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Autonomous assembly : designing for a new era of collective construction
\"We are now on the brink of a new era in construction - that of autonomous assembly. For some time, the widespread adoption of robotic and digital fabrication technologies has made it possible for architects and academic researchers to design non-standard, highly customised structures. These technologies have largely been limited by scalability, focusing mainly on top-down, bespoke fabrication projects, such as experimental pavilions and structures. Autonomous assembly and bottom-up construction techniques hold the promise of greater scalability, adaptability and potentially evolved design possibilities. By capitalising on the advances made in swarm robotics, the collective construction of the animal/insect kingdom, and advances in physical computational, programmable materials or self-assembly, architects and designers are now able to build from the bottom up. This issue presents future scenarios of autonomous assembly by highlighting the viability of decentralised, collective assembly systems, demonstrating the potential to deliver reconfigurable and adaptive solutions.\"--Back cover.
Book
Sphingolipids produced by gut bacteria enter host metabolic pathways impacting ceramide levels
Gut microbes are linked to host metabolism, but specific mechanisms remain to be uncovered. Ceramides, a type of sphingolipid (SL), have been implicated in the development of a range of metabolic disorders from insulin resistance (IR) to hepatic steatosis. SLs are obtained from the diet and generated by de novo synthesis in mammalian tissues. Another potential, but unexplored, source of mammalian SLs is production by Bacteroidetes, the dominant phylum of the gut microbiome. Genomes of
Bacteroides
spp. and their relatives encode serine palmitoyltransfease (SPT), allowing them to produce SLs. Here, we explore the contribution of SL-production by gut
Bacteroides
to host SL homeostasis. In human cell culture, bacterial SLs are processed by host SL-metabolic pathways. In mouse models,
Bacteroides
-derived lipids transfer to host epithelial tissue and the hepatic portal vein. Administration of
B. thetaiotaomicron
to mice, but not an SPT-deficient strain, reduces de novo SL production and increases liver ceramides. These results indicate that gut-derived bacterial SLs affect host lipid metabolism.
Ceramides are a type of sphingolipid (SL) that have been shown to play a role in several metabolic disorders. Here, the authors investigate the effect of SL-production by gut
Bacteroides
on host SL homeostasis and show that microbiome-derived SLs enter host circulation and alter ceramide production.
Journal Article
Decreased sphingolipid synthesis in children with 17q21 asthma–risk genotypes
Risk for childhood asthma is conferred by alleles within the 17q21 locus affecting ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) expression. ORMDL3 inhibits sphingolipid de novo synthesis. Although the effects of 17q21 genotypes on sphingolipid synthesis in human asthma remain unclear, both decreased sphingolipid synthesis and ORMDL3 overexpression are linked to airway hyperreactivity. To characterize the relationship of genetic asthma susceptibility with sphingolipid synthesis, we analyzed asthma-associated 17q21 genotypes (rs7216389, rs8076131, rs4065275, rs12603332, and rs8067378) in both children with asthma and those without asthma, quantified plasma and whole-blood sphingolipids, and assessed sphingolipid de novo synthesis in peripheral blood cells by measuring the incorporation of stable isotope-labeled serine (substrate) into sphinganine and sphinganine-1-phosphate. Whole-blood dihydroceramides and ceramides were decreased in subjects with the 17q21 asthma-risk alleles rs7216389 and rs8076131. Children with nonallergic asthma had lower dihydroceramides, ceramides, and sphingomyelins than did controls. Children with allergic asthma had higher dihydroceramides, ceramides, and sphingomyelins compared with children with nonallergic asthma. Additionally, de novo sphingolipid synthesis was lower in children with asthma compared with controls. These findings connect genetic 17q21 variations that are associated with asthma risk and higher ORMDL3 expression to lower sphingolipid synthesis in humans. Altered sphingolipid synthesis may therefore be a critical factor in asthma pathogenesis and may guide the development of future therapeutics.
Journal Article
A bright cyan-excitable orange fluorescent protein facilitates dual-emission microscopy and enhances bioluminescence imaging in vivo
In vivo
imaging is facilitated by a bright, cyan-excitable orange fluorescent protein that is the basis of an improved bioluminescent protein.
Orange-red fluorescent proteins (FPs) are widely used in biomedical research for multiplexed epifluorescence microscopy with GFP-based probes, but their different excitation requirements make multiplexing with new advanced microscopy methods difficult. Separately, orange-red FPs are useful for deep-tissue imaging in mammals owing to the relative tissue transmissibility of orange-red light, but their dependence on illumination limits their sensitivity as reporters in deep tissues. Here we describe CyOFP1, a bright, engineered, orange-red FP that is excitable by cyan light. We show that CyOFP1 enables single-excitation multiplexed imaging with GFP-based probes in single-photon and two-photon microscopy, including time-lapse imaging in light-sheet systems. CyOFP1 also serves as an efficient acceptor for resonance energy transfer from the highly catalytic blue-emitting luciferase NanoLuc. An optimized fusion of CyOFP1 and NanoLuc, called Antares, functions as a highly sensitive bioluminescent reporter
in vivo
, producing substantially brighter signals from deep tissues than firefly luciferase and other bioluminescent proteins.
Journal Article
Fluorescent indicators for simultaneous reporting of all four cell cycle phases
The far-red fluorescent protein mMaroon1 and a reporter based on stem-loop binding protein enables the generation of Fucci4, a 4-color cell cycle reporter system that can be used to distinguish all phases of the cell cycle. Also online, a paper by Laviv
et al
. uses mMaroon1 as a FRET acceptor for the newly developed CyRFP1.
A robust method for simultaneous visualization of all four cell cycle phases in living cells is highly desirable. We developed an intensiometric reporter of the transition from S to G2 phase and engineered a far-red fluorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal fluorescent indicators that together resolve all cell cycle phases.
Journal Article
Simultaneous dual-color fluorescence lifetime imaging with novel red-shifted fluorescent proteins
Two red fluorescent proteins with long Stokes shift enable simultaneous multicolor 2p imaging. CyRFP1 is well-suited for 2p structural imaging, and FRET sensors made with mCyRFP1 and mMaroon1enable multicolor 2pFLIM in brain slices. Also online, a paper by Bajar
et al
. reports the development of mMaroon1.
We describe a red-shifted fluorescence resonance energy transfer (FRET) pair optimized for dual-color fluorescence lifetime imaging (FLIM). This pair utilizes a newly developed FRET donor, monomeric cyan-excitable red fluorescent protein (mCyRFP1), which has a large Stokes shift and a monoexponential fluorescence lifetime decay. When used together with EGFP-based biosensors, the new pair enables simultaneous imaging of the activities of two signaling molecules in single dendritic spines undergoing structural plasticity.
Journal Article
Association of diabetes severity with cognitive function in US adults: a cross-sectional analysis of the AI-READI multicentre cohort
ObjectivesTo evaluate whether type 2 diabetes mellitus (T2DM) presence and severity are associated with differences in global and domain-specific cognitive function among US adults, using standardised Montreal Cognitive Assessment (MoCA) testing.DesignCross-sectional studySettingThree U.S academic medical centres participating in the Artificial Intelligence–Ready and Equitable Atlas for Diabetes Insights (AI-READI) study.ParticipantsAdults aged ≥40 years enrolled in the AI-READI cross-sectional study at three US academic medical centres were eligible. The study excluded individuals with type 1 diabetes, pregnancy or inability to speak, read and understand English. For this secondary analysis, 1067 participants from the first publicly released AI-READI data set who had MoCA data and assigned glycaemic status were included. Participants were classified into four prespecified glycaemic groups: controls without diabetes (n=371), pre-diabetes (n=239), medication-controlled type 2 diabetes (n=323), and insulin-dependent type 2 diabetes (n=129).Primary and secondary outcome measuresThe primary outcome was global cognitive function measured by the MoCA total score. Secondary outcomes included MoCA domain scores and the prevalence of cognitive impairment, defined as MoCA<26.ResultsSignificant differences in MoCA total scores were observed across glycaemic groups (p<0.001), with the lowest mean score (24.0) among insulin-dependent individuals and highest among controls (25.8), and the significant difference remained in multivariable analyses (p=0.02). Among MoCA subdomains, mean abstraction scores were significantly lower (1.76) in insulin-dependent T2DM group than other glycaemic groups (mean score 1.87–1.89) in multivariable analysis (p=0.03). The prevalence of cognitive impairment increased from 39% in controls to 58% in the insulin-dependent group. In multivariable analysis, increasing diabetes severity was associated with higher risk of cognitive impairment with adjusted OR 1.85 (95% CI: 1.11 to 3.07) for insulin-dependent T2DM and 1.50 (95% CI: 1.05 to 2.15) for medication-controlled T2DM.ConclusionsIndividuals with more advanced T2DM, particularly those on insulin, had significantly higher risk of cognitive impairment. These findings support routine cognitive screening in patients with T2DM, especially those on insulin therapy. Early identification of cognitive impairment may improve diabetes management and cognitive outcomes.
