The native cistrome and sequence motif families of the maize ear.

Elucidating the transcriptional regulatory networks that underlie growth and development requires robust ways to define the complete set of transcription factor (TF) binding sites. Although TF-binding sites are known to be generally located within accessible chromatin regions (ACRs), pinpointing these DNA regulatory elements globally remains challenging. Current approaches primarily identify binding sites for a single TF (e.g. ChIP-seq), or globally detect ACRs but lack the resolution to consistently define TF-binding sites (e.g. DNAse-seq, ATAC-seq). To address this challenge, we developed MNase-defined cistrome-Occupancy Analysis (MOA-seq), a high-resolution (< 30 bp), high-throughput, and genome-wide strategy to globally identify putative TF-binding sites within ACRs. We used MOA-seq on developing maize ears as a proof of concept, able to define a cistrome of 145,000 MOA footprints (MFs). While a substantial majority (76%) of the known ATAC-seq ACRs intersected with the MFs, only a minority of MFs overlapped with the ATAC peaks, indicating that the majority of MFs were novel and not detected by ATAC-seq. MFs were associated with promoters and significantly enriched for TF-binding and long-range chromatin interaction sites, including for the well-characterized FASCIATED EAR4, KNOTTED1, and TEOSINTE BRANCHED1. Importantly, the MOA-seq strategy improved the spatial resolution of TF-binding prediction and allowed us to identify 215 motif families collectively distributed over more than 100,000 non-overlapping, putatively-occupied binding sites across the genome. Our study presents a simple, efficient, and high-resolution approach to identify putative TF footprints and binding motifs genome-wide, to ultimately define a native cistrome atlas.

Bacteriophage steering of Burkholderia cenocepacia toward reduced virulence and increased antibiotic sensitivity.

Antibiotic resistance in bacteria is a growing global concern and has spurred increasing efforts to find alternative therapeutics, such as the use of bacterial viruses, or bacteriophages. One promising approach is to use phages that not only kill pathogenic bacteria but also select phage-resistant survivors that are newly sensitized to traditional antibiotics, in a process called "phage steering." Members of the bacterial genus Burkholderia, which includes various human pathogens, are highly resistant to most antimicrobial agents, including serum immune components, antimicrobial peptides, and polymixin-class antibiotics. However, the application of phages in combination with certain antibiotics can produce synergistic effects that more effectively kill pathogenic bacteria. Herein, we demonstrate that Burkholderia cenocepacia serum resistance is due to intact lipopolysaccharide (LPS) and membranes, and phage-induced resistance altering LPS structure can enhance bacterial sensitivity not only to immune components in serum but also to membrane-associated antibiotics such as colistin. IMPORTANCE Bacteria frequently encounter selection pressure from both antibiotics and lytic phages, but little is known about the interactions between antibiotics and phages. This study provides new insights into the evolutionary trade-offs between phage resistance and antibiotic sensitivity. The creation of phage resistance through changes in membrane structure or lipopolysaccharide composition can simultaneously be a major cause of antibiotic sensitivity. Our results provide evidence of synergistic therapeutic efficacy in phage-antibiotic interactions and have implications for the future clinical use of phage steering in phage therapy applications.

Biocompatible α-Methylenation of Metabolic Butyraldehyde in Living Bacteria.

Small molecule organocatalysts are abundant in all living organisms. However, their use as organocatalysts in cells has been underexplored. Herein, we report that organocatalytic aldol chemistry can be interfaced with living Escherichia coli to enable the α-methylenation of cellular aldehydes using biogenic amines such as L-Pro or phosphate. The biocompatible reaction is mild and can be interfaced with butyraldehyde generated from D-glucose via engineered metabolism to enable the production of 2-methylenebutanal (2-MB) and 2-methylbutanal (2-MBA) by anaerobic fermentation, and 2-methylbutanol (2-MBO) by whole-cell catalysis. Overall, this study demonstrates the combination of non-enzymatic organocatalytic and metabolic reactions in vivo for the sustainable synthesis of valuable non-natural chemicals that cannot be accessed using enzymatic chemistry alone.

Tyramine Derivatives Catalyze the Aldol Dimerization of Butyraldehyde in the Presence of Escherichia coli.

