Mechanisms and regulation of endothelial VEGF receptor signalling.

Vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs) are uniquely required to balance the formation of new blood vessels with the maintenance and remodelling of existing ones, during development and in adult tissues. Recent advances have greatly expanded our understanding of the tight and multi-level regulation of VEGFR2 signalling, which is the primary focus of this Review. Important insights have been gained into the regulatory roles of VEGFR-interacting proteins (such as neuropilins, proteoglycans, integrins and protein tyrosine phosphatases); the dynamics of VEGFR2 endocytosis, trafficking and signalling; and the crosstalk between VEGF-induced signalling and other endothelial signalling cascades. A clear understanding of this multifaceted signalling web is key to successful therapeutic suppression or stimulation of vascular growth.

c-Src-induced vascular malformations require localised matrix degradation at focal adhesions.

Endothelial cells lining the blood vessel wall communicate intricately with the surrounding extracellular matrix, translating mechanical cues into biochemical signals. Moreover, vessels require the capability to enzymatically degrade the matrix surrounding them, to facilitate vascular expansion. c-Src plays a key role in blood vessel growth, with its loss in the endothelium reducing vessel sprouting and focal adhesion signalling. Here, we show that constitutive activation of c-Src in endothelial cells results in rapid vascular expansion, operating independently of growth factor stimulation or fluid shear stress forces. This is driven by an increase in focal adhesion signalling and size, with enhancement of localised secretion of matrix metalloproteinases responsible for extracellular matrix remodelling. Inhibition of matrix metalloproteinase activity results in a robust rescue of the vascular expansion elicited by heightened c-Src activity. This supports the premise that moderating focal adhesion-related events and matrix degradation can counteract abnormal vascular expansion, with implications for pathologies driven by unusual vascular morphologies.

Endothelial c-Src Mediates Neovascular Tuft Formation in Oxygen-Induced Retinopathy.

Vascular retinopathy, characterized by abnormal blood vessel growth in the retina, frequently results in vision impairment or loss. Neovascular tufts, a distinctive pathologic feature of this condition, are highly leaky blood vessel structures, exacerbating secondary complications. Despite their clinical significance, the mechanisms underlying tuft development are not fully elucidated, posing challenges for effective management and treatment of vascular retinopathy. This study investigates the role of c-Src in neovascular tuft formation. Although c-Src has been acknowledged as a pivotal regulator in developmental angiogenesis within the retinal vasculature, its specific role in governing pathologic retinal angiogenesis remains to be fully understood. The oxygen-induced retinopathy model was used for neovascular tuft formation in both Cre-mediated vascular-specific c-Src knockout mice and wild-type littermates. High-resolution imaging and analysis of isolated retinas were conducted. c-Src depletion demonstrated a significant reduction in neovascular tufts within the oxygen-induced retinopathy model. This decrease in tuft formation was observed independently of any alterations in cell death, cell proliferation, or cell adhesion, and the absence of c-Src did not impact tuft pericyte coverage and junctional morphology. These findings underline the critical role of c-Src in the pathogenesis of neovascular tufts in vascular retinopathy. Understanding the molecular mechanisms involving c-Src may offer valuable insights for the development of targeted therapies aimed at mitigating vision-threatening complications associated with retinopathy.

Pre-vaccination frequency of circulatory Tfh is associated with robust immune response to TV003 dengue vaccine.

It has been estimated that more than 390 million people are infected with Dengue virus every year; around 96 millions of these infections result in clinical pathologies. To date, there is only one licensed viral vector-based Dengue virus vaccine CYD-TDV approved for use in dengue endemic areas. While initially approved for administration independent of serostatus, the current guidance only recommends the use of this vaccine for seropositive individuals. Therefore, there is a critical need for investigating the influence of Dengue virus serostatus and immunological mechanisms that influence vaccine outcome. Here, we provide comprehensive evaluation of sero-status and host immune factors that correlate with robust immune responses to a Dengue virus vector based tetravalent vaccine (TV003) in a Phase II clinical cohort of human participants. We observed that sero-positive individuals demonstrate a much stronger immune response to the TV003 vaccine. Our multi-layered immune profiling revealed that sero-positive subjects have increased baseline/pre-vaccination frequencies of circulating T follicular helper (cTfh) cells and the Tfh related chemokine CXCL13/BLC. Importantly, this baseline/pre-vaccination cTfh profile correlated with the vaccinees' ability to launch neutralizing antibody response against all four sero-types of Dengue virus, an important endpoint for Dengue vaccine clinical trials. Overall, we provide novel insights into the favorable cTfh related immune status that persists in Dengue virus sero-positive individuals that correlate with their ability to mount robust vaccine specific immune responses. Such detailed interrogation of cTfh cell biology in the context of clinical vaccinology will help uncover mechanisms and targets for favorable immuno-modulatory agents.

