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Bacteriophages encode auxiliary metabolic genes that support more efficient phage replication. For example, cyanophages carry several genes to maintain host photosynthesis throughout infection, shuttling the energy and reducing power generated away from carbon fixation and into anabolic pathways. Photodamage to the D1/D2 proteins at the core of photosystem II necessitates their continual replacement. Synthesis of functional proteins in bacteria requires co-translational removal of the N-terminal formyl group by a peptide deformylase (PDF). Analysis of marine metagenomes to identify phage-encoded homologs of known metabolic genes found that marine phages carry PDF genes, suggesting that their expression during infection might benefit phage replication. We identified a PDF homolog in the genome of Synechococcus cyanophage S-SSM7. Sequence analysis confirmed that it possesses the three absolutely conserved motifs that form the active site in PDF metalloproteases. Phylogenetic analysis placed it within the Type 1B subclass, most closely related to the Arabidopsis chloroplast PDF, but lacking the C-terminal α-helix characteristic of that group. PDF proteins from this phage and from Synechococcus elongatus were expressed and characterized. The phage PDF is the more active enzyme and deformylates the N-terminal tetrapeptides from D1 proteins more efficiently than those from ribosomal proteins. Solution of the X-ray/crystal structures of those two PDFs to 1.95 A resolution revealed active sites identical to that of the Type 1B Arabidopsis chloroplast PDF. Taken together, these findings show that many cyanophages encode a PDF with a D1 substrate preference that adds to the repertoire of genes used by phages to maintain photosynthetic activities.

The Society of Nuclear Medicine and Molecular Imaging (SNMMI) is a nonprofit, professional, and scientific organization that promotes the science, technology, and practical application of nuclear medicine and molecular imaging. The SNMMI is a leader in advancing, optimizing, and unifying nuclear medicine/molecular imaging, with the ultimate goal of improving human health. It has over 17,000 members worldwide, the majority of which are in the USA. The SNMMI is based in Reston, Virginia, USA.

SUMMARY Background: The 2007 National Institutes of Health incontinence consensus panel emphasised the need for classification and identification of persons at risk for faecal incontinence (FI). Objectives: To explore the prevalence of FI; to characterise severity and ‘bother’; and to identify factors associated with FI in a large sample of community-dwelling women. Design, setting, and participants: A cohort of US women ‡ 45 years old was surveyed via an internet-based questionnaire between September 2009 and April 2010. Main outcome measure: Accidental leakage of liquid or solid stool at least once in the last 12 months. Key results: Eighty-five per cent of those surveyed (5817 ⁄6873) participated and were predominantly white, well educated and insured. The prevalence of FI at least once in the past year was 18.8% (1096 ⁄5817; 95% CI: 17.8‐19.9%) and 97% of these women were bothered by this frequency of leakage. Among 938 respondents with FI, 71.1% (667) preferred the term ‘accidental bowel leakage’ (ABL) over faecal or bowel incontinence. Bowel disorders, urinary incontinence, stroke, age 55‐64, diabetes mellitus and prior vaginal delivery were associated with an increased odds of FI, whereas being married, Black or American Indian ⁄Alaska Native race ⁄ethnicity, and income ‡ $40,000 per year were associated with a decreased odds of FI. Conclusions: Nearly one-fifth of mature US women suffer from bothersome leakage of stool at least yearly and the overwhelming majority prefer the term ‘Accidental Bowel Leakage.’ Those with bowel disorders and urinary incontinence are most likely to experience ABL. Incorporating questions regarding ABL or bowel and bladder disorders into routine screening may aid in identifying silent sufferers of

SUMMARY Background: Accidental bowel leakage (ABL) is associated with negative impact on quality of life (QoL) and many women do not seek care. Objectives: To assess current perspectives and QoL among women with ABL; to identify factors associated with severe impact on condition-specific QoL; and to describe care-seeking for ABL. Design, setting and participants: Sub-analysis of 1096 women with ABL identified through an internet-based, self-administered survey of 5817 US women ‡ 45 years old. Main outcome measure: Severe impact on QoL was defined as response of ‘affects very much’ or ‘greatly’ to any of seven domains within Pelvic Floor Impact Questionnaire. Results: QoL data were available for 85.6% (938 ⁄1096) of women with ABL. Domains relating to frustration, emotional health and participation in social activities demonstrated the greatest negative impact, with 39.2% (95% CI 36.1‐42.4%) having overall severe impact. More frequent ABL, faecal urgency, nocturnal bowel movements, FI without warning, stress FI, weekly urinary incontinence and underlying bowel disorder were associated with severe impact on QoL. Of the 28.6% (268 ⁄938) of women who spoke to a physician about their ABL, the majority did so with a general practitioner or family physician (56.0%, 150 ⁄268). Only 19.0% (51 ⁄268) consulted an internist or gastroenterologist [27.2% (73 ⁄268)]. Conclusions: Nearly 40% of adult women with ABL have severe negative impact on QoL, but less than one-third seek care. More than half of those who seek care do so with their primary care provider. Improved awareness of the prevalence and impact of FI by these providers may decrease barriers and improve QoL. What’s known Nearly one in five mature US women suffers from faecal incontinence or accidental bowel leakage. Although the negative impact of this condition on quality of life has been explored, little is known about emotional well-being, perspectives and coping. Despite its significant negative impact on quality of life, rates of care-seeking for this condition vary widely and remain low, ranging from 8% to 27%.

Structural brain measures are employed as endophenotypes in the search for schizophrenia susceptibility genes. We analyzed two independent structural imaging datasets with voxel-based morphometry and with source-based morphometry, a multivariate, independent components analysis, to determine the stability and heritability of regional gray matter concentration abnormalities in schizophrenia. The samples comprised 209 and 102 patients with schizophrenia and 208 and 96 healthy volunteers, respectively. The second sample additionally included non-ill siblings of participants with and without schizophrenia. A standard voxel-based analysis showed reproducible regional gray matter deficits in the affected participants compared with unrelated, unaffected controls in both datasets: patients showed significant gray matter concentration deficits in cortical frontal, temporal, and insular lobes. Source-based morphometry (SBM) was applied to the gray matter images of the entire sample to determine the effects of diagnosis on networks of covarying structures. The SBM analysis extracted 24 significant sets of covarying regions (components). Four of these components showed significantly lower gray matter concentrations in patients (p < .05). We determined the familiality of the observed SBM components based on 66 sibling pairs (25 discordant for schizophrenia). Two components, one including the medial frontal, insular, inferior frontal, and temporal lobes, and the other including the posterior occipital lobe, showed significant familiality (p < .05). We conclude that structural brain deficits in schizophrenia are replicable, and that SBM can extract unique familial and likely heritable components. SBM provides a useful data reduction technique that can provide measures that may serve as endophenotypes for schizophrenia.