Journal Article
Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A
Factor VIII gene transfer with a single intravenous infusion of valoctocogene roxaparvovec (AAV5-hFVIII-SQ) has demonstrated clinical benefits lasting 5 years to date in people with severe hemophilia A. Molecular mechanisms underlying sustained AAV5-hFVIII-SQ-derived FVIII expression have not been studied in humans. In a substudy of the phase 1/2 clinical trial (
NCT02576795
), liver biopsy samples were collected 2.6–4.1 years after gene transfer from five participants. Primary objectives were to examine effects on liver histopathology, determine the transduction pattern and percentage of hepatocytes transduced with AAV5-hFVIII-SQ genomes, characterize and quantify episomal forms of vector DNA and quantify transgene expression (hFVIII-SQ RNA and hFVIII-SQ protein). Histopathology revealed no dysplasia, architectural distortion, fibrosis or chronic inflammation, and no endoplasmic reticulum stress was detected in hepatocytes expressing hFVIII-SQ protein. Hepatocytes stained positive for vector genomes, showing a trend for more cells transduced with higher doses. Molecular analysis demonstrated the presence of full-length, inverted terminal repeat-fused, circular episomal genomes, which are associated with long-term expression. Interindividual differences in transgene expression were noted despite similar successful transduction, possibly influenced by host-mediated post-transduction mechanisms of vector transcription, hFVIII-SQ protein translation and secretion. Overall, these results demonstrate persistent episomal vector structures following AAV5-hFVIII-SQ administration and begin to elucidate potential mechanisms mediating interindividual variability.
The analysis of liver biopsy samples after AAV gene therapy for hemophilia A reveals normal histology and long-term persistence of the episomal vector, and identifies potential factors contributing to interindividual variability of transgene expression.
Journal Article
Intraocular iron injection induces oxidative stress followed by elements of geographic atrophy and sympathetic ophthalmia
Iron has been implicated in the pathogenesis of age‐related retinal diseases, including age‐related macular degeneration (AMD). Previous work showed that intravitreal (IVT) injection of iron induces acute photoreceptor death, lipid peroxidation, and autofluorescence (AF). Herein, we extend this work, finding surprising chronic features of the model: geographic atrophy and sympathetic ophthalmia. We provide new mechanistic insights derived from focal AF in the photoreceptors, quantification of bisretinoids, and localization of carboxyethyl pyrrole, an oxidized adduct of docosahexaenoic acid associated with AMD. In mice given IVT ferric ammonium citrate (FAC), RPE died in patches that slowly expanded at their borders, like human geographic atrophy. There was green AF in the photoreceptor ellipsoid, a mitochondria‐rich region, 4 h after injection, followed later by gold AF in rod outer segments, RPE and subretinal myeloid cells. The green AF signature is consistent with flavin adenine dinucleotide, while measured increases in the bisretinoid all‐trans‐retinal dimer are consistent with the gold AF. FAC induced formation carboxyethyl pyrrole accumulation first in photoreceptors, then in RPE and myeloid cells. Quantitative PCR on neural retina and RPE indicated antioxidant upregulation and inflammation. Unexpectedly, reminiscent of sympathetic ophthalmia, autofluorescent myeloid cells containing abundant iron infiltrated the saline‐injected fellow eyes only if the contralateral eye had received IVT FAC. These findings provide mechanistic insights into the potential toxicity caused by AMD‐associated retinal iron accumulation. The mouse model will be useful for testing antioxidants, iron chelators, ferroptosis inhibitors, anti‐inflammatory medications, and choroidal neovascularization inhibitors. Intravitreal injection of iron induces photoreceptor oxidative stress, resulting in increased autofluorescence, lipid peroxidation, and myeloid cell infiltration associated with retinal degeneration, geographic atrophy, and choroidal neovascularization.
Journal Article
A red fluorescent protein with improved monomericity enables ratiometric voltage imaging with ASAP3
A ratiometric genetically encoded voltage indicator (GEVI) would be desirable for tracking transmembrane voltage changes in the presence of sample motion. We performed combinatorial multi-site mutagenesis on a cyan-excitable red fluorescent protein to create the bright and monomeric mCyRFP3, which proved to be uniquely non-perturbing when fused to the GEVI ASAP3. The green/red ratio from ASAP3-mCyRFP3 (ASAP3-R3) reported voltage while correcting for motion artifacts, allowing the visualization of membrane voltage changes in contracting cardiomyocytes and throughout the cell cycle of motile cells.
Journal Article