Staphylococcus aureus FtsZ and PBP4 bind to the conformationally dynamic N-terminal domain of GpsB.

In the Firmicutes phylum, GpsB is a membrane associated protein that coordinates peptidoglycan synthesis with cell growth and division. Although GpsB has been studied in several bacteria, the structure, function, and interactome of Staphylococcus aureus GpsB is largely uncharacterized. To address this knowledge gap, we solved the crystal structure of the N-terminal domain of S. aureus GpsB, which adopts an atypical, asymmetric dimer, and demonstrates major conformational flexibility that can be mapped to a hinge region formed by a three-residue insertion exclusive to Staphylococci. When this three-residue insertion is excised, its thermal stability increases, and the mutant no longer produces a previously reported lethal phenotype when overexpressed in Bacillus subtilis. In S. aureus, we show that these hinge mutants are less functional and speculate that the conformational flexibility imparted by the hinge region may serve as a dynamic switch to fine-tune the function of the GpsB complex and/or to promote interaction with its various partners. Furthermore, we provide the first biochemical, biophysical, and crystallographic evidence that the N-terminal domain of GpsB binds not only PBP4, but also FtsZ, through a conserved recognition motif located on their C-termini, thus coupling peptidoglycan synthesis to cell division. Taken together, the unique structure of S. aureus GpsB and its direct interaction with FtsZ/PBP4 provide deeper insight into the central role of GpsB in S. aureus cell division.

Naturally Occurring Mutations of SARS-CoV-2 Main Protease Confer Drug Resistance to Nirmatrelvir.

The SARS-CoV-2 main protease (Mpro) is the drug target of Pfizer's oral drug nirmatrelvir. The emergence of SARS-CoV-2 variants with mutations in Mpro raised the alarm of potential drug resistance. To identify potential clinically relevant drug-resistant mutants, we systematically characterized 102 naturally occurring Mpro mutants located at 12 residues at the nirmatrelvir-binding site, among which 22 mutations in 5 residues, including S144M/F/A/G/Y, M165T, E166 V/G/A, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (kcat/Km < 10-fold change) while being resistant to nirmatrelvir (Ki > 10-fold increase). X-ray crystal structures were determined for six representative mutants with and/or without GC-376/nirmatrelvir. Using recombinant SARS-CoV-2 viruses generated from reverse genetics, we confirmed the drug resistance in the antiviral assay and showed that Mpro mutants with reduced enzymatic activity had attenuated viral replication. Overall, our study identified several drug-resistant hotspots in Mpro that warrant close monitoring for possible clinical evidence of nirmatrelvir resistance, some of which have already emerged in independent viral passage assays conducted by others.

Naturally occurring mutations of SARS-CoV-2 main protease confer drug resistance to nirmatrelvir.

The SARS-CoV-2 main protease (M pro ) is the drug target of Pfizer’s oral drug Paxlovid. The emergence of SARS-CoV-2 variants with mutations in M pro raised the alarm of potential drug resistance. In this study, we identified 100 naturally occurring M pro mutations located at the nirmatrelvir binding site, among which 20 mutants, including S144M/F/A/G/Y, M165T, E166G, H172Q/F, and Q192T/S/L/A/I/P/H/V/W/C/F, showed comparable enzymatic activity to the wild-type (k cat /K m <10-fold change) and resistance to nirmatrelvir (K i >10-fold increase). X-ray crystal structures were determined for seven representative mutants with and/or without GC-376/nirmatrelvir. Viral growth assay showed that M pro mutants with reduced enzymatic activity led to attenuated viral replication. Overall, our study identified several drug resistant hot spots that warrant close monitoring for possible clinical evidence of Paxlovid resistance. One Sentence Summary: Paxlovid resistant SARS-CoV-2 viruses with mutations in the main protease have been identified from clinical isolates.

Mapping of the Sensory Innervation of the Mouse Lung by Specific Vagal and Dorsal Root Ganglion Neuronal Subsets.

