Biliary lipid secretion in the rat. The uncoupling of biliary cholesterol and phospholipid secretion from bile acid secretion by sulfated glycolithocholic acid

Glycolithocholic acid and its sulfated derivative are major metabolites of the secondary bile acid lithocholic acid in man. Both compounds are known to induce cholestasis in experimental animals. We compared the effects of these endogenous hepatotoxins on bile production and biliary lipid composition in rats with chronic biliary drainage. The compounds were administered enterally at relatively low rates (5-50% of the rats' endogenous bile acid secretion in these experiments) to simulate enterohepatic circulation. Both compounds were substantially secreted into bile (more than 90% of dose); sulfated glycolithocholic acid unchanged and glycolithocholic acid after hepatic hydroxylation predominantly in the form of glyco-beta-muricholic acid (cf. Kuipers et al. (1986) Am. J. Physiol. 251, G189-G194). Neither glycolithocholic acid nor its sulfated derivative affected the biliary excretion of endogenous bile acids or bile flow in these experiments. In spite of this, phospholipid and cholesterol secretion were significantly reduced by sulfated glycolithocholic acid but were not altered by glycolithocholic acid. Phospholipid and cholesterol secretion rapidly decreased to 25 and 50% of their initial values, respectively, at biliary output rates of sulfated glycolithocholic acid up to 2 mumol/h, and did not further decrease when this output was increased to 6 mumol/h. Small unilamellar liposomes consisting of cholesterol, [Me-14C]choline-labeled phosphatidylcholine, phosphatidylserine and [3H]cholesteryl oleate in a 5:4:1:0.1 molar ratio were employed to label intrahepatic lipid pools. Administration of sulfated glycolithocholic acid slightly reduced bile acid synthesis from [3H]cholesteryl oleate, but significantly reduced the biliary secretion of [14C]phospholipid. Glycolithocholic acid did not affect the hepatic processing of liposomal lipids. It is concluded that sulfated glycolithocholic acid at low doses causes the uncoupling of biliary lipid secretion from that of bile acids, which might represent in initiating event in sulfated glycolithocholic acid hepatotoxicity.     

Living on the edge: substrate competition explains loss of robustness in mitochondrial fatty-acid oxidation disorders

Background

Defects in genes involved in mitochondrial fatty-acid oxidation (mFAO) reduce the ability of patients to cope with metabolic challenges. mFAO enzymes accept multiple substrates of different chain length, leading to molecular competition among the substrates. Here, we combined computational modeling with quantitative mouse and patient data to investigate whether substrate competition affects pathway robustness in mFAO disorders.

Results

First, we used comprehensive biochemical analyses of wild-type mice and mice deficient for medium-chain acyl-CoA dehydrogenase (MCAD) to parameterize a detailed computational model of mFAO. Model simulations predicted that MCAD deficiency would have no effect on the pathway flux at low concentrations of the mFAO substrate palmitoyl-CoA. However, high concentrations of palmitoyl-CoA would induce a decline in flux and an accumulation of intermediate metabolites. We proved computationally that the predicted overload behavior was due to substrate competition in the pathway. Second, to study the clinical relevance of this mechanism, we used patients’ metabolite profiles and generated a humanized version of the computational model. While molecular competition did not affect the plasma metabolite profiles during MCAD deficiency, it was a key factor in explaining the characteristic acylcarnitine profiles of multiple acyl-CoA dehydrogenase deficient patients. The patient-specific computational models allowed us to predict the severity of the disease phenotype, providing a proof of principle for the systems medicine approach.

Conclusion

We conclude that substrate competition is at the basis of the physiology seen in patients with mFAO disorders, a finding that may explain why these patients run a risk of a life-threatening metabolic catastrophe.