Biogenic amine organocatalysts have transformed the field of synthetic organic chemistry. Yet despite their use in synthesis and to label biomolecules in vitro, amine organocatalysis in vivo has received comparatively little attention - despite the potential of such reactions to be interfaced with living cells and to modify cellular metabolites. Herein we report that biogenic amines derived from L-tyrosine catalyze the self-aldol condensation of butanal to 2-ethylhexenal - a key intermediate in the production of the bulk chemical 2-ethylhexanol - in the presence of living Escherichia coli and outperform many amine organocatalysts currently used in synthetic organic chemistry. Furthermore, we demonstrate that cell lysate from E. coli and the prolific amine overproducer Corynebacterium glutamicum ATCC 13032 catalyze this reaction in vitro, demonstrating the potential for microbial metabolism to be used as a source of organocatalysts for biocompatible reactions in cells.

Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies.

Taxadien-5α-hydroxylase and taxadien-5α-ol O-acetyltransferase catalyze the oxidation of taxadiene to taxadien-5α-ol and subsequent acetylation to taxadien-5α-yl-acetate in the biosynthesis of the blockbuster anticancer drug, paclitaxel (Taxol®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimization of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High-throughput microscale optimization enhanced total oxygenated taxane titer to 39.0 ± 5.7 mg/L and total taxane product titers were comparable at micro and minibioreactor scale at 95.4 ± 18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien-5α-ol isomer titer to 19.2 mg/L, comparable with the 23.8 ± 3.7 mg/L achieved at microscale. A combination of bioprocess optimization and increased gas chromatography-mass spectrometry resolution at 1 L bioreactor scale facilitated taxadien-5α-yl-acetate detection with a final titer of 3.7 mg/L. Total oxygenated taxane titers were improved 2.7-fold at this scale to 78 mg/L, the highest reported titer in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems.

Topologically associating domains are stable units of replication-timing regulation.

Eukaryotic chromosomes replicate in a temporal order known as the replication-timing program. In mammals, replication timing is cell-type-specific with at least half the genome switching replication timing during development, primarily in units of 400-800 kilobases ('replication domains'), whose positions are preserved in different cell types, conserved between species, and appear to confine long-range effects of chromosome rearrangements. Early and late replication correlate, respectively, with open and closed three-dimensional chromatin compartments identified by high-resolution chromosome conformation capture (Hi-C), and, to a lesser extent, late replication correlates with lamina-associated domains (LADs). Recent Hi-C mapping has unveiled substructure within chromatin compartments called topologically associating domains (TADs) that are largely conserved in their positions between cell types and are similar in size to replication domains. However, TADs can be further sub-stratified into smaller domains, challenging the significance of structures at any particular scale. Moreover, attempts to reconcile TADs and LADs to replication-timing data have not revealed a common, underlying domain structure. Here we localize boundaries of replication domains to the early-replicating border of replication-timing transitions and map their positions in 18 human and 13 mouse cell types. We demonstrate that, collectively, replication domain boundaries share a near one-to-one correlation with TAD boundaries, whereas within a cell type, adjacent TADs that replicate at similar times obscure replication domain boundaries, largely accounting for the previously reported lack of alignment. Moreover, cell-type-specific replication timing of TADs partitions the genome into two large-scale sub-nuclear compartments revealing that replication-timing transitions are indistinguishable from late-replicating regions in chromatin composition and lamina association and accounting for the reduced correlation of replication timing to LADs and heterochromatin. Our results reconcile cell-type-specific sub-nuclear compartmentalization and replication timing with developmentally stable structural domains and offer a unified model for large-scale chromosome structure and function.

Isolation and Characterization of the Novel Bacteriophage AXL3 against Stenotrophomonas maltophilia.

The rapid increase in the number of worldwide human infections caused by the extremely antibiotic resistant bacterial pathogen Stenotrophomonas maltophilia is cause for concern. An alternative treatment solution in the post-antibiotic era is phage therapy, the use of bacteriophages to selectively kill bacterial pathogens. In this study, the novel bacteriophage AXL3 (vB_SmaS-AXL_3) was isolated from soil and characterized. Host range analysis using a panel of 29 clinical S. maltophilia isolates shows successful infection of five isolates and electron microscopy indicates that AXL3 is a member of the Siphoviridae family. Complete genome sequencing and analysis reveals a 47.5 kb genome predicted to encode 65 proteins. Functionality testing suggests AXL3 is a virulent phage and results show that AXL3 uses the type IV pilus, a virulence factor on the cell surface, as its receptor across its host range. This research identifies a novel virulent phage and characterization suggests that AXL3 is a promising phage therapy candidate, with future research examining modification through genetic engineering to broaden its host range.

