Temporal control of development is an important aspect of pattern formation that awaits complete molecular analysis. We identified lin-57 as a member of the C. elegans heterochronic gene pathway, which ensures that postembryonic developmental events are appropriately timed. Loss of lin-57 function causes the hypodermis to terminally differentiate and acquire adult character prematurely. lin-57 is hbl-1, revealing a role for the worm hunchback homolog in control of developmental time. Significantly, fly hunchback (hb) temporally specifies cell fates in the nervous system. The hbl-1/lin-57 3'UTR is required for postembryonic downregulation in the hypodermis and nervous system and contains multiple putative binding sites for temporally regulated microRNAs, including let-7. Indeed, we find that hbl-1/lin-57 is regulated by let-7, at least in the nervous system. Examination of the hb 3'UTR reveals potential binding sites for known fly miRNAs. Thus, evolutionary conservation of hunchback genes may include temporal control of cell fate specification and microRNA-mediated regulation.

A chemical synthesis platform with broad applications and flexibility was rationally designed to inhibit biogenesis of adhesive pili assembled by the chaperone-usher pathway in Gram-negative pathogens. The activity of a family of bicyclic 2-pyridones, termed pilicides, was evaluated in two different pilus biogenesis systems in uropathogenic Escherichia coli. Hemagglutination mediated by either type 1 or P pili, adherence to bladder cells, and biofilm formation mediated by type 1 pili were all reduced by Ϸ90% in laboratory and clinical E. coli strains. The structure of the pilicide bound to the P pilus chaperone PapD revealed that the pilicide bound to the surface of the chaperone known to interact with the usher, the outer-membrane assembly platform where pili are assembled. Point mutations in the pilicide-binding site dramatically reduced pilus formation but did not block the ability of PapD to bind subunits and mediate their folding. Surface plasmon resonance experiments confirmed that the pilicide interfered with the binding of chaperone-subunit complexes to the usher. These pilicides thus target key virulence factors in pathogenic bacteria and represent a promising proof of concept for developing drugs that function by targeting virulence factors.antimicrobials ͉ chaperone-usher pathway ͉ pilicide ͉ urinary tract infection

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We report a magic angle spinning (MAS) NMR structure of the drug-resistant S31N mutation of M218–60 from Influenza A. The protein was dispersed in diphytanoyl-sn-glycero-3-phosphocholine lipid bilayers, and the spectra and an extensive set of constraints indicate that M218–60 consists of a dimer of dimers. In particular, ~280 structural constraints were obtained using dipole recoupling experiments that yielded well-resolved 13C–15N, 13C–13C, and 1H–15N 2D, 3D, and 4D MAS spectra, all of which show cross-peak doubling. Interhelical distances were measured using mixed 15N/13C labeling and with deuterated protein, MAS at ωr/2π = 60 kHz, ω0H/2π = 1000 MHz, and 1H detection of methyl–methyl contacts. The experiments reveal a compact structure consisting of a tetramer composed of four transmembrane helices, in which two opposing helices are displaced and rotated in the direction of the membrane normal relative to a four-fold symmetric arrangement, yielding a two-fold symmetric structure. Side chain conformations of the important gating and pH-sensing residues W41 and H37 are found to differ markedly from four-fold symmetry. The rmsd of the structure is 0.7 Å for backbone heavy atoms and 1.1 Å for all heavy atoms. This two-fold symmetric structure is different from all of the previous structures of M2, many of which were determined in detergent and/or with shorter constructs that are not fully active. The structure has implications for the mechanism of H+ transport since the distance between His and Trp residues on different helices is found to be short. The structure also exhibits two-fold symmetry in the vicinity of the binding site of adamantyl inhibitors, and steric constraints may explain the mechanism of the drug-resistant S31N mutation.

The Caenorhabditis elegans heterochronic genes control the relative timing and sequence of many events during postembryonic development, including the terminal differentiation of the lateral hypodermis, which occurs during the final (fourth) molt. Inactivation of the heterochronic gene lin-42 causes hypodermal terminal differentiation to occur precociously, during the third molt. LIN-42 most closely resembles the Period family of proteins from Drosophila and other organisms, proteins that function in another type of biological timing mechanism: the timing of circadian rhythms. Per mRNA levels oscillate with an approximately 24-hour periodicity. lin-42 mRNA levels also oscillate, but with a faster rhythm; the oscillation occurs relative to the approximately 6-hour molting cycles of postembryonic development.

