Investigating best practice for specimen preparation for biological testing of root canal sealers

INTRODUCTION: Biological characterization of root canal sealers is important as it assesses the ability of the root canal sealer to exert antimicrobial properties thus avoiding treatment failures caused by microbial challenge and also assess the cytotoxic effect on the periapical tissues. Assessment of the biological testing of root canal sealers necessitates the sterilisation of the materials prior to evaluation. This study aims to analyse the influence of various sterilisation techniques conducted prior to biological testing on the microstructure and surface properties of endodontic sealers. Assessment of the initial microbial contamination on the material was also undertaken.METHODS: Four commercial sealers were investigated. The sealers were either prepared in a laminar flow cabinet or on a laboratory bench top under ambient conditions. Each group was further divided into 5 groups (n = 3) based on the sterilization technique:1) ethanol-10 mins, 2) ultraviolet-1 h, 3) ethanol-10 mins + ultraviolet-1 h, 4) autoclave, and 5) no sterilisation (control). Microbial levels in the materials were assessed by plate streaking technique. The materials were characterized by scanning electron microscopy and energy dispersive spectroscopy, and Fourier transform infrared spectroscopy, before and after sterilisation, to assess any changes in microstructure and chemical composition.RESULTS: All the materials did not exhibit contamination when prepared in laminar flow chamber in sterile conditions compared with sealers prepared on the bench top. Three of the commercial materials showed changes in microstructure while one (TotalFill) was not affected by the sterilisation. AH Plus and BioRoot RCS exhibited alterations in water and alcohol peaks in FT-IR while the single syringe sealers (TotalFill and BioRoot Flow) showed no changes.CONCLUSIONS: Sterilisation methods cause physical and chemical alterations to sealers. Material preparation should be performed in a laminar flow cabinet and a test for sterility should be performed prior to any biological testing being undertaken. If the materials are not sterile, assessment of the effects of the sterilization methods is recommended.</p

Cardiovascular magnetic resonance findings in non‐hospitalized paediatric patients after recovery from COVID‐19

Aims: Our study aimed to investigate the cardiac involvement with sensitive tissue characterization in non-hospitalized children with coronavirus disease 2019 (COVID-19) infection using cardiovascular magnetic resonance (CMR) imaging. Methods and results: We prospectively enrolled children who recovered from mildly symptomatic COVID-19 infection between November 2020 and January 2021. Patients underwent CMR at 1.5 T (Achieva, Philips Healthcare, Best, the Netherlands) including cine images, native T1 and T2 mapping. Healthy children and paediatric patients with biopsy-proven myocarditis served as control groups. We performed CMR in 18 children with a median (25th-75th percentile) age of 12 (10-15) years, 38 (24-47) days after positive PCR test, and compared them with 7 healthy controls [15 (10-19) years] and 9 patients with myocarditis [10 (4-16) years]. The COVID-19 patients reported no cardiac symptoms. None of the COVID-19 patients showed CMR findings consistent with a myocarditis. Three patients (17%) from the COVID-19 cohort presented with minimal pericardial effusion. CMR parameters of COVID-19 patients, including volumetric and strain values as well as T1 and T2 times, were not significantly different from healthy controls, but from myocarditis patients. These had significantly reduced left ventricular (LV) ejection fraction (P = 0.035), LV global longitudinal strain, and left atrial strain values as well as elevated native T1 values compared with COVID-19 patients (P < 0.001, respectively). Conclusions There was no evidence of myocardial inflammation, fibrosis, or functional cardiac impairment in the studied cohort of children recently. CMR findings were comparable with those of healthy controls. Pericardial effusion suggests a mild pericarditis in a small subgroup. This is pointing to a minor clinical relevance of myocardial involvement in children after mildly symptomatic COVID-19 infections

Surrogates for myocardial power and power efficiency in patients with aortic valve disease