Improved Durability to SARS-CoV-2 Vaccine Immunity following Coimmunization with Molecular Adjuvant Adenosine Deaminase-1.

Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines have demonstrated strong immunogenicity and protection against severe disease, concerns about the duration and breadth of these responses remain. In this study, we show that codelivery of plasmid-encoded adenosine deaminase-1 (pADA) with SARS-CoV-2 spike glycoprotein DNA enhances immune memory and durability in vivo. Coimmunized mice displayed increased spike-specific IgG of higher affinity and neutralizing capacity as compared with plasmid-encoded spike-only-immunized animals. Importantly, pADA significantly improved the longevity of these enhanced responses in vivo. This coincided with durable increases in frequencies of plasmablasts, receptor-binding domain-specific memory B cells, and SARS-CoV-2-specific T follicular helper cells. Increased spike-specific T cell polyfunctionality was also observed. Notably, animals coimmunized with pADA had significantly reduced viral loads compared with their nonadjuvanted counterparts in a SARS-CoV-2 infection model. These data suggest that pADA enhances immune memory and durability and supports further translational studies.

Corrigendum: Commensal bacteria make GPCR ligands that mimic human signalling molecules.

This corrects the article DOI: 10.1038/nature23874.

Cognitive Enhancement, Hyperagency, and Responsibility Explosion.

Hyperagency objections appeal to the risk that cognitive enhancement may negatively impact our well-being by giving us too much control. I charitably formulate and engage with a prominent version of this objection due to Sandel (2009) -viz., that cognitive enhancement may negatively impact our well-being by creating an "explosion" of responsibilities. I first outline why this worry might look prima facie persuasive, and then I show that it can ultimately be defended against. At the end of the day, if we are to resist cognitive enhancement, it should not be based on a Sandel-style hyperagency argument.

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.

Signatures of adaptive decreased virulence of deformed wing virus in an isolated population of wild honeybees (Apis mellifera).

Understanding the ecological and evolutionary processes that drive host-pathogen interactions is critical for combating epidemics and conserving species. The Varroa destructor mite and deformed wing virus (DWV) are two synergistic threats to Western honeybee (Apis mellifera) populations across the globe. Distinct honeybee populations have been found to self-sustain despite Varroa infestations, including colonies within the Arnot Forest outside Ithaca, NY, USA. We hypothesized that in these bee populations, DWV has been selected to produce an avirulent infection phenotype, allowing for the persistence of both host and disease-causing agents. To investigate this, we assessed the titre of viruses in bees from the Arnot Forest and managed apiaries, and assessed genomic variation and virulence differences between DWV isolates. Across groups, we found viral abundance was similar, but DWV genotypes were distinct. We also found that infections with isolates from the Arnot Forest resulted in higher survival and lower rates of symptomatic deformed wings, compared to analogous isolates from managed colonies, providing preliminary evidence to support the hypothesis of adaptive decreased viral virulence. Overall, this multi-level investigation of virus genotype and phenotype indicates that host ecological context can be a significant driver of viral evolution and host-pathogen interactions in honeybees.

PET-RAFT Polymerization of Star Polymers with Folded ortho-Phenylene Cores.

ortho-Phenylenes are one of the simplest classes of aromatic foldamers, adopting helical geometries because of aromatic stacking interactions. The folding and misfolding of ortho-phenylenes are slow on the NMR timescale at or below room temperature, allowing detection of folding states using 1 H NMR spectroscopy. Herein, an ortho-phenylene hexamer is coupled with a RAFT chain transfer agent (CTA) on each repeat unit. A variety of acrylic monomers are polymerized onto the CTA-functionalized ortho-phenylene using PET-RAFT to yield functionalized star polymers with ortho-phenylene cores. The steric bulk of the acrylate monomer units as well as the chain length of each arm of the star polymer is varied. 1 H NMR spectroscopy shows that the folding of the ortho-phenylenes do not vary, providing a robust helical core for star polymer systems.

Commensal bacteria make GPCR ligands that mimic human signalling molecules.

