NF-YB Regulates Spermatogonial Stem Cell Self-Renewal and Proliferation in the Planarian <i>Schmidtea mediterranea</i>

<div><p>Gametes are the source and carrier of genetic information, essential for the propagation of all sexually reproducing organisms. Male gametes are derived from a progenitor stem cell population called spermatogonial stem cells (SSCs). SSCs give rise to male gametes through the coordination of two essential processes: self-renewal to produce more SSCs, and differentiation to produce mature sperm. Disruption of this equilibrium can lead to excessive proliferation of SSCs, causing tumorigenesis, or can result in aberrant differentiation, leading to infertility. Little is known about how SSCs achieve the fine balance between self-renewal and differentiation, which is necessary for their remarkable output and developmental potential. To understand the mechanisms of SSC maintenance, we examine the planarian homolog of Nuclear Factor Y-B (NF-YB), which is required for the maintenance of early planarian male germ cells. Here, we demonstrate that NF-YB plays a role in the self-renewal and proliferation of planarian SSCs, but not in their specification or differentiation. Furthermore, we characterize members of the NF-Y complex in <i>Schistosoma mansoni</i>, a parasitic flatworm related to the free-living planarian. We find that the function of NF-YB in regulating male germ cell proliferation is conserved in schistosomes. This finding is especially significant because fecundity is the cause of pathogenesis of <i>S</i>. <i>mansoni</i>. Our findings can help elucidate the complex relationship between self-renewal and differentiation of SSCs, and may also have implications for understanding and controlling schistosomiasis.</p></div

<i>NF-YB(RNAi)</i> results in progressive loss of male germ cells in <i>S</i>. <i>mediterranea</i> starting from the stem cell population.

<p><b>(A)</b> Schematic of sexual <i>S</i>. <i>mediterranea</i> and magnified section of a planarian testis lobe showing the location of individual cell types and their corresponding markers. Scale bar, 20 μm. Control (RNAi) and <i>NF-YB(RNAi)</i> animals fixed at 14, 23, 32, and 42 days (4, 6, 8, and 10 feedings of double-stranded RNA (dsRNA), 4–5 days between feedings) following the initiation of RNAi, labeled to detect <b>(B)</b> <i>nanos</i> (spermatogonial stem cells or SSCs), <b>(C)</b> <i>gH4</i> (spermatogonia and neoblasts), <b>(D)</b> <i>tkn-1</i> (spermatocytes), and <b>(E)</b> <i>pka</i> (spermatids). The increasing severity of <i>NF-YB(RNAi)</i> phenotype is evident from the initial loss of the least differentiated male germ cells (SSCs and spermatogonia), followed by the more differentiated male germ cells. The primary cell type being affected at each stage is highlighted with a yellow box. Eventually all male germ cells are lost. The remaining <i>gH4</i>^<i>+</i> cells are neoblasts. The numbers on the figure indicate animals with phenotypes similar to the representative image shown. The remaining animals have either a less severe (similar to the image of the previous RNAi time point) or a more severe (similar to the image of the next RNAi time point) phenotype. Scale bars, 50 μm.</p

<i>Sm-NF-YB(RNAi)</i> results in fewer proliferating male germ cells in the parasite <i>S</i>. <i>mansoni</i>.

<p><b>(A)</b> Illustration of male <i>S</i>. <i>mansoni</i> depicting the location of the testes and whole-mount in situ hybridization (WISH) in male schistosomes showing <i>Sm-NF-YB</i> and <i>Sm-nanos-1</i> expression in testes. <b>(B)</b> Magnified view of the <i>Sm-nanos-1</i> expression in control (RNAi) and <i>Sm-NF-YB(RNAi)</i> animals. <i>Sm-nanos-1</i> expression is not detected in <i>Sm-NF-YB(RNAi)</i> animals. Scale bars, 1 mm. <b>(C-E)</b> EdU labeling of control (RNAi), <i>Sm-NF-YB(RNAi)</i>, and <i>Sm-nanos-1(RNAi)</i> in male schistosomes. <b>(C)</b> There are fewer proliferating cells in the testes at early knockdown time points in <i>Sm-NF-YB(RNAi)</i> and <i>Sm-nanos-1(RNAi)</i> animals. Scale bars, 50 μm. <b>(D)</b> At later stages of <i>Sm-NF-YB(RNAi)</i>, the testis structure disintegrates, most likely due to the loss of cycling male germ cells. Scale bars, 50 μm. <b>(E)</b> Whole-mount animals at later stages of RNAi to show distribution of proliferating cells. There are few or no proliferating cells in the testes of <i>Sm-NF-YB(RNAi)</i> and <i>Sm-nanos-1(RNAi)</i> animals. Scale bars, 1 mm.</p

Trisubstituted Imidazoles as <i>Mycobacterium tuberculosis</i> Glutamine Synthetase Inhibitors

<i>Mycobacterium tuberculosis</i> glutamine synthetase (<i>Mt</i>GS) is a promising target for antituberculosis drug discovery. In a recent high-throughput screening study we identified several classes of <i>Mt</i>GS inhibitors targeting the ATP-binding site. We now explore one of these classes, the 2-<i>tert</i>-butyl-4,5-diarylimidazoles, and present the design, synthesis, and X-ray crystallographic studies leading to the identification of <i>Mt</i>GS inhibitors with submicromolar IC₅₀ values and promising antituberculosis MIC values

Structure Guided Lead Generation for <i>M. tuberculosis</i> Thymidylate Kinase (Mtb TMK): Discovery of 3‑Cyanopyridone and 1,6-Naphthyridin-2-one as Potent Inhibitors

<i>M. tuberculosis</i> thymidylate kinase (Mtb TMK) has been shown in vitro to be an essential enzyme in DNA synthesis. In order to identify novel leads for Mtb TMK, we performed a high throughput biochemical screen and an NMR based fragment screen through which we discovered two novel classes of inhibitors, 3-cyanopyridones and 1,6-naphthyridin-2-ones, respectively. We describe three cyanopyridone subseries that arose during our hit to lead campaign, along with cocrystal structures of representatives with Mtb TMK. Structure aided optimization of the cyanopyridones led to single digit nanomolar inhibitors of Mtb TMK. Fragment based lead generation, augmented by crystal structures and the SAR from the cyanopyridones, enabled us to drive the potency of our 1,6-naphthyridin-2-one fragment hit from 500 μM to 200 nM while simultaneously improving the ligand efficiency. Cyanopyridone derivatives containing sulfoxides and sulfones showed cellular activity against <i>M. tuberculosis</i>. To the best of our knowledge, these compounds are the first reports of non-thymidine-like inhibitors of Mtb TMK