A microCT-based platform to quantify drug targeting.

BACKGROUND: Heterotopic ossification (HO) is a frequent and debilitating complication of traumatic musculoskeletal injuries and orthopedic procedures. Prophylactic dosing of botulinum toxin type A (BTxA) holds potential as a novel treatment option if accurately distributed throughout soft-tissue volumes where protection is clinically desired. We developed a high-resolution, microcomputed tomography (microCT)-based imaging strategy to assess drug distribution and validated this platform by quantifying distribution achieved via a prototype delivery system versus a single-bolus injection. METHODS: We injected an iodine-containing contrast agent (iodixanol 320 mg I/mL) into dissected rabbit musculature followed by microCT imaging and analysis. To contrast the performance of distributed versus bolus injections, a three-dimensional (3D) 64-cm3-printed soft-tissue holder was developed. A centered 2-cm3 volume of interest (VOI) was targeted with a single-bolus injection or an equal volume distributed injection delivered via a 3D-printed prototype. VOI drug coverage was quantified as a percentage of the VOI volume that was < 1.0 mm from the injected fluid. RESULTS: The microCT-based approach enabled high-resolution quantification of injection distribution within soft tissue. The distributed dosing prototype provided significantly greater tissue coverage of the targeted VOI (72 ± 3%, mean ± standard deviation) when compared to an equal volume bolus dose (43 ± 5%, p = 0.031) while also enhancing the precision of injection targeting. CONCLUSIONS: A microCT-based imaging technique precisely quantifies drug distribution within a soft-tissue VOI, providing a path to overcome a barrier for clinical translation of prophylactic inhibition of HO by BTxA. RELEVANCE STATEMENT: This platform will facilitate rapid optimization of injection parameters for clinical devices used to effectively and safely inhibit the formation of heterotopic ossification. KEY POINTS: • MicroCT provides high-resolution quantification of soft-tissue drug distribution. • Distributed dosing is required to maximize soft-tissue drug coverage. • Imaging platform will enable rapid screening of 3D-printed drug distribution prototypes.

ExlA: A New Contributor to Pseudomonas aeruginosa Virulence.

ExlA (also called exolysin) is a recently discovered virulence factor secreted by a subset of Pseudomonas aeruginosa strains in which a type 3 secretion system is lacking. exlA-positive strains were identified worldwide in the clinic, causing several types of infectious diseases, and were detected in various locations in the environment. ExlA possesses pore-forming activity and is cytolytic for most human cell types. It belongs to a class of poorly characterized bacterial toxins, sharing a similar protein domain organization and a common secretion pathway. This review summarizes the recent findings regarding ExlA synthesis, its secretion pathway, and its toxic behavior for host cells.

Reconstruction of transmission chains of SARS-CoV-2 amidst multiple outbreaks in a geriatric acute-care hospital: a combined retrospective epidemiological and genomic study.