The peptide wrwycr inhibits Holliday junction resolution and is a potent antimicrobial. To study the physiological effects of wrwycr treatment on Escherichia coli cells, we partially screened the Keio collection of knockout mutants for those with increased sensitivity to wrwycr. Strains lacking part of the ferric-enterobactin (iron-bound siderophore) uptake and utilization system, parts of the enterobactin synthesis pathway, TolC (an outer-membrane channel protein) or Fur (an iron-responsive regulator) were hypersensitive to wrwycr. We provide evidence that the ΔtolC mutant was hypersensitive to wrwycr due to its reduced ability to efflux wrwycr from the cell rather than due to its export of newly synthesized enterobactin. Deleting ryhB, which encodes a small RNA involved in iron regulation, mostly relieved the wrwycr hypersensitivity of the fur and ferric-enterobactin uptake mutants, indicating that the altered regulation of a RyhB-controlled gene was at least partly responsible for the hypersensitivity of these strains. Chelatable iron in the cell, measured by electron paramagnetic resonance spectroscopy, increased dramatically following wrwycr treatment, as did expression of Fur-repressed genes and, to some extent, mutation frequency. These incongruous results suggest that while wrwycr treatment caused accumulation of chelatable iron in the cell, iron was not available to bind to Fur. This is corroborated by the observed induction of the suf system, which assembles iron–sulfur clusters in low-iron conditions. Disruption of iron metabolism by wrwycr, in addition to its effects on DNA repair, may make it a particularly effective antimicrobial in the context of the low-iron environment of a mammalian host.

Prostate cancer is the most common type of cancer in men aside from skin cancer. It is estimated that 233,000 new cases of prostate cancer will occur in the United States in 2014 ([1][1]). Prostate cancer is the second leading cause of cancer death in men. Approximately 29,480 men in the United

Our lab has isolated hexameric peptides that are structure-selective ligands of Holliday junctions (HJ), central intermediates of several DNA recombination reactions. One of the most potent of these inhibitors, WRWYCR, has shown antibacterial activity in part due to its inhibition of DNA repair proteins. To increase the therapeutic potential of these inhibitors, we searched for small molecule inhibitors with similar activities. We screened 11 small molecule libraries comprising over nine million individual compounds and identified a potent N-methyl aminocyclic thiourea inhibitor that also traps HJs formed during site-specific recombination reactions in vitro. This inhibitor binds specifically to protein free HJs and can inhibit HJ resolution by RecG helicase, but only showed modest growth inhibition of bacterial with a hyperpermeable outer membrane; nonetheless, this is an important step in developing a functional analog of the peptide inhibitors.

Eleven Bacillus isolates from the surface and subsurface waters of the Gulf of Mexico were examined for their capacity to sporulate and harbor prophages. Occurrence of sporulation in each isolate was assessed through decoyinine induction, and putative lysogens were identified by prophage induction by mitomycin C treatment. No obvious correlation between ability to sporulate and prophage induction was found. Four strains that contained inducible virus-like particles (VLPs) were shown to sporulate. Four strains did not produce spores upon induction by decoyinine but contained inducible VLPs. Two of the strains did not produce virus-like particles or sporulate significantly upon induction. Isolate B14905 had a high level of virus-like particle production and a high occurrence of sporulation and was further examined by genomic sequencing in an attempt to shed light on the relationship between sporulation and lysogeny. In silico analysis of the B14905 genome revealed four prophage-like regions, one of which was independently sequenced from a mitomycin C-induced lysate. Based on PCR and transmission electron microscopy (TEM) analysis of an induced phage lysate, one is a noninducible phage remnant, one may be a defective phage-like bacteriocin, and two were inducible prophages. One of the inducible phages contained four putative transcriptional regulators, one of which was a SinR-like regulator that may be involved in the regulation of host sporulation. Isolates that both possess the capacity to sporulate and contain temperate phage may be well adapted for survival in the oligotrophic ocean.

When both structural magnetic resonance imaging (sMRI) and functional MRI (fMRI) data are collected they are typically analyzed separately and the joint information is not examined. Techniques that examine joint information can help to find hidden traits in complex disorders such as schizophrenia. The brain is vastly interconnected, and local brain morphology may influence functional activity at distant regions. In this paper we introduce three methods to identify inter-correlations among sMRI and fMRI voxels within the whole brain. We apply these methods to examine sMRI gray matter data and fMRI data derived from an auditory sensorimotor task from a large study of schizophrenia. In Method 1 the sMRI-fMRI cross-correlation matrix is reduced to a histogram and results show that healthy controls (HC) have stronger correlations than do patients with schizophrenia (SZ). In Method 2 the spatial information of sMRI-fMRI correlations is retained. Structural regions in the cerebellum and frontal regions show more positive and more negative correlations, respectively, with functional regions in HC than in SZ. In Method 3 significant sMRI-fMRI inter-regional links are detected, with regions in the cerebellum showing more significant positive correlations with functional regions in HC relative to SZ. Results from all three methods indicate that the linkage between gray matter and functional activation is stronger in HC than SZ. The methods introduced can be easily extended to comprehensively correlate large data sets.

Summary Staphylococcus aureus produces a wide array of viru- lence factors and causes a correspondingly diverse array of infections. Production of these virulence factors is under the control of a complex network of global regulatory elements, one of which is sarA. sarA encodes a DNA binding protein that is considered to function as a transcription factor capable of acting as either a repressor or an activator. Using competitive ELISA assays, we demonstrate that SarA is present at approximately 50 000 copies per cell, which is not characteristic of classical transcription factors. We also demonstrate that SarA is present at all stages of growthinvitroandiscapableofbindingDNAwithhigh affinity but that its binding affinity and pattern of shifted complexes in electrophoretic mobility shift assays is responsive to the redox state. We also show that SarA binds to the bacteriophage lambda (l) attachment site, attL, producing SarA-DNAcomplexes similar to intasomes, which consist of bacteriophage lambda integrase, Escherichia coli integration host factor and attL DNA. In addition, SarA stimulates intramolecular excision recombination in the absence

Peptide inhibitors of phage lambda site-specific recombination were previously isolated by screening synthetic combinatorial peptide libraries. These inhibitors cause the accumulation of complexes between the recombinase and the Holliday junction intermediate of several highly divergent tyrosine recombinases. Peptide WRWYCR and its d -amino acid derivative bind to the center of protein-free junctions and prevent their resolution either by site-specific recombinases or by junction resolvases or helicases. With lesser affinity, the peptides also bind to branched DNA molecules that mimic replication forks. The peptides are bactericidal to both gram-positive and gram-negative bacteria, presumably because they can interfere with DNA repair and with chromosome dimer resolution by the XerC and XerD tyrosine recombinases. In order to test the correspondence between their mechanism in vivo and in vitro, we have tested and shown peptide wrwycr's ability to inhibit the excision of several prophages (lambda, P22, Gifsy-1, Gifsy-2, Fels-1, Fels-2) and to trap Holliday junction intermediates of phage lambda site-specific recombination in vivo. In addition, we found that the peptide inhibits replication of the Salmonella prophage Fels-1 while integrated in the chromosome. These findings further support the proposed mechanistic basis for the antimicrobial activity of the peptide and its use as a tool to dissect strand exchange-dependent DNA repair within cells.

