Biosynthesis of tetrahydropapaverine and semisynthesis of papaverine in yeast.

Tetrahydropapaverine (THP) and papaverine are plant natural products with clinically significant roles. THP is a precursor in the production of the drugs atracurium and cisatracurium, and papaverine is used as an antispasmodic during vascular surgery. In recent years, metabolic engineering advances have enabled the production of natural products through heterologous expression of pathway enzymes in yeast. Heterologous biosynthesis of THP and papaverine could play a role in ensuring a stable supply of these clinically significant products. Biosynthesis of THP and papaverine has not been achieved to date, in part because multiple pathway enzymes have not been elucidated. Here, we describe the development of an engineered yeast strain for de novo biosynthesis of THP. The production of THP is achieved through heterologous expression of two enzyme variants with activity on nonnative substrates. Through protein engineering, we developed a variant of N-methylcoclaurine hydroxylase with activity on coclaurine, enabling de novo norreticuline biosynthesis. Similarly, we developed a variant of scoulerine 9-O-methyltransferase capable of O-methylating 1-benzylisoquinoline alkaloids at the 3' position, enabling de novo THP biosynthesis. Flux through the heterologous pathway was improved by knocking out yeast multidrug resistance transporters and optimization of media conditions. Overall, strain engineering increased the concentration of biosynthesized THP 600-fold to 121 µg/L. Finally, we demonstrate a strategy for papaverine semisynthesis using hydrogen peroxide as an oxidizing agent. Through optimizing pH, temperature, reaction time, and oxidizing agent concentration, we demonstrated the ability to produce semisynthesized papaverine through oxidation of biosynthesized THP.

Current status of undergraduate teaching in forensic & legal medicine in Europe.

The European Council of Legal Medicine (ECLM) is the body established in 1992 to represent practitioners forensic & legal medicine and is composed of delegates of the countries of the European Union (EU) and from other countries which form part of Europe to a current total of 34 member countries. The aims of this study were to determine the current status of undergraduate forensic & legal medicine teaching in the curriculum of medical studies in ECLM countries and to use the results of this study to determine whether it would be appropriate to develop new guidelines and standards for harmonising the content of undergraduate forensic medicine training across ECLM member countries. A detailed questionnaire was sent to all individuals or organisations listed on the ECLM contact database. Responses were received from 21 of 33 countries on the database. These responses showed considerable emphasis on undergraduate teaching of forensic medicine in all countries with the exception of Belgium and the United Kingdom. There was great general consistency in the subjects taught. The data from this survey provide a baseline which should assist in developing a strategy to harmonise forensic & legal medicine undergraduate training in member countries of the ECLM. The ECLM is now in a good position to establish a pan-European working group to coordinate a consensus document identifying an appropriate and modern core undergraduate forensic medicine curriculum that can be presented to the medical education authorities in each country, and which can be adapted for local requirements, based on available personnel, the forensic medicine structure in the country, and most importantly, the needs of the local population.

Complete biosynthesis of the bisbenzylisoquinoline alkaloids guattegaumerine and berbamunine in yeast.

Benzylisoquinoline alkaloids (BIAs) are a diverse class of medicinal plant natural products. Nearly 500 dimeric bisbenzylisoquinoline alkaloids (bisBIAs), produced by the coupling of two BIA monomers, have been characterized and display a range of pharmacological properties, including anti-inflammatory, antitumor, and antiarrhythmic activities. In recent years, microbial platforms have been engineered to produce several classes of BIAs, which are rare or difficult to obtain from natural plant hosts, including protoberberines, morphinans, and phthalideisoquinolines. However, the heterologous biosyntheses of bisBIAs have thus far been largely unexplored. Here, we describe the engineering of yeast strains that produce the Type I bisBIAs guattegaumerine and berbamunine de novo. Through strain engineering, protein engineering, and optimization of growth conditions, a 10,000-fold improvement in the production of guattegaumerine, the major bisBIA pathway product, was observed. By replacing the cytochrome P450 used in the final coupling reaction with a chimeric variant, the product profile was inverted to instead produce solely berbamunine. Our highest titer engineered yeast strains produced 108 and 25 mg/L of guattegaumerine and berbamunine, respectively. Finally, the inclusion of two additional putative BIA biosynthesis enzymes, SiCNMT2 and NnOMT5, into our bisBIA biosynthetic strains enabled the production of two derivatives of bisBIA pathway intermediates de novo: magnocurarine and armepavine. The de novo heterologous biosyntheses of bisBIAs presented here provide the foundation for the production of additional medicinal bisBIAs in yeast.

