2'-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography.

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.

Canadian Plastic Surgery Resident Work Hour Restrictions: Practices and Perceptions of Residents and Program Directors.

BACKGROUND: The impact of resident work hour restrictions on training and patient care remains a highly controversial topic, and to date, there lacks a formal assessment as it pertains to Canadian plastic surgery residents. OBJECTIVE: To characterize the work hour profile of Canadian plastic surgery residents and assess the perspectives of residents and program directors regarding work hour restrictions related to surgical competency, resident wellness, and patient safety. METHODS: An anonymous online survey developed by the authors was sent to all Canadian plastic surgery residents and program directors. Basic summary statistics were calculated. RESULTS: Eighty (53%) residents and 10 (77%) program directors responded. Residents reported working an average of 73 hours in hospital per week with 8 call shifts per month and sleep 4.7 hours/night while on call. Most residents (88%) reported averaging 0 post-call days off per month and 61% will work post-call without any sleep. The majority want the option of working post-call (63%) and oppose an 80-hour weekly maximum (77%). Surgical and medical errors attributed to post-call fatigue were self-reported by 26% and 49% of residents, respectively. Residents and program directors expressed concern about the ability to master surgical skills without working post-call. CONCLUSIONS: The majority of respondents oppose duty hour restrictions. The reason is likely multifactorial, including the desire of residents to meet perceived expectations and to master their surgical skills while supervised. If duty hour restrictions are aggressively implemented, many respondents feel that an increased duration of training may be necessary.

HISTORIQUE: L'effet des restrictions des heures de travail des résidents sur la formation et les soins aux patients est un sujet très controversé. Jusqu'à présent, il n'y a pas d'évaluations officielles de cette réalité chez les résidents canadiens en chirurgie plastique. OBJECTIF: Caractériser le profil des heures de travail des résidents canadiens en chirurgie plastique et évaluer les points de vue des résidents et des directeurs de programme à l'égard de l'effet des restrictions des heures de travail sur la compétence chirurgicale, le bien-être des résidents et la sécurité des patients. MÉTHODOLOGIE: Les auteurs ont préparé un sondage anonyme en ligne qu'ils ont transmis à tous les résidents et les directeurs de programme en chirurgie plastique au Canada. Ils ont synthétisé les statistiques de base. RÉSULTATS: Au total, 80 résidents (53 %) et dix directeurs de programme (77 %) ont répondu au sondage. Les résidents ont déclaré faire une moyenne de 73 heures de travail hospitalier par semaine, faire huit quarts de garde par mois et dormir 4,7 heures par nuit lorsqu'ils sont sur appel. La plupart d'entre eux (88 %) déclarent une moyenne de 0 journée de congé après une garde, et 61 % travaillent ensuite sans avoir dormi. La majorité désire pouvoir travailler après une garde (63 %) et s'oppose à un maximum hebdomadaire de 80 heures (77 %). Par ailleurs, 26 % des résidents précisent avoir fait des erreurs chirurgicales et 49 %, des erreurs médicales qu'ils attribuent à la fatigue accumulée après une garde. Les résidents et les directeurs de programme s'inquiètent de la capacité des résidents à maîtriser les habiletés chirurgicales s'ils ne travaillent pas après les gardes. CONCLUSIONS: La majorité des répondants s'opposent aux restrictions des heures de garde. La raison est probablement multifactorielle, y compris le fait que les résidents souhaitent répondre aux attentes perçues et maîtriser leurs habiletés chirurgicales pendant qu'ils sont sous supervision. Si les restrictions des heures de garde étaient vigoureusement adoptées, de nombreux répondants croient qu'il faudrait allonger la formation.

Increasing the bioavailability of Ru(III) anticancer complexes through hydrophobic albumin interactions.

A series of pyridine-based derivatives of the clinically successful Ru(III)-based complexes indazolium [trans-RuCl4(1H-indazole)2] (KP1019) and sodium [trans-RuCl4(1H-indazole)2] (KP1339) have been synthesized to probe the effect of hydrophobic interactions with human serum albumin (hsA) on anticancer activity. The solution behavior and protein interactions of the new compounds were characterized by using electron paramagnetic resonance (EPR) and UV/Vis spectroscopy. These studies have revealed that incorporation of hydrophobic substituents at the 4'-position of the axial pyridine ligand stabilizes non-coordinate interactions with hsA. As a consequence, direct coordination to the protein is inhibited, which is expected to increase the bioavailability of the complexes, thus potentially leading to improved anticancer activity. By using this approach, the lifetimes of hydrophobic protein interactions were extended from 2 h for the unsubstituted pyridine complex, to more than 24 h for several derivatives. Free complexes were tested for their anticancer activity against the SW480 human colon carcinoma cell line, exhibiting low cytotoxicity. Pre-treatment with hsA improved the solubility of every compound and led to some changes in activity. Particularly notable was the difference in activity between the methyl- and dibenzyl-functionalized complexes. The former shows reduced activity after incubation with hsA, indicating reduced bioavailability due to protein coordination. The latter exhibits little activity on its own but, following treatment with hsA, exhibited significant cytotoxicity, which is consistent with its ability to form non-coordinate interactions with the protein. Overall, our studies demonstrate that non-coordinate interactions with hsA are a viable target for enhancing the activity of Ru(III)-based complexes in vivo.

Pyridine analogues of the antimetastatic Ru(III) complex NAMI-A targeting non-covalent interactions with albumin.

A series of pyridine-based derivatives of the antimetastatic Ru(III) complex imidazolium [trans-RuCl(4)(1H-imidazole)(DMSO-S)] (NAMI-A) have been synthesized along with their sodium-ion compensated analogues. These compounds have been characterized by X-ray crystallography, electron paramagnetic resonance (EPR), NMR, and electrochemistry, with the goal of probing their noncovalent interactions with human serum albumin (hsA). EPR studies show that the choice of imidazolium ligands and compensating ions does not strongly influence the rates of ligand exchange processes in aqueous buffer solutions. By contrast, the rate of formation and persistence of interactions of the complexes with hsA is found to be strongly dependent on the properties of the axial ligands. The stability of noncovalent binding is shown to correlate with the anticipated ability of the various pyridine ligands to interact with the hydrophobic binding domains of hsA. These interactions prevent the oligomerization of the complexes in solution and limit the rate of covalent binding to albumin amino acid side chains. Electrochemical studies demonstrate relatively high reduction potentials for these complexes, leading to the formation of Ru(II) species in aqueous solutions containing biological reducing agents, such as ascorbate. However, EPR measurements indicate that while noncovalent interactions with hsA do not prevent reduction, covalent binding produces persistent mononuclear Ru(III) species under these conditions.