Regional gray matter (GM) abnormalities are well known to exist in patients with chronic schizophrenia. Voxel-based morphometry (VBM) has been previously used on structural magnetic resonance images (MRI) data to characterize these abnormalities. Two multisite schizophrenia studies, the Functional Biomedical Informatics Research Network and the Mind Clinical Imaging Consortium, which include 9 data collection sites, are evaluating the efficacy of pooling structural imaging data across imaging centers. Such a pooling of data could yield the increased statistical power needed to elucidate effects that may not be seen with smaller samples. VBM analyses were performed to evaluate the consistency of patient versus control gray matter concentration (GMC) differences across the study sites, as well as the effects of combining multisite data. Integration of data from both studies yielded a large sample of 503 subjects, including 266 controls and 237 patients diagnosed with schizophrenia, schizoaffective or schizophreniform disorder. The data were analyzed using the combined sample, as well as analyzing each of the 2 multisite studies separately. A consistent pattern of reduced relative GMC in schizophrenia patients compared with controls was found across all study sites. Imaging center-specific effects were evaluated using a region of interest analysis. Overall, the findings support the use of VBM in combined multisite studies. This analysis of schizophrenics and controls from around the United States provides continued supporting evidence for GM deficits in the temporal lobes, anterior cingulate, and frontal regions in patients with schizophrenia spectrum disorders.

The RecG helicase of Escherichia coli unwinds both Holliday junction (HJ) and replication fork DNA substrates. Our lab previously identified and characterized peptides (WRWYCR and KWWCRW) that block the activity of RecG on these substrates. We determined that the peptides bind HJ DNA and prevent the binding of RecG. Herein, we present further evidence that the peptides are competitive inhibitors of RecG binding to its substrates. We have generated structural models of interactions between WRWYCR and a junction substrate. Using the fluorescent probe 2-aminopurine, we show that inhibitors interact with highest affinity with HJs (Kd = 14 nM) and ~4- to 9-fold more weakly with replication fork substrates. The fluorescence assay results agree with the structural model, and predict the molecular basis for interactions between HJ-trapping peptides and branched DNA molecules. Specifically, aromatic amino acids in the peptides stack with bases at the center of the DNA substrates. These interactions are stabilized by hydrogen bonds to the DNA and by intrapeptide interactions. These peptides inhibit several proteins involved in DNA repair in addition to RecG, have been useful as tools to dissect recombination, and possess antibiotic activity. Greater understanding of the peptides’ mechanism of action will further increase their utility.

17O, 15N, 13C, and 1H NMR chemical shieldings are calculated using density functional theory to differentiate among the three primarily helical forms, 310, alpha, and pi in polyalanine peptides under periodic boundary conditions. This study suggests 17O as the best observable, as it has been demonstrated to be sensitive to hydrogen bonding and highly affected by small changes in the polypeptide in helix conformations. This theoretical study seeks to characterize the subtle conformational differences of helical structures by NMR chemical shift observables which may lead to important questions in experimental structure determination on the basis of using chemical shifts to identify protein secondary structures.

Holliday junctions are a central intermediate in diverse pathways of DNA repair and recombination. The isomerization of a junction determines the directionality of the recombination event. Previous studies have shown that the identity of the central sequence of the junction may favor one of the two isomers, in turn controlling the direction of the pathway. Here we demonstrate that, in the absence of DNA sequence-mediated isomer preference, polycations are the major contributor to biasing strand cleavage during junction resolution. In the case of wild-type phage lambda excision junctions, spermidine plays the dominant role in controlling the isomerization state of the junction and increases the rate of junction resolution. Spermidine also counteracts the sequence-imposed bias on resolution. The spermidine-induced bias is seen equally on supercoiled and linear excisive recombination junction intermediates, and thus is not just an artefact of in vitro recombination conditions. The contribution of spermidine requires the presence of accessory factors, and results in the repositioning of Int's core-binding domains on junctions, perhaps due to DNA-spermidine-protein interactions, or by influencing DNA conformation in the core region. Our results lead us to propose that spermidine together with accessory factors promotes the formation of the second junction isomer. We propose that this rearrangement triggers the activation of the second pair of Int active sites necessary to resolve Holliday junctions during phage lambda Int-mediated recombination.

BackgroundManagement of fecal incontinence remains challenging owing to the limited availability of consistently safe, effective, and/or tolerable treatment options.ObjectiveThe aim of this study was to evaluate the efficacy, safety, and tolerability of an anal insert device for the conservative management of fecal incontinence.DesignThis is a multicenter, prospective, open-label study of a single-arm cohort.SettingPatients were recruited between November 2009 and June 2011 from 3 US clinical sites.PatientsSubjects who were ≥18 years old with incontinence severity scores ≥12 of 20, and at least weekly leakage of solid and/or liquid stool, were selected.InterventionsPatients underwent 12 weeks of continuous anal insert device use.Main outcome measuresThe primary outcomes measured were bowel diaries, incontinence severity, satisfaction, and adverse events. The percentage of reduction in leakage frequency and severity was assessed weekly. Sample size calculations predicted that 47 subjects would demonstrate that 70% of subjects would have ≥50% reduction of incontinence frequency. The paired t test and Wilcoxon tests were used as appropriate.ResultsSeventy-seven percent of the 73 completers and 62% of the 91 intent-to-treat subjects achieved a ≥50% reduction in incontinence frequency. Median fecal incontinence frequency was reduced by 82% from 0.9 (mean 1.1 ± 0.9) at baseline to 0.2 (mean 0.3 ± 0.4) episodes of leakage per day at 12 weeks (p < 0.001). Mean fecal incontinence severity scores improved by 32.4% (16.2, ±2.1 vs 10.9, ±4.4 of 20, p < 0.001) and 78% of completers were very or extremely satisfied with the device with no serious adverse events related to device use.LimitationsThis study was limited by the nonvalidated modification of the severity score and the lack of randomization, control comparison group, blinded assessments, and quality-of-life measures.ConclusionsThe anal insert device provides a conservative, safe, and effective management strategy for individuals with fecal incontinence, with high patient satisfaction and low adverse event rates.

