Embarking on a research project…or research for the absolute novice

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Improving the outcomes of carotid endarterectomy: Results of a statewide quality improvement project

AbstractObjective: The purpose of this study was to establish the statewide outcomes for carotid endarterectomy (CEA) and to facilitate improvement in outcomes through feedback, peer discussion, and ongoing process and outcome measurement. Methods: The Medicare Part A claims files were used to identify all Medicare patients undergoing CEA in Iowa during two 12-month time periods (January 1994–December 1994 and June 1995–May 1996). Medical record abstraction was used to obtain surgical indications, perioperative care process, and outcome information. Confidential reports were provided to each hospital (N = 30) where the procedure was performed. Surgeons performing the procedure (N = 79) were invited to meetings to discuss care process variation and outcomes. Voluntary participation was solicited in a standardized program of ongoing hospital-based data collection of CEA process and outcome data. Results: The statewide combined stroke or mortality rate decreased from 7.8% in 1994 to 4.0% in the 1995 to 1996 time period (P <.001). Fourteen hospitals, accounting for 74% of the statewide cases, participated in ongoing data collection. The combined stroke or mortality rate in these hospitals decreased significantly (P <.05) over time from 6.5% (1994) to 3.7% (1995-1996) to 1.8% (June 1997–May 1998). The use of intraoperative assessment of the operative site (20% in 1994, 46% in 1997-1998) and patch angioplasty (14% in 1994, 30% in 1997-1998) increased significantly during this time in the participating hospitals. Conclusions: Confidential feedback of outcome and process data for CEA may lead to change in perioperative care processes and improved outcomes. Standardized community-based outcome analysis should become routine for CEA to ensure that optimum results are being achieved. (J Vasc Surg 2000;31:918-26.

Inhibition of Fungi and Gram-Negative Bacteria by Bacteriocin BacTN635 Produced by Lactobacillus plantarum sp. TN635

The aim of this study was to evaluate 54 lactic acid bacteria (LAB) strains isolated from meat, fermented vegetables and dairy products for their capacity to produce antimicrobial activities against several bacteria and fungi. The strain designed TN635 has been selected for advanced studies. The supernatant culture of this strain inhibits the growth of all tested pathogenic including the four Gram-negative bacteria (Salmonella enterica ATCC43972, Pseudomonas aeruginosa ATCC 49189, Hafnia sp. and Serratia sp.) and the pathogenic fungus Candida tropicalis R2 CIP203. Based on the nucleotide sequence of the 16S rRNA gene of the strain TN635 (1,540 pb accession no FN252881) and the phylogenetic analysis, we propose the assignment of our new isolate bacterium as Lactobacillus plantarum sp. TN635 strain. Its antimicrobial compound was determined as a proteinaceous substance, stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 3 and 11 with an optimum at pH = 7. The BacTN635 was purified to homogeneity by a four-step protocol involving ammonium sulfate precipitation, centrifugal microconcentrators with a 10-kDa membrane cutoff, gel filtration Sephadex G-25, and C18 reverse-phase HPLC. SDS-PAGE analysis of the purified BacTN635, revealed a single band with an estimated molecular mass of approximately 4 kDa. The maximum bacteriocin production (5,000 AU/ml) was recorded after a 16-h incubation in Man, Rogosa, and Sharpe (MRS) medium at 30 °C. The mode of action of the partial purified BacTN635 was identified as bactericidal against Listeria ivanovii BUG 496 and as fungistatic against C. tropicalis R2 CIP203

Changes in chromatin structure during processing of wax-embedded tissue sections

The use of immunofluorescence (IF) and fluorescence in situ hybridisation (FISH) underpins much of our understanding of how chromatin is organised in the nucleus. However, there has only recently been an appreciation that these types of study need to move away from cells grown in culture and towards an investigation of nuclear organisation in cells in situ in their normal tissue architecture. Such analyses, however, especially of archival clinical samples, often requires use of formalin-fixed paraffin wax-embedded tissue sections which need addition steps of processing prior to IF or FISH. Here we quantify the changes in nuclear and chromatin structure that may be caused by these additional processing steps. Treatments, especially the microwaving to reverse fixation, do significantly alter nuclear architecture and chromatin texture, and these must be considered when inferring the original organisation of the nucleus from data collected from wax-embedded tissue sections

Early In Vitro Differentiation of Mouse Definitive Endoderm Is Not Correlated with Progressive Maturation of Nuclear DNA Methylation Patterns

The genome organization in pluripotent cells undergoing the first steps of differentiation is highly relevant to the reprogramming process in differentiation. Considering this fact, chromatin texture patterns that identify cells at the very early stage of lineage commitment could serve as valuable tools in the selection of optimal cell phenotypes for regenerative medicine applications. Here we report on the first-time use of high-resolution three-dimensional fluorescence imaging and comprehensive topological cell-by-cell analyses with a novel image-cytometrical approach towards the identification of in situ global nuclear DNA methylation patterns in early endodermal differentiation of mouse ES cells (up to day 6), and the correlations of these patterns with a set of putative markers for pluripotency and endodermal commitment, and the epithelial and mesenchymal character of cells. Utilizing this in vitro cell system as a model for assessing the relationship between differentiation and nuclear DNA methylation patterns, we found that differentiating cell populations display an increasing number of cells with a gain in DNA methylation load: first within their euchromatin, then extending into heterochromatic areas of the nucleus, which also results in significant changes of methylcytosine/global DNA codistribution patterns. We were also able to co-visualize and quantify the concomitant stochastic marker expression on a per-cell basis, for which we did not measure any correlation to methylcytosine loads or distribution patterns. We observe that the progression of global DNA methylation is not correlated with the standard transcription factors associated with endodermal development. Further studies are needed to determine whether the progression of global methylation could represent a useful signature of cellular differentiation. This concept of tracking epigenetic progression may prove useful in the selection of cell phenotypes for future regenerative medicine applications

