Generation and trapping of ketenes in flow

Ketenes were generated by the thermolysis of alkoxyalkynes under flow conditions, and then trapped with amines and alcohols to cleanly give amides and esters. For a 10 min reaction time, temperatures of 180, 160, and 140 °C were required for >95?% conversion of EtO, iPrO, and tBuO alkoxyalkynes, respectively. Variation of the temperature and flow rate with inline monitoring of the output by IR spectroscopy allowed the kinetic parameters for the conversion of 1-ethoxy-1-octyne to be easily estimated (Ea = 105.4 kJ/mol). Trapping of the in-situ-generated ketenes by alcohols to give esters required the addition of a tertiary amine catalyst to prevent competitive [2+2] addition of the ketene to the alkoxyalkyne precurso

A practical drug discovery project at the undergraduate level

A practical drug discovery project for third-year undergraduates is described. No previous knowledge of medicinal chemistry is assumed. Initial lecture-workshops cover the basic principles; then students are asked to improve the profile of a weakly potent, poorly soluble PI3K inhibitor (1). Compound array design, molecular modelling and screening data analysis are followed by laboratory work in which each student, as part of a team, attempts to synthesise at least two target compounds. The project benefits from significant industrial support, including lectures, student mentoring and consumables. The aim is to make the learning experience as close as possible to real-life industrial situations. Forty-eight target compounds have been prepared, the best of which (5b, 5j, 6b and 6ap) improved the potency and aqueous solubility of the lead compound (1) by 100-1000 fold and 10-fold, respectively

Efficacy of the Enquiring About Tolerance (EAT) study among infants at high risk of developing food allergy.

BACKGROUND: The Enquiring About Tolerance (EAT) study was a randomized trial of the early introduction of allergenic solids into the infant diet from 3 months of age. The intervention effect did not reach statistical significance in the intention-to-treat analysis of the primary outcome. OBJECTIVE: We sought to determine whether infants at high risk of developing a food allergy benefited from early introduction. METHODS: A secondary intention-to-treat analysis was performed of 3 groups: nonwhite infants; infants with visible eczema at enrollment, with severity determined by SCORAD; and infants with enrollment food sensitization (specific IgE ≥0.1 kU/L). RESULTS: Among infants with sensitization to 1 or more foods at enrollment (≥0.1 kU/L), early introduction group (EIG) infants developed significantly less food allergy to 1 or more foods than standard introduction group (SIG) infants (SIG, 34.2%; EIG, 19.2%; P = .03), and among infants with sensitization to egg at enrollment, EIG infants developed less egg allergy (SIG, 48.6%; EIG, 20.0%; P = .01). Similarly, among infants with moderate SCORAD (15-<40) at enrollment, EIG infants developed significantly less food allergy to 1 or more foods (SIG, 46.7%; EIG, 22.6%; P = .048) and less egg allergy (SIG, 43.3%; EIG, 16.1%; P = .02). CONCLUSION: Early introduction was effective in preventing the development of food allergy in specific groups of infants at high risk of developing food allergy: those sensitized to egg or to any food at enrollment and those with eczema of increasing severity at enrollment. This efficacy occurred despite low adherence to the early introduction regimen. This has significant implications for the new national infant feeding recommendations that are emerging around the world

Orally Administered P22 Phage Tailspike Protein Reduces Salmonella Colonization in Chickens: Prospects of a Novel Therapy against Bacterial Infections

One of the major causes of morbidity and mortality in man and economically important animals is bacterial infections of the gastrointestinal (GI) tract. The emergence of difficult-to-treat infections, primarily caused by antibiotic resistant bacteria, demands for alternatives to antibiotic therapy. Currently, one of the emerging therapeutic alternatives is the use of lytic bacteriophages. In an effort to exploit the target specificity and therapeutic potential of bacteriophages, we examined the utility of bacteriophage tailspike proteins (Tsps). Among the best-characterized Tsps is that from the Podoviridae P22 bacteriophage, which recognizes the lipopolysaccharides of Salmonella enterica serovar Typhimurium. In this study, we utilized a truncated, functionally equivalent version of the P22 tailspike protein, P22sTsp, as a prototype to demonstrate the therapeutic potential of Tsps in the GI tract of chickens. Bacterial agglutination assays showed that P22sTsp was capable of agglutinating S. Typhimurium at levels similar to antibodies and incubating the Tsp with chicken GI fluids showed no proteolytic activity against the Tsp. Testing P22sTsp against the three major GI proteases showed that P22sTsp was resistant to trypsin and partially to chymotrypsin, but sensitive to pepsin. However, in formulated form for oral administration, P22sTsp was resistant to all three proteases. When administered orally to chickens, P22sTsp significantly reduced Salmonella colonization in the gut and its further penetration into internal organs. In in vitro assays, P22sTsp effectively retarded Salmonella motility, a factor implicated in bacterial colonization and invasion, suggesting that the in vivo decolonization ability of P22sTsp may, at least in part, be due to its ability to interfere with motility… Our findings show promise in terms of opening novel Tsp-based oral therapeutic approaches against bacterial infections in production animals and potentially in humans

