Circulating SARS-CoV-2 spike N439K variants maintain fitness while evading antibody-mediated immunity.

SARS-CoV-2 can mutate and evade immunity, with consequences for efficacy of emerging vaccines and antibody therapeutics. Here, we demonstrate that the immunodominant SARS-CoV-2 spike (S) receptor binding motif (RBM) is a highly variable region of S and provide epidemiological, clinical, and molecular characterization of a prevalent, sentinel RBM mutation, N439K. We demonstrate N439K S protein has enhanced binding affinity to the hACE2 receptor, and N439K viruses have similar in vitro replication fitness and cause infections with similar clinical outcomes as compared to wild type. We show the N439K mutation confers resistance against several neutralizing monoclonal antibodies, including one authorized for emergency use by the US Food and Drug Administration (FDA), and reduces the activity of some polyclonal sera from persons recovered from infection. Immune evasion mutations that maintain virulence and fitness such as N439K can emerge within SARS-CoV-2 S, highlighting the need for ongoing molecular surveillance to guide development and usage of vaccines and therapeutics.

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

Seoul Virus Associated with Pet Rats, Scotland, UK, 2019.

We describe a case of hemorrhagic fever with renal syndrome caused by Seoul virus in a woman in Scotland, UK. Whole-genome sequencing showed the virus belonged to a lineage characterized by recent international expansion, probably driven by trade in pet rats.

Time-to-response toxicity analysis as a method for drug susceptibility assessment in salmon lice.

The salmon louse Lepeophtheirus salmonis (Krøyer, 1837) is an ectoparasite causing infections of wild and farmed Atlantic salmon (Salmo salar L.) in the Northern hemisphere. While L. salmonis control at commercial mariculture sites increasingly employs non-medicinal approaches, such as cage designs reducing infection rates and biological control through cleaner fish, anti-parasitic drugs are still a requirement for effective fish health care. With only a limited range of salmon delousing agents available, all of which have been in use for more than a decade, drug resistance formation has been reported for different products. Successful resistance management requires reliable susceptibility assessment, which is usually achieved through L. salmonis bioassays. These tests involve the exposure of parasites to different drug concentrations and require significant numbers of suitable L. salmonis stages. The present study reports an alternative bioassay that is based on time-to-response toxicity analyses and can be carried out with limited parasite numbers. The assay determines the median effective time (ET50), i.e., the time required until impaired swimming and/or attachment behaviour becomes apparent in 50% of parasites, by conducting repeated examinations of test animals starting at the time point where exposure to a set drug concentration commences. This experimental approach further allows the estimation of the apparent drug susceptibility of individual L. salmonis by determining their time to response, which may prove useful in experiments designed to elucidate associations between genetic factors and the drug susceptibility phenotype of parasites. Three laboratory strains of L. salmonis differing in susceptibility to emamectin benzoate were characterised using standard 24 h bioassays and time-to-response toxicity assays. While both the median effective concentration (EC50) and the ET50 showed variability between experimental repeats, both types of bioassay consistently discriminated susceptible and drug-resistant L. salmonis laboratory strains. STATEMENT OF RELEVANCE: Infections by sea lice cause significant costs to the global salmon farming industry, which have been estimated to exceed €300 million per year worldwide. Control of sea lice still relies to a significant extent on chemical delousing; however, chemical control is threatened by resistance formation. Resistance can be combated by rotation between different drugs and strategic implementation of non-medicinal strategies. However, resistance management requires reliable and feasible methods of susceptibility assessment. The present study is a technical note introducing a novel approach to susceptibility assessments in sea lice. The method can be applied in susceptibility assessments on farms, where it offers the advantage of a reduced requirement of parasites for testing. In addition, the novel method allows deriving the times of parasite require to show a response after drug treatment has started, thus providing a variable characterizing the drug susceptibility phenotype of individual parasites. Accordingly, the bioassay approach presented here will be useful for studies aiming at unravelling the genetic determinants of drug resistance.

The tangled web of Langer's lines.

Langer's lines are often considered to be guides for elective surgical incisions. Interestingly, Karl Langer was not the first to describe the property of skin that leads to the lines he depicted, nor are these lines in common use today. Rather, it is common that relaxed skin tension lines provide a guide for cosmetically pleasing surgical results. Nevertheless, Langer did conduct extensive studies on human skin that remain as a scholarly body of work.