     

<Emphasis Type="Italic">PLP1</Emphasis> Gene Variation Modulates Leftward and Rightward Functional Hemispheric Asymmetries

Molecular neurobiological factors determining corpus callosum physiology and anatomy have been suggested to be one of the major factors determining functional hemispheric asymmetries. Recently, it was shown that allelic variations in two myelin-related genes, the proteolipid protein 1 gene PLP1 and the contactin 1 gene CNTN1, are associated with differences in interhemispheric integration. Here, we investigated whether three single nucleotide polymorphisms that were associated with interhemispheric integration via the corpus callosum in a previous study also are relevant for functional hemispheric asymmetries. To this end, we tested more than 900 healthy adults with the forced attention dichotic listening task, a paradigm to assess language lateralization and its modulation by cognitive control processes. Moreover, we used the line bisection task, a paradigm to assess functional hemispheric asymmetries in spatial attention. We found that a polymorphism in PLP1, but not CNTN1, was associated with performance differences in both tasks. Both functional hemispheric asymmetries and their modulation by cognitive control processes were affected. These findings suggest that both left and right hemisphere dominant cognitive functions can be modulated by allelic variation in genes affecting corpus callosum structure. Moreover, higher order cognitive processes may be relevant parameters when investigating the molecular basis of hemispheric asymmetries.     

Comparative genomics analysis of Clostridium difficile epidemic strain DH/NAP11/106

Clostridium difficile PCR ribotype 106 (also identified as restriction endonuclease analysis [REA] group DH) recently emerged as the most common strain causing C. difficile infection (CDI) among US adults. We previously identified this strain predominating our pediatric cohort. Pediatric clinical CDI isolates previously characterized by REA underwent antibiotic resistance testing and whole genome sequencing. Of 134 isolates collected from children, 31 (23%) were REA group DH. We performed a comparative genomics analysis to identify DH-associated accessory genes. We identified five DH-associated genes that are associated with virulence in other bacterial species but not previously known to contribute to CDI. These genes are associated with intestinal mucosal adhesion (collagen-binding surface protein), sporulation (sporulation integral membrane protein YtvI), and protection from oxidative stress and foreign DNA (DNA phosphorothioation-dependent restriction proteins, sulfurtransferase, and DNA sulfur modification proteins). The association of these genes was validated in a cohort of 623 publicly available C. difficile sequences, 10 (1.6%) of which were monophyletic to REA group DH through in silico multilocus sequence typing and core genome phylogenetic analysis. Further investigation is required to determine the contribution of these genes to the emergence and virulence of this epidemic strain.     

Self-Enhanced Accumulation of FtsN at Division Sites and Roles for Other Proteins with a SPOR Domain (DamX,DedD, and RlpA) in Escherichia coli Cell Constriction

