A multi-looping chromatin signature predicts dysregulated gene expression in neurons with familial Alzheimer's disease mutations.

Mammalian genomes fold into tens of thousands of long-range loops, but their functional role and physiologic relevance remain poorly understood. Here, using human post-mitotic neurons with rare familial Alzheimer's disease (FAD) mutations, we identify hundreds of reproducibly dysregulated genes and thousands of miswired loops prior to amyloid accumulation and tau phosphorylation. Single loops do not predict expression changes; however, the severity and direction of change in mRNA levels and single-cell burst frequency strongly correlate with the number of FAD-gained or -lost promoter-enhancer loops. Classic architectural proteins CTCF and cohesin do not change occupancy in FAD-mutant neurons. Instead, we unexpectedly find TAATTA motifs amenable to binding by DLX homeodomain transcription factors and changing noncoding RNAPolII signal at FAD-dynamic promoter-enhancer loops. DLX1/5/6 mRNA levels are strongly upregulated in FAD-mutant neurons coincident with a shift in excitatory-to-inhibitory gene expression and miswiring of multi-loops connecting enhancers to neural subtype genes. DLX1 overexpression is sufficient for loop miswiring in wildtype neurons, including lost and gained loops at enhancers with tandem TAATTA arrays and singular TAATTA motifs, respectively. Our data uncover a genome structure-function relationship between multi-loop miswiring and dysregulated excitatory and inhibitory transcriptional programs during lineage commitment of human neurons homozygously-engineered with rare FAD mutations.

Stiffness and Oligomer Content Affect the Initial Adhesion of Staphylococcus aureus to Polydimethylsiloxane Gels.

The growing prevalence of methicillin-resistant Staphylococcus aureus (S. aureus) infections necessitates a greater understanding of their initial adhesion to medically relevant surfaces. In this study, the influence of the mechanical properties and oligomer content of polydimethylsiloxane (PDMS) gels on the initial attachment of Gram-positive S. aureus was explored. Small-amplitude oscillatory shear rheological measurements were conducted to verify that by altering the base to curing (B:C) ratio of the commonly used Sylgard 184 silicone elastomer kit (B:C ratios of 60:1, 40:1, 10:1, and 5:1), PDMS gels could be synthesized with Young's moduli across four distinct regimes: ultrasoft (15 kPa), soft (30 kPa), standard (400 kPa), and stiff (1500 kPa). These as-prepared gels (unextracted) were compared to gels prepared from the same B/C ratios that underwent Soxhlet extraction to remove any unreacted oligomers. While the Young's moduli of unextracted and extracted PDMS gels prepared from the same B:C ratio were statistically equivalent, the associated adhesion failure energy statistically decreased for the ultrasoft gels after extraction (from 25 to 8 J/mm2). The interactions of these eight well-characterized gels with bacteria were tested by using S. aureus SH1000, a commonly studied laboratory strain, as well as S. aureus ATCC 12600, which was isolated from a human lung infection. Increased S. aureus inactivation occurred only when the bacteria were incubated directly on top of the unextracted gels prepared at high B:C ratios (40:1 and 60:1), whereas none of the extracted gels (no unreacted oligomers) had significant levels of inactivated bacteria. S. aureus adhered the least to the stiffest extracted PDMS gels (no unreacted oligomers) and the most to soft, unextracted PDMS gels (with ∼17% unreacted oligomers). These findings suggest that both unreacted oligomers and Young's moduli are important material factors to consider when exploring the attachment behavior of Gram-positive S. aureus to hydrophobic elastomer gels.

Mesoscale Polymer Surfactants: Photolithographic Production and Localization at Droplet Interfaces.

Stabilization of fluid droplets, classically as oil-in-water or water-in-oil emulsions, is typically conducted using molecular surfactants or small particulates that localize at oil-water interfaces. In this paper, we describe a method whereby thin polymer films are converted photolithographically to ribbon-like mesoscale objects, which, in turn, adsorb to fluid interfaces where they extend as appendages, or arms, from the droplet surface. These "mesoscale polymer surfactants", or MPSs, were prepared from thin polymer films containing reactive functional moieties, including coumarin for photo-cross-linking, triphenylsulfonium for photoacid generation, and tert-butyl ester for solubility switching. The resultant MPSs, prepared initially on Si substrates, were released into water to reveal an exquisite shape sensitivity (forming straight, bent, or helical structures) and affinity for droplet interfaces based on their preparation conditions and the properties of the surrounding liquid. Notably, the lithographic techniques employed were amenable to differentiating the wettability of MPS segments, affording access to diblock-like MPSs which adhered to dispersed droplets via their hydrophobic segments, allowing their hydrophilic segments to extend into the continuous phase.

Discovery of a Diazo-Forming Enzyme in Cremeomycin Biosynthesis.

The molecular architectures and potent bioactivities of diazo-containing natural products have attracted the interest of synthetic and biological chemists. Despite this attention, the biosynthetic enzymes involved in diazo group construction have not been identified. Here, we show that the ATP-dependent enzyme CreM installs the diazo group in cremeomycin via late-stage N-N bond formation using nitrite. This finding should inspire efforts to use diazo-forming enzymes in biocatalysis and synthetic biology as well as enable genome-based discovery of new diazo-containing metabolites.

The Human Microbiota, Infectious Disease, and Global Health: Challenges and Opportunities.

