Integrated platform for multiscale molecular imaging and phenotyping of the human brain.

Understanding cellular architectures and their connectivity is essential for interrogating system function and dysfunction. However, we lack technologies for mapping the multiscale details of individual cells and their connectivity in the human organ-scale system. We developed a platform that simultaneously extracts spatial, molecular, morphological, and connectivity information of individual cells from the same human brain. The platform includes three core elements: a vibrating microtome for ultraprecision slicing of large-scale tissues without losing cellular connectivity (MEGAtome), a polymer hydrogel-based tissue processing technology for multiplexed multiscale imaging of human organ-scale tissues (mELAST), and a computational pipeline for reconstructing three-dimensional connectivity across multiple brain slabs (UNSLICE). We applied this platform for analyzing human Alzheimer's disease pathology at multiple scales and demonstrating scalable neural connectivity mapping in the human brain.

Carotid atherosclerotic plaque microcalcification is independently associated with recurrent neurovascular events: a pilot study.

BACKGROUND: Microcalcification and macrocalcification are critical processes in atherosclerotic plaque progression, though how these processes relate to the risk of stroke recurrence in symptomatic carotid atherosclerosis is poorly understood. METHODS: We performed a post-hoc analysis of data from the ICARUSS study, where individuals with acute ischemic stroke originating from ipsilateral carotid stenosis of ≥50% underwent 18F-sodium fluoride-positron emission tomography (NaF-PET) to measure microcalcification. Tracer uptake was quantified using maximum tissue-to-background ratio (TBRmax). Macrocalcification was measured on computed tomography (CT) using Agatston scoring. Patients were followed up for six months for recurrent ipsilateral neurovascular events. RESULTS: Five (27.8%) of 18 individuals had a recurrent ischemic stroke or transient ischemic attack. Ipsilateral carotid plaque NaF uptake at baseline was higher in those with recurrent events compared to those without, and this association remained after adjustment for other vascular risk factors (OR 1.24, 1.03-1.50). Macrocalcification score in the symptomatic artery was also significantly independently associated with ipsilateral recurrence, but the effect size was relatively smaller (OR 1.12, 1.06-1.17 for each 100 unit increase). CONCLUSIONS: Our findings indicate that microcalcification in symptomatic carotid plaques is independently associated with ipsilateral ischemic stroke recurrence. Furthermore, differences in the extent of active microcalcification in macrocalcified plaques may help explain variation in the relationship between calcified carotid plaques and stroke recurrence reported in the literature. Our pilot study indicates that evaluation of carotid artery microcalcification using NaF-PET may be a useful method for risk-stratification of carotid atherosclerosis, though our findings require confirmation in larger cohorts.

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.

Rapid synthesis of hydrogen bond templated handcuff rotaxanes.

The rapid synthesis of hydrogen bond templated handcuff rotaxanes is described. The isolated rotaxanes were characterized by NMR and IR spectroscopies and high resolution mass spectrometry. This report represents a rare demonstration of preparing (2)handcuff [2]rotaxanes by covalently linking separate axles threaded through the rings of a bis-macrocycle by use of the copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction.

Genomic and taxonomic evaluation of 38 Treponema prophage sequences.

BACKGROUND: Despite Spirochetales being a ubiquitous and medically important order of bacteria infecting both humans and animals, there is extremely limited information regarding their bacteriophages. Of the genus Treponema, there is just a single reported characterised prophage. RESULTS: We applied a bioinformatic approach on 24 previously published Treponema genomes to identify and characterise putative treponemal prophages. Thirteen of the genomes did not contain any detectable prophage regions. The remaining eleven contained 38 prophage sequences, with between one and eight putative prophages in each bacterial genome. The prophage regions ranged from 12.4 to 75.1 kb, with between 27 and 171 protein coding sequences. Phylogenetic analysis revealed that 24 of the prophages formed three distinct sequence clusters, identifying putative myoviral and siphoviral morphology. ViPTree analysis demonstrated that the identified sequences were novel when compared to known double stranded DNA bacteriophage genomes. CONCLUSIONS: In this study, we have started to address the knowledge gap on treponeme bacteriophages by characterising 38 prophage sequences in 24 treponeme genomes. Using bioinformatic approaches, we have been able to identify and compare the prophage-like elements with respect to other bacteriophages, their gene content, and their potential to be a functional and inducible bacteriophage, which in turn can help focus our attention on specific prophages to investigate further.

