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Little is known about the peripheral immune cell (PIC) profile of the developing brain despite growing appreciation for these cells in the mature nervous system. To address this gap, the PIC profile, defined as which cells are present, where they are located, and for how long, was examined in the developing rat using spectral flow cytometry. Select regions of the rat brain (cerebellum, hippocampus, and hypothalamus) were examined at embryonic day 20, and postnatal days 0, 7 and 16. At their peak (E20), PICs were most abundant in the cerebellum, then the hippocampus and hypothalamus. Within the PIC pool, monocytes were most prevalent in all regions and time points, and shifted from being majority classical at E20 to non-classical by PN7. T cells increased over time, and shifted from majority cytotoxic to T-helper cells by PN7. This suggests the PIC profile transitions from reactive to adaptive and surveilling in the second postnatal week. NK cells and mast cells increased temporarily, and mast cells were restricted to the hippocampus and hypothalamus, suggesting they may play a specific role in the development of those regions. Mimicking a viral infection by administration of Poly I:C increased the influx of PICs into the neonatal brain, particularly of NK cells and in the case of males only, non-classical monocytes. This work provides a map for researchers as they study immune cell contributions to healthy and pathological brain development.

A protocol specifies interactions between roles, which together constitute a multiagent system (MAS). Enacting a protocol presupposes that agents are bound to the its roles. Existing protocol-based approaches, however, do not adequately treat the practical aspects of how roles bindings come about. Pippi addresses this problem of MAS instantiation. It proposes the notion of a metaprotocol, enacting which instantiates a MAS suitable for enacting a given protocol. Pippi demonstrates the subtleties involved in instantiating MAS arising from protocol composition, correlation, and decentralization. To address these subtleties and further support practical application patterns, we introduce an enhanced protocol language, with support for parameter types (including role and protocol typed parameters, for metaprotocols), interface flexibility, and binding constraints. We discuss the realization of our approach through an extended agent architecture, including the novel concept of a MAS adapter for contact management. We evaluate Pippi's expressiveness by demonstrating common patterns for agent discovery., Comment: 8 pages, to be published in Proc. of the 21st International Conference on Autonomous Agents and Multiagent Systems (AAMAS 2022), Online

Traducción al castellano del intercambio de correspondencia entre Jacques Derrida y Christie V. McDonald, en el otoño de 1981. A partir de las preguntas de McDonald, el filósofo reflexiona en torno a temas como el género, la diferencia sexual, los movimientos feministas, los feminismos, el lugar de la mujer, y el aporte que una perspectiva como la deconstrucción ofrece a su abordaje.

Traducción al castellano del intercambio de correspondencia entre Jacques Derrida y Christie V. McDonald, en el otoño de 1981. A partir de las preguntas de McDonald, el filósofo reflexiona en torno a temas como el género, la diferencia sexual, los movimientos feministas, los feminismos, el lugar de la mujer, y el aporte que una perspectiva como la deconstrucción ofrece a su abordaje.

Summary A large group of E3 ubiquitin ligases is formed by the multisubunit SCF complex, whose core complex (Rbx1/Cul1-Cdc53/Skp1) binds one of many substrate recruiting F-box proteins to form an array of SCF ligases with diverse substrate specificities. It has long been thought that ubiquitylation by SCF ligases is regulated at the level of substrate binding. Here we describe an alternative mechanism of SCF regulation by active dissociation of the F-box subunit. We show that cadmium stress induces selective recruitment of the AAA + ATPase Cdc48/p97 to catalyze dissociation of the F-box subunit from the yeast SCF Met30 ligase to block substrate ubiquitylation and trigger downstream events. Our results not only provide an additional layer of ubiquitin ligase regulation but also suggest that targeted, signal-dependent dissociation of multisubunit enzyme complexes is an important mechanism in control of enzyme function.