Background: There is ongoing uncertainty regarding transmission chains and the respective roles of healthcare workers (HCWs) and elderly patients in nosocomial outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in geriatric settings. Methods: We performed a retrospective cohort study including patients with nosocomial coronavirus disease 2019 (COVID-19) in four outbreak-affected wards, and all SARS-CoV-2 RT-PCR positive HCWs from a Swiss university-affiliated geriatric acute-care hospital that admitted both Covid-19 and non-Covid-19 patients during the first pandemic wave in Spring 2020. We combined epidemiological and genetic sequencing data using a Bayesian modelling framework, and reconstructed transmission dynamics of SARS-CoV-2 involving patients and HCWs, to determine who infected whom. We evaluated general transmission patterns according to case type (HCWs working in dedicated Covid-19 cohorting wards: HCWcovid; HCWs working in non-Covid-19 wards where outbreaks occurred: HCWoutbreak; patients with nosocomial Covid-19: patientnoso) by deriving the proportion of infections attributed to each case type across all posterior trees and comparing them to random expectations. Results: During the study period (1 March to 7 May 2020), we included 180 SARS-CoV-2 positive cases: 127 HCWs (91 HCWcovid, 36 HCWoutbreak) and 53 patients. The attack rates ranged from 10% to 19% for patients, and 21% for HCWs. We estimated that 16 importation events occurred with high confidence (4 patients, 12 HCWs) that jointly led to up to 41 secondary cases; in six additional cases (5 HCWs, 1 patient), importation was possible with a posterior probability between 10% and 50%. Most patient-to-patient transmission events involved patients having shared a ward (95.2%, 95% credible interval [CrI] 84.2%-100%), in contrast to those having shared a room (19.7%, 95% CrI 6.7%-33.3%). Transmission events tended to cluster by case type: patientnoso were almost twice as likely to be infected by other patientnoso than expected (observed:expected ratio 2.16, 95% CrI 1.17-4.20, p=0.006); similarly, HCWoutbreak were more than twice as likely to be infected by other HCWoutbreak than expected (2.72, 95% CrI 0.87-9.00, p=0.06). The proportion of infectors being HCWcovid was as expected as random. We found a trend towards a greater proportion of high transmitters (≥2 secondary cases) among HCWoutbreak than patientnoso in the late phases (28.6% vs. 11.8%) of the outbreak, although this was not statistically significant. Conclusions: Most importation events were linked to HCW. Unexpectedly, transmission between HCWcovid was more limited than transmission between patients and HCWoutbreak. This finding highlights gaps in infection control and suggests the possible areas of improvements to limit the extent of nosocomial transmission. Funding: This study was supported by a grant from the Swiss National Science Foundation under the NRP78 funding scheme (Grant no. 4078P0_198363).

ExlA Pore-Forming Toxin: Localization at the Bacterial Membrane, Regulation of Secretion by Cyclic-Di-GMP, and Detection In Vivo.

ExlA is a highly virulent pore-forming toxin that has been recently discovered in outlier strains from Pseudomonas aeruginosa. ExlA is part of a two-partner secretion system, in which ExlA is the secreted passenger protein and ExlB the transporter embedded in the bacterial outer membrane. In previous work, we observed that ExlA toxicity in a host cell was contact-dependent. Here, we show that ExlA accumulates at specific points of the outer membrane, is likely entrapped within ExlB pore, and is pointing outside. We further demonstrate that ExlA is maintained at the membrane in conditions where the intracellular content of second messenger cyclic-di-GMP is high; lowering c-di-GMP levels enhances ExlB-dependent ExlA secretion. In addition, we set up an ELISA to detect ExlA, and we show that ExlA is poorly secreted in liquid culture, while it is highly detectable in broncho-alveolar lavage fluids of mice infected with an exlA+ strain. We conclude that ExlA translocation is halted at mid-length in the outer membrane and its secretion is regulated by c-di-GMP. In addition, we developed an immunological test able to quantify ExlA in biological samples.

The bacterial toxin ExoU requires a host trafficking chaperone for transportation and to induce necrosis.

Pseudomonas aeruginosa can cause nosocomial infections, especially in ventilated or cystic fibrosis patients. Highly pathogenic isolates express the phospholipase ExoU, an effector of the type III secretion system that acts on plasma membrane lipids, causing membrane rupture and host cell necrosis. Here, we use a genome-wide screen to discover that ExoU requires DNAJC5, a host chaperone, for its necrotic activity. DNAJC5 is known to participate in an unconventional secretory pathway for misfolded proteins involving anterograde vesicular trafficking. We show that DNAJC5-deficient human cells, or Drosophila flies knocked-down for the DNAJC5 orthologue, are largely resistant to ExoU-dependent virulence. ExoU colocalizes with DNAJC5-positive vesicles in the host cytoplasm. DNAJC5 mutations preventing vesicle trafficking (previously identified in adult neuronal ceroid lipofuscinosis, a human congenital disease) inhibit ExoU-dependent cell lysis. Our results suggest that, once injected into the host cytoplasm, ExoU docks to DNAJC5-positive secretory vesicles to reach the plasma membrane, where it can exert its phospholipase activity.

Non-human primate models of human respiratory infections.

