An inducible program of inflammatory gene expression is central to antimicrobial defenses. This response is controlled by a collaboration involving signal-dependent activation of transcription ...factors, transcriptional co-regulators, and chromatin-modifying factors. We have identified a long noncoding RNA (lncRNA) that acts as a key regulator of this inflammatory response. Pattern recognition receptors such as the Toll-like receptors induce the expression of numerous lncRNAs. One of these, lincRNA-Cox2, mediates both the activation and repression of distinct classes of immune genes. Transcriptional repression of target genes is dependent on interactions of lincRNA-Cox2 with heterogeneous nuclear ribonucleoprotein A/B and A2/B1. Collectively, these studies unveil a central role of lincRNA-Cox2 as a broad-acting regulatory component of the circuit that controls the inflammatory response.
Background & Aims The alimentary tract contains a diffuse endocrine system comprising enteroendocrine cells that secrete peptides or biogenic amines to regulate digestion, insulin secretion, food ...intake, and energy homeostasis. Lineage analysis in the stomach revealed that a significant fraction of endocrine cells in the gastric corpus did not arise from Neurogenin3 (Neurog3)-expressing cells, unlike enteroendocrine cells elsewhere in the digestive tract. We aimed to isolate enriched serotonin-secreting and enterochromaffin-like (ECL) cells from the stomach and to clarify their cellular origin. Methods We used Neurogenic differentiation 1 (NeuroD1) and Neurog3 lineage analysis and examined the differentiation of serotonin-producing and ECL cells in stomach tissues of NeuroD1-cre;ROSAtdTom , tryptophan hydroxylase 1 (Tph1)–cyan fluorescent protein (CFP), c-Kitwsh/wsh , and Neurog3Cre;ROSAtdTom mice by immunohistochemistry. We used fluorescence-activated cell sorting to isolate each cell type for gene expression analysis. We also performed RNA sequencing analysis of ECL cells. Results Neither serotonin-secreting nor ECL cells of the corpus arose from cells expressing NeuroD1. Serotonin-secreting cells expressed a number of mast cell genes but not genes associated with endocrine differentiation; they did not develop in c-Kitwsh/wsh mice and were labeled with transplanted bone marrow cells. RNA sequencing analysis of ECL cells revealed high expression levels of many genes common to endocrine cells, including transcription factors, hormones, ion channels, and solute transporters but not markers of bone marrow cells. Conclusions Serotonin-expressing cells of the gastric corpus of mice appear to be bone marrow–derived mucosal mast cells. Gene expression analysis of ECL cells indicated that they are endocrine cells of epithelial origin that do not express the same transcription factors as their intestinal enteroendocrine cell counterparts.
The increasing prevalence of drug-resistant bacterial infections demands the development of new antibacterials that are not subject to existing mechanisms of resistance. Previously, we described ...coumarin-based inhibitors of an underexploited bacterial target, namely the replicative helicase. Here we report the synthesis and evaluation of optimized coumarin-based inhibitors with 9–18-fold increased potency against Staphylococcus aureus (Sa) and Bacillus anthracis (Ba) helicases. Compounds 20 and 22 provided the best potency, with IC50 values of 3 and 1 μM, respectively, against the DNA duplex strand-unwinding activities of both B. anthracis and S. aureus helicases without affecting the single strand DNA-stimulated ATPase activity. Selectivity index (SI = CC50/MIC) values against S. aureus and B. anthracis for compound 20 were 33 and 66 and for compound 22 were 20 and 40, respectively. In addition, compounds 20 and 22 demonstrated potent antibacterial activity against multiple ciprofloxacin-resistant MRSA strains, with MIC values ranging between 0.5 and 4.2 μg/mL.
Natural antisense transcripts (NATs) are a class of long noncoding RNAs (lncRNAs) that are complementary to other protein-coding genes. Although thousands of NATs are encoded by mammalian genomes, ...their functions in innate immunity are unknown. In this study, we identified and characterized a novel NAT, AS-IL1α, which is partially complementary to IL-1α. Similar to IL-1α, AS-IL1α is expressed at low levels in resting macrophages and is induced following infection with Listeria monocytogenes or stimulation with TLR ligands (Pam3CSK4, LPS, polyinosinic-polycytidylic acid). Inducible expression of IL-1α mRNA and protein were significantly reduced in macrophages expressing shRNA that target AS-IL1α. AS-IL1α is located in the nucleus and did not alter the stability of IL-1α mRNA. Instead, AS-IL1α was required for the recruitment of RNA polymerase II to the IL-1α promoter. In summary, our studies identify AS-IL1α as an important regulator of IL-1α transcription during the innate immune response.
