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The common aldehyde dehydrogenase 2 (ALDH2) alcohol flushing variant known as ALDH2*2 affects ∼8% of the world's population. Even in heterozygous carriers, this missense variant leads to a severe loss of ALDH2 enzymatic activity and has been linked to an increased risk of coronary artery disease (CAD). Endothelial cell (EC) dysfunction plays a determining role in all stages of CAD pathogenesis, including early-onset CAD. However, the contribution of ALDH2*2 to EC dysfunction and its relation to CAD are not fully understood. In a large genome-wide association study (GWAS) from Biobank Japan, ALDH2*2 was found to be one of the strongest single-nucleotide polymorphisms associated with CAD. Clinical assessment of endothelial function showed that human participants carrying ALDH2*2 exhibited impaired vasodilation after light alcohol drinking. Using human induced pluripotent stem cell-derived ECs (iPSC-ECs) and CRISPR-Cas9-corrected ALDH2*2 iPSC-ECs, we modeled ALDH2*2-induced EC dysfunction in vitro, demonstrating an increase in oxidative stress and inflammatory markers and a decrease in nitric oxide (NO) production and tube formation capacity, which was further exacerbated by ethanol exposure. We subsequently found that sodium-glucose cotransporter 2 inhibitors (SGLT2i) such as empagliflozin mitigated ALDH2*2-associated EC dysfunction. Studies in ALDH2*2 knock-in mice further demonstrated that empagliflozin attenuated ALDH2*2-mediated vascular dysfunction in vivo. Mechanistically, empagliflozin inhibited Na+/H+-exchanger 1 (NHE-1) and activated AKT kinase and endothelial NO synthase (eNOS) pathways to ameliorate ALDH2*2-induced EC dysfunction. Together, our results suggest that ALDH2*2 induces EC dysfunction and that SGLT2i may potentially be used as a preventative measure against CAD for ALDH2*2 carriers.

The two most clinically important members of the flavivirus genus, Zika virus (ZIKV) and dengue virus (DENV) pose a significant public health challenge. They cause a range of diseases in humans, from hemorrhagic to neurological manifestations, leading to economic and social burden worldwide. Nevertheless, there are no approved antiviral drugs to treat these infections. Zafirlukast is an orally available Food and Drug Administration (FDA) approved drug for the prophylaxis and treatment of chronic asthma. It is a leukotriene receptor antagonist (LTRA) with high selectivity of the cysteinyl leukotriene-1 receptor (CYSLTR1) that acts as an immune modulator. Thus, we evaluated the antiviral potential of Zafirlukast against ZIKV and DENV in SK-N-SH cells. We showed that Zafirlukast exhibited potent antiviral activity against ZIKV, which could be linked to Zafirlukast's immune blockade of TNF signaling pathways and its downstream signaling pathways such as MAPK and ERK1/2. In addition, our results showed that Zafirlukast also counteracts ZIKV-induced changes in key genes involved in cellular lipid metabolism. Thus, these findings highlight the translational potential of optimizing Zafirlukast as a therapeutic agent for the treatment of ZIKV and DENV., (© 2024 Wiley Periodicals LLC.)

Defending against future pandemics requires vaccine platforms that protect across a range of related pathogens. Nanoscale patterning can be used to address this issue. Here, we produce quartets of linked receptor-binding domains (RBDs) from a panel of SARS-like betacoronaviruses, coupled to a computationally designed nanocage through SpyTag/SpyCatcher links. These Quartet Nanocages, possessing a branched morphology, induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented in the vaccine. Equivalent antibody responses are raised to RBDs close to the nanocage or at the tips of the nanoparticle's branches. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increase the strength and breadth of an otherwise narrow immune response. A Quartet Nanocage including the Omicron XBB.1.5 'Kraken' RBD induced antibodies with binding to a broad range of sarbecoviruses, as well as neutralizing activity against this variant of concern. Quartet nanocages are a nanomedicine approach with potential to confer heterotypic protection against emergent zoonotic pathogens and facilitate proactive pandemic protection., (© 2024. The Author(s).)