Commensal bacteria are believed to have important roles in human health. The mechanisms by which they affect mammalian physiology remain poorly understood, but bacterial metabolites are likely to be key components of host interactions. Here we use bioinformatics and synthetic biology to mine the human microbiota for N-acyl amides that interact with G-protein-coupled receptors (GPCRs). We found that N-acyl amide synthase genes are enriched in gastrointestinal bacteria and the lipids that they encode interact with GPCRs that regulate gastrointestinal tract physiology. Mouse and cell-based models demonstrate that commensal GPR119 agonists regulate metabolic hormones and glucose homeostasis as efficiently as human ligands, although future studies are needed to define their potential physiological role in humans. Our results suggest that chemical mimicry of eukaryotic signalling molecules may be common among commensal bacteria and that manipulation of microbiota genes encoding metabolites that elicit host cellular responses represents a possible small-molecule therapeutic modality (microbiome-biosynthetic gene therapy).

Pharmacological cognitive enhancement and the value of achievements: An intervention.

Pharmacological cognitive enhancements nontherapeutically improve cognitive functioning, though recent critics have challenged their use by claiming that cognitive success, aided by the use of cognitive enhancement, is less valuable than otherwise. We criticize two recent responses to this objection, due to Carter and Pritchard and Wang, and propose a different response on behalf of proponents of cognitive enhancement that is shown to be more promising.

Stochastic Local Breakdown of Oxide Film on Ni from Identical-Location Imaging: One Single Site at a Time.

The electrochemical breakdown of a metal oxide film can directly affect the performance of functional electrochemical devices. However, revealing the structural insight into the breakdown sites is challenging because of heterogeneity: different breakdown sites are spatially distributed over the surface. Herein, we combine scanning electrochemical cell microscopy with identical-location microscopies to reveal the heterogeneity in the breakdown of NiO film on Ni in a site-by-site manner. Local critical breakdown potential varies by ∼500 mV, corresponding to an excess energy of 0.02-0.12 J/m2. Correlative composition imaging using time-of-flight secondary ion mass spectrometry shows Ni crystal grains with thinner NiO films are more resistant to breakdown. This high resistance is explained using classical nucleation theory, where the electrical energy is affected by the film thickness through the local interfacial capacitance. The correlative imaging approach overcomes the issue of heterogeneity, providing conclusive insight into the stability of the electrochemical interfaces.

RIPK1 Expression Associates With Inflammation in Early Atherosclerosis in Humans and Can Be Therapeutically Silenced to Reduce NF-κB Activation and Atherogenesis in Mice.

BACKGROUND: Chronic activation of the innate immune system drives inflammation and contributes directly to atherosclerosis. We previously showed that macrophages in the atherogenic plaque undergo RIPK3 (receptor-interacting serine/threonine-protein kinase 3)-MLKL (mixed lineage kinase domain-like protein)-dependent programmed necroptosis in response to sterile ligands such as oxidized low-density lipoprotein and damage-associated molecular patterns and that necroptosis is active in advanced atherosclerotic plaques. Upstream of the RIPK3-MLKL necroptotic machinery lies RIPK1 (receptor-interacting serine/threonine-protein kinase 1), which acts as a master switch that controls whether the cell undergoes NF-κB (nuclear factor κ-light-chain-enhancer of activated B cells)-dependent inflammation, caspase-dependent apoptosis, or necroptosis in response to extracellular stimuli. We therefore set out to investigate the role of RIPK1 in the development of atherosclerosis, which is driven largely by NF-κB-dependent inflammation at early stages. We hypothesize that, unlike RIPK3 and MLKL, RIPK1 primarily drives NF-κB-dependent inflammation in early atherogenic lesions, and knocking down RIPK1 will reduce inflammatory cell activation and protect against the progression of atherosclerosis. METHODS: We examined expression of RIPK1 protein and mRNA in both human and mouse atherosclerotic lesions, and used loss-of-function approaches in vitro in macrophages and endothelial cells to measure inflammatory responses. We administered weekly injections of RIPK1 antisense oligonucleotides to Apoe-/- mice fed a cholesterol-rich (Western) diet for 8 weeks. RESULTS: We find that RIPK1 expression is abundant in early-stage atherosclerotic lesions in both humans and mice. Treatment with RIPK1 antisense oligonucleotides led to a reduction in aortic sinus and en face lesion areas (47.2% or 58.8% decrease relative to control, P<0.01) and plasma inflammatory cytokines (IL-1α [interleukin 1α], IL-17A [interleukin 17A], P<0.05) in comparison with controls. RIPK1 knockdown in macrophages decreased inflammatory genes (NF-κB, TNFα [tumor necrosis factor α], IL-1α) and in vivo lipopolysaccharide- and atherogenic diet-induced NF-κB activation. In endothelial cells, knockdown of RIPK1 prevented NF-κB translocation to the nucleus in response to TNFα, where accordingly there was a reduction in gene expression of IL1B, E-selectin, and monocyte attachment. CONCLUSIONS: We identify RIPK1 as a central driver of inflammation in atherosclerosis by its ability to activate the NF-κB pathway and promote inflammatory cytokine release. Given the high levels of RIPK1 expression in human atherosclerotic lesions, our study suggests RIPK1 as a future therapeutic target to reduce residual inflammation in patients at high risk of coronary artery disease.