Novel Stenotrophomonas maltophilia temperate phage DLP4 is capable of lysogenic conversion.

BACKGROUND: Temperate bacteriophages are capable of lysogenic conversion of new bacterial hosts. This phenomenon is often ascribed to "moron" elements that are acquired horizontally and transcribed independently from the rest of the phage genes. Whereas some bacterial species exhibit relatively little prophage-dependent phenotypic changes, other bacterial species such as Stenotrophomonas maltophilia appear to commonly adopt prophage genetic contributions. RESULTS: The novel S. maltophilia bacteriophage DLP4 was isolated from soil using the highly antibiotic-resistant S. maltophilia strain D1585. Genome sequence analysis and functionality testing showed that DLP4 is a temperate phage capable of lysogenizing D1585. Two moron genes of interest, folA (BIT20_024) and ybiA (BIT20_065), were identified and investigated for their putative activities using complementation testing and phenotypic and transcriptomic changes between wild-type D1585 and the D1585::DLP4 lysogen. The gp24 / folA gene encodes dihydrofolate reductase (DHFR: FolA), an enzyme responsible for resistance to the antibiotic trimethoprim. I-TASSER analysis of DLP4 FolA predicted structural similarity to Bacillus anthracis DHFR and minimum inhibitory concentration experiments demonstrated that lysogenic conversion of D1585 by DLP4 provided the host cell with an increase in trimethoprim resistance. The gp65 / ybiA gene encodes N-glycosidase YbiA, which in E. coli BW25113 is required for its swarming motility phenotype. Expressing DLP4 ybiA in strain ybiA770(del)::kan restored its swarming motility activity to wildtype levels. Reverse transcription-PCR confirmed the expression of both of these genes during DLP4 lysogeny. CONCLUSIONS: S. maltophilia temperate phage DLP4 contributes to the antibiotic resistance exhibited by its lysogenized host strain. Genomic analyses can greatly assist in the identification of phage moron genes potentially involved in lysogenic conversion. Further research is required to fully understand the specific contributions temperate phage moron genes provide with respect to the antibiotic resistance and virulence of S. maltophilia host cells.

Regulated large-scale nucleosome density patterns and precise nucleosome positioning correlate with V(D)J recombination.

We show that the physical distribution of nucleosomes at antigen receptor loci is subject to regulated cell type-specific and lineage-specific positioning and correlates with the accessibility of these gene segments to recombination. At the Ig heavy chain locus (IgH), a nucleosome in pro-B cells is generally positioned over each IgH variable (VH) coding segment, directly adjacent to the recombination signal sequence (RSS), placing the RSS in a position accessible to the recombination activating gene (RAG) recombinase. These changes result in establishment of a specific chromatin organization at the RSS that facilitates accessibility of the genomic DNA for the RAG recombinase. In contrast, in mouse embryonic fibroblasts the coding segment is depleted of nucleosomes, which instead cover the RSS, thereby rendering it inaccessible. Pro-T cells exhibit a pattern intermediate between pro-B cells and mouse embryonic fibroblasts. We also find large-scale variations of nucleosome density over hundreds of kilobases, delineating chromosomal domains within IgH, in a cell type-dependent manner. These findings suggest that developmentally regulated changes in nucleosome location and occupancy, in addition to the known chromatin modifications, play a fundamental role in regulating V(D)J recombination. Nucleosome positioning-which has previously been observed to vary locally at individual enhancers and promoters-may be a more general mechanism by which cells can regulate the accessibility of the genome during development, at scales ranging from several hundred base pairs to many kilobases.

Social Network Gap Analysis Evaluation: A Case Study of the Southeastern Health Equity Council.

Despite the growing emphasis on collaboration in public health, there remains a dearth of literature providing tools for the evaluation of coalitions and councils. This study employed social network gap analysis as an evaluation tool. Survey data collected from the Southeastern Health Equity Council members were used to assess connections among members as a whole, by committee, by state, and by health specialty area. Analysis of how well Southeastern Health Equity Council met the representation outlined in its strategic plan was also conducted. Recommendations for improving the network and opportunities to effectively recruit and advance the work of Southeastern Health Equity Council are discussed.

iSeg: an efficient algorithm for segmentation of genomic and epigenomic data.

