GFP-tagging of extracellular vesicles for rapid process development

Extracellular vesicles (EVs) act as nano-scale molecular messengers owing to their capacity to shuttle functional macromolecular cargo between cells. This intrinsic ability to deliver bioactive cargo has sparked great interest in the use of EVs as novel therapeutic delivery vehicles; investments totaling over $2 billion in 2020 alone were reported for therapeutic EVs. One of the bottlenecks facing the production of EVs is the lack of rapid and high throughput analytics to aid process development. Here CHO cells have been designed and engineered to express GFP-tagged EVs via fusion to CD81. Moreover, this study highlights the importance of parent cell characterization to ensure lack of non-fused GFP for the effective use of this quantitative approach. The fluorescent nature of resulting vesicles allowed for rapid quantification of concentration and yield across the EV purification process. In this manner, the degree of product loss was deduced by mass balance analysis of ultrafiltration processing, reconciled up to 97% of initial feed mass. The use of GFP-tagging allowed for straightforward monitoring of vesicle elution from chromatography separations and detection via western blotting. Collectively, this work illustrates the utility of GFP-tagged EVs as a quantitative and accessible tool for accelerated process development

Endovascular repair of ruptured abdominal aortic aneurysm: A strategy in need of definitive evidence

IntroductionEndovascular strategies have been increasingly used to manage patients with ruptured abdominal aortic aneurysm (AAA) in an attempt to improve patient survival. We analyzed the evidence to support such an approach.MethodsWe performed a systematic literature review of endovascular aneurysm repair (EVAR) of ruptured AAA from 1994 to 2009. The literature analyzed included systematic reviews and population-based studies of ruptured AAA.ResultsSeven systematic reviews were identified, all demonstrating from published data that patients with EVAR of ruptured AAA had significantly reduced mortality compared with controls. Six recently published population-based studies from the United States demonstrated low mortality rates associated with EVAR; however, only a small proportion of ruptured AAAs were treated by EVAR. Systematic reviews and population-based studies both raised concerns about patient selection and publication bias. Two randomized trials are in progress, and one is due to commence 2009.ConclusionsThe outcome of EVAR in a nonselected patient population remains unknown. One or more definitive randomized trials could provide the level I evidence to resolve these issues

Screening of Multiple Potential Control Genes for use in Caste and Body Region Comparisons Using RT-qPCR in Coptotermes formosanus

Formosan subterranean termites, Coptotermes formosanus, are a significant worldwide pest. Molecular gene expression is an important tool for understanding the physiology of organisms. The recent advancement of molecular tools for Coptotermes formosanus is leading to the advancement of the understanding of termite physiology. One of the first steps in analyzing gene expression is the normalization to constant reference genes. Stable reference genes that have constant expression across multiple treatments are important for accurately comparing target genes' expression. The objective of this investigation was to analyze and validate a set of potential reference genes including 8SrRNA; Glyceraldehyde 3-phosphate dehydrogenase (Gadphd); ribosomal protein L7 (RPL); β-actin (BA1); α-tubulin (Atube); α-actin (Aactin); and elongation factor (Elong) as standards for analysis of transcriptional changes in the termite Coptotermes formosanus, across two phenotypic castes, body regions, and colonies. We also compared the expression of hexamerin-1 and 2 using stable and unstable reference genes to demonstrate the importance of consistent control genes. Our results demonstrate that 18S and RPL can serve as reliable expression standards when comparing these different castes and body regions, and we show that C. formosanus Hex-1 and Hex-2 have expression patterns similar to that previously described in R. flavipes

Editor's Choice - Re-interventions After Repair of Ruptured Abdominal Aortic Aneurysm: A Report From the IMPROVE Randomised Trial.

OBJECTIVE/BACKGROUND: The aim was to describe the re-interventions after endovascular and open repair of rupture, and investigate whether these were associated with aortic morphology. METHODS: In total, 502 patients from the IMPROVE randomised trial (ISRCTN48334791) with repair of rupture were followed-up for re-interventions for at least 3 years. Pre-operative aortic morphology was assessed in a core laboratory. Re-interventions were described by time (0-90 days, 3 months-3 years) as arterial or laparotomy related, respectively, and ranked for severity by surgeons and patients separately. Rare re-interventions to 1 year, were summarised across three ruptured abdominal aortic aneurysm trials (IMPROVE, AJAX, and ECAR) and odds ratios (OR) describing differences were pooled via meta-analysis. RESULTS: Re-interventions were most common in the first 90 days. Overall rates were 186 and 226 per 100 person years for the endovascular strategy and open repair groups, respectively (p = .20) but between 3 months and 3 years (mid-term) the rates had slowed to 9.5 and 6.0 re-interventions per 100 person years, respectively (p = .090) and about one third of these were for a life threatening condition. In this latter, mid-term period, 42 of 313 remaining patients (13%) required at least one re-intervention, most commonly for endoleak or other endograft complication after treatment by endovascular aneurysm repair (EVAR) (21 of 38 re-interventions), whereas distal aneurysms were the commonest reason (four of 23) for re-interventions after treatment by open repair. Arterial re-interventions within 3 years were associated with increasing common iliac artery diameter (OR 1.48, 95% confidence interval [CI] 0.13-0.93; p = .004). Amputation, rare but ranked as the worst re-intervention by patients, was less common in the first year after treatment with EVAR (OR 0.2, 95% CI 0.05-0.88) from meta-analysis of three trials. CONCLUSION: The rate of mid-term re-interventions after rupture is high, more than double that after elective EVAR and open repair, suggesting the need for bespoke surveillance protocols. Amputations are much less common in patients treated by EVAR than in those treated by open repair