European Council of Legal Medicine (ECLM) on-site inspection forms for forensic pathology, anthropology, odontology, genetics, entomology and toxicology for forensic and medico-legal scene and corpse investigation: the Parma form.

Further to a previous publication by the European Council of Legal Medicine (ECLM) concerning on-site forensic and medico-legal scene and corpse investigation, this publication provides guidance for forensic medical specialists, pathologists and, where present, coroners' activity at a scene of death inspection and to harmonize the procedures for a correct search, detection, collection, sampling and storage of all elements which may be useful as evidence, and ensure documentation of all these steps. This ECLM's inspection form provides a checklist to be used on-site for the investigation of a corpse present at a crime or suspicious death scene. It permits the collection of all relevant data not only for the pathologist, but also for forensic anthropologists, odontologists, geneticists, entomologists and toxicologists, thus supporting a collaborative work approach. Detailed instructions for the completion of forms are provided.

European council of legal medicine (ECLM) guidelines for the examination of suspected elder abuse.

Article 25 of the Charter of Fundamental Rights of the European Union (adopted in Nice on 7 December 2000) recognizes and respects the rights of older people to lead a life of dignity and independence and to participate in social and cultural life. It also highlights the importance of prevention and recognition of elder abuse, especially since exposure to violence is likely as the population ages, either in familial or in institutional settings. Elder abuse has some issues in common with child abuse but in spite of this fact currently is less recognized. Health professionals have a major role to play in early detection and management of cases of elder abuse. This protocol summarizes some key concepts and approaches to assist in the timely detection and investigation of elder abuse cases by healthcare professionals and forensic practitioners.

Enantioselective Desymmetrization of Methylenedianilines via Enzyme-Catalyzed Remote Halogenation.

Extensive effort has been devoted to engineering flavin-dependent halogenases (FDHs) with improved stability, expanded substrate scope, and altered regioselectivity. Here, we show that variants of rebeccamycin halogenase (RebH) catalyze enantioselective desymmetrization of methylenedianilines via halogenation of these substrates distal to their pro-stereogenic center. Structure-guided engineering was used to increase the conversion and selectivity of these reactions, and the synthetic utility of the halogenated products was shown via conversion of to a chiral α-substituted indole. These results constitute the first reported examples of asymmetric catalysis by FDHs.

Analysis of changes in sexual function in women undergoing pelvic organ prolapse repair with abdominal or vaginal approaches.

INTRODUCTION AND HYPOTHESIS: To examine changes in sexual function after abdominal and transvaginal pelvic organ prolapse repair. METHODS: Women enrolled in our prospective, longitudinal prolapse database with abdominal sacrocolpopexy (ASC) or transvaginal (TVR) pelvic organ prolapse (POP) repair with or without mesh, between 19 December 2008 through 4 June 2014. The Pelvic Organ Prolapse/Urinary Incontinence Sexual Questionnaire (PISQ-12) and the Pelvic Floor Distress Inventory (PFDI -20) were mailed preoperatively, and at 6 and 12 months postoperatively. Patients completed Global Response Assessments to rate their overall satisfaction. RESULTS: Two hundred and four of the 300 women met the inclusion criteria: 74 out of 204 (36 %) had ASC and 130 out of 204 (64 %) had TVR. Seventy-two out of seventy-four ASCs were performed robotically and 2 were open. Baseline demographics were similar except that the ASC patients were significantly younger (60 vs 63, P = 0.019) and had a higher rate of apical repair (77 % vs 55 %). Thirty-six out of seventy-four ASC (48.7 %) and 63 out of 128 TVR patients (49.2 %) were sexually active at baseline (P = 0.94). Sixteen out of thirty-eight ASC (42.1 %) and 18 out 63 TVR patients (28.6 %; P = 0.16) reported dyspareunia at baseline. Seventy-two out of seventy-four ASC (97 %) and 86 out of 130 TVR patients (66 %) had mesh-augmented repairs. There was no difference in sexual activity or dyspareunia between the groups at the 6- or 12-month follow-up. PISQ and PFDI scores improved significantly in both the ASC and TVR groups over time compared with the baseline (p < 0.0001). Most women in the ASC (77.5 %) and TVR (64.8 %) groups were satisfied with the results of prolapse surgery at 12 months. CONCLUSIONS: Sexual function and pelvic floor symptoms improved in a similar manner in patients after abdominal and transvaginal POP surgery.