BACKGROUND: Identification of functional elements of a genome often requires dividing a sequence of measurements along a genome into segments where adjacent segments have different properties, such as different mean values. Despite dozens of algorithms developed to address this problem in genomics research, methods with improved accuracy and speed are still needed to effectively tackle both existing and emerging genomic and epigenomic segmentation problems. RESULTS: We designed an efficient algorithm, called iSeg, for segmentation of genomic and epigenomic profiles. iSeg first utilizes dynamic programming to identify candidate segments and test for significance. It then uses a novel data structure based on two coupled balanced binary trees to detect overlapping significant segments and update them simultaneously during searching and refinement stages. Refinement and merging of significant segments are performed at the end to generate the final set of segments. By using an objective function based on the p-values of the segments, the algorithm can serve as a general computational framework to be combined with different assumptions on the distributions of the data. As a general segmentation method, it can segment different types of genomic and epigenomic data, such as DNA copy number variation, nucleosome occupancy, nuclease sensitivity, and differential nuclease sensitivity data. Using simple t-tests to compute p-values across multiple datasets of different types, we evaluate iSeg using both simulated and experimental datasets and show that it performs satisfactorily when compared with some other popular methods, which often employ more sophisticated statistical models. Implemented in C++, iSeg is also very computationally efficient, well suited for large numbers of input profiles and data with very long sequences. CONCLUSIONS: We have developed an efficient general-purpose segmentation tool and showed that it had comparable or more accurate results than many of the most popular segment-calling algorithms used in contemporary genomic data analysis. iSeg is capable of analyzing datasets that have both positive and negative values. Tunable parameters allow users to readily adjust the statistical stringency to best match the biological nature of individual datasets, including widely or sparsely mapped genomic datasets or those with non-normal distributions.

The spring-loaded genome: nucleosome redistributions are widespread, transient, and DNA-directed.

Nucleosome occupancy plays a key role in regulating access to eukaryotic genomes. Although various chromatin regulatory complexes are known to regulate nucleosome occupancy, the role of DNA sequence in this regulation remains unclear, particularly in mammals. To address this problem, we measured nucleosome distribution at high temporal resolution in human cells at hundreds of genes during the reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV). We show that nucleosome redistribution peaks at 24 h post-KSHV reactivation and that the nucleosomal redistributions are widespread and transient. To clarify the role of DNA sequence in these nucleosomal redistributions, we compared the genes with altered nucleosome distribution to a sequence-based computer model and in vitro-assembled nucleosomes. We demonstrate that both the predicted model and the assembled nucleosome distributions are concordant with the majority of nucleosome redistributions at 24 h post-KSHV reactivation. We suggest a model in which loci are held in an unfavorable chromatin architecture and "spring" to a transient intermediate state directed by DNA sequence information. We propose that DNA sequence plays a more considerable role in the regulation of nucleosome positions than was previously appreciated. The surprising findings that nucleosome redistributions are widespread, transient, and DNA-directed shift the current perspective regarding regulation of nucleosome distribution in humans.

Differential nuclease sensitivity profiling of chromatin reveals biochemical footprints coupled to gene expression and functional DNA elements in maize.

The eukaryotic genome is organized into nucleosomes, the fundamental units of chromatin. The positions of nucleosomes on DNA regulate protein-DNA interactions and in turn influence DNA-templated events. Despite the increasing number of genome-wide maps of nucleosome position, how global changes in gene expression relate to changes in nucleosome position is poorly understood. We show that in nucleosome occupancy mapping experiments in maize (Zea mays), particular genomic regions are highly susceptible to variation introduced by differences in the extent to which chromatin is digested with micrococcal nuclease (MNase). We exploited this digestion-linked variation to identify protein footprints that are hypersensitive to MNase digestion, an approach we term differential nuclease sensitivity profiling (DNS-chip). Hypersensitive footprints were enriched at the 5' and 3' ends of genes, associated with gene expression levels, and significantly overlapped with conserved noncoding sequences and the binding sites of the transcription factor KNOTTED1. We also found that the tissue-specific regulation of gene expression was linked to tissue-specific hypersensitive footprints. These results reveal biochemical features of nucleosome organization that correlate with gene expression levels and colocalize with functional DNA elements. This approach to chromatin profiling should be broadly applicable to other species and should shed light on the relationships among chromatin organization, protein-DNA interactions, and genome regulation.

Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS.

Histone variants are known to play a central role in genome regulation and maintenance. However, many variants are inaccessible by antibody-based methods or bottom-up tandem mass spectrometry due to their highly similar sequences. For many, the only tractable approach is with intact protein top-down tandem mass spectrometry. Here, ultra-high-resolution FT-ICR MS and MS/MS yield quantitative relative abundances of all detected HeLa H2A and H2B isobaric and isomeric variants with a label-free approach. We extend the analysis to identify and relatively quantitate 16 proteoforms from 12 sequence variants of histone H2A and 10 proteoforms of histone H2B from three other cell lines: human embryonic stem cells (WA09), U937, and a prostate cancer cell line LaZ. The top-down MS/MS approach provides a path forward for more extensive elucidation of the biological role of many previously unstudied histone variants and post-translational modifications.

BAF250a Protein Regulates Nucleosome Occupancy and Histone Modifications in Priming Embryonic Stem Cell Differentiation.

The unique chromatin signature of ES cells is fundamental to the pluripotency and differentiation of ES cells. One key feature is the poised chromatin state of master developmental genes that are transcriptionally repressed in ES cells but ready to be activated in response to differentiation signals. Poised chromatin in ES cells contains both H3 Lys-4 trimethylation (H3K4me3) and H3 Lys-27 trimethylation (H3K27me3) methylation, indicating activating and repressing potential. However, the contribution of non-covalent chromatin structure to the poised state is not well understood. To address whether remodeling of nucleosomes is important to the poised state, we characterized the function of BAF250a, a key regulatory subunit of the ES cell ATP-dependent Brahma-associated factor (BAF) chromatin remodeling complex (esBAF). Acute deletion of BAF250a disrupted the differentiation potential of ES cells by altering the expression timing of key developmental genes and pluripotent genes. Our genome-wide nucleosome and histone modification analyses indicated that the disruption of gene expression timing was largely due to changes of chromatin structures at poised genes, particularly those key developmental genes mediated by BAF250a. Specifically, BAF250a deletion caused a nucleosome occupancy increase at H3K4me3- and/or H3K27me3-associated promoters. Moreover, H3K27me3 levels and the number of bivalent promoter genes were reduced in BAF250a KO ES cells. We revealed that BAF250a ablation led to elevated Brg1 but reduced Suz12 recruitment at nucleosome occupancy-increased regions, indicating an unexpected and complicated role of BAF250a in regulating esBAF and Polycomb repressive complex (PRC) activities. Together, our studies identified that BAF250a mediates esBAF and PRC functions to establish the poised chromatin configuration in ES cells, which is essential for the proper differentiation of ES cells.

The isolation and characterization of two Stenotrophomonas maltophilia bacteriophages capable of cross-taxonomic order infectivity.

BACKGROUND: A rapid worldwide increase in the number of human infections caused by the extremely antibiotic resistant bacterium Stenotrophomonas maltophilia is prompting alarm. One potential treatment solution to the current antibiotic resistance dilemma is "phage therapy", the clinical application of bacteriophages to selectively kill bacteria. RESULTS: Towards that end, phages DLP1 and DLP2 (vB_SmaS-DLP_1 and vB_SmaS-DLP_2, respectively) were isolated against S. maltophilia strain D1585. Host range analysis for each phage was conducted using 27 clinical S. maltophilia isolates and 11 Pseudomonas aeruginosa strains. Both phages exhibit unusually broad host ranges capable of infecting bacteria across taxonomic orders. Transmission electron microscopy of the phage DLP1 and DLP2 morphology reveals that they belong to the Siphoviridae family of bacteriophages. Restriction fragment length polymorphism analysis and complete genome sequencing and analysis indicates that phages DLP1 and DLP2 are closely related but different phages, sharing 96.7 % identity over 97.2 % of their genomes. These two phages are also related to P. aeruginosa phages vB_Pae-Kakheti_25 (PA25), PA73, and vB_PaeS_SCH_Ab26 (Ab26) and more distantly related to Burkholderia cepacia complex phage KL1, which together make up a taxonomic sub-family. Phages DLP1 and DLP2 exhibited significant differences in host ranges and growth kinetics. CONCLUSIONS: The isolation and characterization of phages able to infect two completely different species of bacteria is an exciting discovery, as phages typically can only infect related bacterial species, and rarely infect bacteria across taxonomic families, let alone across taxonomic orders.

