Spatial coupling between DNA replication and mismatch repair in Caulobacter crescentus

The DNA mismatch repair (MMR) process detects and corrects replication errors in organisms ranging from bacteria to humans. In most bacteria, it is initiated by MutS detecting mismatches and MutL nicking the mismatch-containing DNA strand. Here, we show that MMR reduces the appearance of rifampicin resistances more than a 100-fold in the Caulobacter crescentus Alphaproteobacterium. Using fluorescently-tagged and functional MutS and MutL proteins, live cell microscopy experiments showed that MutS is usually associated with the replisome during the whole S-phase of the C. crescentus cell cycle, while MutL molecules may display a more dynamic association with the replisome. Thus, MMR components appear to use a 1D-scanning mode to search for rare mismatches, although the spatial association between MutS and the replisome is dispensible under standard growth conditions. Conversely, the spatial association of MutL with the replisome appears as critical for MMR in C. crescentus, suggesting a model where the β-sliding clamp licences the endonuclease activity of MutL right behind the replication fork where mismatches are generated. The spatial association between MMR and replisome components may also play a role in speeding up MMR and/or in recognizing which strand needs to be repaired in a variety of Alphaproteobacteria.     

The Synthesis of 125I-2-Iodohistamine for Radioimmunoassay1

Abstract

Recently radioactively labeled iodohistamines have been claimed to have superior shelf-life to the iodophenols more commonly used in radioimmunoassay of small molecules. This claim is based largely on theoretical considerations; no systematic study has appeared. We found that iodination of histamine on macroscale proceeds rapidly at pH 7–8.4 to yield principally 2-iodohistamine. With large excess of iodine, substantial diiodi-nation can be achieved. In 0.5 N sodium hydroxide solution, tri-iodination produces 1,2,5-triiodohistamine; the N-I bond, how-ever, is somewhat labile. ¹²⁵I-2,5-Diiodohistamine is also some-what unstable, having first order decomposition rate of 1.6 × 10^?3 day^?1 (t^1/2, 182 days), while ¹²⁵I-2-iodohistamine shows barely perceptible change in 60 days (7.5 × 10^?5 day^?1) The assignment of the first iodine introduced to C-2 is based on a comparison of the NMR spectra of monoiodohistamine and histamine. Iodination with carrier-free iodine-125 using the Hunter-Greenwood procedure (chloramine-T) produces a 76% yield of mono- and a 19% yield of diiodo product which are easily isolable by a single TLC using silica gel in the solvent system, ethano1:ethyl ether:water, 5:5:2.

Nothing has appeared on the chemistry of iodination of histamine. Since the proposal of Jeffcoate et al.⁴ that iodohistamines might be more stable than iodophenols, notably iodotyrosine derivates, a number of investigators have used ¹²⁵I-iodohistamines for the radioimmunoassay of estradiol-17β⁵, progesterone^6–9, norethisterone^6,10,11, testosterone⁶ and bile acids¹². Usually an undefined mixture of iodohistamines has been coupled to the assay ligand.

In this paper we report the preparation of standards (2-, 2,5- and l,2,5-iodohistamines) and the effect of experimental parameters on the synthesis of ¹²⁵I-2-iodohistamine and ²⁵¹I-2,5-diiodohistamine.     

Two new species of Pseudorhabdosynochus Yamaguti, 1958 (Monogenea: Diplectanidae) from the deep-sea grouper Epinephelus morrhua (Val.) (Perciformes: Serranidae) off New Caledonia

Three species of monogeneans were collected from Epinephelus morrhua, a deep-sea grouper from the external slope of the coral reef, off New Caledonia, South Pacific, and are the first parasites recorded from this fish species. Pseudorhabdosynochus morrhua n. sp. is characterised by: a sclerotised vagina with small sclerotised chambers and a short secondary canal; squamodiscs with central rows of rodlets which form closed ovals; and a scaly tegument. P. variabilis n. sp. has: a male quadriloculate organ with a characteristic structure; a sclerotised vagina in which the primary canal, secondary canal and accessory structure are very long and the different parts have various arrangements in different specimens; its squamodiscs have central rows of rodlets which form closed circles; and a smooth tegument. A prominent vaginal structure, comparable to that of P. variabilis, has been found only in P. dolicocolpos Neifar & Euzet, 2007, but the species can be distinguished by details of the vagina and other structures. A species of Haliotrema Johnson & Tiegs, 1922 (Ancyrocephalidae) is reported but not described. Specimens of each of the three species were present in similar numbers.     

