An Amplex Red-based fluorometric and spectrophotometric assay for L-asparaginase using its natural substrate.

We report on the development of a sensitive real-time assay for monitoring the activity of L-asparaginase that hydrolyzes L-asparagine to L-aspartate and ammonia. In this method, L-aspartate is oxidized by L-aspartate oxidase to iminoaspartate and hydrogen peroxide (H2O2), and in the detection step horseradish peroxidase uses H2O2 to convert the colorless, nonfluorescent reagent Amplex Red to the red-colored and highly fluorescent product resorufin. The assay was validated in both the absorbance and the fluorescence modes. We show that, due to its high sensitivity and substrate selectivity, this assay can be used to measure enzymatic activity in human serum containing L-asparaginase

Engineering and preclinical evaluation of a human enzyme immune checkpoint inhibitor for cancer therapy

The work presented here builds on the findings that the amino acid kynurenine (L-Kyn) synthesized via the enzymes indoleamine/tryptophan dioxygenase (IDO/TDO) mediates cancer immune suppression in a paracrine fashion and evaluates the hypothesis that systemic elimination of secreted Kyn in the extracellular space using an engineered human kynureninase enzyme (KYNase) to degrade it into inactive metabolites will have pronounced anti-tumor activity and overcome the limitations associated with IDO/TDO inhibitors. The significance of L-Kyn production in cancer is well recognized and has led to the development of inhibitors of IDO and TDO; with at least one IDO inhibitor currently undergoing Phase II/III clinical evaluation. However IDO or TDO inhibition is problematic for cancer therapy because: (1) there are two isoforms of IDO and together with TDO, the inhibition of all possible pathways for Kynurenine generation at present requires the generation of multiple small molecule inhibitors; (2) resistance to inhibition can arise, and (3) as a single agent IDO/TDO inhibitors show very little efficacy. Wild-type human KYNase has a strong preference for the degradation of 3’OH Kynurenine (OH-Kyn) with a kcat/KM = 105 M-1s-1, about 1,000 fold higher than that displayed for L-Kyn degradation (102 M-1s-1). In contrast some bacterial enzymes such as the KYNase from P.fluorescens displays a nearly equal and opposite substrate preference for Kyn over OH-Kyn. Although the P.fluorescens KYNase has ideal kinetics desired in an enzyme therapeutic targeting tumor L-Kyn, its high immunogenicity render it unsuitable as a clinical candidate, necessitating the engineering of a human KYNase with the requisite pharmacological properties. Therefore we undertook a directed evolution campaign coupled with high throughput competitive genetic selections and screening strategies to engineer the human KYNase enzyme, creating variants with \u3e 500 fold increases in catalytic activity towards L-Kyn. Engineered human KYNase enzymes were then PEGylated for long circulatory persistence and administered to mice bearing murine cancer allografts and evaluated for efficacy and PK/PD. We found that administration of PEGylated engineered human KYNase enzymes resulted in lowered systemic L-Kyn levels accompanied by significant tumor growth retardation, extended survival and even complete regressions in a manner similar to that observed with immune checkpoint inhibitors such as anti-PD. Flow cytometric analysis showed a significant increase in the proportion of TCRB+ T cells in the tumor-infiltrating lymphocytes (TILs) that is consistent with significant incorporation of BrdU in CD4+ and CD8+ T cells in the tumor-draining lymph nodes (dLNs) and TILs compared to control mice treated with deactivated enzyme. Additionally, marked elevation of CD8+ and CD4+ T cells expressing granzyme (Gzm)B and interferon (IFNG) was observed in the active-enzyme-treated mice, further highlighting the importance of L-Kyn in tumor evasion of immune surveillance. As a monotherapy, small molecule inhibitors of IDO1 display at most marginal anti-cancer activity in animal models as well as in clinical trials likely due to the redundancy of Kyn biosynthetic pathways; necessitating combinations of IDO1/TDO inhibitors. The therapeutic enzyme approach using engineered human KYNase represents an effective “first in class” drug for restoring T-cell immunity for cancer eradication, without the limitations of IDO1/TDO inhibition. This work further demonstrates the utility of “enzymes as drugs” in targeting aberrantly regulated metabolites in disease states

