and 185 (11·7%) of 1575 patients allocated to usual care alone (incidence rates 3·32 vs 3·92 per 100 person-years, hazard ratio 0·84; 95% CI 0·68-1·05, p=0·12). Although no between-group difference in C-reactive protein (CRP) was observed at baseline, patients treated with colchicine had lower CRP at 28 days and at 1, 2, and 3 years (p<0·05 for all timepoints). The rates of serious adverse events were similar in both groups. INTERPRETATION: Although no statistically significant benefit was observed on the primary intention-to-treat analysis, the findings provide new evidence supporting the rationale for anti-inflammatory therapy in further randomised trials. FUNDING: Health Research Board Ireland, Deutsche Forschungsgemeinschaft (German Research Foundation), and Fonds Wetenschappelijk Onderzoek Vlaanderen (Research Foundation Flanders), Belgium.

Nicotine induces senescence in spermatogonia stem cells by disrupting homeostasis between circadian oscillation and rhythmic mitochondrial dynamics via the SIRT6/Bmal1 pathway.

Infertility is intricately linked with alterations in circadian rhythms along with physiological decline and stem cell senescence. Yet, the direct involvement of circadian mechanisms in nicotine-induced injury to the testes, especially the senescence of spermatogonia stem cells (SSCs), is not well comprehended. This study revealed that nicotine exposure induced testis injury by triggering SSCs senescence along with the upregulation of senescence marker genes and senescence-associated secretory phenotype components. Moreover, nicotine treatment caused mitochondrial hyper-fusion, increased oxidative stress, and DNA damage. Exposure to nicotine was found to suppress the expression of sirtuin 6 (SIRT6), which accelerated the senescence of spermatogonia stem cells (SSCs). This acceleration led to increased acetylation of brain and muscle ARNT-like protein (Bmal1), consequently reducing the expression of Bmal1 protein. Conversely, the overexpression of Bmal1 alleviated mitochondrial hyper-fusion and senescence phenotypes induced by nicotine. Overall, this study unveiled a novel molecular mechanism behind nicotine-induced disorders in spermatogenesis and highlighted the SIRT6/Bmal1 regulatory pathway as a potential therapeutic target for combating nicotine-associated infertility.

Healthcare professional views about a prehospital redirection pathway for stroke thrombectomy: a multiphase deductive qualitative study.

BACKGROUND: Mechanical thrombectomy for stroke is highly effective but time-critical. Delays are common because many patients require transfer between local hospitals and regional centres. A two-stage prehospital redirection pathway consisting of a simple ambulance screen followed by regional centre assessment to select patients for direct admission could optimise access. However, implementation might be challenged by the limited number of thrombectomy providers, a lack of prehospital diagnostic tests for selecting patients and whether finite resources can accommodate longer ambulance journeys plus greater central admissions. We undertook a three-phase, multiregional, qualitative study to obtain health professional views on the acceptability and feasibility of a new pathway. METHODS: Online focus groups/semistructured interviews were undertaken designed to capture important contextual influences. We purposively sampled NHS staff in four regions of England. Anonymised interview transcripts underwent deductive thematic analysis guided by the NASSS (Non-adoption, Abandonment and Challenges to Scale-up, Spread and Sustainability, Implementation) Implementation Science framework. RESULTS: Twenty-eight staff participated in 4 focus groups, 2 group interviews and 18 individual interviews across 4 Ambulance Trusts, 5 Hospital Trusts and 3 Integrated Stroke Delivery Networks (ISDNs). Five deductive themes were identified: (1) (suspected) stroke as a condition, (2) the pathway change, (3) the value participants placed on the proposed pathway, (4) the possible impact on NHS organisations/adopter systems and (5) the wider healthcare context. Participants perceived suspected stroke as a complex scenario. Most viewed the proposed new thrombectomy pathway as beneficial but potentially challenging to implement. Organisational concerns included staff shortages, increased workflow and bed capacity. Participants also reported wider socioeconomic issues impacting on their services contributing to concerns around the future implementation. CONCLUSIONS: Positive views from health professionals were expressed about the concept of a proposed pathway while raising key content and implementation challenges and useful 'real-world' issues for consideration.

FGF18 impairs blastocyst viability, DNA double-strand breaks and maternal recognition of pregnancy genes.

