Everything but the kitchen sink: The use of multiple hypothesis generation methods to investigate an outbreak of Salmonella Enteritidis associated with frozen profiteroles and eclairs.

In December 2018, an outbreak of Salmonella Enteritidis infections was identified in Canada by whole-genome sequencing (WGS). An investigation was initiated to identify the source of the illnesses, which proved challenging and complex. Microbiological hypothesis generation methods included comparisons of Salmonella isolate sequence data to historical domestic outbreaks and international repositories. Epidemiological hypothesis generation methods included routine case interviews, open-ended centralized re-interviewing, thematic analysis of open-ended interview data, collection of purchase records, a grocery store site visit, analytic comparison to healthy control groups, and case-case analyses. Food safety hypothesis testing methods included food sample collection and analysis, and traceback investigations. Overall, 83 cases were identified across seven provinces, with onset dates from 6 November 2018 to 7 May 2019. Case ages ranged from 1 to 88 years; 60% (50/83) were female; 39% (22/56) were hospitalized; and three deaths were reported. Brand X profiteroles and eclairs imported from Thailand were identified as the source of the outbreak, and eggs from an unregistered facility were hypothesized as the likely cause of contamination. This study aims to describe the outbreak investigation and highlight the multiple hypothesis generation methods that were employed to identify the source.

Two Listeria monocytogenes outbreaks in a cancer centre: onsite food premises and their potential health risk to patients.

BACKGROUND: This report describes two L. monocytogenes outbreak investigations that occurred in March and September of 2018 and that linked illness to a food premises located in an Ontario cancer centre. The cancer centre serves patients from across the province. METHODS: In Ontario, local public health agencies follow up with all reported laboratory-confirmed cases of listeriosis to identify possible sources of disease acquisition and to carry out investigations, including at suspected food premises. The Canadian Food Inspection Agency (CFIA) is notified of any Listeria-positive food product collected in relation to a case. The CFIA traces Listeria-positive product through the food distribution system to identify the contamination source and ensure the implicated manufacturing facility implements corrective measures. RESULTS: Outbreaks one and two each involved three outbreak-confirmed listeriosis cases. All six cases were considered genetically related by whole genome sequencing (WGS). In both outbreaks, outbreak-confirmed cases reported consuming meals at a food premises located in a cancer centre (food premises A) before illness onset. Various open deli meat samples and, in outbreak two, environmental swabs (primarily from the meat slicer) collected from food premises A were genetically related to the outbreak-confirmed cases. Food premises A closed as a result of the investigations. CONCLUSIONS: When procuring on-site food premises, healthcare facilities and institutions serving individuals with immuno-compromising conditions should consider the potential health risk of foods available to their patient population.

Cilia proteins are biomarkers of altered flow in the vasculature.

Cilia, microtubule-based organelles that project from the apical luminal surface of endothelial cells (ECs), are widely regarded as low-flow sensors. Previous reports suggest that upon high shear stress, cilia on the EC surface are lost, and more recent evidence suggests that deciliation-the physical removal of cilia from the cell surface-is a predominant mechanism for cilia loss in mammalian cells. Thus, we hypothesized that EC deciliation facilitated by changes in shear stress would manifest in increased abundance of cilia-related proteins in circulation. To test this hypothesis, we performed shear stress experiments that mimicked flow conditions from low to high shear stress in human primary cells and a zebrafish model system. In the primary cells, we showed that upon shear stress induction, indeed, ciliary fragments were observed in the effluent in vitro, and effluents contained ciliary proteins normally expressed in both endothelial and epithelial cells. In zebrafish, upon shear stress induction, fewer cilia-expressing ECs were observed. To test the translational relevance of these findings, we investigated our hypothesis using patient blood samples from sickle cell disease and found that plasma levels of ciliary proteins were elevated compared with healthy controls. Further, sickled red blood cells demonstrated high levels of ciliary protein (ARL13b) on their surface after adhesion to brain ECs. Brain ECs postinteraction with sickle RBCs showed high reactive oxygen species (ROS) levels. Attenuating ROS levels in brain ECs decreased cilia protein levels on RBCs and rescued ciliary protein levels in brain ECs. Collectively, these data suggest that cilia and ciliary proteins in circulation are detectable under various altered-flow conditions, which could serve as a surrogate biomarker of the damaged endothelium.

Stabilizing breathing pattern using local mechanical vibrations: comparison of deterministic and stochastic stimulations in rodent models of apnea of prematurity.