Despite significant advances in treating infectious diseases worldwide, morbidity and mortality associated with pathogen infection remains extraordinarily high and represents a critical scientific and global health challenge. Current strategies to combat these infectious agents include a combination of vaccines, small molecule drugs, increased hygiene standards, and disease-specific interventions. While these approaches have helped to drastically reduce the incidence and number of deaths associated with infection, continued investment in current strategies and the development of novel therapeutic approaches will be required to address these global health threats. Recently, human- and vector-associated microbiotas, the assemblages of microorganisms living on and within their hosts, have emerged as a potentially important factor mediating both infection risk and disease progression. These complex microbial communities are involved in intricate and dynamic interactions with both pathogens as well as the innate and adaptive immune systems of their hosts. Here, we discuss recent findings that have illuminated the importance of resident microbiotas in infectious disease, emphasizing opportunities for novel therapeutic intervention and future challenges for the field. Our discussion will focus on four major global health threats: tuberculosis, malaria, HIV, and enteric/diarrheal diseases. We hope this Perspective will highlight the many opportunities for chemists and chemical biologists in this field as well as inspire efforts to elucidate the mechanisms underlying established disease correlations, identify novel microbiota-based risk factors, and develop new therapeutic interventions.

Heteroatom-Heteroatom Bond Formation in Natural Product Biosynthesis.

Natural products that contain functional groups with heteroatom-heteroatom linkages (X-X, where X = N, O, S, and P) are a small yet intriguing group of metabolites. The reactivity and diversity of these structural motifs has captured the interest of synthetic and biological chemists alike. Functional groups containing X-X bonds are found in all major classes of natural products and often impart significant biological activity. This review presents our current understanding of the biosynthetic logic and enzymatic chemistry involved in the construction of X-X bond containing functional groups within natural products. Elucidating and characterizing biosynthetic pathways that generate X-X bonds could both provide tools for biocatalysis and synthetic biology, as well as guide efforts to uncover new natural products containing these structural features.

The Cremeomycin Biosynthetic Gene Cluster Encodes a Pathway for Diazo Formation.

Diazo groups are found in a range of natural products that possess potent biological activities. Despite longstanding interest in these metabolites, diazo group biosynthesis is not well understood, in part because of difficulties in identifying specific genes linked to diazo formation. Here we describe the discovery of the gene cluster that produces the o-diazoquinone natural product cremeomycin and its heterologous expression in Streptomyces lividans. We used stable isotope feeding experiments and in vitro characterization of biosynthetic enzymes to decipher the order of events in this pathway and establish that diazo construction involves late-stage N-N bond formation. This work represents the first successful production of a diazo-containing metabolite in a heterologous host, experimentally linking a set of genes with diazo formation.

Discovery of the lomaiviticin biosynthetic gene cluster in Salinispora pacifica.

The lomaiviticins are a family of cytotoxic marine natural products that have captured the attention of both synthetic and biological chemists due to their intricate molecular scaffolds and potent biological activities. Here we describe the identification of the gene cluster responsible for lomaiviticin biosynthesis in Salinispora pacifica strains DPJ-0016 and DPJ-0019 using a combination of molecular approaches and genome sequencing. The link between the lom gene cluster and lomaiviticin production was confirmed using bacterial genetics, and subsequent analysis and annotation of this cluster revealed the biosynthetic basis for the core polyketide scaffold. Additionally, we have used comparative genomics to identify candidate enzymes for several unusual tailoring events, including diazo formation and oxidative dimerization. These findings will allow further elucidation of the biosynthetic logic of lomaiviticin assembly and provide useful molecular tools for application in biocatalysis and synthetic biology.

Lomaiviticin biosynthesis employs a new strategy for starter unit generation.

Lomaiviticin biosynthesis is thought to utilize a propionyl starter unit for a type II polyketide synthase (PKS). Discovery of the lomaiviticin (lom) biosynthetic gene cluster suggested an unusual method for starter unit generation involving a bifunctional acyltransferase/decarboxylase (AT/DC) thus far observed only in type I PKS pathways. In vitro biochemical characterization of AT/DC Lom62 confirmed its ability to generate a propionyl-acyl carrier protein (ACP), revealing a new role for this enzymatic activity within natural product biosynthesis.

Ligand mobility modulates immunological synapse formation and T cell activation.

T cell receptor (TCR) engagement induces clustering and recruitment to the plasma membrane of many signaling molecules, including the protein tyrosine kinase zeta-chain associated protein of 70 kDa (ZAP70) and the adaptor SH2 domain-containing leukocyte protein of 76 kDa (SLP76). This molecular rearrangement results in formation of the immunological synapse (IS), a dynamic protein array that modulates T cell activation. The current study investigates the effects of apparent long-range ligand mobility on T cell signaling activity and IS formation. We formed stimulatory lipid bilayers on glass surfaces from binary lipid mixtures with varied composition, and characterized these surfaces with respect to diffusion coefficient and fluid connectivity. Stimulatory ligands coupled to these surfaces with similar density and orientation showed differences in their ability to activate T cells. On less mobile membranes, central supramolecular activation cluster (cSMAC) formation was delayed and the overall accumulation of CD3ζ at the IS was reduced. Analysis of signaling microcluster (MC) dynamics showed that ZAP70 MCs exhibited faster track velocity and longer trajectories as a function of increased ligand mobility, whereas movement of SLP76 MCs was relatively insensitive to this parameter. Actin retrograde flow was observed on all surfaces, but cell spreading and subsequent cytoskeletal contraction were more pronounced on mobile membranes. Finally, increased tyrosine phosphorylation and persistent elevation of intracellular Ca(2+) were observed in cells stimulated on fluid membranes. These results point to ligand mobility as an important parameter in modulating T cell responses.