Nanoscale Colocalized Electrochemical and Structural Mapping of Metal Dissolution Reaction.

Understanding the structure-activity relationship in electrochemical metal dissolution reactions is fundamentally important, from designing higher density batteries to mitigating corrosions. The kinetics of metal dissolution reaction is highly dependent on surface structures, including grain boundaries and local defects. However, directly probing the electrochemical activity at these sites is difficult because the conventional bulk electrochemistry measures an averaged kinetics, obscuring the structure-activity correlation. Herein, we report the colocalized mapping of an electrochemical metal dissolution reaction using Ag as a model system. The local dissolution kinetics is voltammetrically mapped via scanning electrochemical cell microscopy (SECCM), which is correlated with local structures obtained via colocalized electron backscattering diffraction (EBSD). Individual pits of ∼200 nm are formed, and their geometries suggest dissolution is fastest in the direction parallel to the {111} planes. Enhanced dissolution kinetics is observed at the high-angle grain boundaries but not at twin boundaries, which are attributed to the different binding energy of Ag atoms. Furthermore, the faster local dissolution correlates with the geometrically necessary dislocation density. The work demonstrates the importance of nanoscale local electrochemical mapping and colocalized microscopic measurement in obtaining the structure-activity relationship for electrochemical reactions at complex interfaces.

Formation of styrene maleic acid lipid nanoparticles (SMALPs) using SMA thin film on a substrate.

Despite the important role of membrane proteins in biological function and physiology, studying them remains challenging because of limited biomimetic systems for the protein to remain in its native membrane environment. Cryo electron microscopy (Cryo-EM) is emerging as a powerful tool for analyzing the structure of membrane proteins. However, Cryo-EM and other membrane protein analyses are better studied in a native lipid bilayer. Although traditional, mimetic systems have disadvantages that limit their use in the study of membrane proteins. As an alternative, styrene-maleic acid copolymers are used to form nanoparticles with POPC:POPG lipids. Traditional characterization of these styrene maleic acid lipid nanoparticles (SMALPs) includes dynamic light scattering (DLS), electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), and transmission electron microscopy (TEM). In this study a new method was developed that utilizes SMALPs using a styrene-maleic acid copolymer (SMA) thin film on a TEM grid, acting as a substrate. By directly adding POPC:POPG lipid vesicles to the SMA coated grid SMALPs can be formed, visualized, and characterized by TEM without the need to make them in solution prior to imaging. We envision these functionalized grids could aid in single particle specimen preparation, increasing the efficiency of structural biology and biophysical techniques such as Cryo-EM.

Plasma pharmacokinetics, pulmonary disposition, and safety of subcutaneous gamithromycin in alpacas.

The study objective was to determine the disposition of gamithromycin in plasma, peripheral blood polymorphonuclear cells (PMNs), pulmonary epithelial lining fluid (PELF), and bronchoalveolar lavage (BAL) cells in alpacas. A single subcutaneous injection of gamithromycin (6.6 mg/kg) was administered to six healthy adult alpacas. At various time points after administration, gamithromycin concentrations were analyzed via LC-MS/MS in plasma, PMNs, PELF, and BAL cells until Day 14 post-injection. Plasma gamithromycin concentrations were measured in all six alpacas; the remaining three body compartments were analyzed in four alpacas. Gamithromycin rapidly concentrated in blood PMNs, BAL cells, and PELF. Shorter Tmax , and lower Cmax, and AUC were observed in plasma than in the other three compartments. Cmax was highest in BAL cells (26001.80 ± 12400.00 ng/ml) and PMNs (2573.00 ± 963.30 ng/ml) compared to PELF (660.80 ± 413.70 ng/ml) and plasma (452.30 ± 196.20 ng/ml). Mean terminal half-lives were 72.60 ± 14.10 h in plasma, 56.60 ± 10.60 h in PELF, 62.80 ± 85.30 h in PMNs, and 93.60 ± 124.80 h in BAL cells. No injection site reactions occurred. One alpaca developed colic but no other adverse reactions were noted. Overall, gamithromycin was highly concentrated in white blood cells and pulmonary fluids/cells. Clinical utilization of gamithromycin in alpacas should be done with caution until further investigation of potential for colic.

Pharmacokinetic profiles of three dose rates of morphine sulfate following single intravenous, intramuscular, and subcutaneous administration in the goat.