Hilton's law revisited.

Hilton's Law, put forth 150 years ago, is well known and frequently taught in anatomy courses. We critically analyzed the complex description of associated muscular, cutaneous, and articular innervations in order to assess the general applicability of Hilton's Law. We applied rules for interpretation of the Law extrapolated upon but based on Hilton's original writings, and excluded obscure supplementary clauses not considered as part of the Law. We found the Law, as originally written and as we interpreted with some latitude, to be reliable and applicable to all cranial and peripheral nerves. Hilton's Law is a powerful springboard to understand articular anatomy and pathophysiology.

Salmon lice (Lepeophtheirus salmonis) showing varying emamectin benzoate susceptibilities differ in neuronal acetylcholine receptor and GABA-gated chloride channel mRNA expression.

BACKGROUND: Caligid copepods, also called sea lice, are fish ectoparasites, some species of which cause significant problems in the mariculture of salmon, where the annual cost of infection is in excess of €300 million globally. At present, caligid control on farms is mainly achieved using medicinal treatments. However, the continued use of a restricted number of medicine actives potentially favours the development of drug resistance. Here, we report transcriptional changes in a laboratory strain of the caligid Lepeophtheirus salmonis (Krøyer, 1837) that is moderately (~7-fold) resistant to the avermectin compound emamectin benzoate (EMB), a component of the anti-salmon louse agent SLICE® (Merck Animal Health). RESULTS: Suppression subtractive hybridisation (SSH) was used to enrich transcripts differentially expressed between EMB-resistant (PT) and drug-susceptible (S) laboratory strains of L. salmonis. SSH libraries were subjected to 454 sequencing. Further L. salmonis transcript sequences were available as expressed sequence tags (EST) from GenBank. Contiguous sequences were generated from both SSH and EST sequences and annotated. Transcriptional responses in PT and S salmon lice were investigated using custom 15 K oligonucleotide microarrays designed using the above sequence resources. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains, these genes being enriched for functions such as calcium ion binding, chitin metabolism and muscle structure. γ-aminobutyric acid (GABA)-gated chloride channel (GABA-Cl) and neuronal acetylcholine receptor (nAChR) subunits showed significantly lower transcript levels in PT lice compared to S lice. Using RT-qPCR, the decrease in mRNA levels was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR. Salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 µg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice. CONCLUSIONS: Avermectins are believed to exert their toxicity to invertebrates through interaction with glutamate-gated and GABA-gated chloride channels. Further potential drug targets include other Cys-loop ion channels such as nAChR. The present study demonstrates decreased transcript abundances of GABA-Cl and nAChR subunits in EMB-resistant salmon lice, suggesting their involvement in avermectin toxicity in caligids.

Oliver Wendell Holmes, Sr. (1809-1894): physician, jurist, poet, inventor, pioneer, and anatomist.

Oliver Wendell Holmes, Sr. was a physician, dean of the Harvard Medical School, one of the best regarded American poets of the 19th century, father of a future United States Supreme Court Justice, inventor and - unknown to many - an anatomist. His friends included Ralph Waldo Emerson, Henry Wadsworth Longfellow, and Louis Pasteur. He trained with some of the most influential anatomists/surgeons of his day including Lisfranc, Larrey, Velpeau, Bigelow, and Dupuytren. As a teacher of anatomy, he had strong feelings regarding medical curricular reform and to some, was considered one of the best lecturers in the discipline. As dean, he pioneered social reform by admitting both white women and free black men to Harvard Medical School. He coined the term "anesthesia," was the first American to introduce microscopy to a medical curriculum, and made important contributions to the understanding of the spread of infectious disease. Herein, we review the life of this influential American academic and focus on his contributions to the field of anatomy.

Genomic epidemiology of SARS-CoV-2 in a university outbreak setting and implications for public health planning.

