Microbial colonization influences early B-lineage development in the gut lamina propria

The RAG1/RAG2 endonuclease ("RAG") initiates the V(D)J recombination reaction that assembles Ig heavy (IgH) and light (IgL) chain variable region exons from germline gene segments to generate primary antibody repertoires1. IgH V(D)J assembly occurs in progenitor (pro-) B cells followed by that of IgL in precursor (pre-) B cells. Expression of IgH μ and IgL (Igκ or Igλ) chains generates IgM, which is expressed on immature B cells as the B cell antigen-binding receptor ("BCR"). Rag expression can continue in immature B cells2, allowing continued Igκ V(D)J recombination that replaces the initial VκJκ exon with one that generates a new specificity3–5. This “receptor editing” process, which also can lead to Igλ V(D)J recombination and expression3,6,7, provides a mechanism whereby antigen-encounter at the Rag-expressing immature B cell stage helps shape pre-immune BCR repertoires. As the major site of post-natal B cell development, the bone marrow is the principal location of primary Ig repertoire diversification in mice. Here, we report that early B cell development also occurs within the mouse intestinal lamina propria (LP), where the associated V(D)J recombination/receptor editing processes modulate primary LP Ig repertoires. At weanling age in normally housed mice, the LP contains a population of Rag-expressing B lineage cells that harbor intermediates indicative of ongoing V(D)J recombination and which contain cells with pro-B, pre-B, and editing phenotypes. Consistent with LP-specific receptor editing, Rag-expressing LP B-lineage cells have similar VH repertoires, but significantly different Vκ repertoires, compared to those of Rag2-expressing BM counterparts. Moreover, colonization of germ-free mice leads to an increased ratio of Igλ-expressing versus Igκ-expressing B cells specifically in the LP. We conclude that B cell development occurs in the intestinal mucosa, where it is regulated by extra-cellular signals from commensal microbes that influence gut Ig repertoires

Structural basis for native agonist and synthetic inhibitor recognition by the Pseudomonas aeruginosa quorum sensing regulator PqsR (MvfR)

Bacterial populations co-ordinate gene expression collectively through quorum sensing (QS), a cell-to-cell communication mechanism employing diffusible signal molecules. The LysR-type transcriptional regulator (LTTR) protein PqsR (MvfR) is a key component of alkyl-quinolone (AQ)-dependent QS in Pseudomonas aeruginosa. PqsR is activated by 2-alkyl-4-quinolones including the Pseudomonas quinolone signal (PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone), its precursor 2-heptyl-4- hydroxyquinoline (HHQ) and their C9 congeners, 2-nonyl-3-hydroxy-4(1H)-quinolone (C9-PQS) and 2-nonyl-4-hydroxyquinoline (NHQ). These drive the autoinduction of AQ biosynthesis and the up-regulation of key virulence determinants as a function of bacterial population density. Consequently, PqsR constitutes a potential target for novel antibacterial agents which attenuate infection through the blockade of virulence. Here we present the crystal structures of the PqsR co-inducer binding domain (CBD) and a complex with the native agonist NHQ. We show that the structure of the PqsR CBD has an unusually large ligand-binding pocket in which a native AQ agonist is stabilized entirely by hydrophobic interactions. Through a ligand-based design strategy we synthesized and evaluated a series of 50 AQ and novel quinazolinone (QZN) analogues and measured the impact on AQ biosynthesis, virulence gene expression and biofilm development. The simple exchange of two isosteres (OH for NH2) switches a QZN agonist to an antagonist with a concomitant impact on the induction of bacterial virulence factor production. We also determined the complex crystal structure of a QZN antagonist bound to PqsR revealing a similar orientation in the ligand binding pocket to the native agonist NHQ. This structure represents the first description of an LTTR-antagonist complex. Overall these studies present novel insights into LTTR ligand binding and ligand-based drug design and provide a chemical scaffold for further anti-P. aeruginosa virulence drug development by targeting the AQ receptor PqsR

Dimerization of FIR upon FUSE DNA binding suggests a mechanism of c-myc inhibition

c-myc is essential for cell homeostasis and growth but lethal if improperly regulated. Transcription of this oncogene is governed by the counterbalancing forces of two proteins on TFIIH—the FUSE binding protein (FBP) and the FBP-interacting repressor (FIR). FBP and FIR recognize single-stranded DNA upstream of the P1 promoter, known as FUSE, and influence transcription by oppositely regulating TFIIH at the promoter site. Size exclusion chromatography coupled with light scattering reveals that an FIR dimer binds one molecule of single-stranded DNA. The crystal structure confirms that FIR binds FUSE as a dimer, and only the N-terminal RRM domain participates in nucleic acid recognition. Site-directed mutations of conserved residues in the first RRM domain reduce FIR's affinity for FUSE, while analogous mutations in the second RRM domain either destabilize the protein or have no effect on DNA binding. Oppositely oriented DNA on parallel binding sites of the FIR dimer results in spooling of a single strand of bound DNA, and suggests a mechanism for c-myc transcriptional control

Can We Modify the Intrauterine Environment to Halt the Intergenerational Cycle of Obesity?

Child obesity is a global epidemic whose development is rooted in complex and multi-factorial interactions. Once established, obesity is difficult to reverse and epidemiological, animal model, and experimental studies have provided strong evidence implicating the intrauterine environment in downstream obesity. This review focuses on the interplay between maternal obesity, gestational weight gain and lifestyle behaviours, which may act independently or in combination, to perpetuate the intergenerational cycle of obesity. The gestational period, is a crucial time of growth, development and physiological change in mother and child. This provides a window of opportunity for intervention via maternal nutrition and/or physical activity that may induce beneficial physiological alternations in the fetus that are mediated through favourable adaptations to in utero environmental stimuli. Evidence in the emerging field of epigenetics suggests that chronic, sub-clinical perturbations during pregnancy may affect fetal phenotype and long-term human data from ongoing randomized controlled trials will further aid in establishing the science behind ones predisposition to positive energy balance

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead