SUMMARY Although the cellular concentration of miRNAs is critical to their function, how miRNA expression and abundance are regulated during ontogeny is unclear. We applied miRNA-, mRNA-, and ChIP-Seq to characterize the microRNome during lymphopoiesis within the context of the transcriptome and epigenome. We show that lymphocyte-specific miRNAs are either tightly controlled by polycomb group-mediated H3K27me3, or maintained in a semi-activated epigenetic state prior to full expression. Because of miRNA biogenesis, the cellular concentration of mature miRNAs does not typically reflect transcriptional changes. However, we uncover a subset of miRNAs for which abundance is dictated by miRNA gene expression. We confirm that concentration of 5p and 3p miRNA strands depends largely on free energy properties of miRNA duplexes. Unexpectedly, we also find that miRNA strand accumulation can be developmentally regulated. Our data provide a comprehensive map of immunity’s microRNome and reveal the underlying epigenetic and transcriptional forces that shape miRNA homeostasis.

The chemical modification of structurally complex fermentation products, a process known as semisynthesis, has been an important tool in the discovery and manufacture of antibiotics for the treatment of various infectious diseases. However, many of the therapeutics obtained in this way are no longer effective, because bacterial resistance to these compounds has developed. Here we present a practical, fully synthetic route to macrolide antibiotics by the convergent assembly of simple chemical building blocks, enabling the synthesis of diverse structures not accessible by traditional semisynthetic approaches. More than 300 new macrolide antibiotic candidates, as well as the clinical candidate solithromycin, have been synthesized using our convergent approach. Evaluation of these compounds against a panel of pathogenic bacteria revealed that the majority of these structures had antibiotic activity, some efficacious against strains resistant to macrolides in current use. The chemistry we describe here provides a platform for the discovery of new macrolide antibiotics and may also serve as the basis for their manufacture.

A mild and efficient ligand-free Suzuki-Miyaura coupling reaction catalyzed by heterogeneous Pd/C was developed. Aryl bromides and triflates undergo the cross-coupling with aryl boronic acids in excellent yields without the presence of any additives in aqueous media at room temperature. Aryl vinyl boronic acids are also applicable to this coupling reaction and provide the trans-stilbene derivatives in high yields. The application of wet-type Pd/C to the coupling reaction was achieved without any loss of activity under aerobic conditions, and the reuse of Pd/C is feasible for a fifth run without significant loss of activity. Inductively coupled plasma (ICP) mass-spectrometric analysis of the filtrate from the reaction mixture of 4-bromonitrobenzene with phenylboronic acid demonstrated that the palladium metal hardly leached into the solution within the limits of the detector (<1 ppm), thus suggesting that the present Suzuki-Miyaura reaction proceeded by heterogeneous catalysis.

d-Alanine-d-alanine ligase (Ddl) is one of the key enzymes in peptidoglycan biosynthesis and is an important target for drug discovery. The enzyme catalyzes the condensation of two d-Ala molecules using ATP to produce d-Ala-d-Ala, which is the terminal peptide of a peptidoglycan monomer. The structures of five forms of the enzyme from Thermus thermophilus HB8 (TtDdl) were determined: unliganded TtDdl (2.3 Å resolution), TtDdl-adenylyl imidodiphosphate (2.6 Å ), TtDdl-ADP (2.2 Å ), TtDdl-ADP-d-Ala (1.9 Å ) and TtDdl-ATP-d-Ala-d-Ala (2.3 Å ). The central domain rotates as a rigid body towards the active site in a cumulative manner in concert with the local conformational change of three flexible loops depending upon substrate or product binding, resulting in an overall structural change from the open to the closed form through semi-open and semi-closed forms. Reaction-intermediate models were simulated using TtDdlcomplex structures and other Ddl structures previously determined by X-ray methods. The catalytic process accompanied by the cumulative conformational change has been elucidated based on the intermediate models in order to provide new insights regarding the details of the catalytic mechanism.

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We have developed a mild and efficient protocol for the ligand-free and heterogeneous Pd/C-catalyzed hetero Suzuki-Miyaura coupling reaction that allows for the synthesis of both heteroaryl-aryl and heteroaryl-heteroaryl derivatives in good to excellent yields.

A ligand-free and heterogeneous palladium on carbon (Pd/C)-catalyzed hetero-SuzukiMiyaura coupling reaction has been developed. The protocol enables the construction of both heterocyclic-alicyclic and heterocyclic-heterocyclic biaryl derivatives in good to excellent yields. Furthermore, Pd/C could be reused. The time-course study clarified that palladium was leached into the reaction media as the reaction proceeded and then completely deposited on the carbon support.

Plasmodium falciparum thymidylate kinase (PfTMK) can tolerate a range of substrates, which distinguishes it from other thymidylate kinases. The enzyme not only phosphorylates TMP and dUMP but can also tolerate bulkier purines, namely, dGMP, GMP, and dIMP. In order to probe the flexibility of PfTMK in accommodating ligands of various sizes, we developed 6 mutant enzymes and subjected these to thermodynamic, inhibitory and catalytic evaluation. Kinase activity was markedly affected by introducing a larger lysine residue instead of A111. The lack of the hydroxyl group after inducing mutation of Y107F affected enzyme activity, and had a more severe impact on dGMP kinase activity. PfTMK can be inhibited by both purine and pyrimidine nucleosides, raising the possibility of developing highly selective drugs. Thermodynamic analysis revealed that enthalpic forces govern both purine and pyrimidine nucleoside monophosphate binding, and the binding affinity of both substrates was highly comparable. The heat produced due to dGMP binding is lower than that attributable to TMP. This indicates that additional interactions occur with TMP, which may be lost with larger dGMP. Targeting PfTMK not only affects thymidine nucleotide synthesis but may also affect purine nucleotides, and thus the enzyme represents an attractive antimicrobial target.