The underlying threat of new Zika virus (ZIKV) outbreaks remains, as no vaccines or therapies have yet been developed. In vitro research has shown that glycolysis is a key factor to enable sustained ZIKV replication in neuroprogenitors. However, neither in vivo nor clinical investigation of glycolytic modulators as potential therapeutics for ZIKV-related fetal abnormalities has been conducted. Accordingly, we tested the therapeutic potential of metabolic modulators in relevant in vitro systems comprising two pools of neuroprogenitors (NPCs), which resemble early and late stages of pregnancy. Effective doses of metabolic modulators [3.0 μM] dimethyl fumarate (DMF), [3.2 mM] dichloroacetate (DCA), and [6.3 μM] VER-246608 were determined for these cells by their effect on lactate release, pyruvate dehydrogenase (PDH) activity and cell survival. The drugs were used in a 24h pre-treatment and kept throughout ZIKV infection of NPCs. Drug effects and ZIKV replication were assessed at 24- and 56-h post-infection. In early NPCs treated with DMF, DCA and VER-246608, there was a significant reduction in the extracellular release of ZIKV potentially by PDH-mediated increased mitochondrial oxidation of glucose. Out of the three drugs, only DCA was observed to reduce viral replication in late NPCs treated with DCA. Altogether, our findings suggest that reduction of anaerobic glycolysis could be of therapeutic potential against ZIKV-related fetal abnormalities and that clinical translation should consider the use of specific glycolytic modulators over different trimesters., Competing Interests: Declaration of competing interest The authors declare that there is no relevant material or financial interests that relate to the research described in this manuscript. This work was supported by the James and Lillian Martin Centre and from BNC Research Fund, Brasenose College Grant to W.S.J. BV5810 (Sir William Dunn School of Pathology, University of Oxford). At the time of the research, J.G-J., was funded by CIU Research Fund 2022, CIU Trust – Sir Dunn School of Pathology (University of Oxford). L.C.H., is funded by the British Heart Foundation (PG/07/070/23365)., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cyanobacterial harmful algal blooms (cyanoHABs) dominated by Microcystis spp. have significant public health and economic implications in freshwater bodies around the world. These blooms are capable of producing a variety of cyanotoxins, including microcystins, that affect fishing and tourism industries, human and environmental health, and access to drinking water. In this study, we isolated and sequenced the genomes of 21 primarily unialgal Microcystis cultures collected from western Lake Erie between 2017 and 2019. While some cultures isolated in different years have a high degree of genetic similarity (genomic Average Nucleotide Identity >99%), genomic data show that these cultures also represent much of the breadth of known Microcystis diversity in natural populations. Only five isolates contained all the genes required for microcystin biosynthesis while two isolates contained a previously described partial mcy operon. Microcystin production within cultures was also assessed using Enzyme-Linked Immunosorbent Assay (ELISA) and supported genomic results with high concentrations (up to 900 μg L⁻¹) in cultures with complete mcy operons and no or low toxin detected otherwise. These xenic cultures also contained a substantial diversity of bacteria associated with Microcystis, which has become increasingly recognized as an essential component of cyanoHAB community dynamics. These results highlight the genomic diversity among Microcystis strains and associated bacteria in Lake Erie, and their potential impacts on bloom development, toxin production, and toxin degradation. This culture collection significantly increases the availability of environmentally relevant Microcystis strains from temperate North America., Competing Interests: Declaration of Competing Interest The authors declare no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Cortical development consists of an orchestrated process in which progenitor cells exhibit distinct fate restrictions regulated by time-dependent activation of energetic pathways. Thus, the hijacking of cellular metabolism by Zika virus (ZIKV) to support its replication may contribute to damage in the developing fetal brain. Here, we showed that ZIKV replicates differently in two glycolytically distinct pools of cortical progenitors derived from human induced pluripotent stem cells (hiPSCs), which resemble the metabolic patterns of quiescence (early hi-NPCs) and immature brain cells (late hi-NPCs) in the forebrain. This differential replication alters the transcription of metabolic genes in both pools of cortical progenitors but solely upregulates the glycolytic capacity of early hi-NPCs. Analysis using Imagestream® revealed that, during early stages of ZIKV replication, in early hi-NPCs there is an increase in lipid droplet abundance and size. This stage of ZIKV replication significantly reduced the mitochondrial distribution in both early and late hi-NPCs. During later stages of ZIKV replication, late hi-NPCs show reduced mitochondrial size and abundance. The finding that there are alterations of cellular metabolism during ZIKV infection which are specific to pools of cortical progenitors at different stages of maturation may help to explain the differences in brain damage over each trimester., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

In developing a wire-arc directed energy deposition process for superalloys used in high-speed flight environments, Inconel 718 was deposited using a plasma arc process and tested for its high temperature performance. The deposited material was tested in both the as deposited condition and after an age-hardening industry standard heat-treatment for this alloy. Results showed a reduced performance in both deposited conditions, with heat-treated material significantly outperforming as deposited material up to 538 °C. The difference in performance was less significant from 760 to 1000 °C, owing to an in-test aging process which increased the performance of the as deposited material. The microstructure of deposited material showed significant cracking throughout the alloy and formation of secondary phases throughout the matrix, with significantly more precipitation after heat-treating., (© 2023. The Author(s).)

Since its discovery, COVID-19 has rapidly spread across the globe and has had a massive toll on human health, with infection mortality rates as high as 10%, and a crippling impact on the world economy. Despite numerous advances, there remains an urgent need for accurate and rapid point-of-care diagnostic tests and better therapeutic treatment options. To contribute chemically distinct, non-protein-based affinity reagents, we report here the identification of modified DNA-based aptamers that selectively bind to the S1, S2, or receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Several aptamers inhibit the binding of the spike protein to its cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and neutralize authentic SARS-CoV-2 virus in vitro , including all variants of concern. With a high degree of nuclease resistance imparted by the base modifications, these reagents represent a new class of molecules with potential for further development as diagnostics or therapeutics., Competing Interests: A.D.G., C.Z., B.E.R., C.N.C, M.R.O., D.J.S., and N,J, are employees and shareholders of SomaLogic, Inc., (© 2023 The Authors.)

Defending against future pandemics may require vaccine platforms that protect across a range of related pathogens. The presentation of multiple receptor-binding domains (RBDs) from evolutionarily-related viruses on a nanoparticle scaffold elicits a strong antibody response to conserved regions. Here we produce quartets of tandemly-linked RBDs from SARS-like betacoronaviruses coupled to the mi3 nanocage through a SpyTag/SpyCatcher spontaneous reaction. These Quartet Nanocages induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented on the vaccine. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increased the strength and breadth of an otherwise narrow immune response. Quartet Nanocages are a strategy with potential to confer heterotypic protection against emergent zoonotic coronavirus pathogens and facilitate proactive pandemic protection., Competing Interests: Competing interests: M.H. is an inventor on a patent on spontaneous amide bond formation (EP2534484) and a SpyBiotech co-founder and shareholder. M.H. and A.H.K. are inventors on a patent on SpyTag003:SpyCatcher003 (UK Intellectual Property Office 1706430.4). P.J.B. and A.A.C. are inventors on a US patent application filed by the California Institute of Technology that covers the methodology to generate cross-reactive antibodies using mosaic nanoparticles. P.J.B., and A.A.C. are inventors on a US patent application filed by the California Institute of Technology that covers the monoclonal antibodies elicited by vaccination with Mosaic nanoparticles described in this work. P.J.B., A.A.C. and J.R.K. are inventors on a US patent application filed by the California Institute of Technology that covers the methods of isolating cross-reactive antibodies by vaccination with mosaic nanoparticles. All other authors have no competing interests to declare.