Embryonic mortality in cattle is high, reaching 10-40 % in vivo and 60-70 % in vitro. Death of embryos involves reduced expression of genes related to embryonic viability, inhibition of DNA repair and increased DNA damage. In follicular granulosa cells, FGF18 from the theca layer increases apoptosis and DNA damage, so we hypothesized that FGF18 may also affect the oocyte and contribute to early embryonic death. The aims of this study were to identify the effects of FGF18 on cumulus expansion, oocyte maturation and embryo development from cleavage to blastocyst stage using a conventional bovine in vitro embryo production system using ovaries of abattoir origin. Addition of FGF18 during in-vitro maturation did not affect FSH-induced cumulus expansion or rates of nuclear maturation. When FGF18 was present in the culture system, rates of cleavage were not affected however, blastocyst and expanded blastocyst development was substantially inhibited (P < 0.05), indicating a delay of blastulation. The number of phosphorylated histone H2AFX foci per nucleus, a marker of DNA damage, was higher in cleavage-stage embryos cultured with FGF18 than in those from control group (P < 0.05). Furthermore, FGF18 decreased accumulation of PTGS2 and IFNT2 mRNA in blastocysts. In conclusion, these novel findings suggest that FGF18 plays a role in the regulation of embryonic death during the early stages of development by impairing DNA double-strand break repair and expression of genes associated with embryo viability and maternal recognition of pregnancy during the progression from oocyte to expanded blastocysts.

Effects of Glucagon-Like Peptide-1 Receptor Agonists on Gastric Mucosal Visibility and Retained Gastric Contents During Esophagogastroduodenoscopy.

BACKGROUND AND AIMS: Glucagon-like peptide-1 receptor agonists (GLP-1RA) are increasingly utilized in diabetes and obesity management. GLP-1RAs delay gastric emptying; however, their impact on visibility during esophagogastroduodenoscopy (EGD) remains uncertain. METHODS: A 1:1 matched case-control study was conducted. Individuals undergoing EGD on GLP-1RAs were matched to non-users based on demographics and diabetes status. A validated scale (POLPREP) was used to determine gastric mucosal visibility scores. RESULTS: A total of 84 pairs (n=168) were included. GLP-1RA users showed significantly lower visibility scores, with a 2.54 times higher likelihood of lower scores compared to non-users. Additionally, GLP-1RA users had a higher incidence of retained gastric contents (13.1% vs. 4.8%, aOR:4.62, p=0.025) and aborted procedures due to this issue. No anesthesia-related adverse events were observed. CONCLUSIONS: GLP-1RA use at the time of endoscopy exhibited higher odds of lower gastric mucosal visibility scores, retained contents and aborted procedures. Further research is warranted.

A qualitative exploration of ambulance clinician behaviour and decision making to identify factors influencing on-scene times for suspected stroke patients in North East England.

Aims/objectives: Ambulance clinician assessment of suspected stroke patients aims to provide rapid access to specialist care, however regional and national data show increasing pre-hospital times. This study explored paramedic views about factors contributing to on-scene time (OST) for suspected stroke patients, with a view to identifying opportunities for future interventions, to reduce OST. Methods: Views of paramedics from one regional service on factors influencing OST were explored using a qualitative approach. Semi-structured interviews with volunteers were recorded, transcribed and analysed using thematic analysis. Results: Interviews were conducted with 13 paramedics between August and November 2021. Five interlinked themes were identified and described a range of factors influencing OST: 'Initial assessment and sources of information' describes how clinicians make assessments based on initial presentation, influenced by pre-arrival information from ambulance control and family members / bystanders at the scene, and how this influences OST.'Suitability for treatment and interventions' describes how paramedics consider actions such as the face, arms, speech test, cannulation, electrocardiograms and neurological assessments while recognising that pre-hospital interventions for suspected stroke are limited.'The environment' describes the influence of incident setting on OST, including the overall process needed to transport the patient to appropriate care.'Hospital interactions' describes how interactions with hospital staff influenced paramedic actions and OST.'Changing practice' describes the influence of experience and interaction with hospital staff leading to changes in paramedic practice over time. Conclusion: This study provides insight into how UK paramedics spend time on scene with stroke patients. Multiple factors influencing OST were identified which signpost opportunities for interventions designed to reduce OST. Standardising on-scene assessments for stroke patients, refining communication processes between ambulance services and hospital stroke services and increasing availability of stroke continuing professional development for paramedics were all identified as potential targets for improving OST.