Sideromimic Modification of Lactivicin Dramatically Increases Potency against Extensively Drug-Resistant <i>Stenotrophomonas maltophilia </i>Clinical Isolates

Acetamido derivatives of the naturally antibacterial non-β-lactam lactivicin (LTV) have improved activity against their penicillin binding protein targets and reduced hydrolysis by β-lactamases, but penetration into Gram-negative bacteria is still relatively poor. Here we report that modification of the LTV lactone with a catechol-type siderophore increases potency 1,000-fold against Stenotrophomonas maltophilia, a species renowned for its insusceptibility to antimicrobials. The MIC90 of modified lactone compound 17 (LTV17) against a global collection of extensively drug-resistant clinical S. maltophilia isolates was 0.063 μg · ml(-1) Sideromimic modification does not reduce the ability of LTVs to induce production of the L1 and L2 β-lactamases in S. maltophilia and does not reduce the rate at which LTVs are hydrolyzed by L1 or L2. We conclude, therefore, that lactivicin modification with a siderophore known to be preferentially used by S. maltophilia substantially increases penetration via siderophore uptake. LTV17 has the potential to be developed as a novel antimicrobial for treatment of infections by S. maltophilia More generally, our work shows that sideromimic modification in a species-targeted manner might prove useful for the development of narrow-spectrum antimicrobials that have reduced collateral effects

Elective Open Suprarenal Aneurysm Repair in England from 2000 to 2010 an Observational Study of Hospital Episode Statistics

Background: Open surgery is widely used as a benchmark for the results of fenestrated endovascular repair of complex abdominal aortic aneurysms (AAA). However, the existing evidence stems from single-centre experiences, and may not be reproducible in wider practice. National outcomes provide valuable information regarding the safety of suprarenal aneurysm repair. Methods: Demographic and clinical data were extracted from English Hospital Episodes Statistics for patients undergoing elective suprarenal aneurysm repair from 1 April 2000 to 31 March 2010. Thirty-day mortality and five-year survival were analysed by logistic regression and Cox proportional hazards modeling. Results: 793 patients underwent surgery with 14% overall 30-day mortality, which did not improve over the study period. Independent predictors of 30-day mortality included age, renal disease and previous myocardial infarction. 5-year survival was independently reduced by age, renal disease, liver disease, chronic pulmonary disease, and known metastatic solid tumour. There was significant regional variation in both 30-day mortality and 5-year survival after risk-adjustment. Regional differences in outcome were eliminated in a sensitivity analysis for perioperative outcome, conducted by restricting analysis to survivors of the first 30 days after surgery. Conclusions: Elective suprarenal aneurysm repair was associated with considerable mortality and significant regional variation across England. These data provide a benchmark to assess the efficacy of complex endovascular repair of supra-renal aneurysms, though cautious interpretation is required due to the lack of information regarding aneurysm morphology. More detailed study is required, ideally through the mandatory submission of data to a national registry of suprarenal aneurysm repair

Endovascular or open repair strategy for ruptured abdominal aortic aneurysm: 30 day outcomes from IMPROVE randomised trial.

OBJECTIVE: To assess whether a strategy of endovascular repair (if aortic morphology is suitable, open repair if not) versus open repair reduces early mortality for patients with suspected ruptured abdominal aortic aneurysm. DESIGN: Randomised controlled trial. SETTING: 30 vascular centres (29 UK, 1 Canadian), 2009-13. PARTICIPANTS: 613 eligible patients (480 men) with a clinical diagnosis of ruptured aneurysm. INTERVENTIONS: 316 patients were randomised to the endovascular strategy (275 confirmed ruptures, 174 anatomically suitable for endovascular repair) and 297 to open repair (261 confirmed ruptures). MAIN OUTCOME MEASURES: 30 day mortality, with 24 hour and in-hospital mortality, costs, and time and place of discharge as secondary outcomes. RESULTS: 30 day mortality was 35.4% (112/316) in the endovascular strategy group and 37.4% (111/297) in the open repair group: odds ratio 0.92 (95% confidence interval 0.66 to 1.28; P=0.62); odds ratio after adjustment for age, sex, and Hardman index 0.94 (0.67 to 1.33). Women may benefit more than men (interaction test P=0.02) from the endovascular strategy: odds ratio 0.44 (0.22 to 0.91) versus 1.18 (0.80 to 1.75). 30 day mortality for patients with confirmed rupture was 36.4% (100/275) in the endovascular strategy group and 40.6% (106/261) in the open repair group (P=0.31). More patients in the endovascular strategy than in the open repair group were discharged directly to home (189/201 (94%) v 141/183 (77%); P<0.001). Average 30 day costs were similar between the randomised groups, with an incremental cost saving for the endovascular strategy versus open repair of £1186 (€1420; $1939) (95% confidence interval -£625 to £2997). CONCLUSIONS: A strategy of endovascular repair was not associated with significant reduction in either 30 day mortality or cost. Longer term cost effectiveness evaluations are needed to assess the full effects of the endovascular strategy in both men and women. TRIAL REGISTRATION: Current Controlled Trials ISRCTN48334791