Respiratory pathogens represent a great burden for humanity and a potential source of new pandemics, as illustrated by the recent emergence of coronavirus disease 2019 (COVID-19). In recent decades, biotechnological advances have led to the development of numerous innovative therapeutic molecules and vaccine immunogens. However, we still lack effective treatments and vaccines against many respiratory pathogens. More than ever, there is a need for a fast, predictive, preclinical pipeline, to keep pace with emerging diseases. Animal models are key for the preclinical development of disease management strategies. The predictive value of these models depends on their ability to reproduce the features of the human disease, the mode of transmission of the infectious agent and the availability of technologies for monitoring infection. This review focuses on the use of non-human primates as relevant preclinical models for the development of prevention and treatment for human respiratory infections.

[Issues and challenges related to COVID-19 in acute geriatric care: lessons learned from the Geneva experience]. / Enjeux et défis du Covid-19 en gériatrie aiguë: leçons tirées de l'expérience genevoise.

The older patients have been the most affected by the SARS-CoV-2 pandemic. In addition, this infection has been responsible for high mortality rate in this population. In this article we wanted to describe the clinical findings we encountered in older people with COVID-19 and share some of the issues and challenges we faced during the COVID-19 pandemic.

Les personnes âgées ont été les plus touchées par la pandémie de SARS-CoV-2. De plus, cette infection a été responsable d'une mortalité élevée au sein de cette population. Dans cet article, nous avons souhaité décrire les particularités cliniques du Covid-19 que nous avons constatées chez les patients âgés et faire part de plusieurs enjeux et défis auxquels nous avons été confrontés au cours de la pandémie de Covid-19.

Predictors of In-Hospital Mortality in Older Patients With COVID-19: The COVIDAge Study.

OBJECTIVE: To determine predictors of in-hospital mortality related to COVID-19 in older patients. DESIGN: Retrospective cohort study. SETTING AND PARTICIPANTS: Patients aged 65 years and older hospitalized for a diagnosis of COVID-19. METHODS: Data from hospital admission were collected from the electronic medical records. Logistic regression and Cox proportional hazard models were used to predict mortality, our primary outcome. Variables at hospital admission were categorized according to the following domains: demographics, clinical history, comorbidities, previous treatment, clinical status, vital signs, clinical scales and scores, routine laboratory analysis, and imaging results. RESULTS: Of a total of 235 Caucasian patients, 43% were male, with a mean age of 86 ± 6.5 years. Seventy-six patients (32%) died. Nonsurvivors had a shorter number of days from initial symptoms to hospitalization (P = .007) and the length of stay in acute wards than survivors (P < .001). Similarly, they had a higher prevalence of heart failure (P = .044), peripheral artery disease (P = .009), crackles at clinical status (P < .001), respiratory rate (P = .005), oxygen support needs (P < .001), C-reactive protein (P < .001), bilateral and peripheral infiltrates on chest radiographs (P = .001), and a lower prevalence of headache (P = .009). Furthermore, nonsurvivors were more often frail (P < .001), with worse functional status (P < .001), higher comorbidity burden (P < .001), and delirium at admission (P = .007). A multivariable Cox model showed that male sex (HR 4.00, 95% CI 2.08-7.71, P < .001), increased fraction of inspired oxygen (HR 1.06, 95% CI 1.03-1.09, P < .001), and crackles (HR 2.42, 95% CI 1.15-6.06, P = .019) were the best predictors of mortality, while better functional status was protective (HR 0.98, 95% CI 0.97-0.99, P = .001). CONCLUSIONS AND IMPLICATIONS: In older patients hospitalized for COVID-19, male sex, crackles, a higher fraction of inspired oxygen, and functionality were independent risk factors of mortality. These routine parameters, and not differences in age, should be used to evaluate prognosis in older patients.

Inflammasome activation by Pseudomonas aeruginosa's ExlA pore-forming toxin is detrimental for the host.