Of the known essential division proteins in Escherichia coli, FtsN is the last to join the septal ring organelle. FtsN is a bitopic membrane protein with a small cytoplasmic portion and a large periplasmic one. The latter is thought to form an α-helical juxtamembrane region, an unstructured linker, and a C-terminal, globular, murein-binding SPOR domain. We found that the essential function of FtsN is accomplished by a surprisingly small essential domain (^EFtsN) of at most 35 residues that is centered about helix H2 in the periplasm. ^EFtsN contributed little, if any, to the accumulation of FtsN at constriction sites. However, the isolated SPOR domain (^SFtsN) localized sharply to these sites, while SPOR-less FtsN derivatives localized poorly. Interestingly, localization of ^SFtsN depended on the ability of cells to constrict and, thus, on the activity of ^EFtsN. This and other results suggest that, compatible with a triggering function, FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation and that its SPOR domain specifically recognizes some form of septal murein that is only transiently available during the constriction process. SPOR domains are widely distributed in bacteria. The isolated SPOR domains of three additional E. coli proteins of unknown function, DamX, DedD, and RlpA, as well as that of Bacillus subtilis CwlC, also accumulated sharply at constriction sites in E. coli, suggesting that septal targeting is a common property of SPORs. Further analyses showed that DamX and, especially, DedD are genuine division proteins that contribute significantly to the cell constriction process.Bacterial cytokinesis is mediated by a ring-shaped apparatus. Assembly of this septal ring (SR; also called the divisome or septasome) begins at the future site of fission, well before cell constriction initiates, and it remains associated with the leading edge of the invaginating cell envelope until fission is completed. The mature ring in Escherichia coli is made up of at least 10 essential division proteins (FtsA, -B, -I, -K, -L, -N, -Q, -W, and -Z and ZipA), which are each needed to prevent a lethal filamentation phenotype. The first known step in assembly of the division apparatus is polymerization of FtsZ just underneath the cytoplasmic membrane. These polymers are joined by FtsA and ZipA via direct interactions with FtsZ, resulting in an intermediate ring structure (the Z ring), onto which the remaining components assemble in a specific order to form a constriction-competent complex.In addition to the essential SR proteins, a growing number of nonessential proteins that associate with the organelle are being identified. Some of the latter are likely to serve redundant functions, while some may be required only under particular conditions (for reviews on the topic, see references 15, 19, and 25).FtsN belongs to the essential SR proteins and is thought to be the last of this class to join the organelle before the onset of cell constriction (1, 9, 11, 57, 59). It is a type II bitopic transmembrane species of 319 residues with a small cytoplasmic domain (residues 1 to 30), a single transmembrane domain (residues 31 to 54), and a large periplasmic domain (residues 55 to 319) (12) (Fig. ​(Fig.1).1). The periplasmic domain comprises three short regions with an α-helical character that are centered around residues 62 to 67 (H1), 80 to 93 (H2), and 117 to 123 (H3), an unstructured glutamine-rich linker (residues 124 to 242), and a C-terminal globular SPOR domain (residues 243 to 319) that has an affinity for peptidoglycan (55, 60) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.E. coli ftsN locus, FtsN domains, and properties of genetic constructs. Shown are the EZTnKan-2 insertion site in ftsN^slm117 strains and the deletion-replacement in ftsN<>aph (ΔftsN) strains. Numbers refer to the site of insertion (black triangle) or to the base pairs that were replaced with an aph cassette (doubleheaded arrow), counting from the start of ftsN. The domain structure of FtsN is illustrated below the ftsN gene. Indicated are the transmembrane domain (TM; light gray), helices H1, H2, and H3 (black) in the periplasmic juxtamembrane region, and the C-terminal SPOR domain (^SFtsN; dark gray). The small periplasmic peptide that is sufficient for FtsN′s essential function in cell division (^EFtsN [see text]) is indicated with the doubleheaded arrow below the domain structure diagram. Also shown are inserts present on plasmids that produce fusions of various portions of FtsN to GFP or ^TTGFP under the control of the P_lac regulatory region. ^TTGFP-fusions contain the TorA signal peptide (hatched box) that is cleaved upon export to the periplasm via the twin arginine transport (Tat) system. Columns indicate the FtsN residues present in each fusion, whether the fusion could (+) or could not (−) compensate for the absence of native FtsN, and whether it accumulated at constriction sites sharply (+++) or poorly (−−+) or appeared evenly distibuted along the periphery of the cell (−−−).As with most SR proteins, it is unclear what the essential role of FtsN is. The ftsN gene was first identified as a multicopy suppressor of a Ts allele in essential division gene ftsA (11). Elevated levels of FtsN were subsequently found to also suppress some Ts alleles in