Enzymatic digestion does not compromise sliding-mediated cartilage lubrication.

Articular cartilage's remarkable low-friction properties are essential to joint function. In osteoarthritis (OA), cartilage degeneration (e.g., proteoglycan loss and collagen damage) decreases tissue modulus and increases permeability. Although these changes impair lubrication in fully depressurized and slowly slid cartilage, new evidence suggests such relationships may not hold under biofidelic sliding conditions more representative of those encountered in vivo. Our recent studies using the convergent stationary contact area (cSCA) configuration demonstrate that articulation (i.e., sliding) generates interfacial hydrodynamic pressures capable of replenishing cartilage interstitial fluid/pressure lost to compressive loading through a mechanism termed tribological rehydration. This fluid recovery sustains in vivo-like kinetic friction coefficients (µk<0.02 in PBS and <0.005 in synovial fluid) with little sensitivity to mechanical properties in healthy tissue. However, the tribomechanical function of compromised cartilage under biofidelic sliding conditions remains unknown. Here, we investigated the effects of OA-like changes in cartilage mechanical properties, modeled via enzymatic digestion of mature bovine cartilage, on its tribomechanical function during cSCA sliding. We found no differences in sliding-driven tribological rehydration behaviors or µk between naïve and digested cSCA cartilage (in PBS or synovial fluid). This suggests that OA-like cartilage retains sufficient functional properties to support naïve-like fluid recovery and lubrication under biofidelic sliding conditions. However, OA-like cartilage accumulated greater total tissue strains due to elevated strain accrual during initial load application. Together, these results suggest that elevated total tissue strains-as opposed to activity-mediated strains or friction-driven wear-might be the key biomechanical mediator of OA pathology in cartilage. STATEMENT OF SIGNIFICANCE: Osteoarthritis (OA) decreases cartilage's modulus and increases its permeability. While these changes compromise frictional performance in benchtop testing under low fluid load support (FLS) conditions, whether such observations hold under sliding conditions that better represent the joints' dynamic FLS conditions in vivo is unclear. Here, we leveraged biofidelic benchtop sliding experiments-that is, those mimicking joints' native sliding environment-to examine how OA-like changes in mechanical properties effect cartilage's natural lubrication. We found no differences in sliding-mediated fluid recovery or kinetic friction behaviors between naïve and OA-like cartilage. However, OA-like cartilage experienced greater strain accumulation during load application, suggesting that elevated tissue strains (not friction-driven wear) may be the primary biomechanical mediator of OA pathology.

Exosomal or follicular FNDC3A decreases FOLR1 mRNA abundance and progesterone and lactate synthesis in bovine granulosa cells.

In brief: Dairy cattle experience a period of infertility postpartum that is caused in part by the development of IGF1/insulin resistance. This study suggests that an adipokine, FNDC3A, reduces IGF1-dependent glycolysis and may contribute to postpartum infertility. Abstract: Dairy cows go through a period of subfertility after parturition, triggered in part by a disruption of energy homeostasis. The mobilization of body fat alters the secretion of adipokines, which have been shown to impact ovarian function. Fibronectin type III domain-containing 3A (FNDC3A) is a recently discovered adipokine-myokine, and FNDC3A mRNA abundance in subcutaneous adipose tissue is increased postpartum in cattle. In this study, we hypothesized that FNDC3A may compromise granulosa cell function in cattle and investigated this using a well-established in vitro cell culture model. Here, we demonstrate the presence of FNDC3A protein associated with extracellular vesicles in follicular fluid and in plasma, suggesting an endocrine role for this adipokine. FNDC3A protein and mRNA was also detected in the bovine ovary (cortex, granulosa and theca cells, cumulus, oocyte and corpus luteum). Abundance of FNDC3A mRNA in granulosa cells from small follicles was increased by in vitro treatment with the adipokines leptin and TNF but not by visfatin, resistin, adiponectin, chemerin or IGF1. Addition of recombinant FNDC3A at physiological doses (10 ng/mL) to granulosa cells decreased IGF1-dependent progesterone but not estradiol secretion and IGF1-dependent lactate secretion and abundance of GLUT3 and GLUT4 mRNA. This concentration of FNDC3A increased cell viability, abundance of mRNA encoding a putative receptor FOLR1, and increased phosphorylation of Akt. Collectively, these data suggest that FNDC3A may regulate folliculogenesis in cattle by modulating IGF1-dependent granulosa cell steroidogenesis and glucose metabolism.

