No abstract

The US Food and Drug Administration's Mini-Sentinel pilot program is developing an organizational structure as well as principles and policies to govern its operations. These will inform the structure and function of the eventual Sentinel System. Mini-Sentinel is a collaboration that includes 25 participating institutions. We describe the program's current organizational structure and its major principles and policies. The organization includes a coordinating center with program leadership provided by a principal investigator; a planning board and subcommittees; an operations center; and data, methods, and protocol cores. Ad hoc workgroups are created as needed. A privacy panel advises about protection of individual health information. Principles and policies are intended to ensure that Mini-Sentinel conforms to the principles of fair information practices, protects the privacy of individual health information, maintains the security and integrity of data, assures the confidentiality of proprietary information, provides accurate and timely communications, prevents or manages conflicts of interest, and preserves respect for intellectual property rights.

Encapsulins (Enc) are protein nanocompartments which house various cargo enzymes, including a family of decameric ferritin-like proteins. Previously, we elucidated the structure and activity of these ferritin-like proteins (EncFtn) and demonstrated that they must be encapsulated in a nanocompartment for iron storage. Here, we study a recombinant Haliangium ochraceum Enc:EncFtn complex using electron cryo-microscopy (Cryo-EM) and hydrogen/deuterium exchange mass spectrometry (HDX-MS) to gain insight into the structural relationship between Enc and EncFtn. An asymmetric single particle reconstruction reveals four EncFtn decamers in a tetrahedral arrangement within the encapsulin nanocompartment. This leads to a symmetry mismatch between the EncFtn cargo and the icosahedral encapsulin shell. The EncFtn decamers are offset from the interior face of the encapsulin shell and are resolved at a much lower overall resolution in the final reconstruction. This flexibility, and the fixed number of EncFtn decamers sequestered within, implies that the loading of the encapsulin nanocompartment is limited by the steric effect of both engaged and free encapsulin localization sequences. Using a combination of focused refinements and HDX-MS, we observed dynamic behavior of the major five-fold pore, and show the pore opening via the movement of the encapsulin A-domain. These data can accelerate efforts to engineer the sequestration of heterologous cargo proteins and to alter the permeability of the encapsulin shell via pore modifications.

24Retroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-25 transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal 26 structure, integrase gains access to the scissile phosphodiester bonds by lifting DNA off the 27 histone octamer at the site of integration. To clarify the mechanism of DNA looping by 28 integrase, we determined a 3.9 Å resolution structure of the prototype foamy virus intasome 29 engaged with a nucleosome core particle. The structural data along with complementary 30 single-molecule Förster resonance energy transfer measurements reveal twisting and sliding 31 of the nucleosomal DNA arm proximal to the integration site. Sliding the nucleosomal DNA 32 by approximately two base pairs along the histone octamer accommodates the necessary 33 DNA lifting from the histone H2A-H2B subunits to allow engagement with the intasome. 34Thus, retroviral integration into nucleosomes involves the looping-and-sliding mechanism for 35 nucleosomal DNA repositioning, bearing unexpected similarities with chromatin remodelers. 36 37 38 42 function, a multimer of IN assembles on viral DNA (vDNA) ends forming a highly stable 43 nucleoprotein complex, known as the intasome 2-4 . In its first catalytic step, IN resects 3' ends 44 of the vDNA downstream of the invariant CA dinucleotides (3'-processing reaction). It then 45 utilises the freshly released 3'-hydroxyl groups as nucleophiles to attack a pair of 46 phosphodiester bonds on opposing strands of chromosomal DNA, cleaving host DNA and 47 simultaneously joining it to 3' vDNA ends (strand transfer reaction) 5,6 .48 Many important questions pertaining to the nature of the host-virus transactions on 49 chromatin remain unanswered. In particular, it is unclear what role chromatin structure plays 50 in the integration process. Strikingly, although only a fraction of the nucleosomal DNA 51 surface is exposed within the nucleosome core particle (NCP) 7-9 , nucleosomal DNA packing 52 does not impede and rather stimulates integration 10-15 . Because retroviral INs have long 53 been known to prefer bent or distorted DNA targets, DNA bending as it wraps around the 54 histone octamer was thought to facilitate integration into NCPs 12,13 . However, recent 55 structural data revealed that retroviral intasomes require target DNA to adopt a considerably 56 sharper deformation than the smooth bend observed on NCPs 15-19 .57 Intasome structures from several retroviral genera have been determined by X-ray 58 crystallography and cryo-EM 4,17-20 . Despite considerable variability, all intasomes were 59 found to contain the structurally conserved intasomal core assembly minimally comprising 60 four IN subunits synapsing a pair of vDNA ends. Depending on the retroviral species, the 61 core assembly can be decorated by a number of additional IN subunits. The nucleoprotein 62 complex from the prototype foamy virus (PFV) contains only a tetramer of IN, making this 63 well-characterised intasome an ideal model to study the basic mech...