The first step in the encounter between a host and a pathogen is attachment to the host epithelium. For uropathogenic Escherichia coli, these interactions are mediated by type 1 and P adhesive pili, which are long (approximately 1 microm) rods composed of more than 1000 protein subunits arranged in a helical structure. Here we used single-molecule atomic force microscopy to study the mechanical properties of type 1 pili. We found that type 1 pili readily extend under an applied force and that this extensibility is the result of unwinding the pilus rod's helical quaternary structure. The forced unraveling is also reversible, with helical rewinding taking place under considerable forces (approximately 60 pN). These data are similar to those obtained on P pili using optical tweezers, indicating that these are conserved properties of uropathogenic E. coli pili. We also show that our data can readily be reproduced using Monte Carlo simulation techniques based on a two-state kinetic model. This model provides a simple way to extrapolate the mechanical behavior of pili under a wide range of forces. We propose that type 1 pilus unraveling is an essential mechanism for absorbing physiological shear forces encountered during urinary tract infections and probably essential for adhesion and colonization of the bladder epithelium.

Birth defects may influence the risk of childhood cancer development through a variety of mechanisms. The rarity of both birth defects and childhood cancers makes it challenging to study these associations, particularly for the very rare instances of each. To address this limitation, the authors conducted a record linkage-based cohort study among Texas children born between 1996 and 2005. Birth defects in the cohort were identified through the Texas Birth Defects Registry, and children who developed cancer were identified by using record linkage with Texas Cancer Registry data. Over 3 million birth records were included; 115,686 subjects had birth defects, and there were 2,351 cancer cases. Overall, children with a birth defect had a 3-fold increased risk of developing cancer (incidence rate ratio (IRR) = 3.05, 95% confidence interval (CI): 2.65, 3.50), with germ cell tumors (IRR = 5.19, 95% CI: 2.67, 9.41), retinoblastomas (IRR = 2.34, 95% CI: 1.21, 4.16), soft-tissue sarcomas (IRR = 2.12, 95% CI: 1.09, 3.79), and leukemias (IRR = 1.39, 95% CI: 1.09, 1.75) having statistically significant elevated point estimates. All birth defect groups except for musculoskeletal had increased cancer incidence. Untangling the strong relation between birth defects and childhood cancers could lead to a better understanding of the genetic and environmental factors that affect both conditions.

SummaryMembrane cofactor protein (MCP) is a complement regulatory protein that is expressed on human cells and cell lines as two relatively broad species with M, of 58,000-68,000 and 48,000-56,000. The structure of a previously reported cDNA clone indicated that MCP was a type 1 membrane glycoprotein and a member of the regulators of complement activation gene/protein cluster. However, it did not provide an explanation for the unusual phenotypic pattern ofMCP. Therefore, in parallel with an analysis of the gene, additional cDNAs were cloned and characterized . Six different MCP cDNA classes were identified. All encode the same 5' untranslated signal peptide, four SCRs, transmembrane domain, and basic amino acid anchor. However, they differ in the length and composition of an extracellular serine/threonine/proline (STP)-rich area, a site of heavy O-glycosylation, and cytoplasmic tail. Analysis of the MCP gene demonstrated that the variation in cDNA structure was a result of alternative splicing. Peripheral blood cells and cell lines predominantly expressed four of the six isoforms. These varied by the presence or absence of an STP-rich segment of 15 amino acids (STPB) and by the use of one of two cytoplasmic domains . Analysis by polymerase chain reaction, Northern blots, and transfection indicated that the predominance of MCP cDNA isoforms with STPB correlated with the high molecular weight protein phenotype, while the predominance of isoforms without STPB correlated with the lower molecular weight phenotype . The expression in a single cell of four distinct protein species with variable STP-rich regions and cytoplasmic tails represents an interesting example of the use of alternative splicing to provide variability in a mammalian protein .