Molecular Diagnosis of an Ocular Toxocariasis Patient in Vietnam

An ocular Toxocara canis infection is reported for the first time in Vietnam. A 34-year-old man residing in a village of Son La Province, North Vietnam, visited the National Eye Hospital (NEH) in August 2011. He felt a bulge-sticking pain in his left eye and loss of vision occurred over 3 months before visiting the hospital. The eye examination in the hospital showed damage of the left eye, red eye, retinal fibrosis, retinal detachment, inflammation of the eye tissues, retinal granulomas, and a parasitic cyst inside. A larva of Toxocara was collected with the cyst by a medical doctor by surgery. Comparison of 264 nucleotides of internal transcribed spacer 2 (ITS2) of ribosomal DNA was done between our Vietnamese Toxocara canis and other Toxocara geographical isolates, including Chinese T. canis, Japanese T. canis, Sri Lankan T. canis, and Iranian T. canis. The nucleotide homology was 97-99%, when our T. canis was compared with geographical isolates. Identification of a T. canis infection in the eye by a molecular method was performed for the first time in Vietnam.     

Lipid-regulated degradation of HMG-CoA reductase and Insig-1 through distinct mechanisms in insect cells

In mammalian cells, levels of the integral membrane proteins 3-hydroxy-3-methylglutaryl-CoA reductase and Insig-1 are controlled by lipid-regulated endoplasmic reticulum-associated degradation (ERAD). The ERAD of reductase slows a rate-limiting step in cholesterol synthesis and results from sterol-induced binding of its membrane domain to Insig-1 and the highly related Insig-2 protein. Insig binding bridges reductase to ubiquitin ligases that facilitate its ubiquitination, thereby marking the protein for cytosolic dislocation and proteasomal degradation. In contrast to reductase, Insig-1 is subjected to ERAD in lipid-deprived cells. Sterols block this ERAD by inhibiting Insig-1 ubiquitination, whereas unsaturated fatty acids block the reaction by preventing the protein''s cytosolic dislocation. In previous studies, we found that the membrane domain of mammalian reductase was subjected to ERAD in Drosophila S2 cells. This ERAD was appropriately accelerated by sterols and required the action of Insigs, which bridged reductase to a Drosophila ubiquitin ligase. We now report reconstitution of mammalian Insig-1 ERAD in S2 cells. The ERAD of Insig-1 in S2 cells mimics the reaction that occurs in mammalian cells with regard to its inhibition by either sterols or unsaturated fatty acids. Genetic and pharmacologic manipulations coupled with subcellular fractionation indicate that Insig-1 and reductase are degraded through distinct mechanisms that are mediated by different ubiquitin ligase complexes. Together, these results establish Drosophila S2 cells as a model system to elucidate mechanisms through which lipid constituents of cell membranes (i.e., sterols and fatty acids) modulate the ERAD of Insig-1 and reductase.     

Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold “tetramer”. Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance.     

Polychlorinated biphenyls disrupt cell division and tip growth in two species of fucoid algae

Environmental contaminants, including poly‐chlorinated biphenyls (PCBs), are enriched in coastal sediments, and despite a 1977 moratorium by the United States Environmental Protection Agency on the production of PCBs, levels remain high, more so near former industrial plants. The effects of these contaminants on sessile species in the intertidal zone, particularly nonanimal species such as the ubiquitous fucoid brown algae, are not well known. We investigated the developmental effects of chronic PCB treatment beginning at fertilization on two species of marine rockweed, Fucus vesiculosus Linnaeus and Silvetia compressa (J.Agardh) E.Serrão, T.O.Cho, S.M.Boo & Brawley. A mixture of the most widely used PCB congeners, Aroclors 1221, 1242, and 1254, was delivered at concentrations well below levels found in contaminated sediments, and resulted in severely delayed mitosis and cytokinesis in both species. In F. vesiculosus, this delay was accompanied by abnormal spindle morphology. PCB treatment also dramatically slowed or arrested rhizoid growth after 2–4 d, and by 7 d F. vesiculosus embryos were dead; in contrast, polar secretion of adhesive, germination, and photopolar germination were not affected. The dramatic delay in the first cell division and reduction in tip growth within the first week of development are likely to compromise S. compressa's ability to reproduce and establish new generations. Thus, the data presented here suggest that PCBs still present in coastal sediments may be inhibiting recruitment in these species. Moreover, as sediment dredging causes temporary spikes in PCB concentrations, these kinds of bioremediation steps may exacerbate the disruption of fucoid development.     