ftsI, ftsK, and ftsQ (11, 18), and even to allow the propagation of cells with a complete lack of FtsK (22, 26) or of FtsEX (48). Depletion of FtsN allows assembly of all the other known essential components into nonconstricting SRs, but the number of ring structures per unit of cell length in FtsN⁻ filaments is two- to threefold lower than in wild-type (WT) cells (9). Bacterial two-hybrid studies suggest that FtsN interacts with several other SR proteins, including FtsA, FtsI (penicillin-binding protein 3 [PBP3]), FtsQ, FtsW, and MtgA (10, 16, 17, 38). Moreover, it was recently shown that the requirement for FtsN itself can be bypassed in cells producing certain mutant forms of FtsA, which are thought to stabilize the SR to a greater degree than native FtsA (5). These observations are all compatible with a general role of FtsN in stabilizing the ring structure. In addition, it was recently found that FtsN interacts directly with PBP1B, one of the major bifunctional murein synthases in E. coli, and that it can stimulate both its transglycosylase and transpeptidase activities in vitro (46). Thus, in addition to stabilizing the SR, FtsN may have a more specific role in modulating septal murein synthesis. Lastly, based on the fact that FtsN is the last known essential protein to join the SR, it is attractive to speculate the protein plays a role in triggering the constriction phase (10, 25). To what degree any of these proposed functions contribute to the essentiality of FtsN remains unclear.What does seem clear is that the essential activity of FtsN takes place in the periplasm and that residues 139 to 319 are dispensable for its essential function (12, 55). In addition, as residues 1 to 45 are also dispensable for targeting of FtsN to division sites, some portion of the periplasmic domain must also be sufficient to direct the protein to the division apparatus (1).In a genetic screen for synthetic lethality with min (slm) (6, 7), we isolated a mutant strain carrying a transposon insertion in codon 119 of ftsN. The viability of cells containing this severely truncated ftsN^slm117 allele prompted us to better define the functional domains of FtsN, and we did so by studying the properties of fusions between various portions of FtsN to green fluorescent protein (GFP). To sublocalize a subset of these, we took advantage of the ability of the twin arginine transport system (Tat) to export functional and fluorescent GFP fusions into the periplasm, such that their periplasmic localization could be determined in live cells by fluorescence microscopy (6, 8, 50, 54).We show that the essential function of FtsN can be performed by a surprisingly small periplasmic peptide of at most 35 residues that is centered around helix H2 but that this essential domain (^EFtsN) itself is unlikely to contribute much, if anything, to the accumulation of FtsN at constriction sites. On the other hand, the nonessential periplasmic SPOR domain (^SFtsN) localized sharply to these sites by itself, while SPOR-less FtsN derivatives localized poorly, at best. Notably, septal localization of ^SFtsN depended on coproduction of ^EFtsN, in cis or in trans, unless cells were provided with the FtsA^E124A protein (5) to allow constriction to ensue in the complete absence of ^EFtsN. Localization of ^SFtsN also depended on the activity of FtsI (PBP3) and the presence of at least one of the periplasmic murein amidases, AmiA, -B, or -C. The results suggest that FtsN joins the division apparatus in a self-enhancing fashion at the time of constriction initiation, which is compatible with a role of the protein in triggering the constriction phase of the division process. In addition, the results, taken together with earlier biochemical work (44, 46, 55), suggest that ^SFtsN is recruited to some form of septal murein that accumulates only transiently at sites of active constriction.In addition to FtsN, E. coli produces three proteins of unknown function that also bear a C-terminal SPOR domain (PF05036; Pfam 23) (20). Two of these, DamX and DedD, are inner membrane proteins with the same topology as FtsN, while the third, RlpA, is an outer membrane lipoprotein (43, 47, 53). We found that all three also accumulate at septal rings and that each of their SPOR domains act as autonomous septal targeting determinants. Moreover, phenotypes of the mutants indicate that both DamX and DedD contribute to the cell constriction process, leading to classification of these proteins as new nonessential division proteins.A SPOR domain is predicted to be present in at least 1,650 (putative) proteins from over 500 bacterial species (PF05036; Pfam 23) (20), raising the question as to how far SPOR properties have been conserved. We find that the SPOR domain of CwlC, a Bacillus subtilis murein amidase that is active during late stages of sporulation (39, 44), also accumulates sharply at division sites in E. coli.Our results predict that many other bacterial SPOR domain proteins specifically recognize the same or closely related target molecule(s) that accumulates transiently at sites of cell constriction. This is supported by a very recent study showing that SPOR domain proteins from Burkholderia thailandensis, Caulobacter crescentus, and Myxococcus xanthus accumulate at cell constriction sites as well (45).     