The impact of large core and late treatment trials: An update on the modelled annual thrombectomy eligibility of UK stroke patients.

INTRODUCTION: To support decisions about thrombectomy provision, we have previously estimated the annual UK population eligible for treatment as ∼10% of stroke admissions. Since then, eight further randomised trials that could alter the eligibility rate have reported in 2021-23. We updated our estimates of the eligible population from these trials and other recent studies. PATIENTS AND METHODS: An updated decision tree describing the EVT eligible population for UK stroke admissions was produced. Decision criteria were derived from the highest level of evidence available. For nodes where no specific RCT data existed, evidence was obtained from the latest systematic review(s) or the highest quality observational data. RESULTS: We estimate that 15,420 (approximately 15%) of admitted UK stroke patients are now eligible for thrombectomy, or 14,930 if advanced brain imaging using MRI/CT perfusion or collateral assessment were used in all patients. This is a 54% increase in our previous estimate in 2021. Over 50% of LAO strokes are now potentially eligible for thrombectomy. The increase in eligibility is principally due to a much larger cohort of later presenting and/or larger ischaemic core patients. CONCLUSION: Most previously independent LAO stroke patients presenting within 24 h, even in the presence of a large ischaemic core on initial non-contrast CT, should be considered for thrombectomy with use of advanced brain imaging in those presenting beyond 12 h to identify salvageable penumbral brain tissue. Treatment in most patients remains critically time-dependent and our estimates should be interpreted with this in mind.

Exogenous Collagen Crosslinking is Highly Detrimental to Articular Cartilage Lubrication.

Healthy articular cartilage is a remarkable bearing material optimized for near-frictionless joint articulation. Because its limited self-repair capacity renders it susceptible to osteoarthritis (OA), approaches to reinforce or rebuild degenerative cartilage are of significant interest. While exogenous collagen crosslinking (CXL) treatments improve cartilage's mechanical properties and increase its resistance to enzymatic degradation, their effects on cartilage lubrication remain less clear. Here, we examined how the collagen crosslinking agents genipin (GP) and glutaraldehyde (GTA) impact cartilage lubrication using the convergent stationary contact area (cSCA) configuration. Unlike classical configurations, the cSCA sustains biofidelic kinetic friction coefficients (µk) via superposition of interstitial and hydrodynamic pressurization (i.e., tribological rehydration). As expected, glutaraldehyde- and genipin-mediated CXL increased cartilage's tensile and compressive moduli. Although net tribological rehydration was retained after CXL, GP or GTA treatment drastically elevated µk. Both healthy and "OA-like" cartilage (generated via enzymatic digestion) sustained remarkably low µk in saline- (≤0.02) and synovial fluid-lubricated contacts (≤0.006). After CXL, µk increased up to 30-fold, reaching values associated with marked chondrocyte death in vitro. These results demonstrate that mechanical properties (i.e., stiffness) are necessary, but not sufficient, metrics of cartilage function. Furthermore, the marked impairment in lubrication suggests that CXL-mediated stiffening is ill-suited to cartilage preservation or joint resurfacing.

Modeling the selective growth advantage of genetically variant human pluripotent stem cells to identify opportunities for manufacturing process control.

BACKGROUND AIMS: The appearance of genetically variant populations in human pluripotent stem cell (hPSC) cultures represents a concern for research and clinical applications. Genetic variations may alter hPSC differentiation potential or cause phenotype variation in differentiated cells. Further, variants may have properties such as proliferative rate, or response to the culture environment, that differ from wild-type cells. As such, understanding the behavior of these variants in culture, and any potential operational impact on manufacturing processes, will be necessary to control quality of putative hPSC-based products that include a proportion of variant threshold in their quality specification. METHODS: Here we show a computational model that mathematically describes the growth dynamics between commonly occurring genetically variant hPSCs and their counterpart wild-type cells in culture. RESULTS: We show that our model is capable of representing the growth behaviors of both wild-type and variant hPSCs in individual and co-culture systems. CONCLUSIONS: This representation allows us to identify three critical process parameters that drive critical quality attributes when genetically variant cells are present within the system: total culture density, proportion of variant cells within the culture system and variant cell overgrowth. Lastly, we used our model to predict how the variability of these parameters affects the prevalence of both populations in culture.