Macrophage distribution density is tightly regulated within the body, yet the importance of macrophage crowding during in vitro culture is largely unstudied. Using a human induced pluripotent stem cell (iPSC)-derived macrophage model of tissue resident macrophages, we characterize how increasing macrophage culture density changes their morphology and phenotype before and after inflammatory stimulation. In particular, density drives changes in macrophage inflammatory cytokine and chemokine secretion in both resting and activated states. This density regulated inflammatory state is also evident in blood monocyte derived-macrophages, the human monocytic THP-1 immortalized cell line, and iPSC-derived microglia. Density-dependent changes appear to be driven by a transferable soluble factor, yet the precise mechanism remains unknown. Our findings highlight cell plating density as an important but frequently overlooked consideration of in vitro macrophage research relevant to a variety of fields ranging from basic macrophage cell biology to disease studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vaughan-Jackson, Stodolak, Ebrahimi, Johnson, Reardon, Dupont, Zhang, McCullagh and James.)

The SARS-CoV-2 pandemic continues despite the presence of effective vaccines, and novel vaccine approaches may help to reduce viral spread and associated COVID-19 disease. Current vaccine administration modalities are based on systemic needle-administered immunisation which may be suboptimal for mucosal pathogens. Here we demonstrate in a mouse model that small-volume intranasal administration of purified spike (S) protein in the adjuvant polyethylenemine (PEI) elicits robust antibody responses with modest systemic neutralisation activity. Further, we test a heterologous intranasal immunisation regimen, priming with S and boosting with RBD-Fc. Our data identify small volume PEI adjuvantation as a novel platform with potential for protective mucosal vaccine development., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

There is increasing genetic evidence for the role of microglia in neurodegenerative diseases, including Alzheimer's, Parkinson's, and motor neuron disease. Therefore, there is a need to generate authentic in vitro models to study human microglial physiology. Various methods have been developed using human induced Pluripotent Stem Cells (iPSC) to generate microglia, however, systematic approaches to identify which media components are actually essential for functional microglia are mostly lacking. Here, we systematically assess medium components, coatings, and growth factors required for iPSC differentiation to microglia. Using single-cell RNA sequencing, qPCR, and functional assays, with validation across two labs, we have identified several medium components from previous protocols that are redundant and do not contribute to microglial identity. We provide an optimised, defined medium which produces both transcriptionally and functionally relevant microglia for modelling microglial physiology in neuroinflammation and for drug discovery., (© 2022. The Author(s).)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Motor neuron diseases such as amyotrophic lateral sclerosis are primarily characterized by motor neuron degeneration with additional involvement of non-neuronal cells, in particular, microglia. In previous work, we have established protocols for the differentiation of iPSC-derived spinal motor neurons and microglia. Here, we combine both cell lineages and establish a novel co-culture of iPSC-derived spinal motor neurons and microglia, which is compatible with motor neuron identity and function. Co-cultured microglia express key identity markers and transcriptomically resemble primary human microglia, have highly dynamic ramifications, are phagocytically competent, release relevant cytokines and respond to stimulation. Further, they express key amyotrophic lateral sclerosis-associated genes and release disease-relevant biomarkers. This novel and authentic human model system facilitates the study of physiological motor neuron-microglia crosstalk and will allow the investigation of non-cell-autonomous phenotypes in motor neuron diseases such as amyotrophic lateral sclerosis., (© 2022. The Author(s).)

Background: Evaluation of susceptibility to emerging SARS-CoV-2 variants of concern (VOC) requires rapid screening tests for neutralising antibodies which provide protection., Methods: Firstly, we developed a receptor-binding domain-specific haemagglutination test (HAT) to Wuhan and VOC (alpha, beta, gamma and delta) and compared to pseudotype, microneutralisation and virus neutralisation assays in 835 convalescent sera. Secondly, we investigated the antibody response using the HAT after two doses of mRNA (BNT162b2) vaccination. Sera were collected at baseline, three weeks after the first and second vaccinations from older (80-99 years, n  = 89) and younger adults (23-77 years, n  = 310) and compared to convalescent sera from naturally infected individuals (1-89 years, n  = 307)., Results: Here we show that HAT antibodies highly correlated with neutralising antibodies (R = 0.72-0.88) in convalescent sera. Home-dwelling older individuals have significantly lower antibodies to the Wuhan strain after one and two doses of BNT162b2 vaccine than younger adult vaccinees and naturally infected individuals. Moverover, a second vaccine dose boosts and broadens the antibody repertoire to VOC in naïve, not previously infected older and younger adults. Most (72-76%) older adults respond after two vaccinations to alpha and delta, but only 58-62% to beta and gamma, compared to 96-97% of younger vaccinees and 68-76% of infected individuals. Previously infected older individuals have, similarly to younger adults, high antibody titres after one vaccination., Conclusions: Overall, HAT provides a surrogate marker for neutralising antibodies, which can be used as a simple inexpensive, rapid test. HAT can be rapidly adaptable to emerging VOC for large-scale evaluation of potentially decreasing vaccine effectiveness., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2022.)

The trajectories of acquired immunity to severe acute respiratory syndrome coronavirus 2 infection are not fully understood. We present a detailed longitudinal cohort study of UK healthcare workers prior to vaccination, presenting April-June 2020 with asymptomatic or symptomatic infection. Here we show a highly variable range of responses, some of which (T cell interferon-gamma ELISpot, N-specific antibody) wane over time, while others (spike-specific antibody, B cell memory ELISpot) are stable. We use integrative analysis and a machine-learning approach (SIMON - Sequential Iterative Modeling OverNight) to explore this heterogeneity. We identify a subgroup of participants with higher antibody responses and interferon-gamma ELISpot T cell responses, and a robust trajectory for longer term immunity associates with higher levels of neutralising antibodies against the infecting (Victoria) strain and also against variants B.1.1.7 (alpha) and B.1.351 (beta). These variable trajectories following early priming may define subsequent protection from severe disease from novel variants., (© 2022. The Author(s).)

