Gene editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNA

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD. Institutode Investigacio ' n Sanitaria de la Fundacio '

Gene editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNA

TheauthorswouldliketothankMiguelA.MartinforthecarefulmaintenanceofNSGmice,andRebecaSa ́nchezandOmairaAlberquillafortheirtechnicalassistanceinflowcytometry.TheauthorsalsothankFundacio ́n Botı ́n forpromotingtranslationalresearchattheHemato-poieticInnovativeTherapiesDivisionoftheCIEMATPyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD.S

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Holocene geochemical footprint from Semiarid alpine wetlands in southern Spain

Here we provide the geochemical dataset that our research group has collected after 10 years of investigation in the Sierra Nevada National Park in southern Spain. These data come from Holocene sedimentary records from four alpine sites (ranging from ∼2500 to ∼3000 masl): two peatlands and two shallow lakes. Different kinds of organic and inorganic analyses have been conducted. The organic matter in the bulk sediment was characterised using elemental measurements and isotope-ratio mass spectrometry (EA-IRMS). Leaf waxes in the sediment were investigated by means of chromatography with flame-ionization detection and mass spectrometry (GC-FID, GC-MS). Major, minor and trace elements of the sediments were analysed with atomic absorption (AAS), inductively coupled plasma mass spectrometry (ICP-MS), as well as X-ray scanning fluorescence. These data can be reused by environmental researchers and soil and land managers of the Sierra Nevada National Park and similar regions to identify the effect of natural climate change, overprinted by human impact, as well as to project new management policies in similar protected areas.Universidad de Granada. Departamento de Estratigrafía y PaleontologíaJunta de Andalucía: Grupos de investigación RNM190 y RNM309Junta de Andalucía: Proyecto P11-RNM-7332España, Ministerio de Economía y Competitividad: Proyecto CGL2013-47038-RRamón y Cajal Fellowship: RYC-2015-18966Small Research Grant by the Carnegie Trust for the Universities of ScotlandMarie Curie Intra-European Fellowship of the 7th Framework Programme for Research, Technological Development and Demonstration of the European Commission: NAOSIPUK. Grant Number: PIEF-GA-2012-62302

A detailed study of the very-high-energy Crab pulsar emission with the LST-1

Context: There are currently three pulsars firmly detected by imaging atmospheric Cherenkov telescopes (IACTs), two of them reaching TeV energies, challenging models of very-high-energy (VHE) emission in pulsars. More precise observations are needed to better characterize pulsar emission at these energies. The LST-1 is the prototype of the Large-Sized Telescope, that will be part of the Cherenkov Telescope Array Observatory (CTAO). Its improved performance over previous IACTs makes it well suited for studying pulsars. Aims: To study the Crab pulsar emission with the LST-1, improving and complementing the results from other telescopes. These observations can also be used to characterize the potential of the LST-1 to study other pulsars and detect new ones. Methods: We analyzed a total of $\sim$103 hours of gamma-ray observations of the Crab pulsar conducted with the LST-1 in the period from September 2020 to January 2023. The observations were carried out at zenith angles less than 50 degrees. A new analysis of the Fermi-LAT data was also performed, including $\sim$14 years of observations. Results: The Crab pulsar phaseogram, long-term light-curve, and phase-resolved spectra are reconstructed with the LST-1 from 20 GeV to 450 GeV for P1 and up to 700 GeV for P2. The pulsed emission is detected with a significance of 15.2$σ$. The two characteristic emission peaks of the Crab pulsar are clearly detected (>10$σ$), as well as the so-called bridge emission (5.7$σ$). We find that both peaks are well described by power laws, with spectral indices of $\sim$3.44 and $\sim$3.03 respectively. The joint analysis of Fermi-LAT and LST-1 data shows a good agreement between both instruments in the overlapping energy range. The detailed results obtained in the first observations of the Crab pulsar with LST-1 show the potential that CTAO will have to study this type of sources

