An efficient pyrrolysyl-tRNA synthetase for economical production of MeHis-containing enzymes.

Genetic code expansion has emerged as a powerful tool in enzyme design and engineering, providing new insights into sophisticated catalytic mechanisms and enabling the development of enzymes with new catalytic functions. In this regard, the non-canonical histidine analogue Nδ-methylhistidine (MeHis) has proven especially versatile due to its ability to serve as a metal coordinating ligand or a catalytic nucleophile with a similar mode of reactivity to small molecule catalysts such as 4-dimethylaminopyridine (DMAP). Here we report the development of a highly efficient aminoacyl tRNA synthetase (G1PylRSMIFAF) for encoding MeHis into proteins, by transplanting five known active site mutations from Methanomethylophilus alvus (MaPylRS) into the single domain PylRS from Methanogenic archaeon ISO4-G1. In contrast to the high concentrations of MeHis (5-10 mM) needed with the Ma system, G1PylRSMIFAF can operate efficiently using MeHis concentrations of ∼0.1 mM, allowing more economical production of a range of MeHis-containing enzymes in high titres. Interestingly G1PylRSMIFAF is also a 'polyspecific' aminoacyl tRNA synthetase (aaRS), enabling incorporation of five different non-canonical amino acids (ncAAs) including 3-pyridylalanine and 2-fluorophenylalanine. This study provides an important step towards scalable production of engineered enzymes that contain non-canonical amino acids such as MeHis as key catalytic elements.

A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme.

Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical Nδ-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BHMeHis1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BHMeHis1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BHMeHis1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.

Engineering Enzymes for Environmental Sustainability.

The development and implementation of sustainable catalytic technologies is key to delivering our net-zero targets. Here we review how engineered enzymes, with a focus on those developed using directed evolution, can be deployed to improve the sustainability of numerous processes and help to conserve our environment. Efficient and robust biocatalysts have been engineered to capture carbon dioxide (CO2 ) and have been embedded into new efficient metabolic CO2 fixation pathways. Enzymes have been refined for bioremediation, enhancing their ability to degrade toxic and harmful pollutants. Biocatalytic recycling is gaining momentum, with engineered cutinases and PETases developed for the depolymerization of the abundant plastic, polyethylene terephthalate (PET). Finally, biocatalytic approaches for accessing petroleum-based feedstocks and chemicals are expanding, using optimized enzymes to convert plant biomass into biofuels or other high value products. Through these examples, we hope to illustrate how enzyme engineering and biocatalysis can contribute to the development of cleaner and more efficient chemical industry.

Engineering Enzymes for Environmental Sustainability.

The development and implementation of sustainable catalytic technologies is key to delivering our net-zero targets. Here we review how engineered enzymes, with a focus on those developed using directed evolution, can be deployed to improve the sustainability of numerous processes and help to conserve our environment. Efficient and robust biocatalysts have been engineered to capture carbon dioxide (CO2) and have been embedded into new efficient metabolic CO2 fixation pathways. Enzymes have been refined for bioremediation, enhancing their ability to degrade toxic and harmful pollutants. Biocatalytic recycling is gaining momentum, with engineered cutinases and PETases developed for the depolymerization of the abundant plastic, polyethylene terephthalate (PET). Finally, biocatalytic approaches for accessing petroleum-based feedstocks and chemicals are expanding, using optimized enzymes to convert plant biomass into biofuels or other high value products. Through these examples, we hope to illustrate how enzyme engineering and biocatalysis can contribute to the development of cleaner and more efficient chemical industry.

Fellowship training in robotic colorectal surgery within the current hospital setting: an achievable goal?