Cytomegalovirus Vaccine Strain Towne-Derived Dense Bodies Induce Broad Cellular Immune Responses and Neutralizing Antibodies That Prevent Infection of Fibroblasts and Epithelial Cells

Human cytomegalovirus (HCMV), a betaherpesvirus, can cause severe disease in immunosuppressed patients and following congenital infection. A vaccine that induces both humoral and cellular immunity may be required to prevent congenital infection. Dense bodies (DBs) are complex, noninfectious particles produced by HCMV-infected cells and may represent a vaccine option. As knowledge of the antigenicity and immunogenicity of DB is incomplete, we explored characterization methods and defined DB production methods, followed by systematic evaluation of neutralization and cell-mediated immune responses to the DB material in BALB/c mice. DBs purified from Towne-infected cultures treated with the viral terminase inhibitor 2-bromo-5,6-dichloro-1-beta-d-ribofuranosyl benzimidazole riboside (BDCRB) were characterized by nanoparticle tracking analysis (NTA), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), immunoblotting, quantitative enzyme-linked immunosorbent assay, and other methods. The humoral and cellular immune responses to DBs were compared to the immunogenicity of glycoprotein B (gB) administered with the adjuvant AddaVax (gB/AddaVax). DBs induced neutralizing antibodies that prevented viral infection of cultured fibroblasts and epithelial cells and robust cell-mediated immune responses to multiple viral proteins, including pp65, gB, and UL48. In contrast, gB/AddaVax failed to induce neutralizing antibodies that prevented infection of epithelial cells, highlighting a critical difference in the humoral responses induced by these vaccine candidates. Our data advance the potential for the DB vaccine approach, demonstrate important immunogenicity properties, and strongly support the further evaluation of DBs as a CMV vaccine candidate.     

Cloning and Expression of Genes Encoding F107-C and K88-1NT Fimbrial Proteins of Enterotoxigenic Escherichia coli from Piglets

We cloned two genes coding F107-C and K88-1NT fimbrial subunits from strains E. coli C and 1NT isolated from Thua Thien Hue province, Vietnam. The mature peptide of faeG gene from strain E. coli 1NT (called faeG-1NT) is 100 % similarity with faeG gene, while the CDS of fedA gene from strain C (called fedA-C) has a similarity of 97 % with the fedA gene. Expression of the faeG-1NT and fedA-C genes in E. coli BL21 Star™ (DE3) produced proteins of ~31 and 22 kDa, respectively. The effect of IPTG concentration on the K88-1NT and F107-C fimbriae production was investigated. The results showed that 0.5 mM IPTG is suitable for higher expression of K88-1NT subunit, while 0.75 mM IPTG strongly stimulated expression of F107-C subunit. The optimal induction time for expression was also examined. Generally, highest expression of K88-1NT subunit occurred after 6 h of induction, while that of F107-C subunit is after 14 h.     

Hibernation patterns of Turkish hamsters: influence of sex and ambient temperature

Turkish hamsters (Mesocricetus brandti) are a model organism for studies of hibernation, yet a detailed account of their torpor characteristics has not been undertaken. This study employed continuous telemetric monitoring of body temperature (T _b) in hibernating male and female Turkish hamsters at ambient temperatures (T _as) of 5 and 13 °C to precisely characterize torpor bout depth, duration, and frequency, as well as rates of entry into and arousal from torpor. Hamsters generated brief intervals of short (<12 h), shallow test bouts (T _b > 20 °C), followed by deep torpor bouts lasting 4–6 days at T _a = 5 °C and 2–3 days at T _a = 13 °C. Females at T _a = 5 °C had longer bouts than males, but maintained higher torpor T _b; there were no sex differences at T _a = 13 °C. Neither body mass loss nor food intake differed between the two T _as. Hamsters entered torpor primarily during the scotophase (subjective night), but timing of arousals was highly variable. Hamsters at both T _as generated short, shallow torpor bouts between deep bouts, suggesting that this species may be capable of both hibernation and daily torpor.     