There is a general assumption that a more controlled or more focused attentional state is beneficial for most cognitive tasks. However, there has been a growing realization that creative problem ...solving tasks, such as the Remote Associates Task (RAT), may benefit from a less controlled solution approach. To test this hypothesis, in a 2x2 design, we manipulated whether solvers were given the RAT before or after an implicit learning task. We also varied whether they were told to use their gut as part of either initial task. The results suggest that a less analytic approach engendered by a use your gut instruction benefits performance on the RAT for monolingual solvers. The same benefit was not found for bilingual speakers suggesting that more controlled solution processes may be needed when speakers with multiple lexicons perform this task, which relies heavily on accessing common phrases in a particular language.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Gene duplication is a common evolutionary process that leads to the expansion and functional diversification of protein subfamilies. The evolutionary events that cause paralogous proteins to bind ...different protein ligands (functionally diverged interfaces) are investigated and compared to paralogous proteins that bind the same protein ligand (functionally preserved interfaces). We find that functionally diverged interfaces possess more subfamily-specific residues than functionally preserved interfaces. These subfamily-specific residues are usually partially buried at the interface rim and achieve specific binding through optimized hydrogen bond geometries. In addition to optimized hydrogen bond geometries, side-chain modeling experiments suggest that steric effects are also important for binding specificity. Residues that are completely buried at the interface hub are also less conserved in functionally diverged interfaces than in functionally preserved interfaces. Consistent with this finding, hub residues contribute less to free energy of binding in functionally diverged interfaces than in functionally preserved interfaces. Therefore, we propose that protein binding is a delicate balance between binding affinity that primarily occurs at the interface hub and binding specificity that primarily occurs at the interface rim.
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► Functionally diverged interfaces posses unique evolutionary patterns. ► Subfamily-specific residues are typically located at the rim of the interface. ► Subfamily-specific residues have optimized hydrogen bonds. ► Binding energies are reduced at the hub of functionally diverged interfaces.
The type III secretion system (T3SS) is a clinically important virulence mechanism in Pseudomonas aeruginosa that secretes and translocates effector toxins into host cells, impeding the host's rapid ...innate immune response to infection. Inhibitors of T3SS may be useful as prophylactic or adjunctive therapeutic agents to augment the activity of antibiotics in P. aeruginosa infections, such as pneumonia and bacteremia. One such inhibitor, the phenoxyacetamide MBX 1641, exhibits very responsive structure-activity relationships, including striking stereoselectivity, in its inhibition of P. aeruginosa T3SS. These features suggest interaction with a specific, but unknown, protein target. Here, we identify the apparent molecular target by isolating inhibitor-resistant mutants and mapping the mutation sites by deep sequencing. Selection and sequencing of four independent mutants resistant to the phenoxyacetamide inhibitor MBX 2359 identified the T3SS gene pscF, encoding the needle apparatus, as the only locus of mutations common to all four strains. Transfer of the wild-type and mutated alleles of pscF, together with its chaperone and cochaperone genes pscE and pscG, to a ΔpscF P. aeruginosa strain demonstrated that each of the single-codon mutations in pscF is necessary and sufficient to provide secretion and translocation that is resistant to a variety of phenoxyacetamide inhibitor analogs but not to T3SS inhibitors with different chemical scaffolds. These results implicate the PscF needle protein as an apparent new molecular target for T3SS inhibitor discovery and suggest that three other chemically distinct T3SS inhibitors interact with one or more different targets or a different region of PscF.
We previously reported the synthesis and biological activity of a series of cationic bis-indoles with potent, broad-spectrum antibacterial properties. Here, we describe mechanism of action studies to ...test the hypothesis that these compounds bind to DNA and that this target plays an important role in their antibacterial outcome. The results reported here indicate that the bis-indoles bind selectively to DNA at A/T-rich sites, which is correlated with the inhibition of DNA and RNA synthesis in representative Gram-positive (
) and Gram-negative (
) organisms. Further, exposure of
and
to representative bis-indoles resulted in induction of the DNA damage-inducible SOS response. In addition, the bis-indoles were found to be potent inhibitors of cell wall biosynthesis; however, they do not induce the cell wall stress stimulon in
, suggesting that this pathway is inhibited by an indirect mechanism. In light of these findings, the most likely basis for the observed activities of these compounds is their ability to bind to the minor groove of DNA, resulting in the inhibition of DNA and RNA synthesis and other secondary effects.
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