Whole genome sequencing of SARS-CoV-2 has occurred at an unprecedented scale, and can be exploited for characterising outbreak risks at the fine-scale needed to inform control strategies. One setting at continued risk of COVID-19 outbreaks are higher education institutions, associated with student movements at the start of term, close living conditions within residential halls, and high social contact rates. Here we analysed SARS-CoV-2 whole genome sequences in combination with epidemiological data to investigate a large cluster of student cases associated with University of Glasgow accommodation in autumn 2020, Scotland. We identified 519 student cases of SARS-CoV-2 infection associated with this large cluster through contact tracing data, with 30% sequencing coverage for further analysis. We estimated at least 11 independent introductions of SARS-CoV-2 into the student population, with four comprising the majority of detected cases and consistent with separate outbreaks. These four outbreaks were curtailed within a week following implementation of control measures. The impact of student infections on the local community was short-term despite an underlying increase in community infections. Our study highlights the need for context-specific information in the formation of public health policy for higher educational settings.

Genomic epidemiology reveals multiple introductions of SARS-CoV-2 from mainland Europe into Scotland.

Coronavirus disease 2019 (COVID-19) was first diagnosed in Scotland on 1 March 2020. During the first month of the outbreak, 2,641 cases of COVID-19 led to 1,832 hospital admissions, 207 intensive care admissions and 126 deaths. We aimed to identify the source and number of introductions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into Scotland using a combined phylogenetic and epidemiological approach. Sequencing of 1,314 SARS-CoV-2 viral genomes from available patient samples enabled us to estimate that SARS-CoV-2 was introduced to Scotland on at least 283 occasions during February and March 2020. Epidemiological analysis confirmed that early introductions of SARS-CoV-2 originated from mainland Europe (the majority from Italy and Spain). We identified subsequent early outbreaks in the community, within healthcare facilities and at an international conference. Community transmission occurred after 2 March, 3 weeks before control measures were introduced. Earlier travel restrictions or quarantine measures, both locally and internationally, would have reduced the number of COVID-19 cases in Scotland. The risk of multiple reintroduction events in future waves of infection remains high in the absence of population immunity.

Genetic epidemiology of SARS-CoV-2 transmission in renal dialysis units - A high risk community-hospital interface.

OBJECTIVES: Patients requiring haemodialysis are at increased risk of serious illness with SARS-CoV-2 infection. To improve the understanding of transmission risks in six Scottish renal dialysis units, we utilised the rapid whole-genome sequencing data generated by the COG-UK consortium. METHODS: We combined geographical, temporal and genomic sequence data from the community and hospital to estimate the probability of infection originating from within the dialysis unit, the hospital or the community using Bayesian statistical modelling and compared these results to the details of epidemiological investigations. RESULTS: Of 671 patients, 60 (8.9%) became infected with SARS-CoV-2, of whom 16 (27%) died. Within-unit and community transmission were both evident and an instance of transmission from the wider hospital setting was also demonstrated. CONCLUSIONS: Near-real-time SARS-CoV-2 sequencing data can facilitate tailored infection prevention and control measures, which can be targeted at reducing risk in these settings.

The place of anatomy in medical education: AMEE Guide no 41.

This Guide, a combined work by three authors from different countries, provides perspectives into the history of teaching gross anatomy, briefly, from the earliest of times, through to a detailed examination of curricula in both traditional didactic approaches and Problem-Based Learning (PBL) curricula. The delivery of a module within a curriculum in tertiary education is interplay between the content (knowledge and skills) of a subject, the teaching staff involved, the students and their approaches to learning, and the philosophy underpinning the delivery of the learning material. The work is divided into sections that deal with approaches to learning anatomy from the perspective of students, to delivery of the content of the curriculum by lecturers, including the assessment of knowledge, and itemises the topics that could be considered important for an appropriate anatomy module in an integrated course, delivered in a way that emphasises clinical application. The work concludes by looking to the future, and considering what measures may need to be addressed to ensure the continued development of anatomy as a clinically relevant subject in any medical curriculum.

Cross-over: a generalizable phenomenon necessary for secondary intraneural ganglion cyst formation.