Multiwavelength study of the galactic PeVatron candidate LHAASO J2108+5157

Context. Several new ultrahigh-energy (UHE) γ-ray sources have recently been discovered by the Large High Altitude Air Shower Observatory (LHAASO) collaboration. These represent a step forward in the search for the so-called Galactic PeVatrons, the enigmatic sources of the Galactic cosmic rays up to PeV energies. However, it has been shown that multi-TeV γ-ray emission does not necessarily prove the existence of a hadronic accelerator in the source; indeed this emission could also be explained as inverse Compton scattering from electrons in a radiation-dominated environment. A clear distinction between the two major emission mechanisms would only be made possible by taking into account multi-wavelength data and detailed morphology of the source. Aims. We aim to understand the nature of the unidentified source LHAASO J2108+5157, which is one of the few known UHE sources with no very high-energy (VHE) counterpart. Methods. We observed LHAASO J2108+5157 in the X-ray band with XMM-Newton in 2021 for a total of 3.8 hours and at TeV energies with the Large-Sized Telescope prototype (LST-1), yielding 49 hours of good-quality data. In addition, we analyzed 12 years of Fermi-LAT data, to better constrain emission of its high-energy (HE) counterpart 4FGL J2108.0+5155. We used naima and jetset software packages to examine the leptonic and hadronic scenario of the multi-wavelength emission of the source. Results. We found an excess (3.7σ) in the LST-1 data at energies E > 3 TeV. Further analysis of the whole LST-1 energy range, assuming a point-like source, resulted in a hint (2.2σ) of hard emission, which can be described with a single power law with a photon index of Σ = 1.6 ± 0.2 the range of 0.3 - 100 TeV. We did not find any significant extended emission that could be related to a supernova remnant (SNR) or pulsar wind nebula (PWN) in the XMM-Newton data, which puts strong constraints on possible synchrotron emission of relativistic electrons. We revealed a new potential hard source in Fermi-LAT data with a significance of 4σ and a photon index of Σ = 1.9 ± 0.2, which is not spatially correlated with LHAASO J2108+5157, but including it in the source model we were able to improve spectral representation of the HE counterpart 4FGL J2108.0+5155. Conclusions. The LST-1 and LHAASO observations can be explained as inverse Compton-dominated leptonic emission of relativistic electrons with a cutoff energy of 100-30+70 TeV. The low magnetic field in the source imposed by the X-ray upper limits on synchrotron emission is compatible with a hypothesis of a PWN or a TeV halo. Furthermore, the spectral properties of the HE counterpart are consistent with a Geminga-like pulsar, which would be able to power the VHE-UHE emission. Nevertheless, the lack of a pulsar in the neighborhood of the UHE source is a challenge to the PWN/TeV-halo scenario. The UHE γ rays can also be explained as π0 decay-dominated hadronic emission due to interaction of relativistic protons with one of the two known molecular clouds in the direction of the source. Indeed, the hard spectrum in the LST-1 band is compatible with protons escaping a shock around a middle-aged SNR because of their high low-energy cut-off, but the origin of the HE γ-ray emission remains an open question

Star tracking for pointing determination of Imaging Atmospheric Cherenkov Telescopes: Application to the Large-Sized Telescope of the Cherenkov Telescope Array

We present a novel approach to the determination of the pointing of Imaging Atmospheric Cherenkov Telescopes (IACTs) using the trajectories of the stars in their camera s field of view. The method starts with the reconstruction of the star positions from the Cherenkov camera data, taking into account the point spread function of the telescope, to achieve a satisfying reconstruction accuracy of the pointing position. A simultaneous fit of all reconstructed star trajectories is then performed with the orthogonal distance regression (ODR) method. ODR allows us to correctly include the star position uncertainties and use the time as an independent variable. Having the time as an independent variable in the fit makes it better suitable for various star trajectories. This method can be applied to any IACT and requires neither specific hardware nor interface or special data-taking mode. In this paper, we use the Large-Sized Telescope (LST) data to validate it as a useful tool to improve the determination of the pointing direction during regular data taking. The simulation studies show that the accuracy and precision of the method are comparable with the design requirements on the pointing accuracy of the LST (=14''). With the typical LST event acquisition rate of 10 kHz, the method can achieve up to 50 Hz pointing monitoring rate, compared to O(1) Hz achievable with standard techniques. The application of the method to the LST prototype (LST-1) commissioning data shows the stable pointing performance of the telescope