The extent to which immune responses to natural infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and immunization with vaccines protect against variants of concern (VOC) is of increasing importance. Accordingly, here we analyse antibodies and T cells of a recently vaccinated, UK cohort, alongside those recovering from natural infection in early 2020. We show that neutralization of the VOC compared to a reference isolate of the original circulating lineage, B, is reduced: more profoundly against B.1.351 than for B.1.1.7, and in responses to infection or a single dose of vaccine than to a second dose of vaccine. Importantly, high magnitude T cell responses are generated after two vaccine doses, with the majority of the T cell response directed against epitopes that are conserved between the prototype isolate B and the VOC. Vaccination is required to generate high potency immune responses to protect against these and other emergent variants., (© 2021. The Author(s).)

In response to viral infections, the innate immune system rapidly activates expression of several interferon-stimulated genes (ISGs), whose protein and metabolic products are believed to directly interfere with the viral life cycle. Here, we argue that biochemical reactions performed by two specific protein products of ISGs modulate central carbon metabolism to support a broad-spectrum antiviral response. We demonstrate that the metabolites generated by metalloenzymes nitric oxide synthase and the radical S-adenosylmethionine (SAM) enzyme RSAD2 inhibit the activity of the housekeeping and glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We discuss that this inhibition is likely to stimulate a range of metabolic and signalling processes to support a broad-spectrum immune response. Based on these analyses, we propose that inhibiting GAPDH in individuals with deteriorated cellular innate immune response like elderly might help in treating viral diseases such as COVID-19., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Microglia orchestrate neuroimmune responses in several neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Microglia clear up dead and dying neurons through the process of efferocytosis, a specialized form of phagocytosis. The phagocytosis function can be disrupted by environmental or genetic risk factors that affect microglia. This paper presents a rapid and simple in vitro microscopy protocol for studying microglial efferocytosis in an induced pluripotent stem cell (iPSC) model of microglia, using a human neuroblastoma cell line (SH-SY5Y) labeled with a pH-sensitive dye for the phagocytic cargo. The procedure results in a high yield of dead neuroblastoma cells, which display surface phosphatidylserine, recognized as an "eat-me" signal by phagocytes. The 96-well plate assay is suitable for live-cell time-lapse imaging, or the plate can be successfully fixed prior to further processing and quantified by high-content microscopy. Fixed-cell high-content microscopy enables the assay to be scaled up for screening of small molecule inhibitors or assessing the phagocytic function of genetic variant iPSC lines. While this assay was developed to study phagocytosis of whole dead neuroblastoma cells by iPSC-macrophages, the assay can be easily adapted for other cargoes relevant to neurodegenerative diseases, such as synaptosomes and myelin, and other phagocytic cell types.

Human induced Pluripotent Stem Cell (hiPSC) models are a valuable new tool for research into neurodegenerative diseases. Neuroinflammation is now recognized as a key process in neurodegenerative disease and aging, and microglia are central players in this. A plethora of hiPSC-derived microglial models have been published recently to explore neuroinflammation, ranging from monoculture through to xenotransplantation. However, combining physiological relevance, reproducibility, and scalability into one model is still a challenge. We examine key features of the in vitro microglial environment, especially media composition, extracellular matrix, and co-culture, to identify areas for improvement in current hiPSC-microglia models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Hedegaard, Stodolak, James and Cowley.)

Background: TREM2 is a microglial cell surface receptor, with risk mutations linked to Alzheimer's disease (AD), including R47H. TREM2 signalling via SYK aids phagocytosis, chemotaxis, survival, and changes to microglial activation state. In AD mouse models, knockout (KO) of TREM2 impairs microglial clustering around amyloid and prevents microglial activation. The R47H mutation is proposed to reduce TREM2 ligand binding. We investigated cell phenotypes of the R47H mutant and TREM2 KO in a model of human microglia, and compared their transcriptional signatures, to determine the mechanism by which R47H TREM2 disrupts function., Methods: We generated human microglia-like iPSC-macrophages (pMac) from isogenic induced pluripotent stem cell (iPSC) lines, with homozygous R47H mutation or TREM2 knockout (KO). We firstly validated the effect of the R47H mutant on TREM2 surface and subcellular localization in pMac. To assess microglial phenotypic function, we measured phagocytosis of dead neurons, cell morphology, directed migration, survival, and LPS-induced inflammation. We performed bulk RNA-seq, comparing significant differentially expressed genes (DEGs; p < 0.05) between the R47H and KO versus WT, and bioinformatically predicted potential upstream regulators of TREM2-mediated gene expression., Results: R47H modified surface expression and shedding of TREM2, but did not impair TREM2-mediated signalling, or gross phenotypes that were dysregulated in the TREM2 KO (phagocytosis, motility, survival). However, altered gene expression in the R47H TREM2 pMac overlapped by 90% with the TREM2 KO and was characterised by dysregulation of genes involved with immunity, proliferation, activation, chemotaxis, and adhesion. Downregulated mediators of ECM adhesion included the vitronectin receptor αVβ3, and consequently, R47H TREM2 pMac adhered weakly to vitronectin compared with WT pMac. To counteract these transcriptional defects, we investigated TGFβ1, as a candidate upstream regulator. TGFβ1 failed to rescue vitronectin adhesion of pMac, although it improved αVβ3 expression., Conclusions: The R47H mutation is not sufficient to cause gross phenotypic defects of human pMac under standard culture conditions. However, overlapping transcriptional defects with TREM2 KO supports the hypothesised partial loss-of-function effects of the R47H mutation. Furthermore, transcriptomics can guide us to more subtle phenotypic defects in the R47H cells, such as reduced cell adhesion, and can be used to predict targets for therapeutic intervention.