C3orf58, a novel paracrine protein, stimulates cardiomyocyte cell-cycle progression through the PI3K-AKT-CDK7 pathway.

RATIONALE: The regenerative capacity of the heart is markedly diminished shortly after birth, coinciding with overall withdrawal of cardiomyocytes from cell cycle. Consequently, the adult mammalian heart has limited capacity to regenerate after injury. The discovery of factors that can induce cardiomyocyte proliferation is, therefore, of high interest and has been the focus of extensive investigation throughout the past years. OBJECTIVE: We have recently identified C3orf58 as a novel hypoxia and Akt induced stem cell factor (HASF) secreted from mesenchymal stem cells, which can promote cardiac repair through cytoprotective mechanisms. Here, we tested the hypothesis that HASF can also contribute to cardiac regeneration by stimulating cardiomyocyte division and proliferation. METHODS AND RESULTS: Neonatal ventricular cardiomyocytes were stimulated in culture for 7 days with purified recombinant HASF protein. Compared with control untreated cells, HASF-treated neonatal cardiomyocytes exhibited 60% increase in DNA synthesis as measured by bromodeoxyuridine incorporation. These results were confirmed by immunofluorescence confocal microscopy showing a 50% to 100% increase in the number of cardiomyocytes in the mitotic and cytokinesis phases. Importantly, in vivo cardiac overexpression of HASF in a transgenic mouse model resulted in enhanced level of DNA synthesis and cytokinesis in neonatal and adult cardiomyocytes. These proliferative effects were modulated by a phosphoinositide 3-kinase-protein kinase B-cycle-dependent kinase 7 pathway as revealed by the use of phosphoinositide 3-kinase -pathway-specific inhibitors and silencing of the Cdk7 gene. CONCLUSIONS: Our studies support the hypothesis that HASF induces cardiomyocyte proliferation via a phosphoinositide 3-kinase-protein kinase B-cycle-dependent kinase 7 pathway. The implications of this finding may be significant for cardiac regeneration biology and therapeutics.

Directed evolution of RebH for site-selective halogenation of large biologically active molecules.

We recently characterized the substrate scope of wild-type RebH and proceeded to evolve variants of this enzyme with improved stability for biocatalysis. The substrate scopes of both RebH and the stabilized variants, however, are limited primarily to compounds similar in size to tryptophan. A substrate walking approach was used to further evolve RebH variants with expanded substrate scope. Two particularly notable variants were identified: 3-SS, which provides high conversion of tricyclic tryptoline derivatives; and 4-V, which accepts a broad range of large indoles and carbazoles. This constitutes the first reported use of directed evolution to enable the functionalization of substrates not accepted by wild-type RebH and demonstrates the utility of RebH variants for the site-selective halogenation of biologically active compounds.

Trends in less-lethal use of force techniques by police services within England and Wales: 2007-2011.

The aim of this study was to determine the incidence of, and any changes in, usage patterns of the less-lethal forms of Use of Force (UoF) modalities--incapacitant spray, impact rounds, and Taser(R)--between 2007 and 2011 by English and Welsh police services. Additional information regarding the deployment and discharge of firearms was also sought. Two thousand Freedom of Information Act applications were made to 50 police services in England and Wales and related jurisdictions requesting the provision of: (a) the total number of deployments of incapacitant sprays, Taser(R), impact (baton) rounds, and armed response units (ARU); (b) the numbers and types of any resulting medical complications; and (c) the details of any local policies requiring assessment by a healthcare professional following a deployment. Responses were received from 47 police services, with only 10 of these supplying complete data. The remainder supplied incomplete data or refused to supply any data under s12 of the Freedom of Information Act (time and cost restrictions). From 2007 to 2011, the use of incapacitant sprays, Taser, and firearms have increased (incapacitant sprays deployed: 3496, 3976, 6911, 6679, 6853; Taser deployed: 499, 2659, 4560, 6943, 7203; Taser discharged: 15, 85, 161, 338, 461; firearms: 0, 7, 4, 19, 32). Baton rounds and ARU use showed greater variability over the same time period (baton rounds: 1007, 1327, 1123, 1382, 1278; ARUs: 11688, 13652, 13166, 13959, 12090). Only two services could provide details of medical consequences from use of incapacitant sprays, Taser, and baton rounds. No service could provide details of any related medical complications following use of firearms. Data collection and release are variable and inconsistent throughout English and Welsh police services and thus caution is needed in determining trends of UoF techniques. Deaths or injuries inflicted using UoF techniques result in much public scrutiny and the low level of data recorded in these cases is of concern. Common systems for recording use and adverse outcomes of UoF techniques are needed to inform the public and others who have concerns about such techniques.