BACKGROUND: Although currently limited, the requirement for colorectal trainees to attain skills in robotic surgery is likely to increase due to further utilization of robotic platforms globally. The aim of the study is to describe the training programme utilized and assess outcomes of fellowship training in robotic colorectal surgery. METHODS: A structured robotic training programme was generated across a tertiary hospital setting. Review of four prospectively maintained fellow operative logbooks was performed to assess caseload and skill acquisition. Operative and patient-related outcomes were compared with consultant trainer performed cases. Data were analysed using R with a P < 0.05 considered significant. RESULTS: The structured robotic training scheme is a two-tiered system over a 12-month period. The trainer-directed pathway comprised of a robotic console safety course followed by cart-side assisting, a wet lab animal course, dual-console accreditation training course and onsite proctoring, prior to becoming an independent console surgeon. Over 2 years, 265 robotic (n = 143 primary/component surgeon) cases were undertaken with fellows A, B, C and D involved in 63, 77, 75 and 50 robotic colorectal cases, respectively. Individual learning curves revealed independent procedure competency at cases 11, 14, 15 and 12, respectively, for robotic anterior resection. There was no significant difference observed in operative time (P = 0.39), blood loss (P = 0.41), lymph node harvest (P = 0.35), conversion rates (2% versus 4%), anastomotic leaks (1% versus 3%) and R0 resection rates (100% versus 98% colonic, 96% versus 96% rectal, P = 0.48) between surgical fellows and consultant trainers. Clavien-Dindo(III-IV) complications were similar (10% versus 6%,P = 0.25) with no mortalities encountered. CONCLUSION: It is feasible and safe to train fellows in robotic colorectal surgery without compromise of operative- and patient-related outcomes.

Association of Surgical Skill Assessment With Clinical Outcomes in Cancer Surgery.

Importance: Complex surgical interventions are inherently prone to variation yet they are not objectively measured. The reasons for outcome differences following cancer surgery are unclear. Objective: To quantify surgical skill within advanced laparoscopic procedures and its association with histopathological and clinical outcomes. Design, Setting, and Participants: This analysis of data and video from the Australasian Laparoscopic Cancer of Rectum (ALaCaRT) and 2-dimensional/3-dimensional (2D3D) multicenter randomized laparoscopic total mesorectal excision trials, which were conducted at 28 centers in Australia, the United Kingdom, and New Zealand, was performed from 2018 to 2019 and included 176 patients with clinical T1 to T3 rectal adenocarcinoma 15 cm or less from the anal verge. Case videos underwent blinded objective analysis using a bespoke performance assessment tool developed with a 62-international expert Delphi exercise and workshop, interview, and pilot phases. Interventions: Laparoscopic total mesorectal excision undertaken with curative intent by 34 credentialed surgeons. Main Outcomes and Measures: Histopathological (plane of mesorectal dissection, ALaCaRT composite end point success [mesorectal fascial plane, circumferential margin, ≥1 mm; distal margin, ≥1 mm]) and 30-day morbidity. End points were analyzed using surgeon quartiles defined by tool scores. Results: The laparoscopic total mesorectal excision performance tool was produced and shown to be reliable and valid for the specialist level (intraclass correlation coefficient, 0.889; 95% CI, 0.832-0.926; P < .001). A substantial variation in tool scores was recorded (range, 25-48). Scores were associated with the number of intraoperative errors, plane of mesorectal dissection, and short-term patient morbidity, including the number and severity of complications. Upper quartile-scoring surgeons obtained excellent results compared with the lower quartile (mesorectal fascial plane: 93% vs 59%; number needed to treat [NNT], 2.9, P = .002; ALaCaRT end point success, 83% vs 58%; NNT, 4; P = .03; 30-day morbidity, 23% vs 50%; NNT, 3.7; P = .03). Conclusions and Relevance: Intraoperative surgical skill can be objectively and reliably measured in complex cancer interventions. Substantial variation in technical performance among credentialed surgeons is seen and significantly associated with clinical and pathological outcomes.

Esophageal replacement by gastric transposition: A single surgeon's experience from a tertiary pediatric surgical center.

BACKGROUND: Many pediatric surgeons have limited experience of esophageal replacement. This study reports outcomes of esophageal replacement by gastric transposition performed by a single UK-based pediatric surgeon. METHODS: Consecutive patients were identified who underwent esophageal replacement by gastric transposition over a 28 year period. Clinical and demographic data were collected. Weight-for-age Z-scores were calculated for esophageal atresia patients. RESULTS: Nineteen patients were identified. Indication in the majority was long-gap esophageal atresia (n = 17; 10 with tracheoesophageal fistula). At surgery, median age was 8.5 months (range 2-55); median weight was 7.4 kg (range 4.0-17.4 kg). A right-sided thoracotomy or transhiatal approach was used. Median postoperative length of stay was 17.5 days (range 7-130); median intensive care stay was three days (range 1-63). There were no deaths. Anastomotic leak rate at 30 days was 10.5% (n = 2). One patient required early stricture dilatation. Median weight-for-age Z-score increased from -2.17 at one year of age to -1.86, -1.70 and -1.93 at 5, 10 and 15 years. CONCLUSIONS: Esophageal replacement by gastric transposition offers a potentially life-changing treatment; however, it is associated with significant morbidity. The majority of patients eventually achieve full oral feeding and maintenance of weight gain trajectory. A right-sided approach to the esophagus is feasible. TYPE OF STUDY: Treatment Study. LEVEL OF EVIDENCE: IV.