3'-Deoxynucleotides are an important class of drugs because they interfere with the metabolism of nucleotides, and their incorporation into DNA or RNA terminates cell division and viral replication. These compounds are generally produced by multi-step chemical synthesis, and an enzyme with the ability to catalyse the removal of the 3'-deoxy group from different nucleotides has yet to be described. Here, using a combination of HPLC, HRMS and NMR spectroscopy, we demonstrate that a thermostable fungal radical S-adenosylmethionine (SAM) enzyme, with similarity to the vertebrate antiviral enzyme viperin (RSAD2), can catalyse the transformation of CTP, UTP and 5-bromo-UTP to their 3'-deoxy-3',4'-didehydro (ddh) analogues. We show that, unlike the fungal enzyme, human viperin only catalyses the transformation of CTP to ddhCTP. Using electron paramagnetic resonance spectroscopy and molecular docking and dynamics simulations in combination with mutagenesis studies, we provide insight into the origin of the unprecedented substrate promiscuity of the enzyme and the mechanism of dehydration of a nucleotide. Our findings highlight the evolution of substrate specificity in a member of the radical-SAM enzymes. We predict that our work will help in using a new class of the radical-SAM enzymes for the biocatalytic synthesis of 3'-deoxy nucleotide/nucleoside analogues., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The Parkinson's disease-associated gene, LRRK2, is also associated with immune disorders and infectious disease and is expressed in immune subsets. Here, we characterize a platform for interrogating the expression and function of endogenous LRRK2 in authentic human phagocytes using human induced pluripotent stem cell-derived macrophages and microglia. Endogenous LRRK2 is expressed and upregulated by interferon-γ in these cells, including a 187-kDa cleavage product. Using LRRK2 knockout and G2019S isogenic repair lines, we find that LRRK2 is not involved in initial phagocytic uptake of bioparticles but is recruited to LAMP1 + /RAB9 + "maturing" phagosomes, and LRRK2 kinase inhibition enhances its residency at the phagosome. Importantly, LRRK2 is required for RAB8a and RAB10 recruitment to phagosomes, implying that LRRK2 operates at the intersection between phagosome maturation and recycling pathways in these professional phagocytes., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Viperin (RSAD2) is an antiviral radical S-adenosylmethionine (SAM) enzyme highly expressed in different cell types upon viral infection. Recently, it has been reported that the radical-SAM activity of viperin transforms cytidine triphosphate (CTP) to its analogue 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). Based on biochemical studies and cell biological experiments, it was concluded that ddhCTP and its nucleoside form ddhC do not affect the cellular concentration of nucleotide triphosphates and that ddhCTP acts as replication chain terminator. However, our re-evaluation of the reported data and new results indicate that ddhCTP is not an effective viral chain terminator but depletes cellular nucleotide pools and interferes with mitochondrial activity to inhibit viral replication. Our analysis is consistent with a unifying view of the antiviral and radical-SAM activities of viperin., (© 2020 Federation of European Biochemical Societies.)

Radical S-adenosylmethionine (SAM) domain-containing protein 2 (RSAD2; viperin) is a key enzyme in innate immune responses that is highly expressed in response to viral infection and inflammatory stimuli in many cell types. Recently, it was found that RSAD2 catalyses transformation of cytidine triphosphate (CTP) to its analogue 3'-deoxy-3',4'-didehydro-CTP (ddhCTP). The cellular function of this metabolite is unknown. Here, we analysed the extra- and intracellular metabolite levels in human induced pluripotent stem cell (hiPSC)-derived macrophages using high-resolution LC-MS/MS. The results together with biochemical assays and molecular docking simulations revealed that ddhCTP inhibits the NAD + -dependent activity of enzymes including that of the housekeeping enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We propose that ddhCTP regulates cellular metabolism in response to inflammatory stimuli such as viral infection, pointing to a broader function of RSAD2 than previously thought., (© 2020 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

One of the most common complications following external ventricular drain (EVD) placement is infection. Routine cultures of cerebrospinal fluid (CSF) are often used to screen for infection, however several days may pass before infection is discovered. In this study, we compared the predictive value of daily recorded vital sign parameters and peripheral white blood count (WBC) in identifying ventriculostomy-related infections. Patients with EVDs who had CSF cultures for microorganisms performed between January 2011 and July 2017 were assigned to either an infected and/or uninfected study group. Clinical parameters were then compared using t-test, chi squared and multiple logistic regression analyses. Patients of any age and gender were included. One hundred seventy uninfected and 10 infected subjects were included in the study. Nine of the 10 infected patients had an elevated WBC (>10.4 × 10 3 /μL), with a significantly greater WBC (15.9 × 10 3 /μL) than the uninfected group (10.4 × 10 3 /μL) (p-value ≤ 0.0001). Using logistic regression, we found no association between patient vital signs and CSF infection except for WBC (p = .003). As a diagnostic marker for CSF infection, the sensitivity and specificity of WBC elevation greater than 15 × 10 3 /μL was 70% (7/10) and 90.2% (147/163), respectively. This study serves as a 'proof of concept' that WBC could be useful as potential screening tool for early detection of CSF infection post-EVD placement. Future investigation using a large, multicenter prospective study is needed to further assess the applicability of this parameter., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Cavernous malformations of the brain are low-flow vascular lesions that have a propensity to hemorrhage. Extensive surgical approaches are often required for operative cure of deep-seated lesions. A 23-year-old female presented with a cavernous malformation of the left posterior insula with surrounding hematoma measuring up to 3 cm. A minimally invasive (mini-)pterional craniotomy with a transsylvian approach was selected. Endoscopic assistance was utilized to confirm complete resection of the lesion. The minipterional craniotomy is a minimally invasive approach that provides optimal exposure for sylvian fissure dissection and resection of many temporal and insular lesions. The video can be found here: https://youtu.be/9z6&#95;EhU6lxs., Competing Interests: Disclosures The authors report no conflict of interest concerning the materials or methods used in this study or the findings specified in this article., (© 2019, Peyton L. Nisson, Robert T. Wicks, Xiaochun Zhao, Whitney S. James, David Xu, and Peter Nakaji.)