Patterns of DNA Barcode Variation in Canadian Marine Molluscs

Background

Molluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area.

Methodology/Principal Findings

This study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0–26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%–46.5%), and showed a significant positive correlation with nearest neighbour distances.

Conclusions/Significance

DNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.     

Comparison of the pathogenic potentials of environmental and clinical vibrio parahaemolyticus strains indicates a role for temperature regulation in virulence

Although the presence of pathogenic Vibrio spp. in estuarine environments of northern New England has been known for some time (C. H. Bartley and L. W. Slanetz, Appl. Microbiol. 21: 965-966, 1971, and K. R. O'Neil, S. H. Jones, and D. J. Grimes, FEMS Microbiol. Lett. 60:163-167, 1990), their virulence and the relative threat they may pose to human health has yet to be evaluated. In this study, the virulence potential of 33 Vibrio parahaemolyticus isolates collected from the Great Bay Estuary of New Hampshire was assessed in comparison to that of clinical strains. The environmental isolates lack thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), which are encoded by tdh and trh, respectively. Though not hemolytic, they do possess putative virulence factors, such type III secretion system 1, and are highly cytotoxic to human gastrointestinal cells. The expression of known and putative virulence-associated traits, including hemolysin, protease, motility, biofilm formation, and cytotoxicity, by clinical reference isolates correlated with increased temperature from 28°C to 37°C. In contrast, the environmental isolates did not induce their putative virulence-associated traits in response to a temperature of 37°C. We further identified a significant correlation between hemolytic activity and growth phase among clinical strains, whereby hemolysin production decreases with increasing cell density. The introduction of a tdh::gfp promoter fusion into the environmental strains revealed that they regulate this virulence-associated gene appropriately in response to temperature, indicating that their existing regulatory mechanisms are primed to manage newly acquired virulence genes.     

Expression of miRNAs in ovine fetal gonads: potential role in gonadal differentiation

Background  

Gonadal differentiation in the mammalian fetus involves a complex dose-dependent genetic network. Initiation and progression of fetal ovarian and testicular pathways are accompanied by dynamic expression patterns of thousands of genes. We postulate these expression patterns are regulated by small non-coding RNAs called microRNAs (miRNAs). The aim of this study was to identify the expression of miRNAs in mammalian fetal gonads using sheep as a model.     

Community-Based Control of the Brown Dog Tick in a Region with High Rates of Rocky Mountain Spotted Fever, 2012–2013

Rocky Mountain spotted fever (RMSF) transmitted by the brown dog tick (Rhipicephalus sanguineus sensu lato) has emerged as a significant public health risk on American Indian reservations in eastern Arizona. During 2003–2012, more than 250 RMSF cases and 19 deaths were documented among Arizona''s American Indian population. The high case fatality rate makes community-level interventions aimed at rapid and sustained reduction of ticks urgent. Beginning in 2012, a two year pilot integrated tick prevention campaign called the RMSF Rodeo was launched in a ∼600-home tribal community with high rates of RMSF. During year one, long-acting tick collars were placed on all dogs in the community, environmental acaricides were applied to yards monthly, and animal care practices such as spay and neuter and proper tethering procedures were encouraged. Tick levels, indicated by visible inspection of dogs, tick traps and homeowner reports were used to monitor tick presence and evaluate the efficacy of interventions throughout the project. By the end of year one, <1% of dogs in the RMSF Rodeo community had visible tick infestations five months after the project was started, compared to 64% of dogs in Non-Rodeo communities, and environmental tick levels were reduced below detectable levels. The second year of the project focused on use of the long-acting collar alone and achieved sustained tick control with fewer than 3% of dogs in the RMSF Rodeo community with visible tick infestations by the end of the second year. Homeowner reports of tick activity in the domestic and peridomestic setting showed similar decreases in tick activity compared to the non-project communities. Expansion of this successful project to other areas with Rhipicephalus-transmitted RMSF has the potential to reduce brown dog tick infestations and save human lives.