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BackgroundIndwelling pleural catheters (IPCs) offer symptom relief from pleural effusions. Usage is complicated by infection in ~5% of cases.1 IPCs can be colonised when bacteria grow from pleural fluid without infective features.2 The causes of colonisation are undescribed and its relationship with infection unexplored.AimsDescribe the incidence and microbiology of IPC colonisation.Identify clinical features associated with IPC colonisation.Compare asymptomatically colonising bacteria with those from infection.MethodsAll infected and non-infected IPCs were collected from four sites, along with associated metadata. Infected IPCs were identified clinically and non-infected IPCs categorised as ‘colonised’ if bacteria was cultured from them, and ‘clean’ if not. The genomes of all cultured organisms were sequenced, assembled and taxonomically classified. For Staphylococcus aureus isolates, a maximum likelihood phylogenetic tree was constructed from Snippy-core alignment using IQ-Tree, with S. aureus NCTC8325 as reference.ResultsOf 108 IPCs, 54 were ‘clean’, 34 colonised and 20 infected. Infected and colonised IPCs had longer mean insertion durations (197.6 and 181.3 days) compared with ‘clean’ ones (98 days).Of 34 colonised IPCs, 17 were colonised by multiple species. The most common causes were Coagulase-negative Staphylococci (CoNS n=18), Enterobacteriaceae (n=18), Corynebacteria spp (n=4) & S. aureus (n=4).Two of 20 infected IPCs were culture negative. We retrieved the same organism causing infection from 14/18 IPCs. The most common pathogens were S. aureus (n=8), Pseudomonas aeruginosa (n=3), Enterococcus spp (n=2) and Enterobacter cloacae (n=2). Figure 1 demonstrates clustering of S. aureus isolates from deep and superficial infection.Abstract S149 Figure 1Phylogenetic tree of Staphylococcus aureus isolates from colonised and infected IPCs. S. aureus NCTC8325 is the reference strain[Image Omitted. See PDF.]ConclusionA significant proportion of IPCs are colonised. Colonisation and infection are associated with longer insertion duration. Enterobacteriaceae and CoNS are the most common causes of colonisation, whereas S. aureus and Pseudomonas aeruginosa commonly cause infection. S. aureus isolates causing deep infection show higher genetic similarity versus colonising strains. This, and our phylogenetic analysis, suggests colonisation does not cause infection, but rather there is a virulent subset of isolates causing infection. Ongoing work will identify genes and markers associated with infection.ReferencesSundaralingam, et al. Chest 2022;161(5):1407–1425.Sethi, et al. Clinical & Translational Science 2023:1287–1288.

Ubiquitin ligases control the degradation of core clock proteins to govern the speed and resetting properties of the circadian pacemaker. However, few studies have addressed their potential to regulate other cellular events within clock neurons beyond clock protein turnover. Here, we report that the ubiquitin ligase, UBR4/POE, strengthens the central pacemaker by facilitating neuropeptide trafficking in clock neurons and promoting network synchrony. Ubr4 -deficient mice are resistant to jetlag, whereas poe knockdown flies are prone to arrhythmicity, behaviors reflective of the reduced axonal trafficking of circadian neuropeptides. At the cellular level, Ubr4 ablation impairs the export of secreted proteins from the Golgi apparatus by reducing the expression of Coronin 7, which is required for budding of Golgi-derived transport vesicles. In summary, UBR4/POE fulfills a conserved and unexpected role in the vesicular trafficking of neuropeptides, a function that has important implications for circadian clock synchrony and circuit-level signal processing. Although ubiquitin ligases are known to control clock protein degradation, their other roles in clock neurons are unclear. Here the authors report that UBR4 promotes export of neuropeptides from the Golgi for axonal trafficking, which is important for circadian clock synchrony in mice and flies.