Molecular Screening for Digital Dermatitis-Associated Treponemes in Bovine Ischaemic Teat Necrosis Lesions and Milk in Dairy Cattle.

Bovine ischaemic teat necrosis (ITN) is a disease affecting the skin of the teats of dairy cows with an unknown aetiopathogenesis. Digital dermatitis (DD)-associated treponemes have previously been suggested as a potential aetiological agent in ITN, although the sample size was small. The current study, using established PCR techniques, aimed to examine the association with the presence of DD-associated treponemes in a large number of ITN samples from a wider geographical area, and surveyed the potential of milk as an infection reservoir. From 95 ITN lesions, 35.8% (n = 34) were positive for at least one DD-associated treponeme compared with only 5.6% (n = 1) of 18 non-lesioned teats from cows with ITN lesions on a different teat using a nested PCR approach. All 10 age- and production-matched control cows were negative for DD-associated treponemes via PCR. No DD-associated treponemes could be detected from foremilk of cows with (n = 19) and without (n = 31) a DD lesion on the hind feet. DD-associated treponemes could be detected via PCR after incubation in milk for up to 2 h. Therefore, milk does not appear to be a competent reservoir for transmission of DD-associated treponemes. Moreover, in the current study DD-associated treponemes were only detected in a subset of ITN samples, so it is unlikely these opportunistic skin-associated pathogens are the major or sole agent of ITN.

Management of carotid atherosclerosis in stroke.

Internal carotid artery atherosclerosis is a major risk factor for stroke, accounting for 15-20% of ischaemic strokes. Revascularisation procedures-either carotid endarterectomy or carotid artery stenting-can reduce the risk of stroke for those with significant (>50%) luminal stenosis but particularly for those with more severe (70-99%) stenosis. However, advances in medical pharmacotherapy have implications for the relative benefit from surgery for symptomatic carotid atherosclerosis, as well as our approach to asymptomatic disease. This review considers the evidence underpinning the current medical and surgical management of symptomatic carotid atherosclerosis, the importance of factors beyond the degree of luminal stenosis, and developments in therapeutic strategies. We also discuss the importance of non-stenotic but high-risk carotid atherosclerotic plaques on the cause of stroke, and their implications for clinical practice.

Positive selection in the genomes of two Papua New Guinean populations at distinct altitude levels.

Highlanders and lowlanders of Papua New Guinea have faced distinct environmental stress, such as hypoxia and environment-specific pathogen exposure, respectively. In this study, we explored the top genomics regions and the candidate driver SNPs for selection in these two populations using newly sequenced whole-genomes of 54 highlanders and 74 lowlanders. We identified two candidate SNPs under selection - one in highlanders, associated with red blood cell traits and another in lowlanders, which is associated with white blood cell count - both potentially influencing the heart rate of Papua New Guineans in opposite directions. We also observed four candidate driver SNPs that exhibit linkage disequilibrium with an introgressed haplotype, highlighting the need to explore the possibility of adaptive introgression within these populations. This study reveals that the signatures of positive selection in highlanders and lowlanders of Papua New Guinea align closely with the challenges they face, which are specific to their environments.

Brief High-Velocity Motor Skill Training Increases Step Frequency and Improves Length/Frequency Coordination in Slow Walkers With Chronic Motor-Incomplete Spinal Cord Injury.

