Fine mapping of the Schnyder’s crystalline corneal dystrophy locus

Schnyders crystalline corneal dystrophy (SCCD) is a rare autosomal dominant eye disease with a spectrum of clinical manifestations that may include bilateral corneal clouding, arcus lipoides, and anterior corneal crystalline cholesterol deposition. We have previously performed a genome-wide linkage analysis on two large Swede-Finn families and mapped the SCCD locus to a 16-cM interval between markers D1S2633 and D1S228 on chromosome 1p36. We have collected 11 additional families from Finland, Germany, Turkey, and USA to narrow the critical region for SCCD. Here, we have used haplotype analysis with densely spaced microsatellite markers in a total of 13 families to refine the candidate interval. A common disease haplotype was observed among the four Swede-Finn families indicating the presence of a founder effect. Recombination results from all 13 families refined the SCCD locus to 2.32 Mbp between markers D1S1160 and D1S1635. Within this interval, identity-by-state was present in all 13 families for two markers D1S244 and D1S3153, further refining the candidate region to 1.58 Mbp.     

N-terminally extended surfactant protein (SP) C isolated from SP-B-deficient children has reduced surface activity and inhibited lipopolysaccharide binding

In both humans and mice, a deficiency of surfactant protein B (SP-B) is associated with a decreased concentration of mature SP-C and accumulation of a larger SP-C peptide, denoted SP-C(i), which is not observed under normal conditions. Isolation of hydrophobic polypeptides from the lungs of children who died with two different SP-B mutations yielded pure SP-C(i) and showed only trace amounts of mature SP-C. Determination of the SP-C(i) covalent structure revealed a 12-residue N-terminal peptide segment, followed by a 35-residue segment that is identical to mature SP-C. The SP-C(i) structure determined herein is similar to that of a proposed late intermediate in the processing of proSP-C, suggesting that SP-C(i) is the immediate precursor of SP-C. In bronchoalveolar lavage fluid from transgenic mice with a focal deficiency of SP-B, SP-C(i) was detected in the biophysically active, large aggregate fraction and was associated with membrane structures that are typical for a large aggregate surfactant. However, unlike SP-C, SP-C(i) exhibited a very poor ability to promote phospholipid adsorption, gave high surface tension during cyclic film compression, and did not bind lipopolysaccharide in vitro. SP-C(i) is thus capable of associating with surfactant lipids, but its N-terminal dodecapeptide segment must be proteolytically removed to generate a biologically functional peptide. The results of this study indicate that the early postnatal fatal respiratory distress seen in SP-B-deficient children is combined with the near absence of active variants of SP-C.     

Choose Your Weaponry: Selective Storage of a Single Toxic Compound,Latrunculin A,by Closely Related Nudibranch Molluscs

Natural products play an invaluable role as a starting point in the drug discovery process, and plants and animals use many interesting biologically active natural products as a chemical defense mechanism against predators. Among marine organisms, many nudibranch gastropods are known to derive defensive metabolites from the sponges they eat. Here we investigated the putative sequestration of the toxic compound latrunculin A—a 16-membered macrolide that prevents actin polymerization within cellular processes—which has been identified from sponge sources, by five closely related nudibranch molluscs of the genus Chromodoris. Only latrunculin A was present in the rim of the mantle of these species, where storage reservoirs containing secondary metabolites are located, whilst a variety of secondary metabolites were found in their viscera. The species studied thus selectively accumulate latrunculin A in the part of the mantle that is more exposed to potential predators. This study also demonstrates that latrunculin-containing sponges are not their sole food source. Latrunculin A was found to be several times more potent than other compounds present in these species of nudibranchs when tested by in vitro and in vivo toxicity assays. Anti-feedant assays also indicated that latrunculin A was unpalatable to rock pool shrimps, in a dose-dependent manner. These findings led us to propose that this group of nudibranchs has evolved means both to protect themselves from the toxicity of latrunculin A, and to accumulate this compound in the mantle rim for defensive purposes. The precise mechanism by which the nudibranchs sequester such a potent compound from sponges without disrupting their own key physiological processes is unclear, but this work paves the way for future studies in this direction. Finally, the possible occurrence of both visual and chemosensory Müllerian mimicry in the studied species is discussed.     

Conserved regulation of MAP kinase expression by PUF RNA-binding proteins

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression.     

