Imitation of β-lactam binding enables broad-spectrum metallo-β-lactamase inhibitors

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-beta-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential beta-lactamase stable beta-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.Peer reviewe

The genetic architecture of the human cerebral cortex

The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder

MAGIC and H.E.S.S. detect VHE gamma rays from the blazar OT081 for the first time: a deep multiwavelength study

https://pos.sissa.it/395/815/pdfPublished versio

Use of deep learning for structural analysis of computer tomography images of soil samples

Soil samples from several European countries were scanned using medical computer tomography (CT) device and are now available as CT images. The analysis of these samples was carried out using deep learning methods. For this purpose, a VGG16 network was trained with the CT images (X). For the annotation (y) a new method for automated annotation, ‘surrogate’ learning, was introduced. The generated neural networks (NNs) were subjected to a detailed analysis. Among other things, transfer learning was used to check whether the NN can also be trained to other y-values. Visually, the NN was verified using a gradient-based class activation mapping (grad-CAM) algorithm. These analyses showed that the NN was able to generalize, i.e. to capture the spatial structure of the soil sample. Possible applications of the models are discussed

Targeted Repair of p47-CGD in iPSCs by CRISPR/Cas9: Functional Correction without Cleavage in the Highly Homologous Pseudogenes

Mutations in the NADPH oxidase, which is crucial for the respiratory burst in phagocytes, result in chronic granulomatous disease (CGD). The only curative treatment option for CGD patients, who suffer from severe infections, is allogeneic bone marrow transplantation. Over 90% of patients with mutations in the p47phox subunit of the oxidase complex carry the deletion c.75 76delGT (ΔGT). This frequent mutation most likely originates via gene conversion from one of the two pseudogenes NCF1B or NCF1C, which are highly homologous to NCF1 (encodes p47phox) but carry the ΔGT mutation. We applied CRISPR/Cas9 to generate patient-like p47-ΔGT iPSCs for disease modeling. To avoid unpredictable chromosomal rearrangements by CRISPR/Cas9-mediated cleavage in the pseudogenes, we developed a gene-correction approach to specifically target NCF1 but leave the pseudogenes intact. Functional assays revealed restored NADPH oxidase activity and killing of bacteria in corrected phagocytes as well as the specificity of this approach

Targeted Repair of p47-CGD Restores the Ability of iPSC-Derived Macrophages to Kill Bacteria

Chronic granulomatous disease (CGD) is caused by mutations in the NADPH oxidase and is characterized by defective phagocytes. The NAPDH oxidase is important for the production of reactive oxygen species that further regulate the phagosome milieu, such as the release of granules containing antimicrobial proteins, and the killing of bacteria inside the phagosome, which is defective in CGD patients. Only allogenic hematopoietic stem cell transplantation (HSCT) and retroviral gene therapy are curative treatment options for CGD patients. The first gene therapy trials for CGD were complicated by insertional mutagenesis and transgene silencing of the applied LTRdriven gamma-retroviral vector. More promising results were shown by recent studies with lentiviral SIN vectors and a myeloid-specific promoter. However, targeted gene correction using CRISPR-Cas9 would avoid the risk of vector-mediated insertional mutagenesis and silencing of the therapeutic gene. In our study, we developed a gene editing approach to correct p47phox-deficiency, which affects about25% of CGD patients. Over 90% of these patients carry the mutation c.75 76delGT (ΔGT) in exon 2 of NCF1 (encodes p47phox). This highprevalence most likely originates via gene conversion from one of the two pseudogenes NCF1B/C, which are over 99% homologous to NCF1 and carry the aforementioned ΔGT mutation. For proof-of-principle, we corrected p47phox-deficient induced pluripotent stem cells (iPSC)by inserting a minigene donor construct into intron 1 of NCF1. We chose this position, which differs by three additional nucleotides from the pseudogenes, to avoid cleavage in the pseudogenes and, thus, potential chromosomal instability due to multiple DNA doublestrand breaks. To analyze functionality, genetically corrected clones were differentiated into granulocytes and macrophages. Corrected granulocytes expressed p47phoxsimilar to wild type levels, displayedNADPH oxidase activity and were able to form neutrophil extracellular traps, which enable granulocytes to kill microbes extracellularly. Finally, iPSC-derived macrophages were infected with GFP-labeled E. coli. After phagocytosis, corrected macrophages significantly reduced the amount of living bacteria inside their phagosomes as measured by colony-forming units in plated cell lysates compared to uncorrected macrophages. In summary, we demonstrated that the specific insertion of a minigene into intron 1 of NCF1 corrects p47phox-deficiency.Moreover, the pseudogenes remained intact with our strategy, which could be of importance as the pseudogenes might have functional roles. We showed that the corrected iPSC-derived granulocytes and macrophages have all the features necessary to fight infections. In the past, some patients benefited from allogenic granulocyte transfusions. However, due to alloimmunization, this therapy is contraindicative for subsequent HSCT. We propose that autologous iPSC-derived granulocytes and macrophages could be applied therapeutically in the future to combat refractory infections prior to HSCT