OBJECTIVE: To quantify spatiotemporal coordination during overground walking among persons with motor-incomplete spinal cord injury (PwMISCI) by calculating the step length (SL)/step frequency (SF) ratio (ie, the Walk Ratio [WR]) and to examine the effects of motor skill training (MST) on the relationship between changes in these parameters and walking speed (WS). DESIGN: Between-day exploratory analysis. SETTING: Research laboratory in a rehabilitation hospital PARTICIPANTS: PwMISCI (N=26). INTERVENTIONS: 3-day high-velocity MST. MAIN OUTCOME MEASURES: Overground WS, SL, SF, and WR measured during the 10-Meter Walk Test. RESULTS: Among the full sample, MST was associated with increases in WS, SL, SF, and a decrease in the WR. Relative change in WS and SF was higher among slow (ΔWS=↑46%, ΔSF=↑28%) vs fast (ΔWS=↑16%, ΔSF=↑8%) walkers. Change in the WR differed between groups (slow: ΔWR=↓10%; fast: ΔWR=0%). Twenty-six percent of the variability observed in ΔWR among slow walkers could be explained by ΔSF, while ΔSL did not contribute to ΔWR. Among fast walkers, ΔSL accounted for more than twice the observed ΔWR (43%) compared to ΔSF (15%). CONCLUSIONS: On the whole, WR values among PwMISCI are higher than previous reports in other neurologic populations; however, values among fast walkers were comparable to noninjured adults. Slow walkers demonstrated greater variability in the WR, with higher values associated with slower WS. Following MST, increases in WS coincided with a decrease in the WR among slow walkers, mediated primarily through an effect on SF. This finding may point to a specific mechanism by which MST facilitates improvements in WS among PwMISCI with greater mobility deficits.

A pyridine-N-oxide catenane for cation recognition.

The rapid preparation of a pyridine-N-oxide containing [2]catenane is described. The [2]catenane was characterized by NMR spectroscopy, mass spectrometry and X-ray single crystal structure determination. 1H NMR titration experiments reveal the [2]catenane may be reversibly protonated, as well as an ability to bind lithium cations more strongly than sodium cations.

Characterisation of Putative Outer Membrane Proteins from Leptospira borgpetersenii Serovar Hardjo-Bovis Identifies Novel Adhesins and Diversity in Adhesion across Genomospecies Orthologs.

Leptospirosis is a zoonotic bacterial disease affecting mammalian species worldwide. Cattle are a major susceptible host; infection with pathogenic Leptospira spp. represents a public health risk and results in reproductive failure and reduced milk yield, causing economic losses. The characterisation of outer membrane proteins (OMPs) from disease-causing bacteria dissects pathogenesis and underpins vaccine development. As most leptospire pathogenesis research has focused on Leptospira interrogans, this study aimed to characterise novel OMPs from another important genomospecies, Leptospira borgpetersenii, which has global distribution and is relevant to bovine and human diseases. Several putative L. borgpetersenii OMPs were recombinantly expressed, refolded and purified, and evaluated for function and immunogenicity. Two of these unique, putative OMPs (rLBL0972 and rLBL2618) bound to immobilised fibronectin, laminin and fibrinogen, which, together with structural and functional data, supports their classification as leptospiral adhesins. A third putative OMP (rLBL0375), did not exhibit saturable adhesion ability but, together with rLBL0972 and the included control, OmpL1, demonstrated significant cattle milk IgG antibody reactivity from infected cows. To dissect leptospire host-pathogen interactions further, we expressed alleles of OmpL1 and a novel multi-specific adhesin, rLBL2618, from a variety of genomospecies and surveyed their adhesion ability, with both proteins exhibiting divergences in extracellular matrix component binding specificity across synthesised orthologs. We also observed functional redundancy across different L. borgspetersenii OMPs which, together with diversity in function across genomospecies orthologs, delineates multiple levels of plasticity in adhesion that is potentially driven by immune selection and host adaptation. These data identify novel leptospiral proteins which should be further evaluated as vaccine and/or diagnostic candidates. Moreover, functional redundancy across leptospire surface proteins together with identified adhesion divergence across genomospecies further dissect the complex host-pathogen interactions of a genus responsible for substantial global disease burden.

Use of oxygen-loaded nanobubbles to improve tissue oxygenation: Bone-relevant mechanisms of action and effects on osteoclast differentiation.

