The genomic region of the 3' untranslated region (3'UTR) of PHO84, rather than the antisense RNA, promotes gene repression.

PHO84 is a budding yeast gene reported to be negatively regulated by its cognate antisense transcripts both in cis and in trans. In this study, we performed Transient-transcriptome sequencing (TT-seq) to investigate the correlation of sense/antisense pairs in a dbp2Δ strain and found over 700 sense/antisense pairs, including PHO84, to be positively correlated, contrasting the prevailing model. To define what mechanism regulates the PHO84 gene and how this regulation could have been originally attributed to repression by the antisense transcript, we conducted a series of molecular biology and genetics experiments. We now report that the 3' untranslated region (3'UTR) of PHO84 plays a repressive role in sense expression, an activity not linked to the antisense transcripts. Moreover, we provide results of a genetic screen for 3'UTR-dependent repression of PHO84 and show that the vast majority of identified factors are linked to negative regulation. Finally, we show that the PHO84 promoter and terminator form gene loops which correlate with transcriptional repression, and that the RNA-binding protein, Tho1, increases this looping and the 3'UTR-dependent repression. Our results negate the current model for antisense non-coding transcripts of PHO84 and suggest that many of these transcripts are byproducts of open chromatin.

The interaction between the Dbf4 ortholog Chiffon and Gcn5 is conserved in Dipteran insect species.

Chiffon is the sole Drosophila ortholog of Dbf4, the regulatory subunit for the cell-cycle kinase Cdc7 that initiates DNA replication. In Drosophila, the chiffon gene encodes two polypeptides with independent activities. Chiffon-A contains the conserved Dbf4 motifs and interacts with Cdc7 to form the Dbf4-dependent Kinase (DDK) complex, which is essential for a specialized form of DNA replication. In contrast, Chiffon-B binds the histone acetyltransferase Gcn5 to form the Chiffon histone acetyltransferase (CHAT) complex, which is necessary for histone H3 acetylation and viability. Previous studies have shown that the Chiffon-B region is only present within insects. However, it was unclear how widely the interaction between Chiffon-B and Gcn5 was conserved among insect species. To examine this, we performed yeast two-hybrid assays using Chiffon-B and Gcn5 from a variety of insect species and found that Chiffon-B and Gcn5 interact in Diptera species such as Australian sheep blowfly and yellow fever mosquito. Protein domain analysis identified that Chiffon-B has features of acidic transcriptional activators such as Gal4 or VP16. We propose that the CHAT complex plays a critical role in a biological process that is unique to Dipterans and could therefore be a potential target for pest control strategies.

The balancing act of R-loop biology: The good, the bad, and the ugly.

An R-loop is a three-stranded nucleic acid structure that consists of a DNA:RNA hybrid and a displaced strand of DNA. R-loops occur frequently in genomes and have significant physiological importance. They play vital roles in regulating gene expression, DNA replication, and DNA and histone modifications. Several studies have uncovered that R-loops contribute to fundamental biological processes in various organisms. Paradoxically, although they do play essential positive functions required for important biological processes, they can also contribute to DNA damage and genome instability. Recent evidence suggests that R-loops are involved in a number of human diseases, including neurological disorders, cancer, and autoimmune diseases. This review focuses on the molecular basis for R-loop-mediated gene regulation and genomic instability and briefly discusses methods for identifying R-loops in vivo It also highlights recent studies indicating the role of R-loops in DNA double-strand break repair with an updated view of much-needed future goals in R-loop biology.

Antibacterial nanotruffles for treatment of intracellular bacterial infection.