Mechanical stimulation has been shown to reduce apnea of prematurity (AOP), a major concern in preterm infants. Previous work suggested that the underlying mechanism is stochastic resonance, amplification of a subthreshold signal by stochastic stimulation. We hypothesized that the mechanism behind the reduction of apnea length may not be a solely stochastic phenomenon, and suggest that a purely deterministic, non-random mechanical stimulation could be equally as effective. Mice and rats were anesthetized, tracheostomized, and mechanically ventilated to halt spontaneous breathing. Two miniature motors controlled by a microcontroller were attached around the abdomen. Ventilation was paused, stimulations were applied, and the time to the rodent's first spontaneous breath (T) was measured. Six spectrally different signals were compared to one another and the no-stimulation control in mice. The most successful deterministic stimulation (D) at reducing apnea was then compared to a pseudo-random noise (PRN) signal of comparable amplitude and frequency. CO2%, CO2 stabilization time (Ts), O2 saturation (SpO2%), and T were also measured. D significantly reduced T compared to no stimulation for medium and high amplitudes. PRN also reduced T, without  a difference between D and PRN. Furthermore, both stimulations significantly reduced Ts with no significant differences between the respective stimulations. However, there was no effect of D or PRN on SpO2%. The lack of differences between D and PRN led to an additional series of experiment comparing the same D to a band-limited white noise (WN) signal in young rats. Both D and WN were shown to significantly reduce T, with D showing statistical superiority in reduction of apnea. We further speculate that both deterministic and stochastic mechanical stimulations induce some form of mechanotransduction which is responsible for their efficacy, and our findings suggest that mechanical stimulation may be effective in treating AOP. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13534-021-00203-x.

Pedunculopontine tegmentum cholinergic loss leads to a progressive decline in motor abilities and neuropathological changes resembling progressive supranuclear palsy.

Progressive supranuclear palsy (PSP) is the most common atypical Parkinsonism. Although PSP shares some symptomology with Parkinson's disease (PD), PSP has a different underlying pathology characterized by tau aggregation. Furthermore, PSP sufferers respond poorly to PD medications and there are no effective alternative therapeutics. The development of both palliative and disease altering therapeutics has been hampered by the lack of an animal model that displays relevant PSP-like pathology and behavioral deficits. Previously, our lab found that in rats the selective removal of cholinergic pedunculopontine neurons (whose axonal projections overlap with areas of PSP pathology), mimics the extensive loss of cholinergic pedunculopontine neurons seen in PSP, and produces a unique PSP-like combination of deficits in: startle reflex, attention, and motor function. The present study extends those findings by allowing the lesion to incubate for over a year and compares behavioral and post-mortem pathology of pedunculopontine-cholinergic-lesioned and sham-lesioned rats. There was an early startle reflex deficit which did not improve over time. Progressive declines in motor function developed over the course of the year, including an increase in the number of "slips" while navigating various beams and poorly coordinated transitions from an elevated platform into homecages. Histological analysis discovered that the loss off cholinergic pedunculopontine neurons precipitated a significant loss of substantia nigra tyrosine hydroxylase-positive neurons and a significant enlargement of the lateral ventricles. The latter is a distinguishing feature between PSP and PD. This preclinical animal model of PSP has the potential to further our understanding of PSP and aid in the testing of potential therapeutic agents.

Quality of life outcomes in peri-urethral calcium hydroxylapatite injection.

INTRODUCTION AND HYPOTHESIS: Peri-urethral calcium hydroxylapatite injection is an established treatment for patients with stress urinary incontinence. Information is limited regarding calcium hydroxylapatite treatment and quality of life (QOL) outcomes. We hypothesize that patients might improve QOL after peri-urethral calcium hydroxylapatite injection, which was reflected in validated questionnaires. METHODS: The peri-urethral calcium hydroxylapatite injection billing code was used to identify patients who underwent injection from 2011-2013. Female patients who completed the American Urological Association Symptom Score (AUASS), the AUASS QOL and Michigan Incontinence Symptom Index (M-ISI), and the bother score (M-ISI bother), or pad count at baseline and follow-up were included. Change in questionnaire scores and pads were assessed using the paired t test. RESULTS: Sixty patients underwent 1 (30), 2 (63) or 3 (7 %) peri-urethral calcium hydroxylapatite injections performed by a single surgeon. Thirty-seven patients provided questionnaires and 38 provided pad counts, all with a mean age of 75 years. The overall AUASS, AUASS QOL, and overall M-ISI scores improved in 67.6, 54.8, and 61.3 % respectively (4.5 ± 7.9, 1.3 ± 1.7 and 5.5 ± 8.6 respectively). The M-ISI bother score improved in 44.8 % with a mean improvement of 0.5 ± 2.9, but did not reach significance. There was a 1.7 ± 3.7 decrease in the mean number of pads used daily after the procedure (p = 0.006) and 19 % experienced transient urinary retention. CONCLUSIONS: Peri-urethral calcium hydroxylapatite injections can improve urinary QOL scores in patients with initial and recurrent stress urinary incontinence. This short-term retrospective analysis suggests that larger long-term studies focusing on QOL outcomes are needed to evaluate the effect of peri-urethral calcium hydroxylapatite has on incontinence-specific QOL.