Consensus views on the optimum training curriculum for advanced minimally invasive surgery: A delphi study.

INTRODUCTION: There has been a wide uptake in the use of Minimal Invasive Surgery (MIS) globally across different surgical specialties. Whilst evidence exists for a structured training curriculum for basic laparoscopic surgery, there is little agreement on a complete framework for an advanced MIS training curriculum, defining the essential elements of the curriculum including the optimal assessment methods. The aim of this study is to obtain a consensus on the essential elements of a training curriculum for advanced MIS. MATERIALS AND METHODS: A Delphi study was carried out involving 57 international experts in advanced MIS across different surgical specialties. A three round survey was conducted to reach consensus on the essential domains of a curriculum. This included defining the learners, trainers and training centres; curriculum content and competency based assessment. RESULTS: Unanimous agreement was reached for the completion of basic laparoscopic training before entry into advanced training. A trainer should have reached competency in advanced MIS and attended a 'Train the trainer' course. The curriculum should be delivered as modular training, including a multi-modal approach with a structured clinical proctorship programme. Formative assessment was considered as an integral part of learning and should be performed using objective work based assessment tools such as global assessment scale (GAS) forms. Accreditation in advanced MIS can be achieved by objective assessment of technical performance of unedited videos in addition to key clinical performance outcomes. CONCLUSION: A consensus on the framework of an advanced MIS training curriculum has been achieved defining the essential elements of entry criteria, selection of trainers and training units and curriculum content. Multimodal learning, clinical proctorship programme and competency based assessment are integral parts of the curriculum.

Standardization of laparoscopic total mesorectal excision for rectal cancer: a structured international expert consensus.

OBJECTIVE: To establish a structured international expert consensus on a detailed technical description of the laparoscopic total mesorectal excision (TME). BACKGROUND: Laparoscopic TME is a common surgical approach for the treatment of rectal cancer, but there is little agreement on technical details and standards. METHODS: Sixty leading surgical experts from 5 different world regions with a median overall experience of 250 laparoscopic TME participated in this study. Four stages of mixed quantitative and qualitative consensus-finding methods were applied. (1) Semistructured expert interviews were independently analyzed by 2 assessors. (2) Consensus on the interview data was reached using reiterating questionnaires (Delphi method). (3) This was further refined in an interactive workshop. (4) Based on this meeting, a comprehensive text was drafted and final approval was sought by all experts. FINDINGS: Three theme categories were identified in 9 detailed interviews (anatomical landmarks, description of tissue retraction, and operating strategies). Following 2 rounds of a 54-item questionnaire, 29 items achieved very high agreement (A* ≥90%), 14 with good agreement (≥80%), 13 with moderate agreement (≥50%), and 18 with little or no agreement (<50%). In the workshop, areas of agreement were consolidated and conclusions were sought for those with less agreement. The final document was approved after 2 further rounds of surveys by all respondents. CONCLUSIONS: This detailed and agreed technical description of laparoscopic TME may have implications on training, assessment, quality control, and future research.

Methods of quality assurance in multicenter trials in laparoscopic colorectal surgery: a systematic review.