During acute Pseudomonas aeruginosa infection, the inflammatory response is essential for bacterial clearance. Neutrophil recruitment can be initiated following the assembly of an inflammasome within sentinel macrophages, leading to activation of caspase-1, which in turn triggers macrophage pyroptosis and IL-1ß/IL-18 maturation. Inflammasome formation can be induced by a number of bacterial determinants, including Type III secretion systems (T3SSs) or pore-forming toxins, or, alternatively, by lipopolysaccharide (LPS) via caspase-11 activation. Surprisingly, previous studies indicated that a T3SS-induced inflammasome increased pathogenicity in mouse models of P. aeruginosa infection. Here, we investigated the immune reaction of mice infected with a T3SS-negative P. aeruginosa strain (IHMA879472). Virulence of this strain relies on ExlA, a secreted pore-forming toxin. IHMA879472 promoted massive neutrophil infiltration in infected lungs, owing to efficient priming of toll-like receptors, and thus enhanced the expression of inflammatory proteins including pro-IL-1ß and TNF-α. However, mature-IL-1ß and IL-18 were undetectable in wild-type mice, suggesting that ExlA failed to effectively activate caspase-1. Nevertheless, caspase-1/11 deficiency improved survival following infection with IHMA879472, as previously described for T3SS+ bacteria. We conclude that the detrimental effect associated with the ExlA-induced inflammasome is probably not due to hyperinflammation, rather it stems from another inflammasome-dependent process.

The introduction of a standardised national licensing exam as a driver of change in medical education: A qualitative study from Switzerland.

INTRODUCTION: Only a few studies have described the impacts, strengths and needs for further development of national licensing exams (NLE). To gain such insights regarding the Swiss NLE, which includes a multiple-choice and a standardised clinical skills exam, we explored the perceptions of involved experts and stakeholders. METHODS: We explored participants' perceptions in four focus group discussions. The interviews were recorded, transcribed verbatim and qualitatively analysed using a thematic analysis approach. RESULTS: The analysis resulted in five perceived impacts, two strengths and two needs for further developments of the NLE. Perceived impacts were (1) steering students' learning behaviour, (2) supporting teachers and assessors to align teaching to competencies, (3) elevating the importance of the Swiss Catalogue of Learning Objectives, (4) setting incentives for the further development of curricula, and (5) fostering the collaboration between the faculties of medicine. Perceived strengths were the blend of assessment formats, including their competency-based orientation, and the collaborative development approach. Perceived needs lay in the NLE's further development to sustain its fit for purpose and in incentives for people involved. CONCLUSION: According to our study, this NLE had, and has, notable impacts on medical education in Switzerland. Our insights can be useful for others planning a similar undertaking.

Exolysin (ExlA) from Pseudomonas aeruginosa Punctures Holes into Target Membranes Using a Molten Globule Domain.

Bacteria employ several mechanisms, and most notably secretion systems, to translocate effectors from the cytoplasm to the extracellular environment or the cell surface. Pseudomonas aeruginosa widely employs secretion machineries such as the Type III Secretion System to support virulence and cytotoxicity. However, recently identified P. aeruginosa strains that do not express the Type III Secretion System have been shown to express ExlA, an exolysin translocated through a two-partner secretion system, and are the causative agents of severe lung hemorrhage. Sequence predictions of ExlA indicate filamentous hemagglutinin (FHA-2) domains as the prevalent features, followed by a C-terminal domain with no known homologs. In this work, we have addressed the mechanism employed by ExlA to target membrane bilayers by using NMR, small-angle X-ray scattering, atomic force microscopy, and cellular infection techniques. We show that the C-terminal domain of ExlA displays a "molten globule-like" fold that punctures small holes into membranes composed of negatively charged lipids, while other domains could play a lesser role in target recognition. In addition, epithelial cells infected with P. aeruginosa strains expressing different ExlA variants allow localization of the toxin to lipid rafts. ExlA homologs have been identified in numerous bacterial strains, indicating that lipid bilayer destruction is an effective strategy employed by bacteria to establish interactions with multiple hosts.

Targeting of the apical junctional complex by bacterial pathogens.

