Enterococci enhance Clostridioides difficile pathogenesis.

Enteric pathogens are exposed to a dynamic polymicrobial environment in the gastrointestinal tract1. This microbial community has been shown to be important during infection, but there are few examples illustrating how microbial interactions can influence the virulence of invading pathogens2. Here we show that expansion of a group of antibiotic-resistant, opportunistic pathogens in the gut-the enterococci-enhances the fitness and pathogenesis of Clostridioides difficile. Through a parallel process of nutrient restriction and cross-feeding, enterococci shape the metabolic environment in the gut and reprogramme C. difficile metabolism. Enterococci provide fermentable amino acids, including leucine and ornithine, which increase C. difficile fitness in the antibiotic-perturbed gut. Parallel depletion of arginine by enterococci through arginine catabolism provides a metabolic cue for C. difficile that facilitates increased virulence. We find evidence of microbial interaction between these two pathogenic organisms in multiple mouse models of infection and patients infected with C. difficile. These findings provide mechanistic insights into the role of pathogenic microbiota in the susceptibility to and the severity of C. difficile infection.

From Nursery to Nursing Home: Emerging Concepts in Clostridioides difficile Pathogenesis.

Clostridioides difficile is a Gram-positive, spore-forming, anaerobic bacterium that infects the human gastrointestinal tract, causing a wide range of disorders that vary in severity from mild diarrhea to toxic megacolon and/or death. Over the past decade, incidence, severity, and costs associated with C. difficile infection (CDI) have increased dramatically in both the pediatric and adult populations. The factors driving this rapidly evolving epidemiology remain largely unknown but are likely due in part to previously unappreciated host, microbiota, and environmental factors. In this review, we will cover the risks and challenges of CDI in adult and pediatric populations and examine asymptomatic colonization in infants. We will also discuss the emerging role of diet, pharmaceutical drugs, and pathogen-microbiota interactions in C. difficile pathogenesis, as well as the impact of host-microbiota interactions in the manifestation of C. difficile-associated disease. Finally, we highlight new areas of research and novel strategies that may shed light on this complex infection and provide insights into the future of microbiota-based therapeutics for CDI.

Discovery of novel pyrrolidineoxy-substituted heteroaromatics as potent and selective PI3K delta inhibitors with improved physicochemical properties.

In the recent years, PI3Kδ has emerged as a promising target for the treatment of B- and T-cell mediated inflammatory diseases. We present a cellular assay activity analysis for our previously reported 4,6-diaryl quinazoline PI3Kδ inhibitor series that suggests an optimal logP range between 2 and 3. We discovered novel analogues in this lipophilicity space that feature a chiral pyrrolidineoxy-group as a replacement for the position-4 aromatic ring of 4,6-diaryl quinazolines. These Fsp3 enriched derivatives retain potency and selectivity towards PI3Kδ. Compared to 4,6-diaryl quinazolines, their permeability profile is improved and molecular weight as well as PSA are reduced. These modifications offer additional possibilities for derivative generation in a favorable physicochemical property space and thus increase the chances to identify a clinical candidate.

Syk inhibitors with high potency in presence of blood.

We describe two series of Syk inhibitors which potently abrogate Syk kinase function in enzymatic assays, cellular assays and in primary cells in the presence of blood. Introduction of a 7-aminoindole substituent led to derivatives with good kinase selectivity and little or no hERG channel inhibition (3b, 10c).

Platelet adhesion to polyurethane urea under pulsatile flow conditions.

Platelet adhesion to a polyurethane urea surface is a precursor to thrombus formation within blood-contacting cardiovascular devices, and platelets have been found to adhere strongly to polyurethane surfaces below a shear rate of approximately 500 s(-1). The aim of the current work is to determine the properties of platelet adhesion to the polyurethane urea surface as a function of time-varying shear exposure. A rotating disk system was used to study the influence of steady and pulsatile flow conditions (e.g., cardiac inflow and sawtooth waveforms) for platelet adhesion to the biomaterial surface. All experiments were conducted with the same root mean square angular rotation velocity (29.63 rad/s) and waveform period. The disk was rotated in platelet-rich bovine plasma for 2 h, with adhesion quantified by confocal microscopy measurements of immunofluorescently labeled bovine platelets. Platelet adhesion under pulsating flow was found to decay exponentially with increasing shear rate. Adhesion levels were found to depend upon peak platelet flux and shear rate, regardless of rotational waveform. In combination with flow measurements, these results may be useful for predicting regions susceptible to thrombus formation within ventricular assist devices.

Liberation of host heme by Clostridioides difficile-mediated damage enhances Enterococcus faecalis fitness during infection.