Integral abutment bridges: Investigation of seismic soil-structure interaction effects by shaking table testing

In recent years there has been renewed interest on integral abutment bridges (IABs), mainly due to their low construction and maintenance cost. Owing to the monolithic connection between deck and abutments, there is strong soil-structure interaction between the bridge and the backfill under both thermal action and earthquake shaking. Although some of the regions where IABs are adopted qualify as highly seismic, there is limited knowledge as to their dynamic behaviour and vulnerability under strong ground shaking. To develop a better understanding on the seismic behaviour of IABs, an extensive experimental campaign involving over 75 shaking table tests and 4800 time histories of recorded data, was carried out at EQUALS Laboratory, University of Bristol, under the auspices of EU-sponsored SERA project (Seismology and Earthquake Engineering Research Infrastructure Alliance for Europe). The tests were conducted on a 5 m long shear stack mounted on a 3 m × 3 m 6-DOF earthquake simulator, focusing on interaction effects between a scaled bridge model, abutments, foundation piles and backfill soil. The study aims at (a) developing new scaling procedures for physical modelling of IABs, (b) investigating experimentally the potential benefits of adding compressible inclusions (CIs) between the abutment and the backfill and (c) exploring the influence of different types of connection between the abutment and the pile foundation. Results indicate that the CI reduces the accelerations on the bridge deck and the settlements in the backfill, while disconnecting piles from the cap decreases bending near the pile head

Monte-Carlo dosimetry on a realistic cell monolayer geometry exposed to alpha particles

The energy and specific energy absorbed in the main cell compartments (nucleus and cytoplasm) in typical radiobiology experiments are usually estimated by calculations as they are not accessible for a direct measurement. In most of the work, the cell geometry is modelled using the combination of simple mathematical volumes. We propose a method based on high resolution confocal imaging and ion beam analysis (IBA) in order to import realistic cell nuclei geometries in Monte-Carlo simulations and thus take into account the variety of different geometries encountered in a typical cell population. Seventy-six cell nuclei have been imaged using confocal microscopy and their chemical composition has been measured using IBA. A cellular phantom was created from these data using the ImageJ image analysis software and imported in the Geant4 Monte-Carlo simulation toolkit. Total energy and specific energy distributions in the 76 cell nuclei have been calculated for two types of irradiation protocols: a 3 MeV alpha particle microbeam used for targeted irradiation and a 239Pu alpha source used for large angle random irradiation. Qualitative images of the energy deposited along the particle tracks have been produced and show good agreement with images of DNA double strand break signalling proteins obtained experimentally. The methodology presented in this paper provides microdosimetric quantities calculated from realistic cellular volumes. It is based on open-source oriented software that is publicly available

Geminin-Deficient Neural Stem Cells Exhibit Normal Cell Division and Normal Neurogenesis

Neural stem cells (NSCs) are the progenitors of neurons and glial cells during both embryonic development and adult life. The unstable regulatory protein Geminin (Gmnn) is thought to maintain neural stem cells in an undifferentiated state while they proliferate. Geminin inhibits neuronal differentiation in cultured cells by antagonizing interactions between the chromatin remodeling protein Brg1 and the neural-specific transcription factors Neurogenin and NeuroD. Geminin is widely expressed in the CNS during throughout embryonic development, and Geminin expression is down-regulated when neuronal precursor cells undergo terminal differentiation. Over-expression of Geminin in gastrula-stage Xenopus embryos can expand the size of the neural plate. The role of Geminin in regulating vertebrate neurogenesis in vivo has not been rigorously examined. To address this question, we created a strain of Nestin-Cre/Gmnnfl/fl mice in which the Geminin gene was specifically deleted from NSCs. Interestingly, we found no major defects in the development or function of the central nervous system. Neural-specific GmnnΔ/Δ mice are viable and fertile and display no obvious neurological or neuroanatomical abnormalities. They have normal numbers of BrdU+ NSCs in the subgranular zone of the dentate gyrus, and GmnnΔ/Δ NSCs give rise to normal numbers of mature neurons in pulse-chase experiments. GmnnΔ/Δ neurosphere cells differentiate normally into both neurons and glial cells when grown in growth factor-deficient medium. Both the growth rate and the cell cycle distribution of cultured GmnnΔ/Δ neurosphere cells are indistinguishable from controls. We conclude that Geminin is largely dispensable for most of embryonic and adult mammalian neurogenesis