Sensitivity of the Cherenkov Telescope Array to spectral signatures of hadronic PeVatrons with application to Galactic Supernova Remnants

The local Cosmic Ray (CR) energy spectrum exhibits a spectral softening at energies around 3~PeV. Sources which are capable of accelerating hadrons to such energies are called hadronic PeVatrons. However, hadronic PeVatrons have not yet been firmly identified within the Galaxy. Several source classes, including Galactic Supernova Remnants (SNRs), have been proposed as PeVatron candidates. The potential to search for hadronic PeVatrons with the Cherenkov Telescope Array (CTA) is assessed. The focus is on the usage of very high energy $\gamma$-ray spectral signatures for the identification of PeVatrons. Assuming that SNRs can accelerate CRs up to knee energies, the number of Galactic SNRs which can be identified as PeVatrons with CTA is estimated within a model for the evolution of SNRs. Additionally, the potential of a follow-up observation strategy under moonlight conditions for PeVatron searches is investigated. Statistical methods for the identification of PeVatrons are introduced, and realistic Monte--Carlo simulations of the response of the CTA observatory to the emission spectra from hadronic PeVatrons are performed. Based on simulations of a simplified model for the evolution for SNRs, the detection of a $\gamma$-ray signal from in average 9 Galactic PeVatron SNRs is expected to result from the scan of the Galactic plane with CTA after 10 hours of exposure. CTA is also shown to have excellent potential to confirm these sources as PeVatrons in deep observations with $\mathcal{O}(100)$ hours of exposure per source.Comment: 34 pages, 16 figures, Accepted for publication in Astroparticle Physic

Southern African Large Telescope Spectroscopy of BL Lacs for the CTA project

In the last two decades, very-high-energy gamma-ray astronomy has reached maturity: over 200 sources have been detected, both Galactic and extragalactic, by ground-based experiments. At present, Active Galactic Nuclei (AGN) make up about 40% of the more than 200 sources detected at very high energies with ground-based telescopes, the majority of which are blazars, i.e. their jets are closely aligned with the line of sight to Earth and three quarters of which are classified as high-frequency peaked BL Lac objects. One challenge to studies of the cosmological evolution of BL Lacs is the difficulty of obtaining redshifts from their nearly featureless, continuum-dominated spectra. It is expected that a significant fraction of the AGN to be detected with the future Cherenkov Telescope Array (CTA) observatory will have no spectroscopic redshifts, compromising the reliability of BL Lac population studies, particularly of their cosmic evolution. We started an effort in 2019 to measure the redshifts of a large fraction of the AGN that are likely to be detected with CTA, using the Southern African Large Telescope (SALT). In this contribution, we present two results from an on-going SALT program focused on the determination of BL Lac object redshifts that will be relevant for the CTA observatory

Sensitivity of the Cherenkov Telescope Array for probing cosmology and fundamental physics with gamma-ray propagation

The Cherenkov Telescope Array (CTA), the new-generation ground-based observatory for $\gamma$-ray astronomy, provides unique capabilities to address significant open questions in astrophysics, cosmology, and fundamental physics. We study some of the salient areas of $\gamma$-ray cosmology that can be explored as part of the Key Science Projects of CTA, through simulated observations of active galactic nuclei (AGN) and of their relativistic jets. Observations of AGN with CTA will enable a measurement of $\gamma$-ray absorption on the extragalactic background light with a statistical uncertainty below 15% up to a redshift $z=2$ and to constrain or detect $\gamma$-ray halos up to intergalactic-magnetic-field strengths of at least 0.3pG. Extragalactic observations with CTA also show promising potential to probe physics beyond the Standard Model. The best limits on Lorentz invariance violation from $\gamma$-ray astronomy will be improved by a factor of at least two to three. CTA will also probe the parameter space in which axion-like particles could constitute a significant fraction, if not all, of dark matter. We conclude on the synergies between CTA and other upcoming facilities that will foster the growth of $\gamma$-ray cosmology