Gas-loaded nanobubbles have potential as a method of oxygen delivery to increase tumour oxygenation and therapeutically alleviate tumour hypoxia. However, the mechanism(s) whereby oxygen-loaded nanobubbles increase tumour oxygenation are unknown; with their calculated oxygen-carrying capacity being insufficient to explain this effect. Intra-tumoural hypoxia is a prime therapeutic target, at least partly due to hypoxia-dependent stimulation of the formation and function of bone-resorbing osteoclasts which establish metastatic cells in bone. This study aims to investigate potential mechanism(s) of oxygen delivery and in particular the possible use of oxygen-loaded nanobubbles in preventing bone metastasis via effects on osteoclasts. Lecithin-based nanobubbles preferentially interacted with phagocytic cells (monocytes, osteoclasts) via a combination of lipid transfer, clathrin-dependent endocytosis and phagocytosis. This interaction caused general suppression of osteoclast differentiation via inhibition of cell fusion. Additionally, repeat exposure to oxygen-loaded nanobubbles inhibited osteoclast formation to a greater extent than nitrogen-loaded nanobubbles. This gas-dependent effect was driven by differential effects on the fusion of mononuclear precursor cells to form pre-osteoclasts, partly due to elevated potentiation of RANKL-induced ROS by nitrogen-loaded nanobubbles. Our findings suggest that oxygen-loaded nanobubbles could represent a promising therapeutic strategy for cancer therapy; reducing osteoclast formation and therefore bone metastasis via preferential interaction with monocytes/macrophages within the tumour and bone microenvironment, in addition to known effects of directly improving tumour oxygenation.

t-EER: Parameter-Free Tandem Evaluation of Countermeasures and Biometric Comparators.

Presentation attack (spoofing) detection (PAD) typically operates alongside biometric verification to improve reliablity in the face of spoofing attacks. Even though the two sub-systems operate in tandem to solve the single task of reliable biometric verification, they address different detection tasks and are hence typically evaluated separately. Evidence shows that this approach is suboptimal. We introduce a new metric for the joint evaluation of PAD solutions operating in situ with biometric verification. In contrast to the tandem detection cost function proposed recently, the new tandem equal error rate (t-EER) is parameter free. The combination of two classifiers nonetheless leads to a set of operating points at which false alarm and miss rates are equal and also dependent upon the prevalence of attacks. We therefore introduce the concurrent t-EER, a unique operating point which is invariable to the prevalence of attacks. Using both modality (and even application) agnostic simulated scores, as well as real scores for a voice biometrics application, we demonstrate application of the t-EER to a wide range of biometric system evaluations under attack. The proposed approach is a strong candidate metric for the tandem evaluation of PAD systems and biometric comparators.

Gain-of-function research and model organisms in biology.

So-called 'gain-of-function' (GOF) research is virological research that results in a virus substantially more virulent or transmissible than its wild antecedent. GOF research has been subject to ethical analysis in the past, but the methods of GOF research have to date been underexamined by philosophers in these analyses. Here, we examine the typical animal used in influenza GOF experiments, the ferret, and show how despite its longstanding use, it does not easily satisfy the desirable criteria for an animal model We then discuss the limitations of the ferret model, and how those epistemic limitations bear on ethical and policy questions around the risks and benefits of GOF research. We conclude with a reflection on how philosophy of science can contribute to ethical and policy debates around the risks, benefits and relative priority of life sciences research.

Tracking cellular uptake, intracellular trafficking and fate of nanoclay particles in human bone marrow stromal cells.

Clay nanoparticles, in particular synthetic smectites, have generated interest in the field of tissue engineering and regenerative medicine due to their utility as cross-linkers for polymers in biomaterial design and as protein release modifiers for growth factor delivery. In addition, recent studies have suggested a direct influence on the osteogenic differentiation of responsive stem and progenitor cell populations. Relatively little is known however about the mechanisms underlying nanoclay bioactivity and in particular the cellular processes involved in nanoclay-stem cell interactions. In this study we employed confocal microscopy, inductively coupled plasma mass spectrometry and transmission electron microscopy to track the interactions between clay nanoparticles and human bone marrow stromal cells (hBMSCs). In particular we studied nanoparticle cellular uptake mechanisms and uptake kinetics, intracellular trafficking pathways and the fate of endocytosed nanoclay. We found that nanoclay particles present on the cell surface as µm-sized aggregates, enter hBMSCs through clathrin-mediated endocytosis, and their uptake kinetics follow a linear increase with time during the first week of nanoclay addition. The endocytosed particles were observed within the endosomal/lysosomal compartments and we found evidence for both intracellular degradation of nanoclay and exocytosis as well as an increase in autophagosomal activity. Inhibitor studies indicated that endocytosis was required for nanoclay upregulation of alkaline phosphatase activity but a similar dependency was not observed for autophagy. This study into the nature of nanoclay-stem cell interactions, in particular the intracellular processing of nanosilicate, may provide insights into the mechanisms underlying nanoclay bioactivity and inform the successful utilisation of clay nanoparticles in biomaterial design.