IMPORTANCE: Clostridioides difficile and Enterococcus faecalis are two pathogens of great public health importance. Both bacteria colonize the human gastrointestinal tract where they are known to interact in ways that worsen disease outcomes. We show that the damage associated with C. difficile infection (CDI) releases nutrients that benefit E. faecalis. One particular nutrient, heme, allows E. faecalis to use oxygen to generate energy and grow better in the gut. Understanding the mechanisms of these interspecies interactions could inform therapeutic strategies for CDI.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection.

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Physical therapists in integrated palliative care: a qualitative study.

OBJECTIVES: The purpose of this study was to explore the perceptions and experiences of physical therapists (PTs) regarding their role in palliative care (PC) when practising in nations with advanced integration of PC into mainstream healthcare. METHODS: This qualitative study included an electronic demographic survey and semistructured interview. Data analysis included descriptive statistics for demographics and the constant comparative method for interview results. RESULTS: Thirteen PTs from eight nations identified four categories of roles and responsibilities: (1) working with patients and families, (2) being an interdisciplinary team (IDT) member, (3) professional responsibilities beyond direct patient care and (4) factors influencing the role of PTs in PC. Concepts identified were shifting priorities (increased family involvement, emphasis on psychosocial aspects and differences in care philosophy), care across the continuum (accommodating changes in patient status, increasing awareness of PTs' role in varying disease states and working with the IDT) and changing perceptions about PT in PC (perceptions of PTs/others regarding PTs' role in PC and professional responsibilities of the PT in PC). CONCLUSIONS: Based on participant responses, a previously published conceptual framework by Wilson et al in 2017 was updated and included an increased emphasis on patient wishes and dignity, treating breathlessness, patient advocacy within their family and use of technology and networking. Within PC, PTs play a key role on the IDT and can improve quality of life; however, multiple barriers exist to providing PT care within PC. Further advocacy is needed from PTs and professional organisations to integrate these services.

Gigaxonin glycosylation regulates intermediate filament turnover and may impact giant axonal neuropathy etiology or treatment

Gigaxonin (also known as KLHL16) is an E3 ligase adaptor protein that promotes the ubiquitination and degradation of intermediate filament (IF) proteins. Mutations in human gigaxonin cause the fatal neurodegenerative disease giant axonal neuropathy (GAN), in which IF proteins accumulate and aggregate in axons throughout the nervous system, impairing neuronal function and viability. Despite this pathophysiological significance, the upstream regulation and downstream effects of normal and aberrant gigaxonin function remain incompletely understood. Here, we report that gigaxonin is modified by O-linked ß-N-acetylglucosamine (O-GlcNAc), a prevalent form of intracellular glycosylation, in a nutrient- and growth factor­dependent manner. MS analyses of human gigaxonin revealed 9 candidate sites of O-GlcNAcylation, 2 of which ­ serine 272 and threonine 277 ­ are required for its ability to mediate IF turnover in gigaxonin-deficient human cell models that we created. Taken together, the results suggest that nutrient-responsive gigaxonin O-GlcNAcylation forms a regulatory link between metabolism and IF proteostasis. Our work may have significant implications for understanding the nongenetic modifiers of GAN phenotypes and for the optimization of gene therapy for this disease.

An integer minimal principle and triplet sieve method for phasing centrosymmetric structures.

In this paper, a new integer minimal principle model for centrosymmetric structures is presented; one which fully accounts for reciprocal-space phase shifts present in non-symmorphic space groups. Additionally, characterization of false minima of the model is done in terms of even and odd triplets. Based on this characterization, a triplet sieve method is proposed. First, Gaussian elimination using only a subset of reliable triplets is employed for phasing. Triplet subsets are generated using a progressively smaller set of the strongest reflections. Several phase solution sets are generated by enumerating the degrees of freedom present. To facilitate computational evaluation of the quality of these phase solutions, these phase sets are passed into the crystallographic software SnB, which expands the reflection set in two cycles. The final solution is identified via statistics of two crystallographic figures of merit. Computational results are presented for a variety of structures.

Discovery of CDZ173 (Leniolisib), Representing a Structurally Novel Class of PI3K Delta-Selective Inhibitors.