The appearances of intraneural ganglion cysts are being elucidated. We previously introduced the cross-over phenomenon to explain how a fibular (peroneal) or tibial intraneural ganglion cyst arising from the superior tibiofibular joint could give rise to multiple cysts: cyst fluid ascending up the primarily affected nerve could reach the level of the sciatic nerve, fill its common epineurial sheath and spread circumferentially (cross over), at which time pressure fluxes could result in further ascent up the sciatic or descent down the same parent nerve or the opposite, previously unaffected fibular or tibial nerves. In this study, we hypothesized that cross-over could occur in other nerves, potentially leading to the formation of more than one intraneural ganglion cyst in such situations. We analyzed the literature and identified a single case that we could review where proximal extension of an intraneural ganglion cyst involving a nerve at a different site could theoretically undergo cross-over in another major nerve large enough for available magnetic resonance images to resolve this finding. A case of a suprascapular intraneural ganglion cyst previously reported by our group that arose from the glenohumeral joint and extended to the neck was reanalyzed for the presence or absence of cross-over. An injection of dye into the outer epineurium of the suprascapular nerve in a fresh cadaveric specimen was performed to test for cross-over experimentally. Retrospective review of this case of suprascapular intraneural ganglion cyst demonstrated evidence to support previously unrecognized cross-over at the level of the upper trunk, with predominant ascent up the C5 and the C6 nerve roots and subtle descent down the anterior and posterior divisions of the upper trunk as well as the proximal portion of the suprascapular nerve. This appearance gave rise to multiple interconnected intraneural ganglion cysts arising from a single distant connection to the glenohumeral joint. The injection study also demonstrated the cross-over phenomenon and produced a similar pattern as the cyst dissection. This article illustrates that cross-over can occur in another nerve (apart from the prototype fibular nerve). Furthermore, understanding the more complex anatomic nature of the upper trunk cross-over model provides insight into important mechanistic information regarding the bidirectional propagation patterns and formation of primary and secondary intraneural ganglion cysts not afforded by the previously described sciatic nerve cross-over model.

Patterns of intraneural ganglion cyst descent.

On the basis of the principles of the unifying articular theory, predictable patterns of proximal ascent have been described for fibular (peroneal) and tibial intraneural ganglion cysts in the knee region. The mechanism underlying distal descent into the terminal branches of the fibular and tibial nerves has not been previously elucidated. The purpose of this study was to demonstrate if and when cyst descent distal to the articular branch-joint connection occurs in intraneural ganglion cysts to understand directionality of intraneural cyst propagation. In Part I, the clinical records and MRIs of 20 consecutive patients treated at our institution for intraneural ganglion cysts (18 fibular and two tibial) arising from the superior tibiofibular joint were retrospectively analyzed. These patients underwent cyst decompression and disconnection of the articular branch. Five of these patients developed symptomatic cyst recurrence after cyst decompression without articular branch disconnection which was done elsewhere prior to our intervention. In Part II, five additional patients with intraneural ganglion cysts (three fibular and two tibial) treated at other institutions without disconnection of the articular branch were compared. These patients in Parts I and II demonstrated ascent of intraneural cyst to differing degrees (12 had evidence of sciatic nerve cross-over). In addition, all of these patients demonstrated previously unrecognized MRI evidence of intraneural cyst extending distally below the level of the articular branch to the joint of origin: cyst within the proximal most portions of the deep fibular and superficial fibular branches in fibular intraneural ganglion cysts and descending tibial branches in tibial intraneural ganglion cysts. The patients in Part I had complete resolution of their cysts at follow-up MRI examination 1 year postoperatively. The patients in Part II had intraneural recurrences postoperatively within the articular branch, the parent nerve, and the terminal branches, although in three cases they were subclinical. The authors demonstrate that cyst descent distal to the take-off of the articular branch to the joint of origin occurs regularly in patients with fibular and tibial intraneural ganglion cysts. The authors believe that parent terminal branch descent follows ascent up the articular branch from an affected joint of origin. This mechanism for bidirectional flow explains cyst within terminal branches of the fibular and tibial nerves and is dependent on pressure fluxes and resistances. This new pattern is consistent with principles previously described in a unified (articular) theory, is generalizable to other intraneural ganglion cysts arising from joints, and has important implications for pathogenesis and treatment of these intraneural cysts.

Peroneal and tibial intraneural ganglia: correlation between intraepineurial compartments observed on magnetic resonance images and the potential importance of these compartments.