Aneurysms involving the origin of the posterior inferior cerebellar artery (PICA) have a greater incidence of fusiform morphology, intraluminal thrombi, and wall calcifications. At times, a complex treatment strategy with vessel occlusion and revascularization using a bypass graft is necessary for successful obliteration of these aneurysms. The occipital artery (OA) is often the preferred donor graft for lesions of the posterior fossa because of its proximity to the target recipient vessels. However, dissection of an OA can be challenging, given its anatomically tortuous path and the presence of thick surrounding muscles. This video captures the dissection of the OA using an unconventional, "inside-out" harvesting technique and the end-to-side anastomosis of the OA to the PICA at the p3 segment. This was performed in a 58-year-old man who presented with a subarachnoid hemorrhage (Hunt and Hess grade IV) from a ruptured fusiform aneurysm located at the origin of the right PICA. Major steps in Video 1 during this case include 1) dissection and harvesting of the OA using the "inside out" technique, 2) placement of 2 temporary clips occluding the PICA and isolating the P3 segment, 3) end-to-side OA-P3 anastomosis, and 4) removal of the temporary clips and confirmation of the PICA's patency using intraoperative indocyanine green. Due to the potential for infarction of the brainstem, the patient was started on dual antiplatelet therapy postoperatively. The patient tolerated the procedure well and suffered no major complications related to the operation or from being placed on dual antiplatelet therapy. He did experience some mild, posterior neck pain and rigidity at the time of his 3-month follow-up, likely due to nerve injury that occurred while harvesting the OA. Overall, the patient remains in good neurologic status 1 year after the operation. Complex fusiform aneurysms at the origin of PICA can be successfully treated via an OA-PICA bypass.

Objective: Ewing sarcoma (ES) is among the most prevalent of bone sarcomas in young people. Less often, it presents as a primary lesion of the spine (5%-15% of patients with ES)., Methods: A systematic literature search was performed, querying several scientific databases per PRISMA guidelines. Inclusion criteria specified all studies of patients with surgically treated ES located in the spine. Patient age was categorized into three groups: 0-13 years (age group 1), 14-20 years (age group 2), and > 21 (age group 3)., Results: Eighteen studies were included, yielding 28 patients with ES of the spine. Sixty-seven percent of patients experienced a favorable outcome, with laminectomies representing the most common (46%) of surgical interventions. One-, 2-, and 5-year survival rates were 82% (n = 23), 75% (n = 21), and 57% (n = 16), respectively. Patients in age group 2 experienced the greatest mortality rate (75%) compared to age group 1 (9%) and age group 3 (22%). The calculated relative risk score indicated patients in age group 2 were 7.5 times more likely to die than other age groups combined (p = 0.02)., Conclusions: Primary ES of the spine is a rare, debilitating disease in which the role of surgery and its impact on one's quality of life and independence status has not been well described. This study found the majority of patients experienced a favorable outcome with respect to independence status following surgery and adjunctive treatment. An increased risk of recurrence and death was also present among the adolescent age group (14-20 years).

Background: Only a few case reports and case series exist reporting on primary chondrosarcomas of the spine. The objective of this study was to gain a better understanding of this patient population and surgical techniques used for treatment., Methods: A systematic literature search was performed in January 2018 querying several scientific databases, per PRISMA guidelines. Surgery type was categorized into en bloc, piecemeal excision, or non-en bloc or piecemeal excision., Results: In total, 34 records and 3 patients were included in the systematic review, yielding 87 patients with primary chondrosarcoma of the spine. The mean age was 41.5 years, with the tumor most commonly arising in adult patients (90.8%, 79/87); most were male (66.7%, 58/87). Those who underwent piecemeal excision had the highest death rate (56.7%, P ≤ 0.001) and highest rate of recurrence (63.3%, P ≤ 0.001) compared with en bloc and non-en bloc or piecemeal excision. The calculated reduced relative risk (RR) comparing en bloc with the other surgical techniques for recurrence and mortality was 78.8% (RR, 0.21; P ≤ 0.001) and 80.7% (RR, 0.19; P≤ 0.001), respectively. Survival analysis showed patients with a piecemeal excision had 9.4 times hazards ratio for death compared with en bloc (P = 0.001)., Conclusions: CS is a rare lesion that most commonly presents in adult male patients. En bloc surgical resection was associated with a significant decrease in recurrence, mortality, and increased survival compared with the other surgical techniques. In addition, any surgical technique that involved entering the tumor capsule showed a significantly greater risk for recurrence and death., (Copyright © 2018. Published by Elsevier Inc.)

Background Findings of both case control and in vitro investigations suggest that non-steroidal anti-inflammatory drugs (NSAIDs) may play a beneficial role in the occurrence, growth, and subsistence of glioblastoma multiforme (GBM) brain tumor in humans. Objective In the present retrospective cohort study, we assessed the impact of NSAID use on survival in patients diagnosed with and treated for GBM brain tumors. Methods The impact of NSAID use and six other potential prognostic indicators of survival were assessed in 71 patients treated for GBM brain tumors from February 2011 to June 2016. Survival analysis and cross-tabulation analyses were performed to examine the potential relationship between NSAID use and occurrence of intracranial hemorrhage over the course of treatment for GBM. Results Kaplan-Meier analysis revealed no significant difference in survival between patients with and without NSAID use (p = 0.75; 95% CI: 10.12, 18.13). Multiple Cox regression analysis identified only treatment with chemotherapy as imposing any statistically significant effect on survival (Hazard Ratio (HR) = 3.31; p < 0.001; 95% CI: 1.80, 6.07). Cross-tabulation revealed no significant effect of NSAID use on occurrence of hemorrhage during treatment, X 2 (2, N = 71) = 0.65, p 2-Sided = 0.42, (Fisher's Exact Test: p2-sided = 0.56, p1-sided = 0.31). Conclusion These results suggest that history of NSAID use is not a determinant of survival in GBM patients. More rigorous, prospective investigations of the effect of NSAID use on tumor progression are necessary before the utility of this family of drugs in the treatment of GBM can be adequately appraised., Competing Interests: The authors have declared that no competing interests exist.