Imitation of β-lactam binding enables broad-spectrum metallo-β-lactamase inhibitors

Carbapenems are vital antibiotics, but their efficacy is increasingly compromised by metallo-beta-lactamases (MBLs). Here we report the discovery and optimization of potent broad-spectrum MBL inhibitors. A high-throughput screen for NDM-1 inhibitors identified indole-2-carboxylates (InCs) as potential beta-lactamase stable beta-lactam mimics. Subsequent structure-activity relationship studies revealed InCs as a new class of potent MBL inhibitor, active against all MBL classes of major clinical relevance. Crystallographic studies revealed a binding mode of the InCs to MBLs that, in some regards, mimics that predicted for intact carbapenems, including with respect to maintenance of the Zn(II)-bound hydroxyl, and in other regards mimics binding observed in MBL-carbapenem product complexes. InCs restore carbapenem activity against multiple drug-resistant Gram-negative bacteria and have a low frequency of resistance. InCs also have a good in vivo safety profile, and when combined with meropenem show a strong in vivo efficacy in peritonitis and thigh mouse infection models.Peer reviewe

Intensity interferometry with the MAGIC telescopes

Due to their large mirror size, fast response to single photons, sensitivity and telescope baselines in the order of 100 m, Imaging Atmospheric Cherenkov Telescopes are ideally suited to perform intensity interferometry observations. In 2019 a test readout setup was installed in the two 17-m diameter MAGIC telescopes to allow performing interferometry measurements with them. The first on-sky measurements were able to detect correlated intensity fluctuations consistent with the stellar diameters of three different stars: Adhara (n CMa), Benetnasch ([ UMa) and Mirzam (V CMa). After the upgrade of the setup in 2021, MAGIC is now equipped with a high-duty-cycle intensity interferometer, already in operation. A technical description of the interferometer and first performance results obtained by measuring several known stellar diameter are presented

Extreme blazars under the eyes of MAGIC

Extreme high-frequency-peaked BL Lac objects (EHBLs) are the most energetic persistent sources in the universe. This contribution reports on long-term observing campaigns of tens of EHBLs that have been organized by the MAGIC collaboration to enlarge their population at VHE and understand the origin of their extreme properties. EHBLs are characterized by a spectral energy distribution (SED) featuring a synchrotron peak energy above 1 keV. Several EHBLs display a hard spectral index at very high energies (VHE; E > 100 GeV), suggesting a gamma-ray SED component peaking significantly above 1 TeV. Such extreme properties are challenging current standard emission and acceleration mechanisms. Recent studies have also unveiled intriguing disparities in the temporal characteristics of EHBLs. Some sources seem to display a persistent EHBL behaviour, while others belong to the EHBL family only temporarily. Here, we present recent results of the first hard-TeV EHBL catalog. The MAGIC observations are accompanied by an extensive multiwavelength coverage to obtain an optimal determination of the SED. This allow us to investigate leptonic and hadronic scenarios for the emission. We also present the recent detection of the EHBL RX J0812.0+0237 in the VHE band by MAGIC. Finally, we discuss a broad multiwavelength campaign on the BL Lac type object 1ES 2344+514, which showed intermittent EHBL characteristics in August 2016