OBJECT: Previously the authors demonstrated that peroneal and tibial intraneural ganglia arising from the superior tibiofibular joint may occasionally extend proximally within the epineurium to reach the sciatic nerve. The dynamic nature of these cysts, dependent on intraarticular pressures, may give rise to differing clinical and imaging presentations that have remained unexplained until now. To identify the pathogenesis of these unusual cysts and to correlate their atypical magnetic resonance (MR) imaging appearance, the authors retrospectively reviewed their own experience as well as the published literature on these types of intraneural ganglia. METHODS: A careful review of MR images obtained in 22 patients with intraneural ganglia located about the knee region (18 peroneal and four tibial intraneural ganglia) allowed the authors to substantiate three different patterns: outer (epifascicular) epineurial (20 cases); inner (interfascicular) epineurial (one case); and combined outer and inner epineurial (one case). In these cases serial MR images allowed the investigators to track the movement of the cyst within the same layer of the epineurium. All lesions had connections to the superior tibiofibular joint. Nine patients were identified as having lesions with sciatic nerve extension. Seven patients harboring an outer epineurial cyst (six in whom the cyst involved the peroneal nerve and one in whom it involved the tibial nerve) had signs of sciatic nerve cross-over, with the cyst seen in the sciatic nerve and/or other terminal branches. In only two of these cases had the cyst previously been recognized to have sciatic nerve involvement. In contrast, in one case an inner epineurial cyst involving the tibial nerve ascended within the tibial division of the sciatic nerve and did not cross over. A single patient had a combination of both outer and inner epineurial cysts; these were easily distinguished by their distinctive imaging patterns. CONCLUSIONS: This anatomical compartmentalization of intraneural cysts can be used to explain varied clinical and imaging patterns of cleavage planes for cyst formation and propagation. Compartmentalization elucidates the mechanism for cases of outer epineurial cysts in which there are primary ascent, sciatic cross-over, and descent of the lesion down terminal branches; correlates these cysts' atypical MR imaging features; and contrasts a different pattern of inner epineurial cysts in which ascent and descent occur without cross-over. The authors present data demonstrating that the dynamic phases of these intraneural ganglia frequently involve the sciatic nerve. Their imaging features are subtle and serve to explain the underrecognition and underreporting of the longitudinal extension of these cysts. Importantly, cysts extending to the sciatic nerve are still derived from the superior tibiofibular joint. Combined with the authors' previous experimental data, the current observations help the reader understand intraneural ganglia with a different, deeper degree of anatomical detail.

The use of a video interview to enhance gross anatomy students' understanding of professionalism.

There is much room for innovation in teaching medical students professionalism. The goal of this exercise was to enhance first-year Gross Anatomy students' understanding of professionalism, including the attributes of confidentiality, respectful behavior and humanism in medicine through a video interview with a donor family member. Survey results demonstrated that students generally agreed that the video helped them better understand professionalism in the context of the gross anatomy laboratory and gave them a deeper respect for donors. Most students strongly agreed that future medical students would benefit from viewing this video interview.

Leadership and professionalism curriculum in the Gross Anatomy course.

INTRODUCTION: Today's physicians must demonstrate both professionalism and leadership skills in order to succeed in largely team-based healthcare environments. The purpose of this study was to determine if professionalism attributes, leadership style, and leadership style adaptability are associated with academic performance among first-year students early in their medical curriculum. MATERIALS AND METHODS: Students were divided into 4-member dissection groups for the duration of the Gross and Developmental Anatomy course. Leadership responsibility was randomly assigned to a team member on a rotating basis every 5 weeks. After each 5-week block, student performance was measured by written and practical examinations, and each student assessed their leader's or their own professionalism attributes and leadership style using validated survey instruments. RESULTS: Most students demonstrated either a "selling" only (57%) or "participating" only (30%) leadership style with low to moderate leadership adaptability. "Participating" and "delegating" leadership styles have the highest average group written exam scores (89.4%, P <0.008). "Telling" only or "selling" only leaders have the lowest average group exam scores (83.5%, P <0.001). "Selling" and "participating" leaders have significantly lower average group practical exam scores than other styles (81.5%, P <0.007). Positive associations were observed between the written and practical examination scores and the leader's integrity (P = 0.003, P = 0.002) and responsibility (P <0.001, P = 0.037). CONCLUSIONS: This study demonstrates that various situational leadership styles and aspects of professionalism are associated with written and practical examination scores in the Gross and Developmental Anatomy course. Furthermore, it demonstrates that first-year medical students are in need of leadership skill development.