Early experience of thalidomide therapy for high-grade peripheral and facial arteriovenous malformations.

BACKGROUND: Arteriovenous malformations (AVMs) are developmental defects in the vascular system with abnormal connections between arteries and veins. A minority of AVMs are characterized by aggressive growth and continue to proliferate despite maximal surgical and interventional therapy. We report our outcomes with the use of thalidomide as the only UK specialist center adopting this novel approach for the management of AVMs refractory to conventional therapy. METHODS: This was a retrospective case series which included only complex and proliferative AVM lesions (Schobinger grade III and IV). All patients prescribed thalidomide on a compassionate basis between September 2006 and August 2022 after attempts at embolosclerotherapy without satisfactory response were reviewed. RESULTS: Eleven patients were included in our study. The median total duration of thalidomide use was 10 months. Two thirds of patients with pain (six of nine) reported an improvement, three quarters reported a reduction in swelling (six of eight) and all who presented with bleeding reported improvement in overall volume or frequency (four of four). Over the study period, 45% achieved a non-proliferative state with no further target vessel demonstrable on angiography. Mild, tolerable side effects such as fatigue were common (73%). There was only one major adverse reaction (neutropenia) necessitating cessation of therapy. CONCLUSIONS: We can conclude that thalidomide is able to reduce the symptom burden for patients with complex and proliferative AVMs that were refractory to established treatment modalities. Adverse effects are common, but the benefit achieved from taking thalidomide in otherwise treatment resistant cases outweighs the risks, most of which are manageable.

Novel Targets for Molecular Imaging of Inflammatory Processes of Carotid Atherosclerosis: A Systematic Review.

Computed tomography angiography (CTA), magnetic resonance angiography (MRA) and 18F-FDG-PET have proven clinical value when evaluating patients with carotid atherosclerosis. In this systematic review, we will focus on the role of novel molecular imaging tracers in that assessment and their potential strengths to stratify stroke risk. We systematically searched PubMed, Embase, the Web of Science Core Collection, and Cochrane Library for articles reporting on molecular imaging to noninvasively detect or characterize inflammation in carotid atherosclerosis. As our focus was on nonclassical novel targets, we omitted reports solely on 18F-FDG and 18F-NaF. We summarized and mapped the selected studies to provide an overview of the current clinical development in molecular imaging in relation to risk factors, imaging and histological findings, diagnostic and prognostic performance. We identified 20 articles in which the utilized tracers to visualize carotid wall inflammation were somatostatin subtype-2- (SST2-) (n = 5), CXC-motif chemokine receptor 4- (CXCR4-) (n = 3), translocator protein- (TSPO-) (n = 2) and aVß3 integrin-ligands (n = 2) and choline-tracers (n = 2). Tracer uptake correlated with traditional cardiovascular risk factors, that is, age, gender, diabetes, hypercholesterolemia, and hypertension as well as prior cardiovascular disease. We identified discrepancies between tracer uptake and grade of stenosis, plaque calcification, and 18F-FDG uptake, suggesting the importance of alternative characterization of atherosclerosis beyond classical neuroimaging features. Immunohistochemical analysis linked tracer uptake to markers of macrophage infiltration and neovascularization. Symptomatic carotid arteries showed higher uptake compared to asymptomatic (including contralateral, nonculprit) arteries. Some studies demonstrated a potential role of these novel molecular imaging as a specific intermediary (bio)marker for outcome. Several novel tracers show promise for identification of high-risk plaque inflammation. Based on the current evidence we cautiously propose the SST2-ligands and the choline radiotracers as viable candidates for larger prospective longitudinal outcome studies to evaluate their predictive use in clinical practice.