In mammals, circadian entrainment relies on signaling pathways that translate light input into molecular changes within the central pacemaker, the suprachiasmatic nucleus (SCN). Here, using β ‑arrestin1 (ARRB1)-deficient mice, we identify a critical role for β‑arrestin1 in this process, showing that endosomal signaling underlies key steps in clock resetting. We demonstrate that ARRB1 is required for PAC1 receptor internalization and for the activation of endosomal signaling in response to light or PACAP. ARRB1‑dependent PAC1 endosomal signaling activates an ERK1/2-RSK1-S6 cascade that enhances protein translation and contributes to the induction of PER proteins. Transcriptional responses remain intact, underscoring the spatial specificity of ARRB1 function. Our findings position endosomes as critical subcellular hubs for circadian signal transduction and reveal a non-canonical, β-arrestin1-dependent entrainment mechanism that operates through translational control. Together, these results challenge traditional GPCR signaling paradigms and establish endosome-based signaling as a key regulator of circadian timekeeping. β arrestin1-mediated endosomal GPCR signaling is shown to be a crucial component of light-induced entrainment of the central circadian clock in mammals.

β-arrestin1 and β-arrestin2 are proteins of the arrestin family that regulate G-protein coupled receptors (GPCRs), which include receptors of the suprachiasmatic nucleus (SCN) required for responding and entraining to photic signals. Both β-arrestins are ubiquitously expressed and are found in abundant levels in the SCN. No study to date has examined the role of β-arrestins within the mammalian master clock, the SCN. Our research revealed that the absence of β-arrestin1 in mice led to impaired photic response and entrainment, along with an attenuated phase shift and reduced wheel-running activity. At the molecular level, we found disrupted p-ERK induction and modest differences in the basal expression of PER2. No difference was observed in the induction of several immediate early genes. Impaired photic responses led to investigations on melanopsin-expressing cells of the retina; however, no morphological difference was uncovered in the retina or in the expression of melanopsin. We found that the absence of β-arrestin2 established no change in circadian behaviours of our mice. These findings identify β-arrestin1 as a potential regulator of photic entrainment and suggests that β-arrestin2 may be dispensable in circadian regulation. Further experiments are needed to uncover the precise role and mechanism of action of β-arrestins in circadian timekeeping.

Within a few days of beginning her work as the executive director at South Asian Youth Action (SAYA!), Annetta Seecharran was forced to revise her agenda to provide crisis management. This shift was the result of the post––September 11 victimization faced by innumerable members of the South Asian community in New York City. In this paper, Seecharran documents the ways that SAYA!, which had initially functioned as a youth center, quickly expanded its agenda and programs to serve as a clearinghouse. Other changes included the addition of mental health counseling, legal services, and community education programs. SAYA!'s ability, even with its limited resources, to adapt to post––September 11 social landscape shows that the 2001 terrorist attacks, while tragic, also gave birth to new leadership and creativity in the South Asian community.

Our knowledge and understanding of how bacterial pathogens have evolved has been limited by inadequate information on the full ecology of these organisms. Large-scale population genomic analyses have enabled a high-resolution view of variation at the core and accessory genome level, within and between bacterial populations, revealing previously hidden patterns of variation among microorganisms. This sheds light on the evolution and maintenance of ecologically distinct populations of bacteria and has raised the question of whether the same approach can uncover novel information on the ecology of established, clinically important pathogens. Yersinia pseudotuberculosis and Escherichia coli represent 'model' organisms in the study of microbial evolution, but given the high degree of niche overlap in both species, their ecology is largely unknown.In this study, genomic analyses of Y. pseudotuberculosis strains, obtained from various habitats worldwide, revealed a phylogeographic split within the population, with an Asian ancestry and subsequent dispersal of successful clonal lineages across the rest of the world. These lineages were differentiated by CRISPR arrays and we demonstrated that genetic exchange between lineages is restricted. Despite the coexistence of these lineages for thousands of years, the discrete lineage structure of the population is maintained due to the restriction of inter-lineage genetic exchange. The analyses did not identify a role for ecological barriers in defining the distinct lineage structure of the species, suggesting that Y. pseudotuberculosis is a host generalist able to succeed in multiple habitats.The relative abundance of multidrug-resistant extraintestinal pathogenic E. coli (ExPEC) among E. coli inhabiting non-human niches is undetermined, owing to many studies selectively isolating resistant bacteria. To compare the population structure of E. coli from non-human environments with the well-defined population structure of human-clinical E. coli, unbiased sampling of E. coli isolates from river water and retail poultry samples was undertaken. Genomic analysis of isolates revealed a low prevalence of clinically-associated sequence types and potential ExPEC strains among non-human E. coli when contrasted with human-clinical E. coli, suggesting two distinct populations. Comparative genomic analyses further supported this, revealing a noticeable difference in accessory genome content between the two populations and low levels of genetic exchange between closely related strains. This suggests ecological barriers, resulting in gradual genetic isolation, may have contributed to the divergence of these niche-associated populations of E. coli. The investigation concluded that the non-human population of E. coli is unlikely to contribute significantly to the weight of hospital- and community-acquired extraintestinal infections in humans.