Partitioning of oxygen uptake and cost of surfacing during swimming in the air-breathing catfish Pangasianodon hypophthalmus

Though air-breathing has probably evolved mainly as a response to hypoxia, it may provide an important oxygen supplement when metabolism is elevated, as for example during swimming. Due to the increased travelling distance involved when an air-breathing fish swims to and from the surface, and the increased drag when the surface is breached, it can be proposed that air-breathing results in a rise in the apparent cost of transport. In order to investigate this hypothesis, it is necessary to use a fish that is able to swim equally well with and without access to air. The striped catfish Pangasianodon hypophthalmus has been shown to have a sufficiently high capacity for aquatic oxygen uptake in normoxia, to allow for such a comparison. Here, we measured the partitioning of oxygen uptake ( $ \dot{M}{\text{O}}_{2} $ ) during swimming and recovery, and calculated the apparent cost of transport with and without access to air, under normoxic conditions. Aerial $ \dot{M}{\text{O}}_{2} $ constituted 25–40 % of the total $ \dot{M}{\text{O}}_{2} $ during swimming and less than 15 % during recovery. The net cost of transport was 25 % lower in fish that did not air-breathe compared to fish that did, showing that the cost of surfacing can be substantial. This is the first study to measure partitioning in an air-breathing fish during swimming at velocities close to the critical swimming speed.     

Molecular Dynamics Simulations of Homo-oligomeric Bundles Embedded Within a Lipid Bilayer

Using molecular dynamics simulations, we studied the structure, interhelix interactions, and dynamics of transmembrane proteins. Specifically, we investigated homooligomeric helical bundle systems consisting of synthetic α-helices with either the sequence Ac-(LSLLLSL)₃-NH₂ (LS2) or Ac-(LSSLLSL)₃-NH₂ (LS3). The LS2 and LS3 helical peptides are designed to have amphipathic characteristics that form ion channels in membrane. We simulated bundles containing one to six peptides that were embedded in palmitoyl-oleoyl-phosphatidylcholine (POPC) lipid bilayer and placed between two lamellae of water. We aim to provide a fundamental understanding of how amphipathic helical peptides interact with each other and their dynamical behaviors in different homooligomeric states. To understand structural properties, we examined the helix lengths, tilt angles of individual helices and the entire bundle, interhelix distances, interhelix cross-angles, helix hydrophobic-to-hydrophilic vector projections, and the average number of interhelix hydrophilic (serine–serine) contacts lining the pore of the transmembrane channel. To analyze dynamical properties, we calculated the rotational autocorrelation function of each helix and the cross-correlation of the rotational velocity between adjacent helices. The observed structural and dynamical characteristics show that higher order bundles containing four to six peptides are composed of multiple lower order bundles of one to three peptides. For example, the LS2 channel was found to be stable in a tetrameric bundle composed of a “dimer of dimers.” In addition, we observed that there is a minimum of two strong hydrophilic contacts between a pair of adjacent helices in the dimer to tetramer systems and only one strong hydrophilic interhelix contact in helix pairs of the pentamer and hexamer systems. We believe these results are general and can be applied to more complex ion channels, providing insight into ion channel stability and assembly.     

Postembryonic lineages of the Drosophila brain: I. Development of the lineage-associated fiber tracts

Neurons of the Drosophila central brain fall into approximately 100 paired groups, termed lineages. Each lineage is derived from a single asymmetrically-dividing neuroblast. Embryonic neuroblasts produce 1,500 primary neurons (per hemisphere) that make up the larval CNS followed by a second mitotic period in the larva that generates approximately 10,000 secondary, adult-specific neurons. Clonal analyses based on previous works using lineage-specific Gal4 drivers have established that such lineages form highly invariant morphological units. All neurons of a lineage project as one or a few axon tracts (secondary axon tracts, SATs) with characteristic trajectories, thereby representing unique hallmarks. In the neuropil, SATs assemble into larger fiber bundles (fascicles) which interconnect different neuropil compartments. We have analyzed the SATs and fascicles formed by lineages during larval, pupal, and adult stages using antibodies against membrane molecules (Neurotactin/Neuroglian) and synaptic proteins (Bruchpilot/N-Cadherin). The use of these markers allows one to identify fiber bundles of the adult brain and associate them with SATs and fascicles of the larval brain. This work lays the foundation for assigning the lineage identity of GFP-labeled MARCM clones on the basis of their close association with specific SATs and neuropil fascicles, as described in the accompanying paper (Wong et al., 2013. Postembryonic lineages of the Drosophila brain: II. Identification of lineage projection patterns based on MARCM clones. Submitted.).