The Critical Assessment of protein Structure Prediction (CASP) experiment would not have been possible without the prediction targets provided by the experimental structural biology community. In this article, selected crystallographers providing targets for the CASP11 experiment discuss the functional and biological significance of the target proteins, highlight their most interesting structural features, and assess whether these features were correctly reproduced in the predictions submitted to CASP11. Proteins 2016; 84(Suppl 1):34–50. © 2015 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

It has been demonstrated that cytokines play a central role in mediating host responses to endotoxemia. In this study, we examined and compared the effects of E. coli sepsis and the cytokines

Background— Although single-photon emission computed tomography myocardial perfusion imaging (SPECT MPI) has improved the diagnosis and risk stratification of patients with suspected coronary artery disease, it remains a primary source of low-dose radiation exposure for cardiac patients. To determine the biological effects of low-dose radiation from SPECT MPI, we measured the activation of the DNA damage response pathways using quantitative flow cytometry and single-cell gene expression profiling. Methods and Results— Blood samples were collected from patients before and after SPECT MPI (n=63). Overall, analysis of all recruited patients showed no marked differences in the phosphorylation of proteins (H2AX, protein 53, and ataxia telangiectasia mutated) after SPECT. The majority of patients also had either downregulated or unchanged expression in DNA damage response genes at both 24 and 48 hours post-SPECT. Interestingly, a small subset of patients with increased phosphorylation had significant upregulation of genes associated with DNA damage, whereas those with no changes in phosphorylation had significant downregulation or no difference, suggesting that some patients may potentially be more sensitive to low-dose radiation exposure. Conclusions— Our findings showed that SPECT MPI resulted in a variable activation of the DNA damage response pathways. Although only a small subset of patients had increased protein phosphorylation and elevated gene expression postimaging, continued care should be taken to reduce radiation exposure to both the patients and operators.

Introduction: This study was designed to determine the value of fluorodeoxyglucose (FDG) positron emission tomography (PET) in the evaluation of metastatic transitional cell carcinoma (TCC). Methods: Fifty-eight FDG PET scans were performed on 46 consecutive patients with TCC. Results were correlated with radiologic, pathologic, and histologic findings in these patients and the sensitivity of PET for detecting malignancy in untreated TCC patients (n = 48) was compared to the sensitivity in patients who had undergone prior chemotherapy (n = 10). Results: Of 48 scans in patients who had no prior systemic chemotherapy, 10 had increased uptake in proven metastatic TCC lesions and 3 PET studies failed to reveal metastatic TCC (sensitivity 76.9%). In patients free of metastatic disease, 33 revealed no abnormal uptake and 1 study revealed a suspicious area in a patient free of metastases (specificity = 97.1%). However, in 10 patients imaged after receiving chemotherapy, the sensitivity fell to 50% for the detection of histologically confirmed residual/recurrent tumor by PET. Conclusions: FDG PET detects increased metabolic activity. After chemotherapy, viable cancer cells may still be present but with a diminished metabolic rate. As a result, PET imaging is often useful in the evaluation of untreated metastatic TCC metastasis but should be interpreted with caution in patients who have received prior chemotherapy.

Background 18-Fluorodeoxyglucose positron emission tomography (PET) is used clinically to detect recurrent colon cancer after surgical resection, but the sensitivity of PET for premalignant colon lesions and early stage colon cancer is not well defined. Methods In a prospective study, 45 patients with a total of 58 colonic neoplasms, including premalignant polyps, premalignant, flat lesions, and early stage cancers, were evaluated by PET. Results The sensitivity of PET for cancer was 62% (8/13). PET detected 100% (7/7) of cancers 2 cm or larger but only 17% (1/6) of cancers smaller than 2 cm. PET detected 23% (3/13) of flat, premalignant lesions; 70% (7/10) of protruded, premalignant lesions 3 cm or larger; 38% (3/8) of protruded, premalignant lesions between 2 and 2.9 cm; and 14% (2/14) of protruded, premalignant lesions between 1 and 1.9 cm. There was no false-positive PET reading. Conclusions PET has limited sensitivity for flat, premalignant lesions; protruded, premalignant lesions smaller than 3 cm; and colon cancers smaller than 2 cm.

The Keystone Symposium on Bacterial Chromosomes was held between 7 and 12 February 2004 in Santa Fe, New Mexico, USA. The conference was organized by S. Gottesman, N. Kleckner and J. Roth. ![][1] From the study of bacteria we know a great deal about DNA replication, recombination and repair, transcription, translation and gene regulation, and cellular metabolism. However, despite powerful molecular genetics tools, our understanding of the organization and behaviour of bacterial chromosomes has lagged behind, simply because their dynamics have been difficult to observe directly. Bacteria lack a mitotic system, and an equivalent prokaryotic mechanism for the separation of duplicated chromosomes remains mysterious. Confounding the picture is the complexity of the bacterial cell cycle, which is unlike the orderly and interdependent progression of events in eukaryotes. The application of fluorescence microscopy techniques, flow cytometry and cell synchronization has revolutionized our understanding of chromosome dynamics and the cell cycle in bacteria. Much of this conference was devoted to how chromosomes are organized, faithfully replicated and segregated to daughter cells, a few highlights of which are presented here. Dominating the discussion were studies of two model organisms— Bacillus subtilis and Escherichia coli —that diverged from a common ancestor over two billion years ago and are very different beasts. Despite this, these organisms share a number of common features. Both are rod‐shaped cells, about the size of a yeast nucleus, that divide at midcell. Both initiate replication at a single origin ( oriC ) in a circular chromosome, and bidirectional forks converge near a terminus region ( ter ) that has special properties to ensure chromosome segregation to daughter cells. Although there is no true nucleus in bacterial cells, a region of condensed chromosomal DNA (called a ‘nucleoid’) divides and separates before cell fission. In slow‐growing cells, replication is temporally separated from cell division, similar to the … [1]: /embed/graphic-1.gif

Solid-state (1)H, (17)O MAS NMR, (1)H-(93)Nb TRAPDOR NMR, and (1)H double quantum 2D MAS NMR experiments were used to characterize the oxygen, water, and hydroxyl environments in the monoprotonated hexaniobate material, Na(7)[HNb(6)O(19)].15H(2)O. These solid-state NMR experiments demonstrate that the proton is located on the bridging oxygen of the [Nb(6)O(19)](8-) cluster. The solid-state NMR results also show that the NbOH protons are spatially isolated from similar protons, but undergo proton exchange with the water species located in the crystal lattice. On the basis of double quantum (1)H MAS NMR measurements, it was determined that the water species in the crystal lattice have restricted motional dynamics. Two-dimensional (1)H-(17)O MAS NMR correlation experiments show that these restricted waters are preferentially associated with the bridging oxygen. Solution (17)O NMR experiments show that the hydroxyl proton is also attached to the bridging oxygen for the compound in solution. In addition, solution (17)O NMR kinetic studies for the hexaniobate allowed the measurement of relative oxygen exchange rates between the bridging, terminal, and hydroxyl oxygen and the oxygen of the solvent as a function of pH and temperature. These NMR experiments are some of the first investigations into the proton location, oxygen and proton exchange processes, and water dynamics for a base stable polyoxoniobate material, and they provide insight into the chemistry and reactivity of these materials.