LIMLE,a New Molecule Over-Expressed following Activation,Is Involved in the Stimulatory Properties of Dendritic Cells

Dendritic cells are sentinels of the immune system distributed throughout the body, that following danger signals will migrate to secondary lymphoid organs to induce effector T cell responses. We have identified, in a rodent model of graft rejection, a new molecule expressed by dendritic cells that we have named LIMLE (RGD1310371). To characterize this new molecule, we analyzed its regulation of expression and its function. We observed that LIMLE mRNAs were rapidly and strongly up regulated in dendritic cells following inflammatory stimulation. We demonstrated that LIMLE inhibition does not alter dendritic cell maturation or cytokine production following Toll-like-receptor stimulation. However, it reduces their ability to stimulate effector T cells in a mixed leukocyte reaction or T cell receptor transgenic system. Interestingly, we observed that LIMLE protein localized with actin at some areas under the plasma membrane. Moreover, LIMLE is highly expressed in testis, trachea, lung and ciliated cells and it has been shown that cilia formation bears similarities to formation of the immunological synapse which is required for the T cell activation by dendritic cells. Taken together, these data suggest a role for LIMLE in specialized structures of the cytoskeleton that are important for dynamic cellular events such as immune synapse formation. In the future, LIMLE may represent a new target to reduce the capacity of dendritic cells to stimulate T cells and to regulate an immune response.     

Greenhouse gas emissions from dairy manure management: a review of field‐based studies

Livestock manure management accounts for almost 10% of greenhouse gas emissions from agriculture globally, and contributes an equal proportion to the US methane emission inventory. Current emissions inventories use emissions factors determined from small‐scale laboratory experiments that have not been compared to field‐scale measurements. We compiled published data on field‐scale measurements of greenhouse gas emissions from working and research dairies and compared these to rates predicted by the IPCC Tier 2 modeling approach. Anaerobic lagoons were the largest source of methane (368 ± 193 kg CH₄ hd^?1 yr^?1), more than three times that from enteric fermentation (~120 kg CH₄ hd^?1 yr^?1). Corrals and solid manure piles were large sources of nitrous oxide (1.5 ± 0.8 and 1.1 ± 0.7 kg N₂O hd^?1 yr^?1, respectively). Nitrous oxide emissions from anaerobic lagoons (0.9 ± 0.5 kg N₂O hd^?1 yr^?1) and barns (10 ± 6 kg N₂O hd^?1 yr^?1) were unexpectedly large. Modeled methane emissions underestimated field measurement means for most manure management practices. Modeled nitrous oxide emissions underestimated field measurement means for anaerobic lagoons and manure piles, but overestimated emissions from slurry storage. Revised emissions factors nearly doubled slurry CH₄ emissions for Europe and increased N₂O emissions from solid piles and lagoons in the United States by an order of magnitude. Our results suggest that current greenhouse gas emission factors generally underestimate emissions from dairy manure and highlight liquid manure systems as promising target areas for greenhouse gas mitigation.     

Parasite biodiversity in a coral reef fish: twelve species of monogeneans on the gills of the grouper Epinephelus maculatus (Perciformes: Serranidae) off New Caledonia, with a description of eight new species of Pseudorhabdosynochus (Monogenea: Diplectanidae)