Bacterial pathogens residing in host macrophages in intracellular infections are hard to eradicate because traditional antibiotics do not readily enter the cells or get eliminated via efflux pumps. To overcome this challenge, we developed a new particle formulation with a size amenable to selective macrophage uptake, loaded with two antibacterial agents - pexiganan and silver (Ag) nanoparticles. Here, pexiganan was loaded in 600 nm poly(lactic-co-glycolic acid) (PLGA) particles (NP), and the particle surface was modified with an iron-tannic acid supramolecular complex (pTA) that help attach Ag nanoparticles. PLGA particles coated with Ag (NP-pTA-Ag) were taken up by macrophages, but not by non-phagocytic cells, such as fibroblasts, reducing non-specific toxicity associated with Ag nanoparticles. NP-pTA-Ag loaded with pexiganan (Pex@NP-pTA-Ag) showed more potent antibacterial activity against various intracellular pathogens than NP-pTA-Ag or Pex@NP (pexiganan-loaded NP with no Ag), suggesting a collaborative function between pexiganan and Ag nanoparticles. Mouse whole-body imaging demonstrated that, upon intravenous injection, NP-pTA-Ag quickly accumulated in the liver and spleen, where intracellular bacteria tend to reside. These results support that Pex@NP-pTA-Ag is a promising strategy for the treatment of intracellular bacterial infection.

Biphenylthiazole antibiotics with an oxadiazole linker: An approach to improve physicochemical properties and oral bioavailability.

A new series of oxadiazolylbiphenylthiazoles was prepared with the objective of improving the limited solubility of first-generation derivatives while maintaining antibacterial activity against drug-resistant Staphylococcus aureus. Studying the structure-activity relationship at the cationic part provided the piperazine-1-carboximidamide derivative 27 with a MIC (MRSA) value of 1.1 µg/mL, bactericidal mode of action, and a 50-fold improvement in aqueous solubility. Additionally, 27 exhibited a wider safety margin against mammalian cells, and most importantly, a significant improvement in oral bioavailability.

Discovery of a Novel Dibromoquinoline Compound Exhibiting Potent Antifungal and Antivirulence Activity That Targets Metal Ion Homeostasis.

Globally, invasive fungal infections pose a significant challenge to modern human medicine due to the limited number of antifungal drugs and the rise in resistance to current antifungal agents. A vast majority of invasive fungal infections are caused by species of Candida, Cryptococcus, and Aspergillus. Novel antifungal molecules consisting of unexploited chemical scaffolds with a unique mechanism are a pressing need. The present study identifies a dibromoquinoline compound (4b) with broad-spectrum antifungal activity that inhibits the growth of pertinent species of Candida (chiefly C. albicans), Cryptococcus, and Aspergillus at a concentration of as low as 0.5 µg/mL. Furthermore, 4b, at a subinhibitory concentration, interfered with the expression of two key virulence factors (hyphae and biofilm formation) involved in C. albicans pathogenesis. Three yeast deletion strains ( cox17Δ, ssa1Δ, and aft2Δ) related to metal ion homeostasis were found to be highly sensitive to 4b in growth assays, indicating that the compound exerts its antifungal effect through a unique, previously unexploited mechanism. Supplementing the media with either copper or iron ions reversed the strain sensitivity to 4b, further corroborating that the compound targets metal ion homeostasis. 4b's potent antifungal activity was validated in vivo, as the compound enhanced the survival of Caenorhabditis elegans infected with fluconazole-resistant C. albicans. The present study indicates that 4b warrants further investigation as a novel antifungal agent.

Alkynyl-containing phenylthiazoles: Systemically active antibacterial agents effective against methicillin-resistant Staphylococcus aureus (MRSA).

The promising activity of phenylthiazoles against multidrug-resistant bacterial pathogens, in particular MRSA, has been hampered by their limited systemic applicability, due to their rapid metabolism by hepatic microsomal enzymes, resulting in short half-lives. Here, we investigated a series of phenylthiazoles with alkynyl side-chains that were synthesized with the objective of improving stability to hepatic metabolism, extending the utility of phenylthiazoles from topical applications to treatment of a more invasive, systemic MRSA infections. The most promising compounds inhibited the growth of clinically-relevant isolates of MRSA in vitro at concentrations as low as 0.5 µg/mL, and exerted their antibacterial effect by interfering with bacterial cell wall synthesis via inhibition of undecaprenyl diphosphate synthase and undecaprenyl diphosphate phosphatase. We also identified two phenylthiazoles that successfully eradicated MRSA inside infected macrophages. In vivo PK analysis of compound 9 revealed promising stability to hepatic metabolism with a biological half-life of ∼4.5 h. In mice, compound 9 demonstrated comparable potency to vancomycin, and at a lower dose (20 mg/kg versus 50 mg/kg), in reducing the burden of MRSA in a systemic, deep-tissue infection, using the neutropenic mouse thigh-infection model. Compound 9 thus represents a new phenylthiazole lead for the treatment of MRSA infections that warrants further development.