OBJECTIVES: To assess the risk of bias in multicenter randomized controlled trials (RCTs) investigating laparoscopic colorectal cancer surgery and review the use of quality assurance mechanisms to reduce performance bias. BACKGROUND: RCTs represent the criterion standard comparison for health care interventions. For trials investigating interventional techniques, performance bias can arise through variation in delivery of the intervention. METHODS: A comprehensive systematic review was undertaken using MEDLINE and EMBASE databases to identify all large RCTs investigating laparoscopic colorectal cancer surgery. Risk of performance bias was evaluated through assessment of publications and protocols to identify methods used for quality assurance of surgical technique. In addition, the Cochrane Collaboration's "risk of bias" tool was used to evaluate other potential sources of bias. RESULTS: The literature search identified 48 publications, reporting upon 8 individual RCTs. All studies used mechanisms for quality assurance of laparoscopic colorectal surgery. Methods employed included credentialing of surgeons or units through assessment of experience and expertise, standardization of surgical technique, and monitoring. None report the use of structure objective assessment tools for accrediting expertise. All 8 were assessed as low risk of bias using the Cochrane tool. A framework is proposed for use as a model for quality assurance in future surgical trials. CONCLUSIONS: Consideration of risk of performance bias is important when appraising trials investigating an interventional technique. Laparoscopic colorectal surgery RCTs have all employed quality assurance mechanisms to reduce risk of performance bias. Further research is indicated to investigate adopting objective assessment tools for quality assurance within multicenter RCTs.

Timing of surgery after long-course neoadjuvant chemoradiotherapy for rectal cancer: a systematic review of the literature.

BACKGROUND: Neoadjuvant long-course chemoradiotherapy is commonly used to improve the local control and resectability of locally advanced rectal cancer, with surgery performed after an interval of a number of weeks. OBJECTIVE: We report an evidence-based systematic review of published data supporting the optimal time to perform surgical resection after long-course neoadjuvant therapy. DATA SOURCES: A systematic literature search was undertaken of the MEDLINE and Embase electronic databases from 1995 to 2012. STUDY SELECTION: English language articles were included that compared outcomes following rectal cancer surgery performed at different times after a long course of neoadjuvant radiation-based therapy. INTERVENTIONS: : Patients received a long course of neoadjuvant therapy followed by radical surgical resection after an interval period. MAIN OUTCOME MEASURES: The rates of tumor response, R0 resection, sphincter preservation, surgical complications, and disease recurrence were the primary outcomes measured. RESULTS: Fifteen studies were identified: 1 randomized controlled trial, 1 prospective nonrandomized interventional study, and 13 observational studies. Studies compared time intervals that varied between <5 days and >12 weeks, with a large degree of variation in what the standard interval length was considered to be. Four of the 7 studies that reported rates of pathological complete response identified significantly higher rates with an extended interval between chemoradiotherapy and surgery; 3 of 8 studies demonstrated increased primary tumor downstaging with a longer interval. No significant differences have been consistently demonstrated in rates of surgical complications, sphincter preservation, or long-term recurrence and survival. LIMITATIONS: Neoadjuvant regimes, indications for neoadjuvant therapy, and time intervals after chemoradiotherapy were heterogeneous between studies; consequently, meta-analysis could not be performed. CONCLUSIONS: There is limited evidence to support decisions regarding when to resect rectal cancer following chemoradiotherapy. There may be benefits in prolonging the interval between chemoradiotherapy and surgery beyond the 6 to 8 weeks that is commonly practiced. However, outcomes need to be studied further in robust randomized studies.

In patients with ruptured abdominal aortic aneurysm does endovascular repair improve 30-day mortality?

Endovascular aneurysm repair (EVAR) has become widely adopted as the primary treatment modality for abdominal aortic aneurysm in the elective setting. However, equipoise exists regarding the use of this technology for acute ruptured aneurysms. A best evidence topic in cardiovascular surgery was written according to a structured protocol. The question addressed, whether a policy for endovascular repair as the primary mode of treatment for ruptured abdominal aortic aneurysms (rAAAs) would improve outcomes. One thousand three hundred and twenty-eight papers were found using the reported search; of these, 24 presented represent the best evidence to answer the clinical question. The author, journal, date and country of publication, patient group studies, study type, relevant outcomes, results, and study weaknesses of these papers are tabulated. The majority of data available derives from level 2b evidence, with only a single level 1b and no level 1a studies available. Appraisal of theses studies is constrained by limited patient numbers, selection bias and heterogeneity in treatment protocols between the reported series. The sole prospective randomised controlled trial comparing open and endovascular treatments found a 53% mortality amongst patients treated by either modality. This study was, however, underpowered and contrary to numerous cohort series that show reduced mortality with EVAR. The largest body of evidence is found in a co-operative multicentre cohort study spanning 49 institutions that showed superiority of EVAR over open repair in terms of 30-day mortality. We conclude that, within the limitations of the published literature to date, endovascular repair as the primary treatment for rAAA is achievable and appears to be associated with favourable mortality over open repair with appropriate case selection.