Targeting the apical junctional complex during acute bacterial infections can be detrimental for the host in several aspects. First, the rupture of the epithelium or endothelium integrity is toxic in itself. In addition, extracellular bacterial pathogens or bacterial toxins can cross the body's physical barriers using the paracellular route and induce infection or intoxication of distant organs. No single strategy has been developed to disrupt junctional structures, rather each bacterium has its own method, which can be classed in one of the following three categories: (i) proteolysis/perturbation of adhesive proteins involved in tight or adherens junctions by bacterial or toxin-activated eukaryotic proteases, (ii) manipulation of host regulatory pathways leading to weakened intercellular adhesion, or (iii) delocalization of the junctional complex to open the gateway toward the subepithelial compartment. In this review, examples of each of these mechanisms are provided to illustrate how creative bacteria can be when seeking to disrupt cell-cell junctions.

Neuromuscular dysfunction, independent of gait dysfunction, modulates trabecular bone homeostasis in mice.

OBJECTIVES: To clarify the effects of neuromuscular dysfunction on hindlimb loading, muscle atrophy, and bone homeostasis. METHODS: We quantified changes to hindlimb loading, muscle atrophy, and bone morphology following either Botulinum toxin A (BTxA) induced muscle paralysis or peripheral nerve injury (PNI) in mice; two in vivo models that we anticipated would differently alter gait and mechanical loading patterns due to their distinct effects on neuromuscular signaling. To confirm the expected behavioral effects of PNI, we assessed mechanical allodynia of the ipsilateral hindlimb using von Frey testing and activity (distance traveled and speed) was monitored in both groups using open field testing. Peak vertical ground reaction forces (GRF) and ankle and knee kinematics during normal locomotion were quantified and used to estimate peak mid-diaphyseal normal strains. Muscle atrophy and trabecular and cortical bone morphology were assessed via high-resolution microCT imaging. RESULTS: BTxA-induced calf paralysis caused severe muscle atrophy and altered gait kinetics and kinematics and reduced gait-induced normal strains. PNI increased mechanical allodynia but did not alter gait, nor did it cause muscle atrophy. We observed that muscle paralysis and PNI both led to severe trabecular bone loss but only BTxA-induced paralysis increased cortical bone resorption. CONCLUSIONS: While mechanical stimuli clearly have essential functions in bone development and adaptation, these data emphasize that neuromuscular signaling, independent of load-induced mechanical strains, may modulate trabecular bone homeostasis in normal and disease states.

What is the profile of older drivers considered medically unfit to drive? A cross-sectional survey in Western Switzerland.

OBJECTIVE: We aimed to assess how often and for what reasons general practitioners (GPs) consider older drivers medically unfit to drive. METHODS: All GPs certified to carry out fitness-to-drive assessments in Geneva (medical assessors, n = 69), as well as a random sample of 500 GPs practising in Vaud, Neuchatel and Jura, were asked to complete a questionnaire about the mean number of assessments per week, the number of negative decisions in the previous year and the main reason for the most recent negative decision. RESULTS: Completed questionnaires were returned by 268 respondents (45 medical assessors and 223 other GPs, participation rate: 47%). The mean proportion of drivers with a negative decision was 2.2% (standard deviation [SD] 3.3). The proportion was slightly lower among medical assessors (1.1%, SD 1.3) compared to other GPs (2.3%, SD 3.3, p <0.001). The main reasons for being considered medically unfit to drive were cognitive (64%) and visual acuity impairments (18%). CONCLUSIONS: GPs in this survey reported considering approximately 2% of older drivers as medically unfit to drive, mainly because of cognitive and visual acuity impairments. Further research should identify how GPs decide if older drivers are fit or unfit, and assess the effectiveness of medical screening in reducing car crashes involving older drivers.

Functional Consequences of Calcium Influx Promoted by Bacterial Pore-Forming Toxins.

Bacterial pore-forming toxins induce a rapid and massive increase in cytosolic Ca2+ concentration due to the formation of pores in the plasma membrane and/or activation of Ca2+-channels. As Ca2+ is an essential messenger in cellular signaling, a sustained increase in Ca2+ concentration has dramatic consequences on cellular behavior, eventually leading to cell death. However, host cells have adapted mechanisms to protect against Ca2+ intoxication, such as Ca2+ efflux and membrane repair. The final outcome depends upon the nature and concentration of the toxin and on the cell type. This review highlights the repercussions of Ca2+ overload on the induction of cell death, repair mechanisms, cellular adhesive properties, and the inflammatory response.

Adherence to guidelines when evaluating fitness-to-drive in the elderly: a practice review of Swiss physicians.

OBJECTIVE: We aimed to explore the extent to which general practitioners (GPs) in Western Switzerland adhere to Swiss recommendations when assessing fitness-to-drive in the elderly. METHODS: A random sample of 500 GPs practicing in Vaud, Neuchatel and Jura, and all GPs certified to conduct fitness-to-drive assessments in Geneva ("experts", n = 69) were invited to participate. They were asked how often they performed twenty procedures (recommended in Swiss guidelines developed by experts in traffic medicine) when assessing older drivers during the previous year, scored on a five-point Likert scale ranging from "never" to "always performed". The GPs were considered to be adhering to the recommended procedure if they performed it often or always. We computed the proportion of GPs adhering to each procedure, and compared GPs with or without specialised expertise. RESULTS: A total of 268 GPs completed the questionnaire (participation rate 47%). The most frequently reported procedures were asking for current medication (96%), cardiovascular (94%) and neurological diseases (91%), and screening for visual acuity impairment (93%), whereas the least frequently reported procedures were screening for cognitive impairment in drivers aged between 70 and 80 years (44%) and for mood disorder (31%), asking for a history of driving license withdrawal (38%), and interviewing close relatives (10%). Six procedures were statistically significantly more frequently performed by the experts than by the other GPs. In general, GPs reported using validated tools, except when screening for at-risk drinking and mood disorder (tools used by 26 and 28%, respectively). CONCLUSIONS: Many Swiss GPs seem not to systematically follow the current Swiss recommendations. Although several important procedures appear to routinely be part of older drivers' assessment, others are infrequently performed. Further research should identify how GPs select the recommended items to which they adhere and those they never apply, and how to facilitate the use of recommended procedures to help them decide if a person is fit, unfit or requiring further evaluation.

Corrigendum: Pseudomonas aeruginosa Takes a Multi-Target Approach to Achieve Junction Breach.

[This corrects the article on p. 532 in vol. 7, PMID: 29379773.].

CLIQ-BID: A method to quantify bacteria-induced damage to eukaryotic cells by automated live-imaging of bright nuclei.

Pathogenic bacteria induce eukaryotic cell damage which range from discrete modifications of signalling pathways, to morphological alterations and even to cell death. Accurate quantitative detection of these events is necessary for studying host-pathogen interactions and for developing strategies to protect host organisms from bacterial infections. Investigation of morphological changes is cumbersome and not adapted to high-throughput and kinetics measurements. Here, we describe a simple and cost-effective method based on automated analysis of live cells with stained nuclei, which allows real-time quantification of bacteria-induced eukaryotic cell damage at single-cell resolution. We demonstrate that this automated high-throughput microscopy approach permits screening of libraries composed of interference-RNA, bacterial strains, antibodies and chemical compounds in ex vivo infection settings. The use of fluorescently-labelled bacteria enables the concomitant detection of changes in bacterial growth. Using this method named CLIQ-BID (Cell Live Imaging Quantification of Bacteria Induced Damage), we were able to distinguish the virulence profiles of different pathogenic bacterial species and clinical strains.

Exolysin Shapes the Virulence of Pseudomonas aeruginosa Clonal Outliers.

Bacterial toxins are important weapons of toxicogenic pathogens. Depending on their origin, structure and targets, they show diverse mechanisms of action and effects on eukaryotic cells. Exolysin is a secreted 170 kDa pore-forming toxin employed by clonal outliers of Pseudomonas aeruginosa providing to some strains a hyper-virulent behaviour. This group of strains lacks the major virulence factor used by classical strains, the Type III secretion system. Here, we review the structural features of the toxin, the mechanism of its secretion and the effects of the pore formation on eukaryotic cells.