BE [(1,4)-a-D-glucan:(I,4)-a-D-glucan 6-glucosyltransferase, EC 2.4.1.18] catalyses a transglycosylation reaction where a branch-point is created by the cleavage of an a-l,4 glycosidic bond to form an a-l,6 glycosidic bond. Branching enzyme (BE) from baker's yeast was purified to near homogeneity by chromatography on DEAE-cellulose, Sephacryl S-200 and Protein Pak Q. Electrophoresis on SDS-PAGE revealed one major band of molecular weight 74 kDa. Three distinct methods for determining BE activity (Phosphorylase Stimulation, Iodine- Binding and Branch-Linkage Assays) were used to characterise the purified protein. The enzyme displayed a temperature optimum between 15-25°C and a broad pH optimum of 6.5-7.5 with maximum activity occurring in phosphate buffer. The enzyme was fully stable after incubation at 20°C for 5 hours. A Km value of 1474 Jlg/ ml for amylose was obtained. Primary structural analysis involving N-terminal sequencing and amino acid composition suggested that yeast BE may share some homology with BEs isolated from other sources. Immunological comparisons between yeast, maize (BEll) and Escherichia coli BE using yeast polyclonal antiserum indicated that the enzymes may share antigenic determinants. However, similar comparisons between yeast BE and E.coli antiserum revealed that the antibody only recognised yeast BE in its denatured conformation. Yeast BE was used to modify potato amylose and amylopectin and wheat starch. The enzyme was capable of introducing additional branch points to these substrates resulting in a displacement of the iodine Amax from 629 nm to 568 nm, from 543 nm to 411 nm and from 632 nm to 568 nm for amylose, amylopectin and wheat starch, respectively. HPAEC-PAD analysis of the branched products produced by yeast BE revealed that predominantly short chains of dp 2 to I? were transferred. At least three BE fractions of higher specific activities were isolated from brewer's yeast hatyested at the late exponential phase, suggesting the expression of more than one BE in Saccharomyces cerevisiae.

Yersinia pseudotuberculosis is a Gram negative intestinal pathogen of humans and has been responsible for several nation-wide gastro-intestinal outbreaks. Large-scale population genomic studies have been performed on the other human pathogenic Yersinia, Y. pestis and Y. enterocolitica allowing a high-resolution understanding of the ecology, evolution and dissemination of these pathogens. However, to date no large-scale global population genomic analysis of Y. pseudotuberculosis has been performed. Here we present analyses of the genomes of 134 strains of Y. pseudotuberculosis isolated from around the world, from multiple ecosystems since 1960s. Our data display a phylogeographic split within the population, with an Asian ancestry and subsequent dispersal of successful clonal lineages into Europe and the rest of the world. These lineages can be differentiated by CRISPR cluster arrays, and we show that the lineages are limited with respect to inter-lineage genetic exchange. This restriction of genetic exchange maintains the discrete lineage structure in the population despite co-existence of lineages for thousands of years in multiple countries. Our data highlights how CRISPR can be informative of the evolutionary trajectory of bacterial lineages, and merits further study across bacteria.