The airways are densely innervated by sensory afferent nerves, whose activation regulates respiration and triggers defensive reflexes (e.g., cough, bronchospasm). Airway innervation is heterogeneous, and distinct afferent subsets have distinct functional responses. However, little is known of the innervation patterns of subsets within the lung. A neuroanatomical map is critical for understanding afferent activation under physiological and pathophysiological conditions. Here, we quantified the innervation of the mouse lung by vagal and dorsal root ganglion (DRG) sensory subsets defined by the expression of Pirt (all afferents), 5HT3 (vagal nodose afferents), Tac1 (tachykinergic afferents), and transient receptor potential vanilloid 1 channel (TRPV1; defensive/nociceptive afferents) using Cre-mediated reporter expression. We found that vagal afferents innervate almost all conducting airways and project into the alveolar region, whereas DRG afferents only innervate large airways. Of the two vagal ganglia, only nodose afferents project into the alveolar region, but both nodose and jugular afferents innervate conducting airways throughout the lung. Many afferents that project into the alveolar region express TRPV1. Few DRG afferents expressed TRPV1. Approximately 25% of blood vessels were innervated by vagal afferents (many were Tac1+). Approximately 10% of blood vessels had DRG afferents (some were Tac1+), but this was restricted to large vessels. Lastly, innervation of neuroepithelial bodies (NEBs) correlated with the cell number within the bodies. In conclusion, functionally distinct sensory subsets have distinct innervation patterns within the conducting airways, alveoli and blood vessels. Physiologic (e.g., stretch) and pathophysiological (e.g., inflammation, edema) stimuli likely vary throughout these regions. Our data provide a neuroanatomical basis for understanding afferent responses in vivo.

Pathways to 'recovery' and social reintegration: The experiences of long-term clients of methadone maintenance treatment in an Irish drug treatment setting.

INTRODUCTION: This paper examines the experiences of long-term clients of methadone maintenance treatment (MMT) in one area of Dublin in the context of a recent emphasis on rehabilitation and recovery in Irish drug policy. METHODS: In-depth qualitative interviews were conducted with 25 long-term clients of methadone maintenance treatment (MMT). All participants had first enrolled in methadone treatment at least ten years prior to participating in the research and a majority (n = 16) had first accessed MMT more than 20 years previously. RESULTS: While acknowledging several beneficial aspects of methadone treatment, research participants saw themselves as passive recipients of a clinical regime that offered no opportunity to exercise agency in relation to their ongoing treatment. Rather than perceiving themselves as progressing along a pathway to recovery, the treatment experience was depicted in terms of stasis or confinement. Neither did participants report any progress in attaining the kind of social reintegration that is commonly presented as a key aspect of addiction recovery and which, in the Irish context, is a central plank of drug policy discourse. DISCUSSION: The findings highlight a disconnect between policies that ostensibly aim to promote social reintegration and recovery and the experiences of individuals who are long-term clients of MMT. Irish policy aspirations of facilitating opiate-dependent clients to progress along a pathway to recovery are difficult, if not impossible, to realise given the marginal status of addiction services within the health system and the difficulties involved in securing ongoing cooperation from other public service sectors.

Identification and Characterization of an Aspergillus niger Amine Oxidase that Detoxifies Intact Fumonisins.

Fumonisin contamination of maize damaged by Fusarium verticillioides and related species is a major problem when it is grown under warm and dry conditions. Consumption of fumonisin contaminated food and feed is harmful to both humans and livestock. Novel tools for reducing or eliminating fumonisin toxicity may be useful to the agri-feed sector to deal with this worldwide problem. Enzymes capable of catabolizing fumonisins have been identified from microorganisms that utilize fumonisins as an energy source. However, fumonisin detoxifying enzymes produced by the very species that biosynthesize the toxin have yet to be reported. Here we describe the identification and characterization of a novel amine oxidase synthesized by the fumonisin-producing fungus Aspergillus niger. We have recombinantly expressed this A. niger enzyme in E. coli and demonstrated its ability to oxidatively deaminate intact fumonisins without requiring exogenous cofactors. This enzyme, termed AnFAO (A. niger fumonisin amine oxidase), displays robust fumonisin deamination activity across a broad range of conditions, has a high native melting temperature, and can be purified to >95% homogeneity at high yield in a one-step enrichment. AnFAO is a promising tool to remediate fumonisin-contaminated feed including maize destined for ethanol production.

AIDS, Sexual Health, and the Catholic Church in 1980s Ireland: A Public Health Paradox?

Our aim was to explore the paradoxical role of the Catholic Church during the early AIDS era in establishing a policy network committed to the principles of public health, even in instances in which these principles contradicted Catholic moral teaching. This article is rooted in a study that explored the transformational effects of AIDS on Irish sexual health policy during the initial decade of the epidemic; that is, as it unfolded in Ireland between 1982 and 1992. Our source material, consisting of 20 documents that had been unexplored for more than 25 years, was obtained from the Dublin AIDS Alliance Ltd., an Irish civil society organization that supports HIV-positive people. Our archival research was supported by 18 semistructured key informant interviews with contemporaneous politicians, representatives of the Catholic Church, clinical personnel, and AIDS activists. We examined the critical role of a Catholic priest in developing a coherent national policy response that reflected the public health principles advocated by the World Health Organization at a time when the moral authority of the Roman Catholic Church had a significant influence on successive Irish governments. (Am J Public Health. 2018;108:908-913.).

Coolmine Therapeutic Community, Dublin: a 40-year history of Ireland's first voluntary drug treatment service.