Bacterial toxin RelE: a highly efficient ribonuclease with exquisite substrate specificity using atypical catalytic residues.

The toxin RelE is a ribosome-dependent endoribonuclease implicated in diverse cellular processes, including persistence. During amino acid starvation, RelE inhibits translation by cleaving ribosomal A-site mRNA. Although RelE is structurally similar to other microbial endoribonucleases, the active-site amino acid composition differs substantially and lacks obvious candidates for general acid-base functionality. Highly conserved RelE residues (Lys52, Lys54, Arg61, Arg81, and Tyr87) surround the mRNA scissile phosphate, and specific 16S rRNA contacts further contribute to substrate positioning. We used a single-turnover kinetic assay to evaluate the catalytic importance of individual residues in the RelE active site. Within the context of the ribosome, RelE rapidly cleaves A-site mRNA at a rate similar to those of traditional ribonucleases. Single-turnover rate constants decreased between 10(2)- and 10(6)-fold for the RelE active-site mutants of Lys52, Lys54, Arg61, and Arg81. RelE may principally promote catalysis via transition-state charge stabilization and leaving-group protonation, in addition to achieving in-line substrate positioning in cooperation with the ribosome. This kinetic analysis complements structural information to provide a foundation for understanding the molecular mechanism of this atypical endoribonuclease.

Addressing the gap between public health emergency planning and incident response: Lessons learned from the 2009 H1N1 outbreak in San Diego County.

Objectives: Since 9/11, Incident Command System (ICS) and Emergency Operations Center (EOC) are relatively new concepts to public health, which typically operates using less hierarchical and more collaborative approaches to organizing staff. This paper describes the 2009 H1N1 influenza outbreak in San Diego County to explore the use of ICS and EOC in public health emergency response. Methods: This study was conducted using critical case study methodology consisting of document review and 18 key-informant interviews with individuals who played key roles in planning and response. Thematic analysis was used to analyze data. Results: Several broad elements emerged as key to ensuring effective and efficient public health response: 1) developing a plan for emergency response; 2) establishing the framework for an ICS; 3) creating the infrastructure to support response; 4) supporting a workforce trained on emergency response roles, responsibilities, and equipment; and 5) conducting regular preparedness exercises. Conclusions: This research demonstrates the value of investments made and that effective emergency preparedness requires sustained efforts to maintain personnel and material resources. By having the infrastructure and experience based on ICS and EOC, the public health system had the capability to surge-up: to expand its day-to-day operation in a systematic and prolonged manner. None of these critical actions are possible without sustained funding for the public health infrastructure. Ultimately, this case study illustrates the importance of public health as a key leader in emergency response.

Genomic analysis of the hydrocarbon-producing, cellulolytic, endophytic fungus Ascocoryne sarcoides.

The microbial conversion of solid cellulosic biomass to liquid biofuels may provide a renewable energy source for transportation fuels. Endophytes represent a promising group of organisms, as they are a mostly untapped reservoir of metabolic diversity. They are often able to degrade cellulose, and they can produce an extraordinary diversity of metabolites. The filamentous fungal endophyte Ascocoryne sarcoides was shown to produce potential-biofuel metabolites when grown on a cellulose-based medium; however, the genetic pathways needed for this production are unknown and the lack of genetic tools makes traditional reverse genetics difficult. We present the genomic characterization of A. sarcoides and use transcriptomic and metabolomic data to describe the genes involved in cellulose degradation and to provide hypotheses for the biofuel production pathways. In total, almost 80 biosynthetic clusters were identified, including several previously found only in plants. Additionally, many transcriptionally active regions outside of genes showed condition-specific expression, offering more evidence for the role of long non-coding RNA in gene regulation. This is one of the highest quality fungal genomes and, to our knowledge, the only thoroughly annotated and transcriptionally profiled fungal endophyte genome currently available. The analyses and datasets contribute to the study of cellulose degradation and biofuel production and provide the genomic foundation for the study of a model endophyte system.