Ab initio molecular orbital calculations (Hartree–Fock, HF and density functional theory, DFT) of the 17 O NMR electrical field gradient (EFG) parameters for the bridging oxygen (O B ) in the model cluster H 4 P 2 O 7 are presented. The variation of the quadrupolar coupling constant ( C Q ) and asymmetry parameter ( η Q ) for the EFG of the bridging oxygen was shown to be a function of both the bridging P–O B –P bond angle and the OP–O B –PO conformation. These ab initio calculations allowed empirical relationships between the 17 O NMR EFG parameters and the P–O B –P bond angle to be tabulated for a wide range of OP–O B –PO conformations. For experimentally realistic ranges of the P–O B –P bond angle, the 17 O NMR EFG parameters are dominated by variations in the P–O B –P bond angle, with minor changes due to the OP–O B –PO conformation. These calculations also demonstrated systematic differences in the predicted 17 O EFG parameters obtained using HF and DFT level of theory.

Summary We have investigated the fluidity of the Salmonella chromosome architecture using the phage lambda site-specific recombination system as a probe. We determined how chromosome position affects the extent of integrase-mediated recombination between pairs of inversely oriented att sites at various loci. We also investigated the accessibility of each chromo- somal att site to an extrachromosomal partner carried on a low-copy plasmid. Recombination events were assayed by semi-quantitative polymerase chain reac- tion of the attP product. The extent of recombination between the chromosome and the plasmid was gen- erally higher than intrachromosomal recombination except for two loci, araA :: attL and galT :: attL , which gave no detectable recombination with any other locus. Based on 20 intervals, we found that chromo- somal locations are not equally accessible to each other. Although multiple factors probably affect accessibility, the most important is the specific com- bination of the end-points used. Neither the size of the intervals nor the accessibility of individual end- points to extrachromosomal sequences is as impor- tant. These results suggest that the chromosome is not completely fluid but rather organized in some way, with barriers that limit the movement of DNA within the cell. The nature of the barriers involved in chro- mosomal organization remains to be determined.

The bacteriophage lambda integrase catalyzes four site-specific recombination pathways with distinct protein and DNA requirements and nucleoprotein intermediates. Some of these intermediates are very transient and difficult to obtain in significant amounts, due to the high efficiency and processivity of integrase, the lack of requirements for external energy factors or metal ions, and the highly reversible nature of each of the intermediates. We have previously used mixture-based combinatorial libraries to identify hexapeptides that trap 40-60% of recombination substrates at the Holliday junction stage of the reaction. These inhibitors discriminate between the four pathways, blocking one of them (bent-L recombination) more severely than the others and blocking the excision pathway least. We presume that these differences reflect specific conformational differences of the nucleoprotein intermediates in each pathway. We have now identified new inhibitors of the excision pathway. One of these, WRWYCR, is over 50-fold more potent at inhibiting excision than the previously identified peptides. This peptide stably traps Holliday junction complexes in all recombination pathways mediated by integrase as well as Cre. This finding and other data presented indicate that the peptide's target is a common feature shared by the Holliday junction complexes assembled by tyrosine recombinases. We have taken advantage of reversible inhibition by the active peptides to develop a new assay for Holliday junction resolution. This assay is particularly useful for determining junction resolution rates in cases where complexes directly assembled on junction substrates undergo little or no catalysis.

Two bacteriophages of an environmental isolate of Vibrio parahaemolyticus were isolated and sequenced. The VP16T and VP16C phages were separated from a mixed lysate based on plaque morphology and exhibit 73 to 88% sequence identity over about 80% of their genomes. Only about 25% of their predicted open reading frames are similar to genes with known functions in the GenBank database. Both phages have cos sites and open reading frames encoding proteins closely related to coliphage lambda's terminase protein (the large subunit). Like in coliphage lambda and other siphophages, a large operon in each phage appears to encode proteins involved in DNA packaging and capsid assembly and presumably in host lysis; we refer to this as the structural operon. In addition, both phages have open reading frames closely related to genes encoding DNA polymerase and helicase proteins. Both phages also encode several putative transcription regulators, an apparent polypeptide deformylase, and a protein related to a virulence-associated protein, VapE, of Dichelobacter nodosus . Despite the similarity of the proteins and genome organization, each of the phages also encodes a few proteins not encoded by the other. We did not identify genes closely related to genes encoding integrase proteins belonging to either the tyrosine or serine recombinase family, and we have no evidence so far that these phages can lysogenize the V. parahaemolyticus strain 16 host. Surprisingly for active lytic viruses, the two phages have a codon usage that is very different than that of the host, suggesting the possibility that they may be relative newcomers to growth in V. parahaemolyticus . The DNA sequences should allow us to characterize the lifestyles of VP16T and VP16C and the interactions between these phages and their host at the molecular level, as well as their relationships to other marine and nonmarine phages.

F-18 fluorodeoxyglucose (FDG) is typically administered intravenously for positron emission tomography. The authors present a case of oral administration of FDG for evaluation of a pulmonary nodule and review the limited literature on this subject. Oral administration of FDG is a useful alternative to intravenous administration in patients with difficult intravenous access when the alimentary tract is not involved in the clinical diagnosis.

Background: Ultraprofound hypothermia may have a place in trauma rescue and resuscitation. We describe resuscitation of dogs after asanguhteous perfusion and circulatory arrest of 2 hours at 2° to 4°C. Methods: Nine dogs were cooled using a bypass apparatus and their circulating blood replaced with bicarbonated Hextend (Abbott, North Chicago, IL). Perfusion was continued to 2° to 4°C, and 60 mL of 2 mol/L KCl and 20 mL of 50% MgSO 4 .7H 2 O were infused intra-arterially, and circulation was arrested for 2 hours. The dogs were then rewarmed, transfused, defibrillated, weaned from bypass, and allowed to awaken. Preoperative and postoperative biochemistry and hematology were compared. Results: Six dogs recovered fully. One of these dogs died of an infection 2 weeks later. Three other dogs never recovered because of technical or procedural difficulties. Biochemical and hematologic parameters were normal by 3 weeks. Conclusion: Hypothermic blood substitution with Hextend allows resuscitation after 2 hours of ice-cold circulatory arrest in dogs.

