Substrate elucidation and mutasynthesis: Characterisation of the biosynthetic pathway of a tripyrrolic secondary metabolite, prodigiosin

A wide variety of biological activity can be found in the realm of secondary metabolites. The tripyrrole prodigiosin illustrates this perfectly with activities ranging from antimicrobial to immunosuppressive and anticancer. Microorganisms producing the red pigment prodigiosin were first isolated more than 150 years ago. Yet, its structure was only elucidated in the 1960s and the biosynthetic pathway remained mainly unknown until the 2000s. This secondary metabolite results from a bifurcated pathway where monopyrrole 2-methyl-3-pentyl-1H-pyrrole (MAP) and bipyrrole 4-methoxy-1H,1'H-2,2'-bipyrrole (MBC) are produced independently before condensation. As MBC is an intermediate in the synthesis of other natural products, the enzymes involved in its formation have been well characterised. In contrast, the three enzymes – PigD, PigE and PigB – involved in the formation of MAP are specific to the biosynthesis of prodigiosin and less is known about them. This thesis focuses on the latter two enzymes. PigE was first described as a transaminase catalysing the transformation of 3-acetyloctanal into 3-acetyloctylamine (which cyclises to form dihydroMAP) and this activity has been confirmed by feeding intermediates to various gene-knockout strains. However, in vitro studies have demonstrated that 3-acetyloctanal could not be the product of PigD. In addition, bioinformatics analysis of its amino acid sequence showed that PigE has two domains: a transaminase Cterminal moiety and an unspecified N-terminal one, which we propose is a thioester reductase that converts a 3-acetyloctanoyl thioester to 3-acetyloctanal. Attempted chemical complementation of a pigD-knockout strain of Serratia using synthetic thioester, carboxylic acid and aldehyde substrates showed that both the thioester and the aldehyde can be used for pigment production, indicating that a thioester reductase is involved in prodigiosin biosynthetic pathway. Furthermore, the PigE protein was expressed in a heterogeneous host, purified and submitted to a number of activity and kinetic assays, which demonstrated that it can reduce a 3-acetyloctanoyl thioester substrate. The oxidation of dihydroMAP to MAP had previously been shown to be catalysed by an FADdependent oxidase PigB. The kinetics of HapB, a homologue of PigB had been studied by a previous group member. To take this project further we studied the substrate flexibility of the enzyme and used it to form new analogues of prodigiosin by mutasynthesis. Ten analogues of dihydroMAP with modifications either in the C2 or C3 positions were synthesised. Both extensions and truncations in the length of the chain at the C3 position could be accepted, whereas alkyl chains longer than 3 carbons on the C2 position could not be accommodated. Similar results were found in vivo when the analogues were fed to a pigD-knockout strain of Serratia, showing that PigB and the condensing enzyme PigC shared similar flexibility. Eight analogues of prodigiosin were hence obtained and their antimicrobial activities against Gram-positive and Gram-negative bacteria were assessed.BBSRC Frances and Augustus Newman Fundatio

Revision in the first steps of the biosynthesis of the red antibiotic prodigiosin: use of a synthetic thioester to validate a new intermediate.

Funder: Frances and Augustus Newman FoundationFunder: Emmanuel College, University of CambridgeFunder: Cambridge Commonwealth TrustA biosynthetic pathway for the red-antibiotic, prodigiosin, was proposed over a decade ago but not all the suggested intermediates could be detected experimentally. Here we show that a thioester that was not originally included in the pathway is an intermediate. In addition, the enzyme PigE was originally described as a transaminase but we present evidence that it also catalyses the reduction of the thioester intermediate to its aldehyde substrate

Fast Autofocusing using Tiny Transformer Networks for Digital Holographic Microscopy

The numerical wavefront backpropagation principle of digital holography confers unique extended focus capabilities, without mechanical displacements along z-axis. However, the determination of the correct focusing distance is a non-trivial and time consuming issue. A deep learning (DL) solution is proposed to cast the autofocusing as a regression problem and tested over both experimental and simulated holograms. Single wavelength digital holograms were recorded by a Digital Holographic Microscope (DHM) with a 10$\mathrm{x}$ microscope objective from a patterned target moving in 3D over an axial range of 92 $\mu$m. Tiny DL models are proposed and compared such as a tiny Vision Transformer (TViT), tiny VGG16 (TVGG) and a tiny Swin-Transfomer (TSwinT). The experiments show that the predicted focusing distance $Z_R^{\mathrm{Pred}}$ is accurately inferred with an accuracy of 1.2 $\mu$m in average in comparison with the DHM depth of field of 15 $\mu$m. Numerical simulations show that all tiny models give the $Z_R^{\mathrm{Pred}}$ with an error below 0.3 $\mu$m. Such a prospect would significantly improve the current capabilities of computer vision position sensing in applications such as 3D microscopy for life sciences or micro-robotics. Moreover, all models reach state of the art inference time on CPU, less than 25 ms per inference