We aimed to assess surrogate markers for left ventricular (LV) myocardial power and efficiency in patients with isolated aortic stenosis (AS) and combined stenosis/regurgitation (AS/AR). In AS (n = 59), AS/AR (n = 21) and controls (n = 14), surrogates for LV myocardial power and circulatory/external myocardial efficiency were obtained from cardiac MRI. Median surrogate LV myocardial power was increased in AS, 7.7 W/m2 (interquartile range 6.0-10.2; p = 0.010) and AS/AR, 10.8 W/m2 (8.9-13.4; p < 0.001) when compared to controls, 5.4 W/m2 (4.2-6.5), and was lower in AS than AS/AR (p < 0.001). Surrogate circulatory efficiency was decreased in AS, 8.6% (6.8-11.1; p < 0.001) and AS/AR, 5.4% (4.1-6.2; p < 0.001) when compared to controls, 11.8% (9.8-16.9). Surrogate external myocardial efficiency was higher in AS, 15.2% (11.9-18.6) than in AS/AR, 12.2% (10.1-14.2; p = 0.031) and was significantly lower compared to controls, 12.2% (10.7-18.1) in patients with reduced ejection fraction (EF), 9.8% (8.1-11.7; p = 0.025). In 16% of all cases, left ventricular mass/volume indices and EF were within normal ranges, wheras surrogate LV myocardial power was elevated and patients were symptomatic. Although influenced by pressure/volume load, the myocardium is additionally affected by remodelling processes. Surrogates for circulatory efficiency and LV myocardial power gradually reflect alterations in patients with AS and AS/AR, even when surrogate external myocardial efficiency, EF, mass and volume indices still remain compensated

Clostridium difficile modulates host innate immunity via toxin-independent and dependent mechanism(s)

Clostridium difficile infection (CDI) is the leading cause of hospital and community-acquired antibiotic-associated diarrhoea and currently represents a significant health burden. Although the role and contribution of C. difficile toxins to disease pathogenesis is being increasingly understood, at present other facets of C. difficile-host interactions, in particular, bacterial-driven effects on host immunity remain less studied. Using an ex-vivo model of infection, we report that the human gastrointestinal mucosa elicits a rapid and significant cytokine response to C. difficile. Marked increase in IFN-γ with modest increase in IL-22 and IL-17A was noted. Significant increase in IL-8 suggested potential for neutrophil influx while presence of IL-12, IL-23, IL-1β and IL-6 was indicative of a cytokine milieu that may modulate subsequent T cell immunity. Majority of C. difficile-driven effects on murine bone-marrow-derived dendritic cell (BMDC) activation were toxin-independent; the toxins were however responsible for BMDC inflammasome activation. In contrast, human monocyte-derived DCs (mDCs) released IL-1β even in the absence of toxins suggesting host-specific mediation. Infected DC-T cell crosstalk revealed the ability of R20291 and 630 WT strains to elicit a differential DC IL-12 family cytokine milieu which culminated in significantly greater Th1 immunity in response to R20291. Interestingly, both strains induced a similar Th17 response. Elicitation of mucosal IFN-γ/IL-17A and Th1/Th17 immunity to C. difficile indicates a central role for this dual cytokine axis in establishing antimicrobial immunity to CDI

Generation of a fully erythromycin-sensitive strain of Clostridioides difficile using a novel CRISPR-Cas9 genome editing system

© 2019, The Author(s). Understanding the molecular pathogenesis of Clostridioides difficile has relied on the use of ermB-based mutagens in erythromycin-sensitive strains. However, the repeated subcultures required to isolate sensitive variants can lead to the acquisition of ancillary mutations that affect phenotype, including virulence. CRISPR-Cas9 allows the direct selection of mutants, reducing the number of subcultures and thereby minimising the likelihood of acquiring additional mutations. Accordingly, CRISPR-Cas9 was used to sequentially remove from the C. difficile 630 reference strain (NCTC 13307) two ermB genes and pyrE. The genomes of the strains generated (630Δerm* and 630Δerm*ΔpyrE, respectively) contained no ancillary mutations compared to the NCTC 13307 parental strain, making these strains the preferred option where erythromycin-sensitive 630 strains are required. Intriguingly, the cas9 gene of the plasmid used contained a proximal frameshift mutation. Despite this, the frequency of mutant isolation was high (96% and 89% for ermB and pyrE, respectively) indicating that a functional Cas9 is still being produced. Re-initiation of translation from an internal AUG start codon would produce a foreshortened protein lacking a RuvCI nucleolytic domain, effectively a ‘nickase’. The mutation allowed cas9 to be cloned downstream of the strong Pthl promoter. It may find application elsewhere where the use of strong, constitutive promoters is preferred