Met30 is the substrate recognition subunit of the essential ubiquitin ligase SCF(Met30). The essential function of Met30 is the inactivation of the Saccharomyces cerevisiae transcription factor Met4, because fully activated Met4 induces a cell cycle arrest. Met4 regulates expression of genes involved in the sulfur assimilation pathway and coordinates the transcriptional program and cell cycle progression in response to cadmium and arsenic stress. Met4 lacks DNA binding activity and requires either Cbf1 or one of the two homologous proteins Met31 and Met32 for promoter association. Accordingly, met4 mutants, cbf1 mutants, and met31 met32 double mutants are methionine auxotroph. We isolated a truncated version of Met32 (Met32(Delta145-192)) as a dominant suppressor of the cell cycle defect of met30 mutants. Expression of Met32(Delta145-192) significantly reduced induction of Met4-regulated genes. Interestingly, both Cbf1- and Met31/32-dependent genes were affected by Met32(Delta145-192). Mechanistically, Met32(Delta145-192) prevented recruitment of Met4 to both Cbf1 and Met31/32-dependent promoters. We further demonstrated that Met32 is part of the Cbf1-Met4 complex bound to Cbf1-recruiting promoter elements and that Met31/32 are required for formation of a stable Met4-Cbf1 transcription complex. These results suggest a regulatory role of Met32 as part of the Cbf1-Met4 complex and provide molecular insight into coordination of cell cycle response and modulation of gene expression programs.

The phage-encoded Xis protein is the major determinant controlling the direction of recombination in phage lambda. Xis is a winged-helix DNA binding protein that cooperatively binds to the attR recombination site to generate a curved microfilament, which promotes assembly of the excisive intasome but inhibits formation of an integrative intasome. We find that lambda synthesizes surprisingly high levels of Xis immediately upon prophage induction when excision rates are maximal. However, because of its low sequence-specific binding activity, exemplified by a 1.9 Å co-crystal structure of a nonspecifically bound DNA complex, Xis is relatively ineffective at promoting excision in vivo in the absence of the host Fis protein. Fis binds to a segment in attR that almost entirely overlaps one of the Xis binding sites. Instead of sterically excluding Xis binding from this site, as has been previously believed, we show that Fis enhances binding of all three Xis protomers to generate the microfilament. A specific Fis-Xis interface is supported by the effects of mutations within each protein, and relaxed, but not completely sequence-neutral, binding by the central Xis protomer is supported by the effects of DNA mutations. We present a structural model for the 50 bp curved Fis-Xis cooperative complex that is assembled between the arm and Holliday junction Int binding sites whose trajectory places constraints on models for the excisive intasome structure.

The DNA architectural protein Xis regulates the construction of higher-order nucleoprotein intasomes that integrate and excise the genome of phage lambda from the Escherichia coli chromosome. Xis modulates the directionality of site-specific recombination by stimulating phage excision 10 6 -fold, while simultaneously inhibiting phage reintegration. Control is exerted by cooperatively assembling onto a ≈35-bp DNA regulatory element, which it distorts to preferentially stabilize an excisive intasome. Here, we report the 2.6-Å crystal structure of the complex between three cooperatively bound Xis proteins and a 33-bp DNA containing the regulatory element. Xis binds DNA in a head-to-tail orientation to generate a micronucleoprotein filament. Although each protomer is anchored to the duplex by a similar set of nonbase specific contacts, malleable protein–DNA interactions enable binding to sites that differ in nucleotide sequence. Proteins at the ends of the duplex sequence specifically recognize similar binding sites and participate in cooperative binding via protein–protein interactions with a bridging Xis protomer that is bound in a less specific manner. Formation of this polymer introduces ≈72° of curvature into the DNA with slight positive writhe, which functions to connect disparate segments of DNA bridged by integrase within the excisive intasome.

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein that binds two different classes of DNA-binding sites within its recombination target sites. The several functions of Int are apportioned between a large carboxy-terminal domain that cleaves and ligates DNA at each of its four "core-type" DNA-binding sites and a small amino-terminal domain, whose primary function is binding to each of its five "arm-type" DNA sites, which are distant from the core region. Int bridges between the two classes of binding sites are facilitated by accessory DNA-bending proteins that along with Int comprise higher-order recombinogenic complexes. We show here that although the 64 amino-terminal residues of Int bind efficiently to a single arm site, this protein cannot form doubly bound complexes on adjacent arm sites. However, 1-70 Int does show the same cooperative binding to adjacent arm sites as the full length protein. We also found that 1-70 Int specifies cooperative interactions with the accessory protein Xis when the two are bound to their adjacent cognate sites P2 and X1, respectively. To complement the finding that these two amino-terminal domain functions (along with arm DNA binding) are all specified by residues 1-70, we determined that Thr75 is the first residue of the minimal carboxy-terminal domain, thereby identifying a specific interdomain linker region. We have measured the affinity constants for Int binding to each of the five arm sites and the cooperativity factors for Int binding to the two pairs of adjacent arm sites, and we have identified several DNA structural features that contribute to the observed patterns of Int binding to arm sites. Taken together, the results highlight several interesting features of arm DNA binding that invite speculation about additional levels of complexity in the regulation of lambda site-specific recombination.