Substrate flexibility of the flavin-dependent dihydropyrrole oxidases PigB and HapB involved in antibiotic prodigiosin biosynthesis

In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin-dependent oxidase responsible for formation of 2-methyl-3-amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5a (resulting from transamination of the aldehyde of 3-acetyloctanal) and 6 (resulting from transamination of the ketone), were synthesised. Only 5a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane-associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colorimetric assays for PigB and HapB were developed, and the HapB-catalysed reaction was kinetically characterised. Ten analogues of 5a were synthesised, varying in the C2 and C3 side-chains, and tested as substrates of HapB in vitro and for restoration of pigment production in Serratia ΔpigD in vivo. All lengths of side-chain tested at C3 were accepted but only short side-chains at C2 were accepted. The knowledge that 5a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5a makes a range of prodigiosin analogues readily available by mutasynthesis.We acknowledge the Frances and Augustus Newman foundation, the Cambridge Commonwealth Trust, Emmanuel College, Cambridge, and the B.B.S.R.C. (award codesBB/N008081/1 and BB/K001833/1) for funding this research

Literature review: Alzheimer’s disease policies in OECD countries

Alzheimer's disease (AD) and other major neuro-cognitive disorders (NCDs) pose significant challenges as the number of people living with Alzheimer's disease is rapidly increasing. Alzheimer's disease is incurable and has biopsychosocial implications for those affected. This obviously raises a multitude of issues for persons with AD and their families, but from a societal point of view, these issues require developing collective solutions by transforming communities, developing better organizational practices and adopting renewed public policies. It is in this spirit that since the early 2000s some governments have adopted action plans for these diseases. We conducted a literature review with the objective of understanding the social dynamics that led to the adoption of public policies, identifying their main areas of action, and exploring how these measures are actually implemented. This report presents the results in four main sections: Section 1. The literature review strategy. We used three types of documentary data: 1) 57 scientific articles that focused on the process of recognizing the social problem of AD and NCDs, the political agenda, content, implementation and effects; 2) two reports published by international organizations; and 3) action plans for these diseases which have been adopted by eight of the ten Canadian provinces. We analyzed these documents using a grid based on a public policy analysis model, and the main areas of public policy as identified by the World Health Organization (WHO). Section 2. How ad and NCD’s came to appear on the political agenda, their emergence as a social problem and the adoption of dedicated public policies. From 1900 to 1970, AD and NCDs were mainly perceived as a state of madness associated with old age. The boom in medical research in this area in the following decades changed the social representations and contributed to the recognition of the pathological nature. The dissociation of normal aging from cognitive disorders has contributed to the development of associative movements along with increased media attention. Although AD and NCDs emerged as a social problem in the second half of the 20th century, the importance of the psychosocial needs of those living with their impact was diminished in public policy and in the care provided. However, recognition of these needs has grown since the early 2000s, and many governments have adopted action plans. Several scientific articles propose that the participation of people with Alzheimer's disease, their families and the organizations that make their voices heard in the policy making process, is a particularly favourable condition for the recognition of their needs and their expertise. Governments must, however, develop mechanisms that effectively support the ability of these actors to act, rather than simply delegating responsibilities to them. Section 3. Actions plans – comparison and identification of predominant trends. In a report by the WHO, seven main areas were identified : 1)dementia as a public health priority; 2) dementia awareness and friendliness; 3)dementia risk reduction; 4) dementia diagnosis, treatment, care and support; 5) support for dementia carers; 6) information systems for dementia; and 7) dementia research and innovation (World Health Organization, 2017). These areas transcend action plans for AD and other major NCDs, and are thus broadly convergent, although WHO recommends that governments operationalize them in concrete measures adapted to their political, sociosanitary, population and territorial realities. We observed that measures to improve early phases of the care and service trajectory, such as improved diagnosis, are the focus of the action plans. Improved diagnosis is usually put in place too late, constituting a major obstacle to the implementation of follow-up adapted for people living with the repercussions of these diseases. This explains why diagnostic measures are almost universally promoted in public policies, under various conceptual arrangements. However, several action plans have not given the same importance to the development of care and services, following diagnosis, and this can generate feelings of helplessness. A holistic approach to the needs of people living with AD and NCDs, requires public policies to reflect the same intensity in all of the main areas. This can be achieved using the concepts of dementia capable, dementia friendly and dementia positive. These shared concepts are useful in functional components, in transforming the physical and social environment, and in recognizing that people with Alzheimer's disease deserve to live a fulfilling life. This approach is key for people living with these diseases, and their loved ones, to fully exercise their remaining abilities and live with dignity. Section 4. Policy implementation process. The first characteristic of effective implementation of an action plan is the adequate investment of financial resources. The second determinant is the development of a national steering committee, which serves to coordinate numerous departments and partners in various levels of government involved in the implementation of change. The committee helps plan activities implemented, address issues encountered, and monitor and follow up on changes made. The third determinant is the development of a strategy to support change. Several governments have developed projects that experiment with change in practices. Projects that stand out are identified and the conditions of change and implementation are understood, with a view to scaling up. Based on learnings from the literature review, governments need to consider four major aspects when engaging in this type of strategy: 1) mobilizing local actors is crucial to rooting change in local realities; 2) change needs to be flagged and actors must be supported to fully achieve the fundamental objectives of public policy; 3)experimental projects must be rigorously evaluated to draw meaningful learning from them; and 4) large-scale dissemination of innovative practices must mobilize the same type of implementation strategy and maintain the importance of the conceptual foundations on which the experimental projects were based. The policy implementation process must be a priority for governments since the real action to effectively improve the quality of life of people living with the repercussions of AD and NCDs can be realized only by the effective implementation of the measures proposed in the action plans