Effects of incapacitant spray deployed in the restraint and arrest of detainees in the Metropolitan Police Service area, London, UK: a prospective study.

PURPOSE: In the United Kingdom (UK) police restraint and control of detainees is undertaken by assorted means. Two types of incapacitant spray (IS) are approved by the UK Home Office for use: CS (o-chlorobenzylidine malononitrile, dissolved in an organic solvent--methyl iso-butyl ketone and pelargonic acid vanillyamide (PAVA). The aim of this study was to document the effects of incapacitant sprays, by symptom assessment and medical examination, within a few hours of deployment. METHODS: A detailed proforma was produced to explore the nature of the arrest, the nature of exposure to the incapacitant spray, the type of incapacitant spray, the symptoms experienced and the medical findings. RESULTS: 99 proformas were completed. 74 % were completed by detainees and 26 % were completed by police officers. 88 % were exposed to CS spray, the remainder to PAVA spray. The mean time of assessment after exposure was 2.8 ± 2.33 h (mean ± SD). The most frequent sites of IS contact were the face and scalp (n = 78), and exposure to the left and right eyes (n = 32). The most common symptoms were: painful eyes (n = 68); red eyes (n = 58); runny nose (n = 59); lacrimation (n = 55); nasal discomfort (n = 52); skin irritation (n = 49); and skin burning (n = 45). The most common medical findings were: conjunctival erythema (n = 34); skin erythema (n = 21); and rhinorrhea (n = 20). CONCLUSIONS: Symptoms and signs of exposure to IS lasted longer than was expected (a mean of 2.8 h). Approximately 30 % of those exposed had ocular effects and 20 % had skin effects. The findings of this study will enable the guidelines on the expected effects and duration of symptoms resulting from exposure to incapacitant sprays to be reviewed and suggestions for their management to be refined.

Guidelines for photography of cutaneous marks and injuries: a multi-professional perspective.

Investigators assessing the likelihood of physical abuse, must make a decision as to whether the injury seen matches the explanation given. In some instances the pattern of these injuries can give the investigator a possible link to the cause of the injury. Thus, matching an injury pattern to an implement or weapon used has forensic implications. The current method of capturing patterned injuries together with poor scale placement often result in some form of distortion that causes a change to the shape of the patterned injury. The aim of this guideline is to assist individuals dealing with the capture of photographic evidence for the investigation of suspected non-accidental patterned cutaneous injuries (PCI), and to ensure high standards of image quality are met for both evidential records and for forensic analysis. The technical equipment specified within these guidelines are recommended by the authors as a basic requirement for imaging best practice, due to their ability to capture detailed and critical data. For precise pattern matching analysis, it is vital that both the injury and the suspected implement are photographed in accordance with this guidance.

The role of a Medical Examiner Service in an acute NHS hospital: Perspectives from doctors and next of kin.

A new Medical Examiner system was introduced in England and Wales in 2019 to scrutinise all non-coronial deaths. The three key roles of independent Medical Examiner scrutiny are to establish accurate causes of death, determine whether coronial referral is required and identify any care concerns. This is the first published service evaluation exploring the views of doctors and next of kin with whom Medical Examiner Services interact. The aims were to understand whether the Medical Examiner Service was achieving its three main roles. Surveys were sent electronically to the qualified attending practitioners, and by post to the next of kin, of a consecutive series of deceased patients reviewed by an acute NHS hospital Medical Examiner Service in the East of England. Recruitment took place over a five month period in 2023. Results are based on 100 returned surveys from doctors (response rate 35%) and 179 completed by next of kin (response rate 65%). Findings suggest the Medical Examiner Service was successfully achieving its three key roles and well received by both doctors and next of kin. Service user feedback is clearly important as Medical Examiner Services continue to develop into the statutory phase during 2024, when they are anticipated to review approximately 400,000 deaths per annum in England and Wales. This study demonstrates such feedback is not only useful for service development, but also eminently possible.