Effect of detection heterogeneity in occupancy‐detection models: an experimental test of time‐to‐first‐detection methods

Imperfect detection can bias estimates of site occupancy in ecological surveys but can be corrected by estimating detection probability. Time‐to‐first‐detection (TTD) occupancy models have been proposed as a cost–effective survey method that allows detection probability to be estimated from single site visits. Nevertheless, few studies have validated the performance of occupancy‐detection models by creating a situation where occupancy is known, and model outputs can be compared with the truth. We tested the performance of TTD occupancy models in the face of detection heterogeneity using an experiment based on standard survey methods to monitor koala Phascolarctos cinereus populations in Australia. Known numbers of koala faecal pellets were placed under trees, and observers, uninformed as to which trees had pellets under them, carried out a TTD survey. We fitted five TTD occupancy models to the survey data, each making different assumptions about detectability, to evaluate how well each estimated the true occupancy status. Relative to the truth, all five models produced strongly biased estimates, overestimating detection probability and underestimating the number of occupied trees. Despite this, goodness‐of‐fit tests indicated that some models fitted the data well, with no evidence of model misfit. Hence, TTD occupancy models that appear to perform well with respect to the available data may be performing poorly. The reason for poor model performance was unaccounted for heterogeneity in detection probability, which is known to bias occupancy‐detection models. This poses a problem because unaccounted for heterogeneity could not be detected using goodness‐of‐fit tests and was only revealed because we knew the experimentally determined outcome. A challenge for occupancy‐detection models is to find ways to identify and mitigate the impacts of unobserved heterogeneity, which could unknowingly bias many models.     

HIV Shedding from Male Circumcision Wounds in HIV-Infected Men: A Prospective Cohort Study

BackgroundA randomized trial of voluntary medical male circumcision (MC) of HIV—infected men reported increased HIV transmission to female partners among men who resumed sexual intercourse prior to wound healing. We conducted a prospective observational study to assess penile HIV shedding after MC.ConclusionPenile HIV shedding is significantly reduced after healing of MC wounds. Lower plasma VL is associated with decreased frequency and quantity of HIV shedding from MC wounds. Starting ART prior to MC should be considered to reduce male-to-female HIV transmission risk. Research is needed to assess the time on ART required to decrease shedding, and the acceptability and feasibility of initiating ART at the time of MC.     

Investigation of an F-18 oxytocin receptor selective ligand via PET imaging

The compound 1-(1-(2-(2-(2-fluoroethoxy)-4-(piperidin-4-yloxy)phenyl)acetyl)piperidin-4-yl)-3,4-dihydroquinolin-2(1H)-one (1) was synthesized and positively evaluated in vitro for high potency and selectivity with human oxytocin receptors. The positron emitting analogue, [F-18]1, was synthesized and investigated in vivo via PET imaging using rat and cynomolgus monkey models. PET imaging studies in female Sprague–Dawley rats suggested [F-18]1 reached the brain and accumulated in various regions of the brain, but washed out too rapidly for adequate quantification and localization. In vivo PET imaging studies in a male cynomolgus monkey suggested [F-18]1 had limited brain penetration while specific uptake of radioactivity significantly accumulated within the vasculature of the cerebral ventricles in areas representative of the choroid plexus.     

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.     

Haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26: X-ray crystallographic studies of dehalogenation of brominated substrates

The haloalkane dehalogenases are detoxifying enzymes that convert a broad range of halogenated substrates to the corresponding alcohols. Complete crystal structures of haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), and complexes of LinB with 1,2-propanediol/1-bromopropane-2-ol and 2-bromo-2-propene-1-ol, products of debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, were determined from 1.8 A resolution X-ray diffraction data. Published structures of native LinB and its complex with 1,3-propanediol [Marek et al. (2000) Biochemistry 39, 14082-14086] were reexamined. The full and partial debromination of 1,2-dibromopropane and 2,3-dibromopropene, respectively, conformed to the observed general trend that the sp(3)-hybridized carbon is the predominant electrophilic site for the S(N)2 bimolecular nucleophilic substitution in dehalogenation reaction. The 2-bromo-2-propene-1-ol product of 2,3-dibromopropene dehalogenation in crystal was positively identified by the gas chromatography-mass spectroscopy (GC-MS) technique. The 1,2-propanediol and 1-bromopropane-2-ol products of 1,2-dibromopropane dehalogenation in crystal were also supported by the GC-MS identification. Comparison of native LinB with its complexes showed high flexibility of residues 136-157, in particular, Asp146 and Glu147, from the cap domain helices alpha(4) and alpha(5)('). Those residues were shifted mainly in direction toward the ligand molecules in the complex structures. It seems the cap domain moves nearer to the core squeezing substrate into the active center closer to the catalytic triad. This also leads to slight contraction of the whole complex structures. The flexibility detected by crystallographic analysis is in remarkable agreement with flexibility observed by molecular dynamic simulations.