Les droits disciplinaires des fonctions publiques : « unification », « harmonisation » ou « distanciation ». A propos de la loi du 26 avril 2016 relative à la déontologie et aux droits et obligations des fonctionnaires

The production of tt‾ , W+bb‾ and W+cc‾ is studied in the forward region of proton–proton collisions collected at a centre-of-mass energy of 8 TeV by the LHCb experiment, corresponding to an integrated luminosity of 1.98±0.02 fb−1 . The W bosons are reconstructed in the decays W→ℓν , where ℓ denotes muon or electron, while the b and c quarks are reconstructed as jets. All measured cross-sections are in agreement with next-to-leading-order Standard Model predictions.The production of $t\overline{t}$, $W+b\overline{b}$ and $W+c\overline{c}$ is studied in the forward region of proton-proton collisions collected at a centre-of-mass energy of 8 TeV by the LHCb experiment, corresponding to an integrated luminosity of 1.98 $\pm$ 0.02 \mbox{fb}^{-1}. The $W$ bosons are reconstructed in the decays $W\rightarrow\ell\nu$, where $\ell$ denotes muon or electron, while the $b$ and $c$ quarks are reconstructed as jets. All measured cross-sections are in agreement with next-to-leading-order Standard Model predictions

Multidifferential study of identified charged hadron distributions in ZZ-tagged jets in proton-proton collisions at s=\sqrt{s}=13 TeV

Jet fragmentation functions are measured for the first time in proton-proton collisions for charged pions, kaons, and protons within jets recoiling against a $Z$ boson. The charged-hadron distributions are studied longitudinally and transversely to the jet direction for jets with transverse momentum 20 $< p_{\textrm{T}} < 100$ GeV and in the pseudorapidity range $2.5 < \eta < 4$. The data sample was collected with the LHCb experiment at a center-of-mass energy of 13 TeV, corresponding to an integrated luminosity of 1.64 fb$^{-1}$. Triple differential distributions as a function of the hadron longitudinal momentum fraction, hadron transverse momentum, and jet transverse momentum are also measured for the first time. This helps constrain transverse-momentum-dependent fragmentation functions. Differences in the shapes and magnitudes of the measured distributions for the different hadron species provide insights into the hadronization process for jets predominantly initiated by light quarks.Comment: All figures and tables, along with machine-readable versions and any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-013.html (LHCb public pages

Study of the B−→Λc+Λˉc−K−B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-} decay