Bacteriophage λ integrase (Int) catalyzes site-specific recombination between pairs of attachment (att) sites. Theattsites contain weak Int-binding sites called core-type sites that are separated by a 7-bp overlap region, where cleavage and strand exchange occur. We have characterized a number of mutant Int proteins with substitutions at positions S282 (S282A, S282F, and S282T), S286 (S286A, S286L, and S286T), and R293 (R293E, R293K, and R293Q). We investigated the core- and arm-binding properties and cooperativity of the mutant proteins, their ability to catalyze cleavage, and their ability to form and resolve Holliday junctions. Our kinetic analyses have identified synapsis as the rate-limiting step in excisive recombination. The IntS282 and IntS286 mutants show defects in synapsis in the bent-L and excisive pathways, respectively, while the IntR293 mutants exhibit synapsis defects in both the excision and bent-L pathways. The results of our study support earlier findings that the catalytic domain also serves a role in binding to core-type sites, that the core contacts made by this domain are important for both synapsis and catalysis, and that Int contacts core-type sites differently among the four recombination pathways. We speculate that these residues are important for the proper positioning of the catalytic residues involved in the recombination reaction and that their positions differ in the distinct nucleoprotein architectures formed during each pathway. Finally, we found that not all catalytic events in excision follow synapsis: theattLsite probably undergoes several rounds of cleavage and ligation before it synapses and exchanges DNA withattR.

[reaction: see text] Described are the syntheses of eight macrocyclic peptides designed to trap Holliday junctions in bacteria, thereby inhibiting bacterial growth. These macrocycles were designed from linear dimerized hexapeptides that bind to the C-2 symmetrical Holliday junction. They were synthesized from three monomers using a combinatorial-like strategy that permits elucidation of the monomer role in accumulation of Holliday junctions and antibiotic activity. These macrocycles are an important step in designing and synthesizing a new class of antibiotics.

© The Author 2014. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved.Analyses of gray matter concentration (GMC) deficits in patients with schizophrenia (Sz) have identified robust changes throughout the cortex. We assessed the relationships between diagnosis, overall symptom severity, and patterns of gray matter in the largest aggregated structural imaging dataset to date. We performed both sourcebased morphometry (SBM) and voxel-based morphometry (VBM) analyses on GMC images from 784 Sz and 936 controls (Ct) across 23 scanning sites in Europe and the United States. After correcting for age, gender, site, and diagnosis by site interactions, SBM analyses showed 9 patterns of diagnostic differences. They comprised separate cortical, subcortical, and cerebellar regions. Seven patterns showed greater GMC in Ct than Sz, while 2 (brainstem and cerebellum) showed greater GMC for Sz. The greatest GMC deficit was in a single pattern comprising regions in the superior temporal gyrus, inferior frontal gyrus, and medial frontal cortex, which replicated over analyses of data subsets. VBM analyses identified overall cortical GMC loss and one small cluster of increased GMC in Sz, which overlapped with the SBM brainstem component. We found no significant association between the component loadings and symptom severity in either analysis. This mega-analysis confirms that the commonly found GMC loss in Sz in the anterior temporal lobe, insula, and medial frontal lobe form a single, consistent spatial pattern even in such a diverse dataset. The separation of GMC loss into robust, repeatable spatial patterns across multiple datasets paves the way for the application of these methods to identify subtle genetic and clinical cohort effects.

Viruses are the most common biological entities in the oceans by an order of magnitude. However, very little is known about their diversity. Here we report a genomic analysis of two uncultured marine viral communities. Over 65% of the sequences were not significantly similar to previously reported sequences, suggesting that much of the diversity is previously uncharacterized. The most common significant hits among the known sequences were to viruses. The viral hits included sequences from all of the major families of dsDNA tailed phages, as well as some algal viruses. Several independent mathematical models based on the observed number of contigs predicted that the most abundant viral genome comprised 2–3% of the total population in both communities, which was estimated to contain between 374 and 7,114 viral types. Overall, diversity of the viral communities was extremely high. The results also showed that it would be possible to sequence the entire genome of an uncultured marine viral community.

s: To assess the affordability of health care to poor rural households in Vietnam under conditions of transition from a planned to a market economy and, in light of other transitional experience, inform policy on increasing access of the poor to affordable care of acceptable quality.Observational study by cross sectional socioeconomic survey, longitudinal healthcare seeking survey, and qualitative semi-structured interviews and focus group discussions; qualitative follow up over six years.Four rural communes in north of Vietnam between 1992 and 1998. SURVEY PARTICIPANTS: 656 households (2995 people) selected by systematic random sampling.Compared with non-poor households, poor households had significantly lower average per capita rates of healthcare consultation and expenditure (p0.01 in both cases). Poor households delayed and minimised healthcare seeking, especially of expensive hospital services. Two thirds of average healthcare spending by poor households was on relatively inexpensive but frequent acts of local ambulatory care. The poor restrained their healthcare seeking but not in proportion to income: for households reporting illness, the average proportion of income devoted to health care was 21.9% for the poor compared with 8.2% for the non-poor (p0.01). To meet healthcare costs, many poor households reduced essential consumption, sold assets and incurred debt, threatening their future livelihood.In the short-term the poor need exemption from public sector user fees in both primary and hospital care. In the longer run the government budget and prepayment schemes should replace direct user charges in healthcare finance. Transitional economies like Vietnam should preserve the public health services built up under the planned economy. Market reforms that stimulate growth in the economy appear inappropriate to reform of social sectors.

Accurate assessment of myocardial viability is critical for identifying patients likely to benefit from coronary revascularization. Positron emission tomography (PET) has several advantages over single photon emission computed tomography (SPECT), including higher sensitivity and specificity, as well as the ability to measure myocardial blood flow and myocardial metabolism in absolute terms, which is important in understanding the pathophysiology of ischemic cardiomyopathy. The most commonly used PET tracer is [18F]2-fluoro-2deoxy-D-glucose (FDG). The dependence of ischemic myocardium on glucose metabolism makes FDG an ideal tracer in this setting. Studies have shown positive and negative predictive values for the detection of viable myocardium in the range of 48-94%, and 73-96%, respectively. FDG is superior to SPECT using thallium or technetium myocardial perfusion agents, as well as echocardiography with dobutamine infusion. FDG PET also provides important prognostic information. Patients with evidence of myocardial viability by FDG PET have fewer cardiac events and survive longer if revascularized compared to patients who are treated medically. This article will review myocardial metabolism, PET procedures and interpretive criteria, as well as problems and limitations. Data from the literature regarding diagnostic and prognostic information will also be summarized.