Hyperspectral Mapping of Human Primary and Stem Cells at Cell-Matrix Interfaces.

Extracellular matrices interface with cells to promote cell growth and tissue development. Given this critical role, matrix mimetics are introduced to enable biomedical materials ranging from tissue engineering scaffolds and tumor models to organoids for drug screening and implant surface coatings. Traditional microscopy methods are used to evaluate such materials in their ability to support exploitable cell responses, which are expressed in changes in cell proliferation rates and morphology. However, the physical imaging methods do not capture the chemistry of cells at cell-matrix interfaces. Herein, we report hyperspectral imaging to map the chemistry of human primary and embryonic stem cells grown on matrix materials, both native and artificial. We provide the statistical analysis of changes in lipid and protein content of the cells obtained from infrared spectral maps to conclude matrix morphologies as a major determinant of biochemical cell responses. The study demonstrates an effective methodology for evaluating bespoke matrix materials directly at cell-matrix interfaces.

Prehospital transdermal glyceryl trinitrate for ultra-acute ischaemic stroke: data from the RIGHT-2 randomised sham-controlled ambulance trial.

BACKGROUND: The effect of transdermal glyceryl trinitrate (GTN, a nitrovasodilator) on clinical outcome when administered before hospital admission in suspected stroke patients is unclear. Here, we assess the safety and efficacy of GTN in the prespecified subgroup of patients who had an ischaemic stroke within the Rapid Intervention with Glyceryl trinitrate in Hypertensive stroke Trial-2 (RIGHT-2). METHODS: RIGHT-2 was an ambulance-based multicentre sham-controlled blinded-endpoint study with patients randomised within 4 hours of onset. The primary outcome was a shift in scores on the modified Rankin scale (mRS) at day 90. Secondary outcomes included death; a global analysis (Wei-Lachin test) containing Barthel Index, EuroQol-5D, mRS, telephone interview for cognitive status-modified and Zung depression scale; and neuroimaging-determined 'brain frailty' markers. Data were reported as n (%), mean (SD), median [IQR], adjusted common OR (acOR), mean difference or Mann-Whitney difference (MWD) with 95% CI. RESULTS: 597 of 1149 (52%) patients had a final diagnosis of ischaemic stroke; age 75 (12) years, premorbid mRS>2 107 (18%), Glasgow Coma Scale 14 (2) and time from onset to randomisation 67 [45, 108] min. Neuroimaging 'brain frailty' was common: median score 2 [2, 3] (range 0-3). At day 90, GTN did not influence the primary outcome (acOR for increased disability 1.15, 95% CI 0.85 to 1.54), death or global analysis (MWD 0.00, 95% CI -0.10 to 0.09). In subgroup analyses, there were non-significant interactions suggesting GTN may be associated with more death and dependency in participants randomised within 1 hour of symptom onset and in those with more severe stroke. CONCLUSIONS: In patients who had an ischaemic stroke, ultra-acute administration of transdermal GTN in the ambulance did not improve clinical outcomes in a population with more clinical and radiological frailty than seen in previous in-hospital trials.

DUSP1 mRNA modulation during porcine circovirus type 2 and porcine reproductive and respiratory syndrome virus co-infection regulates viruses replication.

The effects of porcine circovirus type 2b (PCV2b) and porcine reproductive and respiratory syndrome virus (PRRSV) co-infection in epithelial cells of the swine respiratory tract is unknown. In the present study, the newborn pig trachea cell line NPTr-CD163, which is permissive to both viruses, was persistently infected with PCV2b and then with PRRSV. Viral replication, cell viability, cytokines' mRNA expression, and modulation of cellular genes expression were evaluated in infected cells. In NPTr-CD163 co-infection model, PCV2b replication was enhanced while PRRSV replication was suppressed. Cell viability was significantly decreased during PCV2b single infection and co-infection compared to mock-infected and PRRSV single infected cells. However, no difference was observed in cell viability between PCV2b and PCV2b/PRRSV infected cells. The IL6, IL8 and IL10 mRNA expression was significantly higher in co-infected cells compared to PCV2b and PRRSV single infected cells. Moreover, the IFN-α/ß expression was significantly reduced in co-infected cells compared to PCV2b infected cells whereas it remained higher compared to PRRSV infected cells. The differential gene expression analysis revealed that the mRNA expression level of the cellular gene DUSP1 was significantly higher in all PRRSV infection models compared to PCV2b single infected cells. Knockdown of DUSP1 expression in co-infected cells significantly reduced PCV2b replication, suggesting a role for DUSP1 in PCV2b/PRRSV pathogenesis.

