Phosphorylation in the Ser/Arg-rich region of the nucleocapsid of SARS-CoV-2 regulates phase separation by inhibiting self-association of a distant helix.

The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently folded monomeric N-terminal domain that is the primary site for RNA binding and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation, we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino-acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA-binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA-binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid-liquid phase separation of the entire protein.

Intimidation or harassment among family medicine residents in Saskatchewan: a cross-sectional survey.

Introduction: Up to 98% of practicing family physicians, and over 75% of resident physicians in Canada experience abusive incidents. Despite the negative consequences of abusive incidents, few residents report these events to their supervisors or institution. We sought to estimate the prevalence of abusive incidents experienced or witnessed by Saskatchewan family medicine residents (FMRs) and identify their responses to these events. Methods: Anonymous survey invitations were emailed to all 110 Saskatchewan FMRs in Saskatchewan in November and December 2020. Demographic characteristics, frequency of witnessed and experienced abusive incidents, sources of incidents and residents' responses were collected. Incidents were classified as minor, major, severe, or as racial discrimination based on a previously published classification system. Results: The response rate was 34.5% (38/110). Ninety-two percent (35/38) of residents witnessed a minor incident and 91.7% (32/36) of residents experienced a minor incident. Seventy-one percent (27/38) of residents witnessed racial discrimination while 19.4% (7/36) of residents experienced racial discrimination. Patients were the most common source of abusive incidents. Twenty-nine percent of residents reported abusive incidents to their supervisors. Most residents were aware of institutional reporting policies. Conclusions: Most Saskatchewan FMRs experienced or witnessed abusive incidents, but few were reported. This study provided the opportunity to reassess policies on abusive incidents, which should consider sources of abuse, confidence in reporting, and education.

Introduction: Jusqu'à 98 % des médecins de famille en exercice et plus de 75 % des médecins résidents au Canada sont victimes d'incidents de violence. Malgré le préjudice subi, peu de résidents signalent ces incidents à leurs superviseurs ou à l'établissement. Nous avons tenté d'estimer la prévalence des incidents de violence dont ont été victimes ou témoins les résidents en médecine familiale (RMF) en Saskatchewan et de connaître leurs réactions face à ces situations. Méthodes: Des invitations à participer à un sondage anonyme ont été envoyées par courriel aux 110 RMF de la Saskatchewan en novembre et décembre 2020. Nous avons recueilli des données portant sur les caractéristiques démographiques des résidents, sur la fréquence des incidents de violence dont ils ont été témoins ou victimes, sur les sources des incidents et sur leurs réactions aux incidents. Ces derniers ont été classés comme mineurs, majeurs, graves ou comme actes de discrimination raciale sur la base d'un système de classification existant. Résultats: Le taux de réponse a été de 34,5 % (38/110). Quatre-vingt-douze pour cent (35/38) des résidents ont été témoins d'un incident mineur et 91,7 % (32/36) en ont vécu un eux-mêmes Soixante et onze pour cent (27/38) des résidents ont été témoins de discrimination raciale, tandis que 19,4 % (7/36) en ont été victimes. Le plus souvent, les auteurs de comportements violents étaient des patients. Vingt-neuf pour cent des résidents ont signalé l'incident à leur superviseur. La plupart des résidents connaissaient la politique de signalement de la violence de l'établissement. Conclusions: La plupart des RMF de la Saskatchewan ont vécu des incidents violents ou en ont été témoins, mais peu d'entre eux les ont signalés. Cette étude ouvre la voie à une réévaluation des politiques en matière de prévention de la violence, qui devraient tenir compte des sources de la violence et de la confiance des victimes envers le processus de signalement, et prévoir de la formation.

RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of SARS-CoV-2 nucleocapsid.

The nucleocapsid (N) protein of SARS-CoV-2 binds viral RNA, condensing it inside the virion, and phase separating with RNA to form liquid-liquid condensates. There is little consensus on what differentiates sequence-independent N-RNA interactions in the virion or in liquid droplets from those with specific genomic RNA (gRNA) motifs necessary for viral function inside infected cells. To identify the RNA structures and the N domains responsible for specific interactions and phase separation, we use the first 1,000 nt of viral RNA and short RNA segments designed as models for single-stranded and paired RNA. Binding affinities estimated from fluorescence anisotropy of these RNAs to the two-folded domains of N (the NTD and CTD) and comparison to full-length N demonstrate that the NTD binds preferentially to single-stranded RNA, and while it is the primary RNA-binding site, it is not essential to phase separation. Nuclear magnetic resonance spectroscopy identifies two RNA-binding sites on the NTD: a previously characterized site and an additional although weaker RNA-binding face that becomes prominent when binding to the primary site is weak, such as with dsRNA or a binding-impaired mutant. Phase separation assays of nucleocapsid domains with double-stranded and single-stranded RNA structures support a model where multiple weak interactions, such as with the CTD or the NTD's secondary face promote phase separation, while strong, specific interactions do not. These studies indicate that both strong and multivalent weak N-RNA interactions underlie the multifunctional abilities of N.

Total knee arthroplasty in multiple sclerosis.

We present a case report of total knee arthroplasty complicated by spasticity and contractures in a patient with multiple sclerosis (MS). Four previous case reports in the literature describe adverse outcomes after total knee arthroplasty in persons with MS secondary to severe spasticity. Preoperative, intraoperative, and postoperative considerations for persons with MS, which may help to improve functional outcomes, are discussed. Prospective research is needed among persons with MS to help determine the timing and selection of persons for arthroplasty and to minimize complications related to spasticity.