Summary Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryo-EM, we show that the binding of target double-stranded DNA (dsDNA) to a Type I-F Csy surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally-encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveal mechanistic details underlying target recognition and inhibition.

Background Tocilizumab, an interleukin-6 receptor blocker, has been used in the inflammatory phase of COVID-19, but its impact independent of corticosteroids remains unclear in patients with severe disease. Methods In this retrospective analysis of patients with COVID-19 admitted between March 2 and April 14, 2020 to a large academic medical center in New York City, we describe outcomes associated with tocilizumab 400 mg (without methylprednisolone) compared to a propensity-matched control. The primary endpoints were change in a 7-point ordinal scale of oxygenation and ventilator free survival, both at days 14 and 28. Secondary endpoints include incidence of bacterial superinfections and gastrointestinal perforation. Primary outcomes were evaluated using t-test. Results We identified 33 patients who received tocilizumab and matched 74 controls based on demographics and health measures upon admission. After adjusting for illness severity and baseline ordinal scale, we failed to find evidence of an improvement in hypoxemia based on an ordinal scale at hospital day 14 in the tocilizumab group (OR 2.2; 95% CI, 0.7–6.5; p = 0.157) or day 28 (OR 1.1; 95% CI, 0.4–3.6; p = 0.82). There also was no evidence of an improvement in ventilator-free survival at day 14 (OR 0.8; 95% CI, 0.18–3.5; p = 0.75) or day 28 (OR 1.1; 95% CI, 0.1–1.8; p = 0.23). There was no increase in secondary bacterial infection rates in the tocilizumab group compared to controls (OR 0.37; 95% CI, 0.09–1.53; p = 0.168). Conclusions There was no evidence to support an improvement in hypoxemia or ventilator-free survival with use of tocilizumab 400 mg in the absence of corticosteroids. No increase in secondary bacterial infections was observed in the group receiving tocilizumab.

The Salmonella typhimurium trans-membrane FliF MS ring templates assembly of the rotary bacterial flagellar motor, which also contains a cytoplasmic C-ring. A full-frame fusion of FliF with the rotor protein FliG assembles rings in non-motile expression hosts. 3D electron microscopy reconstructions of these FliFFliG rings show three high electron-density sub-volumes. 3D-classification revealed heterogeneity of the assigned cytoplasmic volume consistent with FliG lability. We used residue coevolution to construct homodimer building blocks for ring assembly, with X-ray crystal structures from other species and injectisome analogs. The coevolution signal validates folds and, importantly, indicates strong homodimer contacts for three ring building motifs (RBMs), initially identified in injectisome structures. It also indicates that the cofolded domains of the FliG N-terminal domain (FliG_N) with embedded α-helical FliF carboxy-terminal tail homo-oligomerize. The FliG middle and C-terminal domains (FliG_MC) have a weak signal for homo-dimerization but have coevolved to conserve their stacking contact. The homodimers and their ring models fit well into the 3D reconstruction. We hypothesize that a stable FliF periplasmic hub provides a platform for FliG ring self-assembly, but the FliG_MC ring has only limited stability without the C-ring. We also present a mechanical model for torque transmission in the FliFFliG ring.