Immunological and pathological outcomes of SARS-CoV-2 challenge following formalin-inactivated vaccine in ferrets and rhesus macaques

There is an urgent requirement for safe and effective vaccines to prevent COVID-19. A concern for the development of new viral vaccines is the potential to induce vaccine-enhanced disease (VED). This was reported in several preclinical studies with both SARS-CoV-1 and MERS vaccines but has not been reported with SARS-CoV-2 vaccines. We have used ferrets and rhesus macaques challenged with SARS-CoV-2 to assess the potential for VED in animals vaccinated with formaldehyde-inactivated SARS-CoV-2 (FIV) formulated with Alhydrogel, compared to a negative control vaccine. We showed no evidence of enhanced disease in ferrets or rhesus macaques given FIV except for mild transient enhanced disease seen 7 days after infection in ferrets. This increased lung pathology was observed at day 7 but was resolved by day 15. We also demonstrate that formaldehyde treatment of SARS-CoV-2 reduces exposure of the spike receptor binding domain providing a mechanistic explanation for suboptimal immunity

Convalescent plasma in patients admitted to hospital with COVID-19 (RECOVERY): a randomised controlled, open-label, platform trial

Background: Many patients with COVID-19 have been treated with plasma containing anti-SARS-CoV-2 antibodies. We aimed to evaluate the safety and efficacy of convalescent plasma therapy in patients admitted to hospital with COVID-19. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]) is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. The trial is underway at 177 NHS hospitals from across the UK. Eligible and consenting patients were randomly assigned (1:1) to receive either usual care alone (usual care group) or usual care plus high-titre convalescent plasma (convalescent plasma group). The primary outcome was 28-day mortality, analysed on an intention-to-treat basis. The trial is registered with ISRCTN, 50189673, and ClinicalTrials.gov, NCT04381936. Findings: Between May 28, 2020, and Jan 15, 2021, 11558 (71%) of 16287 patients enrolled in RECOVERY were eligible to receive convalescent plasma and were assigned to either the convalescent plasma group or the usual care group. There was no significant difference in 28-day mortality between the two groups: 1399 (24%) of 5795 patients in the convalescent plasma group and 1408 (24%) of 5763 patients in the usual care group died within 28 days (rate ratio 1·00, 95% CI 0·93–1·07; p=0·95). The 28-day mortality rate ratio was similar in all prespecified subgroups of patients, including in those patients without detectable SARS-CoV-2 antibodies at randomisation. Allocation to convalescent plasma had no significant effect on the proportion of patients discharged from hospital within 28 days (3832 [66%] patients in the convalescent plasma group vs 3822 [66%] patients in the usual care group; rate ratio 0·99, 95% CI 0·94–1·03; p=0·57). Among those not on invasive mechanical ventilation at randomisation, there was no significant difference in the proportion of patients meeting the composite endpoint of progression to invasive mechanical ventilation or death (1568 [29%] of 5493 patients in the convalescent plasma group vs 1568 [29%] of 5448 patients in the usual care group; rate ratio 0·99, 95% CI 0·93–1·05; p=0·79). Interpretation: In patients hospitalised with COVID-19, high-titre convalescent plasma did not improve survival or other prespecified clinical outcomes. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research

Tocilizumab in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Background: In this study, we aimed to evaluate the effects of tocilizumab in adult patients admitted to hospital with COVID-19 with both hypoxia and systemic inflammation. Methods: This randomised, controlled, open-label, platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing several possible treatments in patients hospitalised with COVID-19 in the UK. Those trial participants with hypoxia (oxygen saturation &lt;92% on air or requiring oxygen therapy) and evidence of systemic inflammation (C-reactive protein ≥75 mg/L) were eligible for random assignment in a 1:1 ratio to usual standard of care alone versus usual standard of care plus tocilizumab at a dose of 400 mg–800 mg (depending on weight) given intravenously. A second dose could be given 12–24 h later if the patient's condition had not improved. The primary outcome was 28-day mortality, assessed in the intention-to-treat population. The trial is registered with ISRCTN (50189673) and ClinicalTrials.gov (NCT04381936). Findings: Between April 23, 2020, and Jan 24, 2021, 4116 adults of 21 550 patients enrolled into the RECOVERY trial were included in the assessment of tocilizumab, including 3385 (82%) patients receiving systemic corticosteroids. Overall, 621 (31%) of the 2022 patients allocated tocilizumab and 729 (35%) of the 2094 patients allocated to usual care died within 28 days (rate ratio 0·85; 95% CI 0·76–0·94; p=0·0028). Consistent results were seen in all prespecified subgroups of patients, including those receiving systemic corticosteroids. Patients allocated to tocilizumab were more likely to be discharged from hospital within 28 days (57% vs 50%; rate ratio 1·22; 1·12–1·33; p&lt;0·0001). Among those not receiving invasive mechanical ventilation at baseline, patients allocated tocilizumab were less likely to reach the composite endpoint of invasive mechanical ventilation or death (35% vs 42%; risk ratio 0·84; 95% CI 0·77–0·92; p&lt;0·0001). Interpretation: In hospitalised COVID-19 patients with hypoxia and systemic inflammation, tocilizumab improved survival and other clinical outcomes. These benefits were seen regardless of the amount of respiratory support and were additional to the benefits of systemic corticosteroids. Funding: UK Research and Innovation (Medical Research Council) and National Institute of Health Research