OBJECTIVE Cushing disease is caused by a pituitary micro- or macroadenoma that hypersecretes adrenocorticotropic hormone (ACTH), resulting in hypercortisolemia. For decades, transsphenoidal resection (TSR) has been an efficacious treatment but with certain limitations, namely precise tumor localization and complete excision. The authors evaluated the novel use of a double-antibody sandwich assay for the real-time quantitation of ACTH in resected pituitary specimens with the goals of augmenting pathological diagnosis and ultimately improving long-term patient outcome. METHODS This study involved a retrospective review of records and an analysis of assay values, pathology slides, and MRI studies of patients with Cushing disease who had undergone TSR in the period from 2009 to 2014 and had at least 1 year of follow-up in coordination with an endocrinologist. In the operating room, biopsy specimens from the patients had been analyzed for tissue ACTH concentration. Additional samples were simultaneously sent for frozen-section pathological analysis. The ACTH assay performance was compared against pathology assessments of surgical tumor samples using receiver operating characteristic (ROC) analysis and against pre- and postoperative MRI studies. RESULTS Fourteen patients underwent TSR with guidance by ACTH-antibody assay and pathological assessment of 127 biopsy samples and were followed up for an average of 3 years. The ACTH threshold for discriminating adenomatous from normal tissue was 290,000 pg/mg of tissue, based on jointly maximized sensitivity (95.0%) and specificity (71.3%). Lateralization discordance between preoperative MRI studies and surgical visualization was noted in 3 patients, confirming the impression that MRI alone may not achieve optimal localization. A majority of the patients (85.7%) attained long-term disease remission based on urinary free cortisol levels, plasma cortisol levels, and long-term corticosteroid therapy. Comparisons of patient-months of remission and treatment failure showed that the remission rate in the study sample statistically exceeds the rate in historical controls (71.9%; p = 0.0007, Fisher's exact test). Long-term unexpected hormonal deficiencies were statistically similar between study patients (29%) and those in a meta-analysis (25%; p = 0.7596, Fisher's exact test). CONCLUSIONS These preliminary findings reflect the promising potential of tissue-based ACTH-antibody-guided assay for improving the cure rates of Cushing disease patients undergoing TSR. Further studies with larger sample sizes, further refinements of assay interpretation, and longer-term follow-ups are needed.

A recent gamma-retroviral clinical Chronic Granulomatous Disease (CGD) gene therapy (GT) trial achieved proof-of-concept but was accompanied by activation of oncogenes and transgene silencing. The ubiquitous chromatin opening element (UCOE) comprises the sequences of two divergently oriented house-keeping gene promoters and is known to have anti-silencing properties. In a screen we identified two novel UCOE constructs that prevent adjacent promoter methylation in P 19 cells. Experiments were continued with the shorter UCOE constructs in induced pluripotent stem cells (iPSC) derived from a p47phox-deficient CGD patient. The iPSC line was transduced with the lentiviral GT vectors expressing P47 under the constitutively active SFFV promoter with UCOE element (UCOE&#95;SF) and without UCOE element (SF) adjacent to the SFFV promoter. The iPSC were expanded before propagation towards neutrophils. 20 days after transduction the UCOE&#95;SF vector was protected from methylation in iPSC as previously shown in P 19 cells, whereas the SF vector was heavily methylated in iPSC. The UCOE&#95;SF vector maintained stable transgene expression in iPSC, macrophages and neutrophils, whereas the SF vector was strongly silenced. The UCOE&#95;SF vector stably restored ROS production in neutrophils, whereas for the SF vector the count of ROS producing cells was marginal. To conclude, we have shown that the prevention of transgene silencing by UCOE is functionally and mechanistically preserved upon terminal neutrophil differentiation.

To examine the pathogenic role of α-synuclein (αS) in Parkinson's Disease, we have generated induced Pluripotent Stem Cell lines from early onset Parkinson's Disease patients with SNCA A53T and SNCA Triplication mutations, and in this study have differentiated them to PSC-macrophages (pMac), which recapitulate many features of their brain-resident cousins, microglia. We show that SNCA Triplication pMac, but not A53T pMac, have significantly increased intracellular αS versus controls and release significantly more αS to the medium. SNCA Triplication pMac, but not A53T pMac, show significantly reduced phagocytosis capability and this can be phenocopied by adding monomeric αS to the cell culture medium of control pMac. Fibrillar αS is taken up by pMac by actin-rearrangement-dependent pathways, and monomeric αS by actin-independent pathways. Finally, pMac degrade αS and this can be arrested by blocking lysosomal and proteasomal pathways. Together, these results show that macrophages are capable of clearing αS, but that high levels of exogenous or endogenous αS compromise this ability, likely a vicious cycle scenario faced by microglia in Parkinson's disease.