It has been stated that certain chains of biological reactions can go to near completion in both directions as needed without any exterior driving force. This claim represents a thermodynamic impossibility. Yet, this impossibility is of a type not usually covered by the traditional list of impossibility devices known as perpetual motion machines. Rather, it represents a perpetual motion machine of the third kind (PM3) that becomes impossible only in finite time. At non-vanishing rate the chain of biological reactions above would constitute a finite-time perpetual motion machine. The resolution of this quandary leads directly to a notion of staging free energy: the free energy invested in choreographing all the actors of a biochemical reaction directing them to the needed places at the needed times. We conclude by noting that biological systems make ample use of invertible degrees of freedom that often operate near PM3 limits.

Coxsackievirus B3 (CVB3), a member of the picornavirus family and enterovirus genus, causes viral myocarditis, aseptic meningitis, and pancreatitis in humans. We genetically engineered a unique molecular marker, “fluorescent timer” protein, within our infectious CVB3 clone and isolated a high-titer recombinant viral stock (Timer-CVB3) following transfection in HeLa cells. “Fluorescent timer” protein undergoes slow conversion of fluorescence from green to red over time, and Timer-CVB3 can be utilized to track virus infection and dissemination in real time. Upon infection with Timer-CVB3, HeLa cells, neural progenitor and stem cells (NPSCs), and C2C12 myoblast cells slowly changed fluorescence from green to red over 72 hours as determined by fluorescence microscopy or flow cytometric analysis. The conversion of “fluorescent timer” protein in HeLa cells infected with Timer-CVB3 could be interrupted by fixation, suggesting that the fluorophore was stabilized by formaldehyde cross-linking reactions. Induction of a type I interferon response or ribavirin treatment reduced the progression of cell-to-cell virus spread in HeLa cells or NPSCs infected with Timer-CVB3. Time lapse photography of partially differentiated NPSCs infected with Timer-CVB3 revealed substantial intracellular membrane remodeling and the assembly of discrete virus replication organelles which changed fluorescence color in an asynchronous fashion within the cell. “Fluorescent timer” protein colocalized closely with viral 3A protein within virus replication organelles. Intriguingly, infection of partially differentiated NPSCs or C2C12 myoblast cells induced the release of abundant extracellular microvesicles (EMVs) containing matured “fluorescent timer” protein and infectious virus representing a novel route of virus dissemination. CVB3 virions were readily observed within purified EMVs by transmission electron microscopy, and infectious virus was identified within low-density isopycnic iodixanol gradient fractions consistent with membrane association. The preferential detection of the lipidated form of LC3 protein (LC3 II) in released EMVs harboring infectious virus suggests that the autophagy pathway plays a crucial role in microvesicle shedding and virus release, similar to a process previously described as autophagosome-mediated exit without lysis (AWOL) observed during poliovirus replication. Through the use of this novel recombinant virus which provides more dynamic information from static fluorescent images, we hope to gain a better understanding of CVB3 tropism, intracellular membrane reorganization, and virus-associated microvesicle dissemination within the host., Author Summary Enteroviruses are significant human pathogens, causing myocarditis, aseptic meningitis and encephalitis. The mechanisms of enterovirus dissemination in the host and cell-to-cell spread may be critical factors influencing viral pathogenesis. Here, we have generated a recombinant coxsackievirus expressing “fluorescence timer” protein (Timer-CVB3) which assists in following the progression of infection within the host. Unexpectedly, we observed the shedding of microvesicles containing virus in partially-differentiated progenitor cells infected with Timer-CVB3. These extracellular microvesicles (EMVs) were released in high levels following cellular differentiation, and may play a role in virus dissemination. Timer-CVB3 will be a valuable tool in monitoring virus spread in the infected host.

The role of Positron Emission Tomography (PET) using 18F-fluorodeoxyglucose (FDG) in the management of thyroid cancer is discussed. It is important to ensure that patients are relaxed because uptake of FDG in tense or active muscles in the neck and larynx can be misinterpreted as metastases. The major role for PET is in patients where the stage of disease is uncertain, usually the result of discordant negative 131I scan and a positive serum thyroglobulin (Tg) values. PET identifies the source of Tg production in 50-80% of patients. PET scan can be negative in well differentiated cancers which retain the ability to trap iodine. This can result in a 'flip/flop', with negative PET, positive radio-iodine scan, or positive PET, negative radioiodine scan. PET is also valuable in identifying the source of calcitonin production in patients with medullary thyroid cancer. When focal uptake is seen in the thyroid of patients who are scanned for non thyroidal reasons, the likelihood of primary thyroid cancer is high. In contrast diffuse uptake of FDG in the thyroid is usually the result of auto-immune thyroid disorders.

Site-specific recombination is involved in processes ranging from resolution of bacterial chromosome dimers to adeno-associated viral integration and is a versatile tool for mammalian genetics. The bacteriophage λ-encoded site-specific recombinase integrase (Int) is one of the best studied site-specific recombinases and mediates recombination via four distinct pathways. We have characterized a mutant version of λ Int, IntT236I; this mutant can perform the bent-L pathway only, whereas the corresponding IntT236A mutant can perform bent-L, excision and integration pathways. Experiments with both IntT236I and IntT236A show that the hydroxyl group of threonine is necessary for wild-type recombination. Substitution of the threonine by serine leads to nearly complete rescue of the mutant phenotypes. In addition, our data show that the IntT236I mutant is defective partially due to obstructive steric interactions. Comparisons of crystal structures reveal that the threonine at residue 236 may play an important role in stabilizing recombination intermediates through solvent-mediated protein-DNA interactions at the core-binding sites and that the hydroxyl group is important for effective cleavage and Holliday junction formation. Our data also indicate that Int contacts the core sites differently in intermediates assembled in excisive versus bent-L recombination.