Aryl-alkyl-lysines: Membrane-Active Fungicides That Act against Biofilms of Candida albicans.

Mortality due to pathogenic fungi has been exacerbated by the rapid development of resistance to frontline antifungal drugs. Fungicidal compounds with novel mechanisms of action are urgently needed. Aryl-alkyl-lysines, which are membrane-active small molecules, were earlier shown to be broad-spectrum antibacterial agents with potency in vitro and in vivo. Herein, we report the antifungal properties of aryl-alkyl-lysines. After identifying the most active compound (NCK-10), we tested its activity against a panel of clinically relevant pathogenic fungi and examined NCK-10's effect against immature and mature biofilms of Candida albicans. NCK-10 was capable of inhibiting the growth of various species of fungi (including Candida spp., Cryptococcus spp., and Aspergillus fumigatus) at concentrations similar to those of antifungal drugs used clinically. It was observed that polarization and permeability of the fungal cell membrane were compromised upon addition of NCK-10, indicating its mechanism is disruption of the fungal cell membrane. In addition to interfering with the growth of planktonic fungi, NCK-10 demonstrated the ability to both inhibit biofilm formation and reduce the metabolic activity of cells in C. albicans biofilm. Additionally, our compound was capable of crossing the blood-brain barrier in an in vitro model, expanding the potential antifungal applications for NCK-10. Overall, aryl-alkyl-lysines were found to be excellent compounds that warrant further investigation as novel antifungal agents.

Investigating the Antibacterial Activity of Biphenylthiazoles against Methicillin- and Vancomycin-Resistant Staphylococcus aureus (MRSA and VRSA).

Phenylthiazoles were reported previously as a new scaffold with antibacterial activity against an array of multidrug-resistant staphylococci. However, their promising antibacterial activity was hampered in large part by their short half-life due to excessive hepatic clearance. Close inspection of the structure-activity-relationships (SARs) of the phenylthiazoles revealed two important structural features necessary for antibacterial activity (a nitrogenous and a lipophilic component). Incorporating the nitrogenous part within a pyrimidine ring resulted in analogues with a prolonged half-life, while the biphenyl moiety revealed the most potent analogue 1b. In this study, advantageous moieties have been combined to generate a new hybrid scaffold of 5-pyrimidinylbiphenylthiazole with the objective of enhancing both anti-MRSA activity and drug-like properties. Among the 37 tested biphenylthiazoles, piperazinyl-containing derivatives 10, 30, and 36 were the most potent analogues with MIC values as low as 0.39 µg/mL. Additionally, 36 exhibited significant improvement in stability to hepatic metabolism.

Arylthiazole antibiotics targeting intracellular methicillin-resistant Staphylococcus aureus (MRSA) that interfere with bacterial cell wall synthesis.

The promising antibacterial potency of arylthiazole antibiotics is offset by their limited activity against intracellular bacteria (namely methicillin-resistant Staphylococcus aureus (MRSA)), similar to many clinically-approved antibiotics. The failure to target these hidden pathogens is due to the compounds' lack of proper characteristics to accumulate intracellularly. Fine tuning of the size and polar-surface-area of the linking heteroaromatic ring provided a new series of 5-thiazolylarylthiazoles with balanced properties that allow them to sufficiently cross and accumulate inside macrophages infected with MRSA. The most promising compound 4i exhibited rapid bactericidal activity, good metabolic stability and produced over 80% reduction of intracellular MRSA in infected macrophages.