A prospective study of MRI biomarkers in the brain and lower limb muscles for prediction of lower limb motor recovery following stroke.

The aim of this prospective observational longitudinal study was to explore and decipher the predictive value of prospective MRI biomarkers in the brain and lower limb muscles for 3-month lower limb motor recovery following stroke. In the brain, we measured the integrity of the corticospinal tract (fractional anisotropy/"FA"). In the muscles, we measured volume, fatty replacement (fat fraction analysis and proton spectroscopy) and oedema. Measurements were taken at two time points: (1) within 4 weeks of stroke (baseline measurement, clinical and imaging) and (2) 3 months following stroke (follow up measurement, clinical only). Clinical measurements consisted of assessments of functional ability and strength (Fugl-Meyer score, motor NIHSS, Functional Ambulation Category/"FAC", and muscle dynamometry). Twenty-three patients completed imaging and clinical assessments at baseline and follow-up; five patients had partial imaging assessment. The results provided some evidence that damage to the corticospinal tract would result in less motor recovery: recovery of the Fugl-Meyer score and dynamometric ankle plantarflexion, ankle dorsiflexion, and knee extension correlated positively and significantly with fractional anisotropy (0.406-0.457; p = 0.034-p = 0.016). However, fractional anisotropy demonstrated a negative correlation with recovery of the Functional Ambulation Category (-0.359, p = 0.046). For the muscle imaging, significant inverse correlation was observed between vastus lateralis fat fraction vs. NIHSS recovery (-0.401, p = 0.04), and a strong positive correlation was observed between ratio of intra- to extra-myocellular lipid concentrations and the recovery of knee flexion (0.709, p = 0.007). This study supports previous literature indicating a positive correlation between the integrity of the corticospinal tract and motor recovery post-stroke, expanding the limited available literature describing this relationship specifically for the lower limb. However, recovery of functional ambulation behaved differently to other clinical recovery markers by demonstrating an inverse relationship with corticospinal tract integrity. The study also introduces some muscle imaging biomarkers as potentially valuable in the prediction of 3-month lower limb motor recovery following stroke.

Targeted Synthesis of End-On Dinitrogen-Bridged Lanthanide Metallocenes and Their Reactivity as Divalent Synthons.

High-yield syntheses of the lanthanide dinitrogen complexes [(Cp2tttM)2(µ-1,2-N2)] (1M, M = Gd, Tb, Dy; Cpttt = 1,2,4-C5tBu3H2), in which the [N2]2- ligands solely adopt the rare end-on or 1,2-bridging mode, are reported. The bulk of the tert-butyl substituents and the smaller radii of gadolinium, terbium, and dysprosium preclude formation of the side-on dinitrogen bonding mode on steric grounds. Elongation of the nitrogen-nitrogen bond relative to N2 is observed in 1M, and their Raman spectra show a major absorption consistent with N═N double bonds. Computational analysis of 1Gd identifies that the local symmetry of the metallocene units lifts the degeneracy of two 5dπ orbitals, leading to differing overlap with the π* orbitals of [N2]2-, a consequence of which is that the dinitrogen ligand occupies a singlet ground state. Magnetic measurements reveal antiferromagnetic exchange in 1M and single-molecule magnet (SMM) behavior in 1Dy. Ab initio calculations show that the magnetic easy axis in the ground doublets of 1Tb and 1Dy align with the {M-N═N-M} connectivity, in contrast to the usual scenario in dysprosium metallocene SMMs, where the axis passes through the cyclopentadienyl ligands. The [N2]2- ligands in 1M allow these compounds to be regarded as two-electron reducing agents, serving as synthons for divalent gadolinium, terbium, and dysprosium. Proof of principle for this concept is obtained in the reactions of 1M with 2,2'-bipyridyl (bipy) to give [Cp2tttM(κ2-bipy)] (2M, M = Gd, Tb, Dy), in which the lanthanide is ligated by a bipy radical anion, with strong metal-ligand direct exchange coupling.