Changes in the nature and outcome of notifications to HM Coroner from the Norfolk and Norwich University Hospital, UK, before and after the introduction of a medical examiner service: 2018 versus 2022.

A medical examiner (ME) system was introduced to England and Wales in 2019 intended to ensure appropriate notification of cases to HM Coroner (HMC). The aim of the study is to determine and compare: (a) the nature of notifications to HMC for Norfolk from the Norfolk and Norwich University Hospital (NNUH) in 2018 compared with 2022; (b) to determine the outcome of those notifications and (c) to establish patterns of change in the number and nature of such notifications. HMC and ME datasets were interrogated to determine differences between notifications to HMC and outcomes in 2018 compared with 2022. From deaths at NNUH (2018 - n = 2605; 2022 - n = 2969), there were significantly fewer HMC notifications in 2022 compared with 2018 (25.3% vs. 17.6%). A decrease in notifications was noted for persons undergoing any 'treatment or procedure of a medical or similar nature' (24.0% vs. 16.2%) p < 0.0014. An increase in notifications was noted for neglect, including self-neglect (3.3% vs. 12.2%) p < 0.001. Of the coronial outcomes, there were significant increases in the numbers of post-mortem (PM) examinations (29.3% vs. 35.5%) p = 0.0276 and inquests (26.0% vs. 31.4%) p = 0.0485). There was a significant decrease in no further action by HMC (5.7 vs. 2.3) p = 0.0485. The study shows that the introduction of the medical examiner service has resulted in significant change in the nature of HMC notification categories. The notifications appear to be more appropriate, with an increased proportion of inquests and PM examinations and with a reduction in 100 A or 'no further action' outcomes.

Provision of forensic healthcare services for police custodial settings in England, Wales and Northern Ireland: Current practice and implications for other services?

Provision of forensic healthcare services may affect patient safety and criminal justice outcomes. We reviewed models of delivery for services in police custody in terms of cost, types of healthcare professionals and their minimum required experience, training and qualification. Relevant information was requested under the Freedom of Information Act from all police services in England, Wales and Northern Ireland. Additional information was sought from the London Ambulance Service and the Metropolitan Police Service. A third of respondent police services refused to provide the requested information and only a small minority answered the questions in their entirety. Many police services cited excessive cost and commercial interests as reasons for not providing the information. A marked variation in models of forensic healthcare provision across police services which responded was identified. London Ambulance Service call-outs to Metropolitan Police Service custody suites for those arrested varied from 0% to 3.8%. There is substantial inconsistency and variability of information on forensic healthcare services in police custody. A standardised national dataset of all aspects of police custodial healthcare (irrespective of by whom such services are provided) should be established. We advise that the Association of Police & Crime Commissioners, College of Policing the National Police Chiefs' Council and NHS England Health and Justice engage on these matters and work with the Faculty of Forensic & Legal Medicine, the United Kingdom Association of Forensic Nurses and Paramedics, and the College of Paramedics to restart the transfer of all police custodial healthcare services to the National Health Service.

The workload of a medical examiner service at an acute National Health Service hospital during the COVID-19 pandemic: The Norfolk & Norwich University Hospital experience.

The COVID-19 pandemic commenced in March 2020. In May 2019, a new Medical Examiner system was introduced to scrutinise deaths of patients dying within acute National Health Service Trusts. The Coronavirus Act 2020 which came into force in March 2020 modified certification of death requirements. Newly formed Medical Examiner Services were advised they could suspend scrutiny during the pandemic. The Norfolk & Norwich University Hospital Medical Examiner Service (NNUH MES) continued to scrutinise patient deaths throughout. This study summarises the workload of the NNUH MES from 1st June 2020 to 31st May 2021 over which period 2856 deaths were recorded and 2687 scrutinised by the Medical Examiners.

Needs assessment of essential anatomy: The perspective of adult primary care resident physicians.