Molecular Biology ProgramSloan-Kettering Institute, NewYork, NY 10021, USAThe study of biochemical pathways requires the isolation and characteriz-ation of each and every intermediate in the pathway. For the site-specificrecombination reactions catalyzed by the bacteriophage l tyrosine recom-binase integrase (Int), this has been difficult because of the high level ofefficiency of the reaction, the highly reversible nature of certain reactionsteps, and the lack of requirements for high-energy cofactors or metals.By screening synthetic peptide combinatorial libraries, we have identifiedtwo related hexapeptides, KWWCRW and KWWWRW, that block thestrand-cleavage activity of Int but not the assembly of higher-order inter-mediates. Although the peptides bind DNA, their inhibitory activityappears to be more specifically targeted to the Int-substrate complex,insofar as inhibition is resistant to high levels of non-specific competitorDNA and the peptides have higher levels of affinity for the Int-DNA sub-strate complex than for DNA alone. The peptides inhibit the four path-ways of Int-mediated recombination with different potencies, suggestingthat the interactions of the Int enzyme with its DNA substrates differsamong pathways. The KWWCRW and KWWWRW peptides also inhibitvaccinia virus topoisomerase, a type IB enzyme, which is mechanisticallyand structurally related to Int. The peptides differentially affect the for-ward and reverse DNA transesterification steps of the vaccinia topoi-somerase. They block formation of the covalent vaccinia topoisomerase-DNA intermediate, but have no apparent effect on DNA religation bypreformed covalent complexes. The peptides also inhibit Escherichia colitopoisomerase I, a type IA enzyme. Finally, the peptides inhibit thebacteriophage T4 type II topoisomerase and several restriction enzymeswith 2000-fold lower potency than they inhibit integrase in the bent-Lpathway.

Bacteriophage lambda integrase (Int) catalyzes at least four site-specific recombination pathways between pairs of attachment ( att ) sites. Protein-protein contacts between monomers of Int are presumed to be important for these site-specific recombination events for several reasons: Int binds to the att sites cooperatively, catalytic Int mutants can complement each other for strand cleavage, and crystal structures for two other recombinases in the Int family (Cre from phage P1 and Int from Haemophilus influenzae phage HP1) show extensive protein-protein contacts between monomers. We have begun to investigate interactions between Int monomers by three approaches. First, using a genetic assay, we show that regions of protein-protein interactions occur throughout Int, including in the amino-terminal domain. This domain was previously thought to be important only for high-affinity protein-DNA interactions. Second, we have found that an amino-terminal His tag reduces cooperative binding to DNA. This disruption in cooperativity decreases the stable interaction of Int with core sites, where catalysis occurs. Third, using protein-protein cross-linking to investigate the multimerization of Int during recombination, we show that Int predominantly forms dimers, trimers, and tetramers. Moreover, we show that the cysteine at position 25 is present at or near the interface between monomers that is involved in the formation of dimers and tetramers. Our evidence indicates that the amino-terminal domain of Int is involved in protein-protein interactions that are likely to be important for recombination.

Cardiac positron emission tomography (PET) is an accurate method for assessing myocardial perfusion and metabolism in the evaluation of coronary heart disease. PET allows more accurate detection of myocardial ischemia than single photon emission tomography (SPECT). In addition, PET has higher spatial resolution and allows attenuation correction and the quantification of various physiologic parameters. PET with 2-(fluorine-18) fluoro-2-deoxy-D-glucose is considered the standard of reference for predicting improvement in regional or global left ventricular function after revascularization by identifying hibernating viable myocardium that shows diminished perfusion and preserved metabolism. Other less commonly used clinical applications of cardiac PET include assessment of myocardial oxygen consumption and fatty acid metabolism. The use of PET in myocardial imaging is expected to increase in the near future with the regional distribution of positron-emitting radiotracers and the emergence of relatively low-cost PET systems.

In solution, B-form DNA is isotropically flexible only when it vastly exceeds its persistence length (150 bp; reviewed in reference 8). Nevertheless, DNA binding proteins often impose an anomalous structure onto their DNA targets despite typically contacting fewer than 30 bp. These deformations can be dramatic, however, such that the generated bend itself has a functional consequence. Since these DNA bending proteins generally require no coupled energy source for binding, the deformations are probably derived from the favorable thermodynamics of protein-DNA interactions. Proteins accomplish this feat by using various mechanisms, for example, charge neutralization of phosphates and/or destabilization of DNA base-stacking interactions (25, 30). What role does the DNA sequence play in its own distortion? While specific protein-DNA interactions are most often mediated via specific hydrogen bonding between DNA bases and amino acid residues, there is an ever-growing class of proteins that select their target site based on indirect readout, i.e., a structure or subset of structures that are specifically recognized by a protein (34). The bacterial protein integration host factor (IHF) epitomizes such proteins. It is a small heterodimeric protein consisting of homologous subunits, α and β, that binds and bends DNA specifically. Although known DNA targets are selected over bulk DNA by at least 1,000-fold (40), there is an inherent degeneracy of sequence in the target selection. IHF was originally discovered as a protein required for efficient integration of the bacteriophage lambda into the Escherichia coli chromosome (36). Subsequently, IHF has been shown to participate in virtually every type of nucleoprotein system (e.g., transcription, replication, and recombination). While its role is always that of an accessory factor, its involvement can be anything from a nominal 2-fold influence, as in promoter activation (23), to as great as a 10,000-fold effect, as in lambda integrative recombination. It is the complex of IHF with DNA that makes it unique among bending proteins; the recently solved cocrystal structure of IHF bound to one of its natural binding sites shows that the DNA is bent by 180° into a virtual U-turn (24, 25). IHF typically binds to a 30- to 35-bp sequence which can be divided into at least two domains. The 3′ region is significantly conserved, and sites share the consensus WATCAANNNNTTR (where W is A or T, R is purine, and N is any base). The lone cytosine base is conserved in every known natural site. Unlike the 3′ region, the 5′ region seems almost random; natural sites are typically AT rich, but no obvious patterns of sequence emerge as conserved. How does the IHF binding site accommodate specific binding and bending? Of particular interest is the 5′ binding domain, since it appears variant in each sequence. Recent evidence suggests that this domain is most successful at binding when an appropriately positioned run of adenines, or an A-tract, is present (11). A-tracts typically consist of three to six consecutive adenines that, in the proper sequence context, create an intrinsically rigid structure with a narrow minor groove; adjacent sequences typically possess an anisotropic bend. What facets of A-tracts, if any, attract IHF for preferred binding? We have attempted to answer these questions by performing a systematic evolution of ligands by exponential enrichment (SELEX) analysis. Selective pressure in vitro for high-affinity binding was applied to a population of IHF binding sites where the wild-type 3′ domain was held constant and the 5′ domain was randomized. Individual sequences were then compared for binding affinity, gross structure of nucleoprotein complexes, and the ability to function in bacteriophage lambda site-specific recombination. We found that the 5′ region can vary the affinity for IHF at least 100-fold. Although comprehensive rules for these sequence determinants could not be deduced, this region contributes significantly to the structure but little to the function of these nucleoprotein complexes. Finally, we found that while the base composition of this region is skewed in native sites, there appears to be no gross base composition advantage for either affinity or function.