Coral reefs are known for their high level of biodiversity, but parasite biodiversity has not been evaluated. Cases such as Epinephelus maculatus, described here, show that the numerical estimation of parasite biodiversity in coral reefs could reach more than ten times the number of fish species; consequently, the extinction of certain fish species from endangered coral reefs would result in the co-extinction of at least ten times the number of parasite species. E. maculatus is a grouper of intermediate size (1-2 kg) and common in the coral reefs of New Caledonia, South Pacific. Based on the examination of more than 800 monogenean specimens, 12 species of monogeneans (ten diplectanids and two ancyrocephalids) were differentiated on the gills. These species of diplectanids have not been found in other epinephelines in the same area and thus are considered as specific to this host. In addition, three species of copepods, and isopod larvae, are present on the gills; E. maculatus thus has a total of 16 species of gill ectoparasites, which can be found together on a single individual fish. Diplectanids include Laticola dae Journo & Justine, 2006, which is the most abundant species representing about 50% of the specimens, and nine species which are rare, each representing 2-7% of the specimens: Diplectanum uitoe n. sp. and eight species of Pseudorhabdosynochus Yamaguti, 1958. D. uitoe, provisionally attributed to Diplectanum Diesing, 1858, is characterised by a small conical penis with internal walls. Pseudorhabdosynochus auitoe n. sp., P. buitoe n. sp., P. cuitoe n. sp., P. duitoe n. sp., P. euitoe n. sp. and P. fuitoe n. sp. are differentiated on the basis of the morphology of the sclerotised vagina, but are very similar in other characteristics; P. guitoe n. sp. is characterised by a quadriloculate organ with very thick walls and a very small sclerotised vagina; and P. huitoe n. sp. is characterised by its sclerotised vagina and by very long ventral and dorsal haptoral bars. Two rare (2-3% of specimens) ancyrocephalids, Haliotrema epinepheli Young, 1969 and Haliotrema sp., are briefly described in relation to the male copulatory organs and haptoral bars; H. epinepheli is apparently a generalist species found in various epinephelines and other fish species. A table of the 50 species of diplectanids (Pseudorhabdosynochus, Laticola Yang et al., 2006, Echinoplectanum Justine & Euzet, 2006 and Diplectanum) from serranids is provided.     

Cryptic species of <Emphasis Type="Italic">Euryakaina</Emphasis> n. g. (Digenea: Cryptogonimidae) from sympatric lutjanids in the Indo-West Pacific

A survey of the endohelminth fauna of Indo-West Pacific Lutjanidae (Perciformes) revealed the presence of the species Siphoderina manilensis (Velasquez, 1961) Miller & Cribb, 2008 and S. marina (Hafeezullah & Siddiqi, 1970) Miller & Cribb, 2008 in seven Lutjanus spp. from sites off the Great Barrier Reef, the Maldives, New Caledonia and Ningaloo Reef, Western Australia. A combination of morphological and ribosomal DNA analyses of these cryptogonimids prompted the transfer of these taxa to a new genus, Euryakaina n. g., as E. manilensis n. comb. and E. marina n. comb., based on comparative analysis with other cryptogonimid taxa. Euryakaina n. g. is distinguished from all other cryptogonimid genera by the combination of a fusiform body, the few relatively small, widely spaced oral spines (sometimes absent), a highly lobed ovary, opposite to slightly oblique testes, vitelline follicles that extend from the anterior margin of the testes to slightly posterior to the intestinal bifurcation, and an excretory vesicle that bifurcates dorsal to the ovary and reunites briefly slightly posterior to the intestinal bifurcation. Morphometric analysis of these taxa alone suggests they should be reduced to synonymy, but DNA sequence analyses and ecological niche partitioning provide evidence that they form a cryptic species complex in sympatric lutjanids in the Indo-West Pacific. The secondary structure of the ITS2 rDNA for species of Euryakaina was also modelled and analysed for the presences of compensatory base changes (CBCs) or hemi-CBCs in order to explore the usefulness of these changes as a tool to help elucidate the taxonomy of this complex system. We also report what we interpret here as intraspecific variation in the ITS2 rDNA between individuals of E. manilensis from Lutjanus vitta recovered off the Great Barrier Reef and New Caledonia.     

Interaction of Crohn's disease susceptibility genes in an Australian paediatric cohort

Genetic susceptibility is an important contributor to the pathogenesis of Crohn''s disease (CD). We investigated multiple CD susceptibility genes in an Australian paediatric onset CD cohort. Newly diagnosed paediatric onset CD patients (n = 72) and controls (n = 98) were genotyped for 34 single nucleotide polymorphisms (SNPs) in 18 genetic loci. Gene-gene interaction analysis, gene-disease phenotype analysis and genetic risk profiling were performed for all SNPs and all genes. Of the 34 SNPs analysed, four polymorphisms on three genes (NOD2, IL23R, and region 3p21) were significantly associated with CD status (p<0.05). All three CD specific paediatric polymorphisms on PSMG1 and TNFRSF6B showed a trend of association with p<0.1. An additive gene-gene interaction involving TLR4, PSMG1, TNFRSF6B and IRGM was identified with CD. Genes involved in microbial processing (TLR4, PSMG1, NOD2) were significantly associated either at the individual level or in gene-gene interactive roles. Colonic disease was significantly associated with disease SNP rs7517847 (IL23R) (p<0.05) and colonic and ileal/colonic disease was significantly associated with disease SNP rs125221868 (IBD5) and SLC22A4 & SLC22A4/5 variants (p<0.05). We were able to demonstrate genetic association of several genes to CD in a paediatric onset cohort. Several of the observed associations have not been reported previously in association with paediatric CD patients. Our findings demonstrate that CD genetic susceptibility in paediatric patients presents as a complex interaction between numerous genes.     