Objective: This study aims to establish a new classification system for proximal anterior cerebral artery (A1) aneurysms and to offer treatment strategies, based on the lessons learned by studying the case reports of 41 patients with A1 aneurysms., Methods: A total of 2332 consecutive patients with intracranial aneurysms were treated at a single medical center between June 2005 and May 2015. Forty-one patients with 42 A1 aneurysms were treated by surgical clipping or endovascular coiling. Clinical data, radiographic results, treatments, and patient outcomes were later collected and analyzed., Results: Of 2332 intracranial aneurysm patients, 1.76% (n = 41) had a total of 42 A1 aneurysms. Twenty-six of these (62%) were proximal A1 segment aneurysms, 9 (21%) were distal A1 segment aneurysms, and 7 (17%) were fusiform A1 aneurysms. A1 aneurysms can be classified into 3 main types. Types IA and IB originate from the posterior wall of the proximal A1 segment. Type IA projects posterior-inferiorly, whereas type IB projects posterior-superiorly. Type IIA originates from the distal trunk of the A1 artery. Type IIB originates from an angle of an abnormal cortical branch or a ring of an A1 arterial fenestration. Type III consists of fusiform or dissecting aneurysms located anywhere along the A1 segment. After studying the range of treatments and outcomes, when treating these complex morphologies, we recommend clipping type I and II A1 aneurysms and embolizing type III A1 aneurysms., Conclusions: A1 artery aneurysms are a rare type of aneurysm with unique characteristics. The classification system proposed here accurately summarizes these characteristics to better guide treatment strategies., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Lipocalin-2 (also known as NGAL) levels are elevated in obesity and diabetes yet relatively little is known regarding effects on the heart. We induced pressure overload (PO) in mice and found that lipocalin-2 knockout (LKO) mice exhibited less PO-induced autophagy and NLRP3 inflammasome activation than Wt. PO-induced mitochondrial damage was reduced and autophagic flux greater in LKO mice, which correlated with less cardiac dysfunction. All of these observations were negated upon adenoviral-mediated restoration of normal lipocalin-2 levels in LKO. Studies in primary cardiac fibroblasts indicated that lipocalin-2 enhanced priming and activation of NLRP3-inflammasome, detected by increased IL-1β, IL-18 and Caspase-1 activation. This was attenuated in cells isolated from NLRP3-deficient mice or upon pharmacological inhibition of NLRP3. Furthermore, lipocalin-2 induced release of HMGB1 from cells and NLRP3-inflammasome activation was attenuated by TLR4 inhibition. We also found evidence of increased inflammasome activation and reduced autophagy in cardiac biopsy samples from heart failure patients. Overall, this study provides new mechanistic insight on the detrimental role of lipocalin-2 in the development of cardiac dysfunction., Competing Interests: None.

Microglia are increasingly implicated in brain pathology, particularly neurodegenerative disease, with many genes implicated in Alzheimer's, Parkinson's, and motor neuron disease expressed in microglia. There is, therefore, a need for authentic, efficient in vitro models to study human microglial pathological mechanisms. Microglia originate from the yolk sac as MYB-independent macrophages, migrating into the developing brain to complete differentiation. Here, we recapitulate microglial ontogeny by highly efficient differentiation of embryonic MYB-independent iPSC-derived macrophages then co-culture them with iPSC-derived cortical neurons. Co-cultures retain neuronal maturity and functionality for many weeks. Co-culture microglia express key microglia-specific markers and neurodegenerative disease-relevant genes, develop highly dynamic ramifications, and are phagocytic. Upon activation they become more ameboid, releasing multiple microglia-relevant cytokines. Importantly, co-culture microglia downregulate pathogen-response pathways, upregulate homeostatic function pathways, and promote a more anti-inflammatory and pro-remodeling cytokine response than corresponding monocultures, demonstrating that co-cultures are preferable for modeling authentic microglial physiology., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mutations in the leucine-rich repeat kinase 2 ( LRRK2 ) gene are found in familial and idiopathic cases of Parkinson's disease (PD), but are also associated with immune-related disorders, notably Crohn's disease and leprosy. Although the physiological function of LRRK2 protein remains largely elusive, increasing evidence suggests that it plays a role in innate immunity, a process that also has been implicated in neurodegenerative diseases, including PD. Innate immunity involves macrophages and microglia, in which endogenous LRRK2 expression is precisely regulated and expression is strongly up-regulated upon cell activation. This brief report discusses the current understanding of the involvement of LRRK2 in innate immunity particularly in relation to PD, critically examining its role in myeloid cells, particularly macrophages and microglia., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Phagocytosis of pathogens, apoptotic cells and debris is a key feature of macrophage function in host defense and tissue homeostasis. Quantification of macrophage phagocytosis in vitro has traditionally been technically challenging. Here we report the optimization and validation of the IncuCyte ZOOM® real time imaging platform for macrophage phagocytosis based on pHrodo® pathogen bioparticles, which only fluoresce when localized in the acidic environment of the phagolysosome. Image analysis and fluorescence quantification were performed with the automated IncuCyte™ Basic Software. Titration of the bioparticle number showed that the system is more sensitive than a spectrofluorometer, as it can detect phagocytosis when using 20× less E. coli bioparticles. We exemplified the power of this real time imaging platform by studying phagocytosis of murine alveolar, bone marrow and peritoneal macrophages. We further demonstrate the ability of this platform to study modulation of the phagocytic process, as pharmacological inhibitors of phagocytosis suppressed bioparticle uptake in a concentration-dependent manner, whereas opsonins augmented phagocytosis. We also investigated the effects of macrophage polarization on E. coli phagocytosis. Bone marrow-derived macrophage (BMDM) priming with M2 stimuli, such as IL-4 and IL-10 resulted in higher engulfment of bioparticles in comparison with M1 polarization. Moreover, we demonstrated that tolerization of BMDMs with lipopolysaccharide (LPS) results in impaired E. coli bioparticle phagocytosis. This novel real time assay will enable researchers to quantify macrophage phagocytosis with a higher degree of accuracy and sensitivity and will allow investigation of limited populations of primary phagocytes in vitro., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chronic granulomatous disease (CGD) is a rare genetic disease characterized by severe and persistent childhood infections. It is caused by the lack of an antipathogen oxidative burst, normally performed by phagocytic cells to contain and clear bacterial and fungal growth. Restoration of immune function can be achieved with heterologous bone marrow transplantation; however, autologous bone marrow transplantation would be a preferable option. Thus, a method is required to recapitulate the function of the diseased gene within the patient's own cells. Gene therapy approaches for CGD have employed randomly integrating viruses with concomitant issues of insertional mutagenesis, inaccurate gene dosage, and gene silencing. Here, we explore the potential of the recently described clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 site-specific nuclease system to encourage repair of the endogenous gene by enhancing the levels of homologous recombination. Using induced pluripotent stem cells derived from a CGD patient containing a single intronic mutation in the CYBB gene, we show that footprintless gene editing is a viable option to correct disease mutations. Gene correction results in restoration of oxidative burst function in iPS-derived phagocytes by reintroduction of a previously skipped exon in the cytochrome b-245 heavy chain (CYBB) protein. This study provides proof-of-principle for a gene therapy approach to CGD treatment using CRISPR-Cas9., (Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)