This study aimed to evaluate pharmacokinetic profiles of morphine in goats following a single dose administered intravenously, intramuscularly, or subcutaneously at 0.1 mg/kg, 0.25 mg/kg, and 0.4 mg/kg. Study population included eight healthy adult goats in a randomized cross-over study. Serial plasma samples were collected and morphine was quantified using high-performance liquid chromatography/mass spectrometry. Data fit a two-compartment model following intravenous administration and a non-compartmental model following both intramuscular and subcutaneous administration. Plasma elimination half-life was 2.88 ± 1.13 h (0.1 mg/kg), 2.30 ± 0.49 h (0.25 mg/kg), and 2.67 ± 0.82 h (0.4 mg/kg) following IV morphine. Intramuscular Cmax values were 13.4 ± 2.77 ng/ml (0.1 mg/kg), 34 ± 11.50 ng/ml (0.25 mg/kg), and 68.9 ± 24.5 ng/ml (0.4 mg/kg). Intramuscular Tmax f(h) or IM dosing (in hrs) was 0.19 ± 0.14 (0.1 mg/kg), 0.24 ± 0.24 (0.25 mg/kg), and 0.21 ± 0.24 (0.4 mg/kg). Subcutaneous Cmax values were 9.88 ± 3.31 ng/ml (0.1 mg/kg), 28.5 ± 11.6 ng/ml (0.25 mg/kg), and 39.4 ± 14.3 ng/ml (0.4 mg/kg). Subcutaneous Tmax (h) values for SC dosing were 0.36 ± 0.21 (0.1 mg/kg), 0.31 ± 0.17 (0.25 mg/kg), and 0.4 ± 0.13 (0.4 mg/kg). Intramuscular bioavailability values were 153.77 ± 12.60% (0.4 mg/kg), 104.8 ± 25.12% (0.25 mg/kg), and 100.7 ± 29.57% (0.1 mg/kg). Subcutaneous bioavailability values were 130.58 ± 19.07% (0.4 mg/kg), 116.6 ± 27.03% (0.25 mg/kg), and 111.6 ± 23.24% (0.1 mg/kg). No adverse effects were observed. Assuming plasma concentration required to induce analgesia is 16 ± 9 ng/ml in goats, as demonstrated in humans, it is suggested to administer morphine intramuscularly at 0.4 mg/kg every 3-4 h or SC every 2-3 h. This is a speculative conclusion therefore further studies evaluating pharmacodynamics and plasma analgesic threshold in goats is recommended.

Cognitive enhancement and authenticity: moving beyond the Impasse.

In work on the ethics of cognitive enhancement use, there is a pervasive concern that such enhancement will-in some way-make us less authentic (e.g., Bublitz and Merkel 2009; Juth 2011). Attempts to clarify what this concern amounts to and how to respond to it often lead to debates on the nature of the "true self" (e.g., Maslen et al. 2014) and what constitutes "genuine human activity" (e.g., Kass 2003). This paper shows that a new and effective way to make progress on whether certain cases of cognitive enhancement problematically undermine authenticity is to make use of considerations from the separate debate on the nature of authentic emotion. Drawing in particular on Wasserman and Liao (2008), the present paper offers new conditions that can help us assess the impact of cognitive enhancements on authenticity.

MAFB modulates the maturation of lymphatic vascular networks in mice.

BACKGROUND: Lymphatic vessels play key roles in tissue fluid homeostasis, immune cell trafficking and in diverse disease settings. Lymphangiogenesis requires lymphatic endothelial cell (LEC) differentiation, proliferation, migration, and co-ordinated network formation, yet the transcriptional regulators underpinning these processes remain to be fully understood. The transcription factor MAFB was recently identified as essential for lymphangiogenesis in zebrafish and in cultured human LECs. MAFB is activated in response to VEGFC-VEGFR3 signaling and acts as a downstream effector. However, it remains unclear if the role of MAFB in lymphatic development is conserved in the mammalian embryo. RESULTS: We generated a Mafb loss-of-function mouse using CRISPR/Cas9 gene editing. Mafb mutant mice presented with perinatal lethality associated with cyanosis. We identify a role for MAFB in modifying lymphatic network morphogenesis in the developing dermis, as well as developing and postnatal diaphragm. Furthermore, mutant vessels displayed excessive smooth muscle cell coverage, suggestive of a defect in the maturation of lymphatic networks. CONCLUSIONS: This work confirms a conserved role for MAFB in murine lymphatics that is subtle and modulatory and may suggest redundancy in MAF family transcription factors during lymphangiogenesis.