Curricular development and modification involve first identifying a problem and then performing a needs assessment, which can guide the design of curricular components. Pedagogical changes, coupled with reductions in curricular time for gross anatomy, pose challenges and impose restrictions within medical school curricula. In order to make anatomy education effective and efficient, it is important to determine the anatomy considered essential for medical education through a targeted needs assessment. In this study, 50 adult primary care resident physicians in family medicine (FM) and internal medicine (IM) were surveyed to assess the importance of 907 anatomical structures, or groups of structures, across all anatomical regions from a curated list based on the boldface terms in four primary anatomy texts. There were no statistically significant differences in the ratings of structures between the two groups for any anatomical region. In total, 17.0% of structures, or groups of structures, were classified as essential, 58.0% as more important, 24.4% as less important, and 0.7% as not important. FM residents rated tissues classified as skeleton, nerves, fasciae, anatomical spaces, blood vessels, lymphatics, and surface anatomy (p < 0.0001) significantly higher than IM residents, but there were no differences in the rating of muscles or organs (p > 0.0056). It was notable that 100.0% of cranial nerves were classified as essential, and 94.5% of surface anatomy structures were classified as essential or more important. It is proposed that results of this study can serve to inform curricular development and revision.

Quality of photographic images provided for injury interpretation: room for improvement?

The aim of the study was to determine the quality and nature of photographic images submitted to the National Injuries Database (part of the National Policing Improvement Agency) in the United Kingdom, for expert assessment and interpretation. A prospective analysis of cases (n = 50) were assessed for the number, origin, type and quality of images. Of 1,332 images in 50 cases submitted to the National Injuries Database, only 24% were appropriately labeled with an index; 5% were unfocussed; 64% had no rules or measurement scales; 0% had color charts or bars; 1.3% were of unidentifiable parts of the anatomy. The images sent to the National Injuries Database for interpretation originated from a variety of sources with no consistency in presentation. In particular, features such as rules or scales, color charts and appropriate labels with indexes, were absent in a variable number of cases, although these factors may at times be crucial to interpretation. It would be appropriate for standards to be developed and used for consistency in image capture, presentation and reproduction for interpretation for court settings in order to improve the quality of evidence available.

Complaints against health care professionals providing police custodial and forensic medical/health care services in England, Wales and Northern Ireland - what do we know?

Awareness of the nature and frequency of complaints against health care professionals working in police custodial health care services could provide opportunities to improve patient safety. To explore this freedom of information requests were sent to police services in England, Wales and Northern Ireland, to professional regulatory bodies and to the Independent Office for Police Conduct. Eighty-seven percent of police services responded but only a minority provided complete responses, with data not being held, or not being held in an easily retrievable format, being provided as reasons. The nature and frequency of complaints were similar to a previous 2017 study, suggesting a failure to learn lessons from the investigation of complaints and implement change in clinical practice. No evidence of an accessible complaints handling and recording procedure was provided across the police services surveyed. Regulatory bodies provided some information on the nature of complaints made against doctors and nurses working in police custodial settings, but that for paramedics was unable to do so. It is recommended that the communication loop between police services, those bodies providing health care and forensic medical services and regulatory bodies needs to be closed. A common reporting system or the application of established complaints handling procedures and reporting structures, which could be achieved by transferring these services to the National Health Service, may enhance patient safety in police custody.

Convergence of per capita carbon dioxide emissions among developing countries: evidence from stochastic and club convergence tests.

This exploratory study extends the literature on the convergence of per capita carbon dioxide emissions in analyzing stochastic and club convergence within a panel framework for developing countries. The results from Pesaran (Journal of Applied Econometrics, 22(2), 265-312, 2007) and Bai and Carrion-i-Silvestre (Review of Economic Studies, 76(2), 471-501, 2009) panel unit root tests with allowance for cross-sectional dependence confirm stochastic convergence for low-income, lower middle-income, and combined country panels. Further analysis using the nonlinear time-varying factor model of Phillips and Sul (Econometrica, 75(6), 1771-1855, 2007; Journal of Applied Econometrics, 24(7), 1153-1185, 2009) to test for convergence reveals the emergence of multiple convergence clubs within each of the three country panels examined. We observe geographic proximity among many of the countries within the respective convergence clubs.