The predominant expression of phosphoinositide 3-kinase δ (PI3Kδ) in leukocytes and its critical role in B and T cell functions led to the hypothesis that selective inhibitors of this isoform would have potential as therapeutics for the treatment of allergic and inflammatory disease. Targeting specifically PI3Kδ should avoid potential side effects associated with the ubiquitously expressed PI3Kα and ß isoforms. We disclose how morphing the heterocyclic core of previously discovered 4,6-diaryl quinazolines to a significantly less lipophilic 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine, followed by replacement of one of the phenyl groups with a pyrrolidine-3-amine, led to a compound series with an optimal on-target profile and good ADME properties. A final lipophilicity adjustment led to the discovery of CDZ173 (leniolisib), a potent PI3Kδ selective inhibitor with suitable properties and efficacy for clinical development as an anti-inflammatory therapeutic. In vitro, CDZ173 inhibits a large spectrum of immune cell functions, as demonstrated in B and T cells, neutrophils, monocytes, basophils, plasmocytoid dendritic cells, and mast cells. In vivo, CDZ173 inhibits B cell activation in rats and monkeys in a concentration- and time-dependent manner. After prophylactic or therapeutic dosing, CDZ173 potently inhibited antigen-specific antibody production and reduced disease symptoms in a rat collagen-induced arthritis model. Structurally, CDZ173 differs significantly from the first generation of PI3Kδ and PI3Kγδ-selective clinical compounds. Therefore, CDZ173 could differentiate by a more favorable safety profile. CDZ173 is currently in clinical studies in patients suffering from primary Sjögren's syndrome and in APDS/PASLI, a disease caused by gain-of-function mutations of PI3Kδ.

Discovery and Pharmacological Characterization of Novel Quinazoline-Based PI3K Delta-Selective Inhibitors.

Inhibition of the lipid kinase PI3Kδ is a promising principle to treat B and T cell driven inflammatory diseases. Using a scaffold deconstruction-reconstruction strategy, we identified 4-aryl quinazolines that were optimized into potent PI3Kδ isoform selective analogues with good pharmacokinetic properties. With compound 11, we illustrate that biochemical PI3Kδ inhibition translates into modulation of isoform-dependent immune cell function (human, rat, and mouse). After oral administration of compound 11 to rats, proximal PD markers are inhibited, and dose-dependent efficacy in a mechanistic plaque forming cell assay could be demonstrated.

Discovery and profiling of a selective and efficacious Syk inhibitor.

We describe the discovery of selective and potent Syk inhibitor 11, which exhibited favorable PK profiles in rat and dog and was found to be active in a collagen-induced arthritis model in rats. Compound 11 was selected for further profiling, but, unfortunately, in GLP toxicological studies it showed liver findings in rat and dog. Nevertheless, 11 could become a valuable tool compound to investigate the rich biology of Syk in vitro and in vivo.

Design and synthesis of trans-3-(2-(4-((3-(3-(5-methyl-1,2,4-oxadiazolyl))- phenyl)carboxamido)cyclohexyl)ethyl)-7-methylsulfonyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SB-414796): a potent and selective dopamine D3 receptor antagonist.

At their clinical doses, current antipsychotic agents share the property of both dopamine D(2) and D(3) receptor blockade. However, a major disadvantage of many current medications are the observed extrapyramidal side-effects (EPS), postulated to arise from D(2) receptor antagonism. Consequently, a selective dopamine D(3) receptor antagonist could offer an attractive antipsychotic therapy, devoid of the unwanted EPS. Using SAR information gained in two previously reported series of potent and selective D(3) receptor antagonists, as exemplified by the 2,3,4,5-tetrahydro-1H-3-benzazepine 10 and the 2,3-dihydro-1H-isoindoline 11, a range of 7-sulfonyloxy- and 7-sulfonylbenzazepines has been prepared. Compounds of this type combined a high level of D(3) affinity and selectivity vs D(2) with an excellent pharmacokinetic profile in the rat. Subsequent optimization of this series to improve selectivity over a range of receptors and reduce cytochrome P450 inhibitory potential gave trans-3-(2-(4-((3-(3-(5-methyl-1,2,4-oxidiazolyl))phenyl)carboxamido)cyclohexyl)ethyl)-7-methylsulfonyl-2,3,4,5-tetrahydro-1H-3-benzazepine (58, SB-414796). This compound is a potent and selective dopamine D(3) receptor antagonist with high oral bioavailability and is CNS penetrant in the rat. Subsequent evaluation in the rat has shown that 58 preferentially reduces firing of dopaminergic cells in the ventral tegmental area (A10) compared to the substantia nigra (A9), an observation consistent with a prediction for atypical antipsychotic efficacy. In a separate study, 58 has been shown to block expression of the conditioned place preference (CPP) response to cocaine in male rats, suggesting that it may also have a role in the treatment of cue-induced relapse in drug-free cocaine addicts.

SnB version 2.3: triplet sieve phasing for centrosymmetric structures.

A new version of the direct-methods program SnB has been developed. This version incorporates the triplet sieve method for phasing centrosymmetric structures in a way that is transparent to users. The triplet sieve procedure may decrease significantly the time required to achieve a solution for such structures.

Studies towards the identification of a new generation of atypical antipsychotic agents.

A rational structure-activity relationship study around compound (1) is reported. The lead optimisation programme led to the identification of sulfonamide (25), a molecule combining dopamine D2/D3 receptor antagonism with serotonin 5-HT2A, 5-HT2C, 5-HT6 receptor antagonism for an effective treatment of schizophrenia. Compound (25) was shown to possess the required in vivo activity with no EPS liability.