Geminin Is Required for Zygotic Gene Expression at the Xenopus Mid-Blastula Transition

In many organisms early development is under control of the maternal genome and zygotic gene expression is delayed until the mid-blastula transition (MBT). As zygotic transcription initiates, cell cycle checkpoints become activated and the tempo of cell division slows. The mechanisms that activate zygotic transcription at the MBT are incompletely understood, but they are of interest because they may resemble mechanisms that cause stem cells to stop dividing and terminally differentiate. The unstable regulatory protein Geminin is thought to coordinate cell division with cell differentiation. Geminin is a bi-functional protein. It prevents a second round of DNA replication during S and G2 phase by binding and inhibiting the essential replication factor Cdt1. Geminin also binds and inhibits a number of transcription factors and chromatin remodeling proteins and is thought to keep dividing cells in an undifferentiated state. We previously found that the cells of Geminin-deficient Xenopus embryos arrest in G2 phase just after the MBT then disintegrate at the onset of gastrulation. Here we report that they also fail to express most zygotic genes. The gene expression defect is cell-autonomous and is reproduced by over-expressing Cdt1 or by incubating the embryos in hydroxyurea. Geminin deficient and hydroxyurea-treated blastomeres accumulate DNA damage in the form of double stranded breaks. Bypassing the Chk1 pathway overcomes the cell cycle arrest caused by Geminin depletion but does not restore zygotic gene expression. In fact, bypassing the Chk1 pathway by itself induces double stranded breaks and abolishes zygotic transcription. We did not find evidence that Geminin has a replication-independent effect on transcription. We conclude that Geminin is required to maintain genome integrity during the rapid cleavage divisions, and that DNA damage disrupts zygotic gene transcription at the MBT, probably through activation of DNA damage checkpoint pathways

Geographical and temporal distribution of SARS-CoV-2 clades in the WHO European Region, January to June 2020

We show the distribution of SARS-CoV-2 genetic clades over time and between countries and outline potential genomic surveillance objectives. We applied three available genomic nomenclature systems for SARS-CoV-2 to all sequence data from the WHO European Region available during the COVID-19 pandemic until 10 July 2020. We highlight the importance of real-time sequencing and data dissemination in a pandemic situation. We provide a comparison of the nomenclatures and lay a foundation for future European genomic surveillance of SARS-CoV-2.Peer reviewe

Autoantibodies against type I IFNs in patients with life-threatening COVID-19

Interindividual clinical variability in the course of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is vast. We report that at least 101 of 987 patients with life-threatening coronavirus disease 2019 (COVID-19) pneumonia had neutralizing immunoglobulin G (IgG) autoantibodies (auto-Abs) against interferon-w (IFN-w) (13 patients), against the 13 types of IFN-a (36), or against both (52) at the onset of critical disease; a few also had auto-Abs against the other three type I IFNs. The auto-Abs neutralize the ability of the corresponding type I IFNs to block SARS-CoV-2 infection in vitro. These auto-Abs were not found in 663 individuals with asymptomatic or mild SARS-CoV-2 infection and were present in only 4 of 1227 healthy individuals. Patients with auto-Abs were aged 25 to 87 years and 95 of the 101 were men. A B cell autoimmune phenocopy of inborn errors of type I IFN immunity accounts for life-threatening COVID-19 pneumonia in at least 2.6% of women and 12.5% of men

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

Life-threatening `breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS- CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals ( age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto- Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-a2 and IFN-., while two neutralized IFN-omega only. No patient neutralized IFN-ss. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population