A general protein O-glycosylation system within the Burkholderia cepacia complex is involved in motility and virulence.

Bacteria of the Burkholderia cepacia complex (Bcc) are pathogens of humans, plants, and animals. Burkholderia cenocepacia is one of the most common Bcc species infecting cystic fibrosis (CF) patients and its carriage is associated with poor prognosis. In this study, we characterized a general O-linked protein glycosylation system in B. cenocepacia K56-2. The PglLBc O-oligosaccharyltransferase (O-OTase), encoded by the cloned gene bcal0960, was shown to be capable of transferring a heptasaccharide from the Campylobacter jejuni N-glycosylation system to a Neisseria meningitides-derived acceptor protein in an Escherichia coli background, indicating that the enzyme has relaxed specificities for both the sugar donor and protein acceptor. In B cenocepacia K56-2, PglLBc is responsible for the glycosylation of 23 proteins involved in diverse cellular processes. Mass spectrometry analysis revealed that these proteins are modified with a trisaccharide HexNAc-HexNAc-Hex, which is unrelated to the O-antigen biosynthetic process. The glycosylation sites that were identified existed within regions of low complexity, rich in serine, alanine, and proline. Disruption of bcal0960 abolished glycosylation and resulted in reduced swimming motility and attenuated virulence towards both plant and insect model organisms. This study demonstrates the first example of post-translational modification in Bcc with implications for pathogenesis.

Burkholderia cepacia complex Phage-Antibiotic Synergy (PAS): antibiotics stimulate lytic phage activity.

The Burkholderia cepacia complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect Burkholderia cenocepacia strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum. B. cenocepacia K56-2-infected Galleria mellonella larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing.

Chromatin-interaction compartment switch at developmentally regulated chromosomal domains reveals an unusual principle of chromatin folding.

Several 400- to 800-kb murine chromosome domains switch from early to late replication during loss of pluripotency, accompanied by a stable form of gene silencing that is resistant to reprogramming. We found that, whereas enhanced nuclease accessibility correlated with early replication genome-wide, domains that switch replication timing during differentiation were exceptionally inaccessible even when early-replicating. Nonetheless, two domains studied in detail exhibited substantial changes in transcriptional activity and higher-order chromatin unfolding confined to the region of replication timing change. Chromosome conformation capture (4C) data revealed that in the unfolded state in embryonic stem cells, these domains interacted preferentially with the early-replicating chromatin compartment, rarely interacting even with flanking late-replicating domains, whereas after differentiation, these same domains preferentially associated with late-replicating chromatin, including flanking domains. In both configurations they retained local boundaries of self-interaction, supporting the replication domain model of replication-timing regulation. Our results reveal a principle of developmentally regulated, large-scale chromosome folding involving a subnuclear compartment switch of inaccessible chromatin. This unusual level of regulation may underlie resistance to reprogramming in replication-timing switch regions.

Aerosol phage therapy efficacy in Burkholderia cepacia complex respiratory infections.

Phage therapy has been suggested as a potential treatment for highly antibiotic-resistant bacteria, such as the species of the Burkholderia cepacia complex (BCC). To address this hypothesis, experimental B. cenocepacia respiratory infections were established in mice using a nebulizer and a nose-only inhalation device. Following infection, the mice were treated with one of five B. cenocepacia-specific phages delivered as either an aerosol or intraperitoneal injection. The bacterial and phage titers within the lungs were assayed 2 days after treatment, and mice that received the aerosolized phage therapy demonstrated significant decreases in bacterial loads. Differences in phage activity were observed in vivo. Mice that received phage treatment by intraperitoneal injection did not demonstrate significantly reduced bacterial loads, although phage particles were isolated from their lung tissue. Based on these data, aerosol phage therapy appears to be an effective method for treating highly antibiotic-resistant bacterial respiratory infections, including those caused by BCC bacteria.