Upon induction of a bacteriophage λ lysogen, a site-specific recombination reaction excises the phage genome from the chromosome of its bacterial host. A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which functions both as a DNA architectural factor and by cooperatively recruiting integrase to an adjacent binding site specifically required for excision. Here we present the three-dimensional structure of Xis and the results of a structure-based mutagenesis study to define the molecular basis of its function. Xis adopts an unusual “winged”-helix motif that is modeled to interact with the major- and minor-grooves of its binding site through a single α-helix and loop structure (“wing”), respectively. The C-terminal tail of Xis, which is required for cooperative binding with integrase, is unstructured in the absence of DNA. We propose that asymmetric bending of DNA by Xis positions its unstructured C-terminal tail for direct contacts with the N-terminal DNA-binding domain of integrase and that an ensuing disordered to ordered transition of the tail may act to stabilize the formation of the tripartite integrase–Xis–DNA complex required for phage excision.

Traducción al castellano del intercambio de correspondencia entre Jacques Derrida y Christie V. McDonald, en el otoño de 1981. A partir de las preguntas de McDonald, el filósofo reflexiona en torno a temas como el género, la diferencia sexual, los movimientos feministas, los feminismos, el lugar de la mujer, y el aporte que una perspectiva como la deconstrucción ofrece a su abordaje., Translation to Spanish of the correspondence between Jacques Derrida and Christie V. McDonald during the autumn of 1981. Considering McDonald’s inquiries, the philosopher reflects on subjects such as gender, sexual difference, feminist movements, feminisms, the woman’s place, and the contribution that a perspective such as deconstruction offers to these matters.

Traducción al castellano del intercambio de correspondencia entre Jacques Derrida y Christie V. McDonald, en el otoño de 1981. A partir de las preguntas de McDonald, el filósofo reflexiona en torno a temas como el género, la diferencia sexual, los movimientos feministas, los feminismos, el lugar de la mujer, y el aporte que una perspectiva como la deconstrucción ofrece a su abordaje.

Traducción al castellano del intercambio de correspondencia entre Jacques Derrida y Christie V. McDonald, en el otoño de 1981. A partir de las preguntas de McDonald, el filósofo reflexiona en torno a temas como el género, la diferencia sexual, los movimientos feministas, los feminismos, el lugar de la mujer, y el aporte que una perspectiva como la deconstrucción ofrece a su abordaje. Translation to Spanish of the correspondence between Jacques Derrida and Christie V. McDonald during the autumn of 1981. Considering McDonald’s inquiries, the philosopher reflects on subjects such as gender, sexual difference, feminist movements, feminisms, the woman’s place, and the contribution that a perspective such as deconstruction offers to these matters.

Traducción al castellano del intercambio de correspondencia entre Jacques Derrida y Christie V. McDonald, en el otoño de 1981. A partir de las preguntas de McDonald, el filósofo reflexiona en torno a temas como el género, la diferencia sexual, los movimientos feministas, los feminismos, el lugar de la mujer, y el aporte que una perspectiva como la deconstrucción ofrece a su abordaje.

Traducción al castellano del intercambio de correspondencia entre Jacques Derrida y Christie V. McDonald, en el otoño de 1981. A partir de las preguntas de McDonald, el filósofo reflexiona en torno a temas como el género, la diferencia sexual, los movimientos feministas, los feminismos, el lugar de la mujer, y el aporte que una perspectiva como la deconstrucción ofrece a su abordaje., Translation to Spanish of the correspondence between Jacques Derrida and Christie V. McDonald during the autumn of 1981. Considering McDonald’s inquiries, the philosopher reflects on subjects such as gender, sexual difference, feminist movements, feminisms, the woman’s place, and the contribution that a perspective such as deconstruction offers to these matters.

Traducción al castellano del intercambio de correspondencia entre Jacques Derrida y Christie V. McDonald, en el otoño de 1981. A partir de las preguntas de McDonald, el filósofo reflexiona en torno a temas como el género, la diferencia sexual, los movimientos feministas, los feminismos, el lugar de la mujer, y el aporte que una perspectiva como la deconstrucción ofrece a su abordaje.