The decay $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ is studied in proton-proton collisions at a center-of-mass energy of $\sqrt{s}=13$ TeV using data corresponding to an integrated luminosity of 5 $\mathrm{fb}^{-1}$ collected by the LHCb experiment. In the $\Lambda_{c}^+ K^{-}$ system, the $\Xi_{c}(2930)^{0}$ state observed at the BaBar and Belle experiments is resolved into two narrower states, $\Xi_{c}(2923)^{0}$ and $\Xi_{c}(2939)^{0}$, whose masses and widths are measured to be $$m(\Xi_{c}(2923)^{0}) = 2924.5 \pm 0.4 \pm 1.1 \,\mathrm{MeV}, \\ m(\Xi_{c}(2939)^{0}) = 2938.5 \pm 0.9 \pm 2.3 \,\mathrm{MeV}, \\ \Gamma(\Xi_{c}(2923)^{0}) = \phantom{000}4.8 \pm 0.9 \pm 1.5 \,\mathrm{MeV},\\ \Gamma(\Xi_{c}(2939)^{0}) = \phantom{00}11.0 \pm 1.9 \pm 7.5 \,\mathrm{MeV},$$ where the first uncertainties are statistical and the second systematic. The results are consistent with a previous LHCb measurement using a prompt $\Lambda_{c}^{+} K^{-}$ sample. Evidence of a new $\Xi_{c}(2880)^{0}$ state is found with a local significance of $3.8\,\sigma$, whose mass and width are measured to be $2881.8 \pm 3.1 \pm 8.5\,\mathrm{MeV}$ and $12.4 \pm 5.3 \pm 5.8 \,\mathrm{MeV}$, respectively. In addition, evidence of a new decay mode $\Xi_{c}(2790)^{0} \to \Lambda_{c}^{+} K^{-}$ is found with a significance of $3.7\,\sigma$. The relative branching fraction of $B^{-} \to \Lambda_{c}^{+} \bar{\Lambda}_{c}^{-} K^{-}$ with respect to the $B^{-} \to D^{+} D^{-} K^{-}$ decay is measured to be $2.36 \pm 0.11 \pm 0.22 \pm 0.25$, where the first uncertainty is statistical, the second systematic and the third originates from the branching fractions of charm hadron decays.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-028.html (LHCb public pages

Measurement of the ratios of branching fractions R(D∗)\mathcal{R}(D^{*}) and R(D0)\mathcal{R}(D^{0})

The ratios of branching fractions $\mathcal{R}(D^{*})\equiv\mathcal{B}(\bar{B}\to D^{*}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(\bar{B}\to D^{*}\mu^{-}\bar{\nu}_{\mu})$ and $\mathcal{R}(D^{0})\equiv\mathcal{B}(B^{-}\to D^{0}\tau^{-}\bar{\nu}_{\tau})/\mathcal{B}(B^{-}\to D^{0}\mu^{-}\bar{\nu}_{\mu})$ are measured, assuming isospin symmetry, using a sample of proton-proton collision data corresponding to 3.0 fb${ }^{-1}$ of integrated luminosity recorded by the LHCb experiment during 2011 and 2012. The tau lepton is identified in the decay mode $\tau^{-}\to\mu^{-}\nu_{\tau}\bar{\nu}_{\mu}$. The measured values are $\mathcal{R}(D^{*})=0.281\pm0.018\pm0.024$ and $\mathcal{R}(D^{0})=0.441\pm0.060\pm0.066$, where the first uncertainty is statistical and the second is systematic. The correlation between these measurements is $\rho=-0.43$. Results are consistent with the current average of these quantities and are at a combined 1.9 standard deviations from the predictions based on lepton flavor universality in the Standard Model.Comment: All figures and tables, along with any supplementary material and additional information, are available at https://cern.ch/lhcbproject/Publications/p/LHCb-PAPER-2022-039.html (LHCb public pages

Measurement of the J/ψ pair production cross-section in pp collisions at s=13 \sqrt{s}=13 TeV

The production cross-section of J/ψ pairs is measured using a data sample of pp collisions collected by the LHCb experiment at a centre-of-mass energy of $\sqrt{s}=13$ TeV, corresponding to an integrated luminosity of 279 ±11 pb$^{−1}$. The measurement is performed for J/ψ mesons with a transverse momentum of less than 10 GeV/c in the rapidity range 2.0 < y < 4.5. The production cross-section is measured to be 15.2 ± 1.0 ± 0.9 nb. The first uncertainty is statistical, and the second is systematic. The differential cross-sections as functions of several kinematic variables of the J/ψ pair are measured and compared to theoretical predictions.The production cross-section of $J/\psi$ pairs is measured using a data sample of $pp$ collisions collected by the LHCb experiment at a centre-of-mass energy of $\sqrt{s} = 13 \,{\mathrm{TeV}}$, corresponding to an integrated luminosity of $279 \pm 11 \,{\mathrm{pb^{-1}}}$. The measurement is performed for $J/\psi$ mesons with a transverse momentum of less than $10 \,{\mathrm{GeV}}/c$ in the rapidity range $2.0<y<4.5$. The production cross-section is measured to be $15.2 \pm 1.0 \pm 0.9 \,{\mathrm{nb}}$. The first uncertainty is statistical, and the second is systematic. The differential cross-sections as functions of several kinematic variables of the $J/\psi$ pair are measured and compared to theoretical predictions