Evasion of IFN-γ Signaling by Francisella novicida Is Dependent upon Francisella Outer Membrane Protein C

Francisella tularensis is a Gram-negative facultative intracellular bacterium and the causative agent of the lethal disease tularemia. An outer membrane protein (FTT0918) of F. tularensis subsp. tularensis has been identified as a virulence factor. We generated a F. novicida (F. tularensis subsp. novicida) FTN_0444 (homolog of FTT0918) fopC mutant to study the virulence-associated mechanism(s) of FTT0918.The ΔfopC strain phenotype was characterized using immunological and biochemical assays. Attenuated virulence via the pulmonary route in wildtype C57BL/6 and BALB/c mice, as well as in knockout (KO) mice, including MHC I, MHC II, and µmT (B cell deficient), but not in IFN-γ or IFN-γR KO mice was observed. Primary bone marrow derived macrophages (BMDM) prepared from C57BL/6 mice treated with rIFN-γ exhibited greater inhibition of intracellular ΔfopC than wildtype U112 strain replication; whereas, IFN-γR KO macrophages showed no IFN-γ-dependent inhibition of ΔfopC replication. Moreover, phosphorylation of STAT1 was downregulated by the wildtype strain, but not the fopC mutant, in rIFN-γ treated macrophages. Addition of NG-monomethyl-L-arginine, an NOS inhibitor, led to an increase of ΔfopC replication to that seen in the BMDM unstimulated with rIFN-γ. Enzymatic screening of ΔfopC revealed aberrant acid phosphatase activity and localization. Furthermore, a greater abundance of different proteins in the culture supernatants of ΔfopC than that in the wildtype U112 strain was observed.F. novicida FopC protein facilitates evasion of IFN-γ-mediated immune defense(s) by down-regulation of STAT1 phosphorylation and nitric oxide production, thereby promoting virulence. Additionally, the FopC protein also may play a role in maintaining outer membrane stability (integrity) facilitating the activity and localization of acid phosphatases and other F. novicida cell components

A practical drug discovery project at the undergraduate level

Teaser&apos;You make the compounds you design&apos;: this article describes a new way for chemistry undergraduates to learn about drug discovery. A practical drug discovery project at the undergraduate level In this article, we describe a practical drug discovery project for third-year undergraduates. No previous knowledge of medicinal chemistry is assumed. Initial lecture workshops cover the basic principles; then students, in teams, seek to improve the profile of a weakly potent, insoluble phosphatidylinositide 3-kinase delta (PI3Kd) inhibitor (1) through compound array design, molecular modelling, screening data analysis and the synthesis of target compounds in the laboratory. The project benefits from significant industrial support, including lectures, student mentoring and consumables. The aim is to make the learning experience as close as possible to real-life industrial situations. In total, 48 target compounds were prepared, the best of which (5b, 5j, 6b and 6ap) improved the potency and aqueous solubility of the lead compound (1) by 100-1000 fold and !tenfold, respectively. This article is an account of a &apos;hands-on&apos; drug discovery course that has been running for the past 3 years at the University of Nottingham. The purpose is fivefold: (i) to teach students, who are in the third year of a 4-year MSci degree course, how new medicines are discovered; (ii) to give an appreciation of the role of the chemist in that process; (iii) to give students practice in compound design and data interpretation; (iv) to use industry-standard equipment and methods in the laboratory; and (v) to develop communication, team-working and interpersonal skills. Key aspects of the course included the participation of scientists from GlaxoSmithKline (GSK) as lecturers and workshop mentors and, above all, in the practical application of drug discovery principles in the laboratory

An original phylogenetic approach identified mitochondrial haplogroup T1a1 as inversely associated with breast cancer risk in BRCA2 mutation carriers

Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers. Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals. Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk. Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.Peer reviewe

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference