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The crystal and molecular structures of 5e-androstan-3fl-ol-17-one (epiandrosterone, C19H3002) and 5fl-androstane-30ql7fl-diol (C19H3202) have been determined from three-dimensional X-ray data. The initial phases for these structures were obtained by a least-squares analysis of the structure invariants cos (~oa+~2+~03). Both substances crystallize in space group P2a with two molecules in the unit cell. The unit-cell dimensions are a= 6.596, b=21.521, c= 6.313 A, and fl= 109.36 ° for epiandrosterone and a= 11.875, b = 7.157, c= 10.960 A, and fl= 114.70 ° for 5fl-androstane-3e,17fl-diol. In crystals of these two substances and of androsterone, the C(3) epimer of epiandrosterone, hydrogen bonds form spirals of molecules parallel to the symmetry axes and cause the packing and orientation of the epiandrosterone molecules within the unit cell to be different from the molecular stacking in androsterone and 5/?-androstane-3e,17fl-diol crystals. A detailed discussion of steroids having unsaturated side-chain linkage to the D ring at C(17) is presented.

Thousands of long noncoding RNAs are encoded in mammalian genomes, yet most remain uncharacterized. In this study, we functionally characterized a mouse long noncoding RNA named U90926. Analysis of U90926 RNA levels revealed minimal expression across multiple tissues at steady state. However, the expression of this gene was highly induced in macrophages and dendritic cells by TLR activation, in a p38 MAPK- and MyD88-dependent manner. To study the function of U90926, we generated U90926-deficient (U9-KO) mice. Surprisingly, we found minimal effects of U90926 deficiency in cultured macrophages. Given the lack of macrophage-intrinsic effect, we investigated the subcellular localization of U90926 transcript and its protein-coding potential. We found that U90926 RNA localizes to the cytosol, associates with ribosomes, and contains an open reading frame that encodes a novel glycosylated protein (termed U9-ORF), which is secreted from the cell. An in vivo model of endotoxic shock revealed that, in comparison with wild type mice, U9-KO mice exhibited increased sickness responses and mortality. Mechanistically, serum levels of IL-6 were elevated in U9-KO mice, and IL-6 neutralization improved endotoxemia outcomes in U9-KO mice. Taken together, these results suggest that U90926 expression is protective during endotoxic shock, potentially mediated by the paracrine and/or endocrine actions of the novel U9-ORF protein secreted by activated myeloid cells.

The nosocomial pathogen, Clostridioides difficile, is a spore-forming obligate anaerobe that depends on its aerotolerant spore form to transmit infections. Functional spore formation depends on the assembly of a proteinaceous layer known as the coat around the developing spore. In C. difficile, coat assembly depends on the conserved spore protein, SpoIVA, and the clostridial-specific spore protein, SipL, which directly interact. Mutations that disrupt their interaction cause coat to mislocalize and impair spore formation. In B. subtilis, SpoIVA is an ATPase that uses ATP hydrolysis to drive its polymerization around the forespore. Loss of SpoIVA ATPase activity impairs B. subtilis SpoIVA encasement of the forespore and activates a quality control mechanism that eliminates these defective cells. Since this mechanism is lacking in C. difficile, we tested whether mutations in C. difficile's SpoIVA ATPase motifs impact functional spore formation. Disrupting C. difficile SpoIVA ATPase motifs resulted in phenotypes that were typically >104 less severe than the equivalent mutations in B. subtilis. Interestingly, mutation of ATPase motif residues predicted to abrogate SpoIVA binding to ATP decreased SpoIVA-SipL interaction, whereas mutation of ATPase motif residues predicted to disrupt ATP hydrolysis but maintain ATP binding enhanced SpoIVA-SipL interaction. When a sipL mutation known to reduce binding to SpoIVA was combined with a spoIVA mutation predicted to prevent SpoIVA binding to ATP, spore formation was severely exacerbated. Since this phenotype is allele-specific, our data implies that SipL recognizes the ATP-bound form of SpoIVA and highlights the importance of this interaction for functional C. difficile spore formation. Importance The major pathogen Clostridioides difficile depends on its spore form to transmit disease. However, the mechanism by which C. difficile assembles spores remains poorly characterized. We previously showed that binding between the spore morphogenetic proteins, SpoIVA and SipL, regulates assembly of the protective coat layer around the forespore. In this study, we determined that mutations in C. difficile SpoIVA's ATPase motifs result in relatively minor defects in spore formation in contrast with Bacillus subtilis. Nevertheless, our data suggest that SipL preferentially recognizes the ATP-bound form of SpoIVA and identify a specific residue in SipL's C-terminal LysM domain that is critical for recognizing the ATP-bound form of SpoIVA. These findings advance our understanding of how SpoIVA-SipL interactions regulate C. difficile spore assembly.

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