Dynamic Conduction and Repolarisation Changes in Early Arrhythmogenic Right Ventricular Cardiomyopathy versus Benign Outflow Tract Ectopy Demonstrated by High Density Mapping & Paced Surface ECG Analysis

Aims

The concealed phase of arrhythmogenic right ventricular cardiomyopathy (ARVC) may initially manifest electrophysiologically. No studies have examined dynamic conduction/repolarization kinetics to distinguish benign right ventricular outflow tract ectopy (RVOT ectopy) from ARVC''s early phase. We investigated dynamic endocardial electrophysiological changes that differentiate early ARVC disease expression from RVOT ectopy.

Methods

22 ARVC (12 definite based upon family history and mutation carrier status, 10 probable) patients without right ventricular structural anomalies underwent high-density non-contact mapping of the right ventricle. These were compared to data from 14 RVOT ectopy and 12 patients with supraventricular tachycardias and normal hearts. Endocardial & surface ECG conduction and repolarization parameters were assessed during a standard S₁-S₂ restitution protocol.

Results

Definite ARVC without RV structural disease could not be clearly distinguished from RVOT ectopy during sinus rhythm or during steady state pacing. Delay in Activation Times at coupling intervals just above the ventricular effective refractory period (VERP) increased in definite ARVC (43±20 ms) more than RVOT ectopy patients (36±14 ms, p = 0.03) or Normals (25±16 ms, p = 0.008) and a progressive separation of the repolarisation time curves between groups existed. Repolarization time increases in the RVOT were also greatest in ARVC (definite ARVC: 18±20 ms; RVOT ectopy: 5±14, Normal: 1±18, p<0.05). Surface ECG correlates of these intracardiac measurements demonstrated an increase of greater than 48 ms in stimulus to surface ECG J-point pre-ERP versus steady state, with an 88% specificity and 68% sensitivity in distinguishing definite ARVC from the other groups. This technique could not distinguish patients with genetic predisposition to ARVC only (probable ARVC) from controls.

Conclusions

Significant changes in dynamic conduction and repolarization are apparent in early ARVC before detectable RV structural abnormalities, and were present to a lesser degree in probable ARVC patients. Investigation of dynamic electrophysiological parameters may be useful to identify concealed ARVC in patients without disease pedigrees by using endocardial electrogram or paced ECG parameters.     

Exosomal Lipids Impact Notch Signaling and Induce Death of Human Pancreatic Tumoral SOJ-6 Cells

Exosomes are of increasing interest as alternative mode of cell-to-cell communication. We previously reported that exosomes secreted by human SOJ-6 pancreatic tumor cells induce (glyco)protein ligand-independent cell death and inhibit Notch-1 pathway, this latter being particularly active during carcinogenesis and in cancer stem cells. Therefore, we asked whether exosomal lipids were key-elements for cell death and hypothesized that cholesterol-rich membrane microdomains were privileged sites of exosome interactions with tumor cells. To address these questions and based on the lipid composition of exosomes from SOJ-6 cells (Ristorcelli et al. (2008) FASEB J. 22; 3358–3369) enriched in cholesterol and sphingomyelin (lipids forming liquid-ordered phase, Lo) and depleted in phospholipids (lipids forming liquid-disordered phase, Ld), we designed Synthetic Exosome-Like Nanoparticles (SELN) with ratios Lo/Ld from 3.0 to 6.0 framing that of SOJ-6 cell exosomes. SELN decreased tumor cell survival, the higher the Lo/Ld ratio, the lower the cell survival. This decreased survival was due to activation of cell death with inhibition of Notch pathway. FRET analyses indicated fusions/exchanges of SELN with cell membranes. Fluorescent SELN co-localized with the ganglioside GM1 then with Rab5A, markers of lipid microdomains and of early endosomes, respectively. These interactions occurred at lipid microdomains of plasma and/or endosome membranes where the Notch-1 pathway matures. We thus demonstrated a major role for lipids in interactions between SELN and tumor cells, and in the ensued cell death. To our knowledge this is the first report on such effects of lipidic nanoparticles on tumor cell behavior. This may have implications in tumor progression.