Gene editing enables rapid engineering of complex antibiotic assembly lines

From Springer Nature via Jisc Publications RouterHistory: received 2021-09-09, accepted 2021-11-02, registration 2021-11-08, pub-electronic 2021-11-25, online 2021-11-25, collection 2021-12Publication status: PublishedFunder: RCUK | Biotechnology and Biological Sciences Research Council (BBSRC); doi: https://doi.org/10.13039/501100000268; Grant(s): BB/L013754/1, BB/N023536/1Abstract: Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized

Loss-of-function mutations in UDP-Glucose 6-Dehydrogenase cause recessive developmental epileptic encephalopathy

AbstractDevelopmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids. Consistent with being loss-of-function alleles, we show using patients’ primary fibroblasts and biochemical assays, that these mutations either impair UGDH stability, oligomerization, or enzymatic activity. In vitro, patient-derived cerebral organoids are smaller with a reduced number of proliferating neuronal progenitors while mutant ugdh zebrafish do not phenocopy the human disease. Our study defines UGDH as a key player for the production of extracellular matrix components that are essential for human brain development. Based on the incidence of variants observed, UGDH mutations are likely to be a frequent cause of recessive epileptic encephalopathy.</jats:p

Speculative futures on ChatGPT and generative artificial intelligence (AI): a collective reflection from the educational landscape

While ChatGPT has recently become very popular, AI has a long history and philosophy. This paper intends to explore the promises and pitfalls of the Generative Pre-trained Transformer (GPT) AI and potentially future technologies by adopting a speculative methodology. Speculative future narratives with a specific focus on educational contexts are provided in an attempt to identify emerging themes and discuss their implications for education in the 21st century. Affordances of (using) AI in Education (AIEd) and possible adverse effects are identified and discussed which emerge from the narratives. It is argued that now is the best of times to define human vs AI contribution to education because AI can accomplish more and more educational activities that used to be the prerogative of human educators. Therefore, it is imperative to rethink the respective roles of technology and human educators in education with a future-oriented mindset

Loss-of-function mutations in UDP-Glucose 6-Dehydrogenase cause recessive developmental epileptic encephalopathy

Developmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids. Consistent with being loss-of-function alleles, we show using patients’ primary fibroblasts and biochemical assays, that these mutations either impair UGDH stability, oligomerization, or enzymatic activity. In vitro, patient-derived cerebral organoids are smaller with a reduced number of proliferating neuronal progenitors while mutant ugdh zebrafish do not phenocopy the human disease. Our study defines UGDH as a key player for the production of extracellular matrix components that are essential for human brain development. Based on the incidence of variants observed, UGDH mutations are likely to be a frequent cause of recessive epileptic encephalopathy

Search for new particles in events with energetic jets and large missing transverse momentum in proton-proton collisions at root s=13 TeV

A search is presented for new particles produced at the LHC in proton-proton collisions at root s = 13 TeV, using events with energetic jets and large missing transverse momentum. The analysis is based on a data sample corresponding to an integrated luminosity of 101 fb(-1), collected in 2017-2018 with the CMS detector. Machine learning techniques are used to define separate categories for events with narrow jets from initial-state radiation and events with large-radius jets consistent with a hadronic decay of a W or Z boson. A statistical combination is made with an earlier search based on a data sample of 36 fb(-1), collected in 2016. No significant excess of events is observed with respect to the standard model background expectation determined from control samples in data. The results are interpreted in terms of limits on the branching fraction of an invisible decay of the Higgs boson, as well as constraints on simplified models of dark matter, on first-generation scalar leptoquarks decaying to quarks and neutrinos, and on models with large extra dimensions. Several of the new limits, specifically for spin-1 dark matter mediators, pseudoscalar mediators, colored mediators, and leptoquarks, are the most restrictive to date.Peer reviewe

Combined searches for the production of supersymmetric top quark partners in proton-proton collisions at root s=13 TeV

A combination of searches for top squark pair production using proton-proton collision data at a center-of-mass energy of 13 TeV at the CERN LHC, corresponding to an integrated luminosity of 137 fb(-1) collected by the CMS experiment, is presented. Signatures with at least 2 jets and large missing transverse momentum are categorized into events with 0, 1, or 2 leptons. New results for regions of parameter space where the kinematical properties of top squark pair production and top quark pair production are very similar are presented. Depending on themodel, the combined result excludes a top squarkmass up to 1325 GeV for amassless neutralino, and a neutralinomass up to 700 GeV for a top squarkmass of 1150 GeV. Top squarks with masses from 145 to 295 GeV, for neutralino masses from 0 to 100 GeV, with a mass difference between the top squark and the neutralino in a window of 30 GeV around the mass of the top quark, are excluded for the first time with CMS data. The results of theses searches are also interpreted in an alternative signal model of dark matter production via a spin-0 mediator in association with a top quark pair. Upper limits are set on the cross section for mediator particle masses of up to 420 GeV

Probing effective field theory operators in the associated production of top quarks with a Z boson in multilepton final states at root s=13 TeV

Peer reviewe