Volatile organic compound production by organisms in the genus Ascocoryne and a re-evaluation of myco-diesel production by NRRL 50072.

The Patagonian fungal endophyte NRRL 50072 is reported to produce a variety of medium-chain and highly branched volatile organic compounds (VOCs) that have been highlighted for their potential as fuel alternatives and are collectively termed myco-diesel. To assess the novelty of this observation, we determined the extent to which ten closely related Ascocoryne strains from commercial culture collections possess similar VOC production capability. DNA sequencing established a high genetic similarity between NRRL 50072 and each Ascocoryne isolate, consistent with its reassignment as Ascocoryne sarcoides. The Ascocoryne strains did not produce highly branched medium-chain-length alkanes, and efforts to reproduce the branched alkane production of NRRL 50072 were unsuccessful. However, we confirmed the production of 30 other products and expanded the list of VOCs for NRRL 50072 and members of the genus Ascocoryne. VOCs detected from the cultures consisted of short- and medium-chain alkenes, ketones, esters and alcohols and several sesquiterpenes. Ascocoryne strains NRRL 50072 and CBS 309.71 produced a more diverse range of volatiles than the other isolates tested. CBS 309.71 also showed enhanced production compared with other strains when grown on cellulose agar. Collectively, the members of the genus Ascocoryne demonstrated production of over 100 individual compounds, with a third of the short- and medium-chain compounds also produced when cultures were grown on a cellulose substrate. This comparative production analysis could facilitate future studies to identify and manipulate the biosynthetic machinery responsible for production of individual VOCs, including several that have a potential application as biofuels.

Ward round--Late presentation of acute compartment syndrome in the thigh.

Acute compartment syndrome of the thigh is rare but has been described as a result of femur fracture and also thigh contusion in sports injury. Emergency fasciotomy has routinely been the recommended treatment. We describe a patient with a closed femur fracture, initially without any syndrome whilst on traction, and required surgical intervention. He was found to have a large haematoma associated with significant muscle damage in the posterior compartment. This case is unusual in that symptoms started 12 days after injury possibly after manipulation of the leg whilst on traction. Diagnosis is mainly clinical with an earliest alarming sign being disproportional increasing pain on passive stretch of the group of muscles. A high index of suspicion and prompt intervention are required to diagnose and treat compartment syndrome and prevent irreversible damage.

The production of myco-diesel hydrocarbons and their derivatives by the endophytic fungus Gliocladium roseum (NRRL 50072).

An endophytic fungus, Gliocladium roseum (NRRL 50072), produced a series of volatile hydrocarbons and hydrocarbon derivatives on an oatmeal-based agar under microaerophilic conditions as analysed by solid-phase micro-extraction (SPME)-GC/MS. As an example, this organism produced an extensive series of the acetic acid esters of straight-chained alkanes including those of pentyl, hexyl, heptyl, octyl, sec-octyl and decyl alcohols. Other hydrocarbons were also produced by this organism, including undecane, 2,6-dimethyl; decane, 3,3,5-trimethyl; cyclohexene, 4-methyl; decane, 3,3,6-trimethyl; and undecane, 4,4-dimethyl. Volatile hydrocarbons were also produced on a cellulose-based medium, including heptane, octane, benzene, and some branched hydrocarbons. An extract of the host plant, Eucryphia cordifolia (ulmo), supported the growth and hydrocarbon production of this fungus. Quantification of volatile organic compounds, as measured by proton transfer mass spectrometry (PTR-MS), indicated a level of organic substances in the order of 80 p.p.m.v. (parts per million by volume) in the air space above the oatmeal agar medium in an 18 day old culture. Scaling the PTR-MS profile the acetic acid heptyl ester was quantified (at 500 p.p.b.v.) and subsequently the amount of each compound in the GC/MS profile could be estimated; all yielded a total value of about 4.0 p.p.m.v. The hydrocarbon profile of G. roseum contains a number of compounds normally associated with diesel fuel and so the volatiles of this fungus have been dubbed 'myco-diesel'. Extraction of liquid cultures of the fungus revealed the presence of numerous fatty acids and other lipids. All of these findings have implications in energy production and utilization.