AIM: To document the evolution over 40 years (from 1973 to 2013) of Coolmine Therapeutic Community (Ireland's first voluntary drug treatment service) against a background of broader drug policy developments in the Republic of Ireland and elsewhere during this period. METHODS: Data were gathered by means of archival research within Coolmine, complemented by semi-structured interviews with former clients, current and former Coolmine management and staff, and representatives of outsider stakeholder interests. RESULTS: Coolmines's history has three phases: (1) an early and uncontentious phase, in which external authorities provided financial support for Coolmine without questioning its work practices or outcomes; (2) a middle, controversial phase, in which Coolmine struggled for survival in an external policy environment now dominated by harm reduction strategies; and (3) a final phase in which, through the use of conventional corporate governance, Coolmine management sought to repair its damaged reputation by introducing evidence-based clinical practices. CONCLUSIONS: Coolmine Therapeutic Community was established when drug treatment services in Ireland were in their infancy, and its changing fortunes over subsequent decades reflected changing perceptions of what constitutes appropriate addiction treatment-and in particular the role to be played by former addicts within addiction treatment systems-as well as changing perceptions of funding relationships between statutory authorities and voluntary providers of health and social services.

Oxytetracycline interactions at the soil-water interface: effects of environmental surfaces on natural transformation and growth inhibition of Azotobacter vinelandii.

The mechanism of oxytetracycline (OTC) adsorption to a silty clay loam soil was investigated using sorption isotherm experiments, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction spectroscopy (XRD). Sorption data fit well to a cation-exchange capacity sorption model. Spectroscopic data indicate that the interactions between oxytetracycline and silty clay loam soil were primarily through electrostatic interactions between the protonated dimethylamino group of OTC and the negatively charged moieties on the surface of the soil. Based on XRD results, OTC adsorption appeared to inhibit the ethylene glycol solvation of the expandable clay minerals, suggesting that OTC had diffused into the clay interlayer space. The presence of adsorbed OTC did not significantly affect the transformation frequency of the soil bacterium Azotobacter vinelandii with plasmid DNA (soil alone 3 × 10(6) ± 4 × 10(6) and soil with adsorbed OTC 4 × 10(6) ± 0.5 × 10(6) ). Growth was inhibited by adsorbed OTC, although a greater mass of adsorbed OTC was required to achieve the same degree of inhibition as the system of dissolved OTC alone. These results suggest that the interactions of tetracyclines at the soil-water interface will affect the growth of sensitive microorganisms in soil microbial communities.

Alcoholics Anonymous in Ireland: AA's first European experience.

AIMS: To trace the evolution of Alcoholics Anonymous in Ireland from its establishment there in 1946, focusing on the efforts of early members to publicize the fellowship and negotiate a role for themselves in relation to existing religious and healthcare institutions. METHODS: Archival research, drawing mainly on primary sources in AA archives in New York and Dublin. RESULTS: Anticipated tensions between this fellowship, which had its roots in Evangelical Protestantism, and the politically powerful Roman Catholic Church in Ireland were skillfully avoided; initial hostility from the medical profession quickly dissipated; and AA distanced itself from policy debate on the wider topic of alcoholism as disease. CONCLUSIONS: The relatively smooth introduction of AA to Ireland, the first European country in which it was established, may be attributed to the essentially pragmatic nature of the fellowship and the strategic abilities of its early members.

Cocaine use in Europe - a multi-centre study: patterns of use in different groups.

AIM: The study investigates patterns of cocaine powder and crack cocaine use of different groups in nine European cities. DESIGN, SETTING, PARTICIPANTS: Multi-centre cross-sectional study conducted in Barcelona, Budapest, Dublin, Hamburg, London, Paris, Rome, Vienna, and Zurich. Data were collected by structured face-to-face interviews. The sample comprises 1,855 cocaine users out of three subgroups: 632 cocaine users in addiction treatment, mainly maintenance treatment; 615 socially marginalized cocaine users not in treatment, and 608 socially integrated cocaine users not in treatment. MEASUREMENTS: Use of cocaine powder, crack cocaine and other substances in the last 30 days, routes of administration, and lifetime use of cocaine powder and crack cocaine. FINDINGS: The marginalized group showed the highest intensity of cocaine use, the highest intensity of heroin use and of multiple substance use. 95% of the integrated group snorted cocaine powder, while in the two other groups, injecting was quite prevalent, but with huge differences between the cities. 96% of all participants had used at least one other substance in addition to cocaine in the last 30 days. CONCLUSIONS: The use of cocaine powder and crack cocaine varies widely between different groups and between cities. Nonetheless, multiple substance use is the predominating pattern of cocaine use, and the different routes of administration have to be taken into account.