Chloroplast NADPH-Dependent Thioredoxin Reductase from Chlorella vulgaris Alleviates Environmental Stresses in Yeast Together with 2-Cys Peroxiredoxin

Chloroplast NADPH-dependent thioredoxin reductase (NTRC) catalyzes the reduction of 2-Cys peroxiredoxin (2-Cys Prx) and, thus, probably functions as an antioxidant system. The functions of the enzyme in oxidative and salt stresses have been reported previously. We have previously identified and characterized NTRC in Chlorella vulgaris. In the present study, we isolated a full-length cDNA clone encoding 2-Cys Prx from C. vulgaris and investigated the involvement of Chlorella NTRC/2-Cys Prx system in several environmental stress tolerances by using yeast as a eukaryotic model. Deduced Chlorella 2-Cys Prx was homologous to those of chloroplast 2-Cys Prxs from plants, and two conserved cysteine residues were found in the deduced sequence. Enzyme assay showed that recombinant mature C. vulgaris NTRC (mCvNTRC) transferred electrons from NADPH to recombinant mature C. vulgaris 2-Cys Prx (mCvPrx), and mCvPrx decomposed hydrogen peroxide, tert-butyl hydroperoxide, and peroxynitrite by cooperating with mCvNTRC. Based on the results, the mCvNTRC/mCvPrx antioxidant system was identified in Chlorella. The antioxidant system genes were expressed in yeast separately or coordinately. Stress tolerances of yeast against freezing, heat, and menadione-induced oxidative stresses were significantly improved by expression of mCvNTRC, and the elevated tolerances were more significant when both mCvNTRC and mCvPrx were co-expressed. Our results reveal a novel feature of NTRC: it functions as an antioxidant system with 2-Cys Prx in freezing and heat stress tolerances.     

Chicken egg yolk antibodies (IgY) for detecting circulating antigens of Schistosoma japonicum

BackgroundIgY isolated from egg yolk has been widely used in immunodiagnostic tests, including tests to detect circulating antigen (soluble egg antigen or SEA) of Schistosoma japonicum.ResultsA sandwich ELISA was established using a combination of anti-S. japonicum SEA-IgY polyclonal antibodies and IgM monoclonal antibodies. To explore sensitivity and specificity of the sandwich ELISA, serum samples from 43 patients infected with S. japonicum were tested. All acute cases and 91.3% of the chronic cases showed a positive reaction. Only 5% of the control sera from healthy persons gave a positive response. Cross-reactions with antibodies to nine other parasites were rare.ConclusionThe developed immunoassay is reasonably sensitive and specific. It could be used for field research and treatment efficacy assessments.     

Glial Activation and Segmental Upregulation of Interleukin-1β (IL-1β) in the Rat Spinal Cord after Surgical Incision

The present study investigated the expression patterns of glial cells and interleukin-1β (IL-1β) in the rat spinal cord after a surgical incision, which is closely related with clinical postoperative pain. Microglia and astrocytes became activated in the spinal cord following incision. Real-time polymerase chain reaction (PCR) and immunohistochemisty showed that IL-1β mRNA and protein level in the spinal cord was transiently upregulated after surgical incision. The increased IL-1β-immunoreactivity (IR) was mainly localized in neurons but not the activated microglia or astrocytes. Although obvious increase in IL-1β-IR could be observed in the lumbar segments of the spinal cord ipsilateral to a hind paw incision, significant upregulation of IL-1β was not detected in the lumbar segments following thoracic incision. The present study indicated that surgical incision could induce glial activation and segmental upregulation of IL-1β in the spinal cord. The activated glial cells and upregulated IL-1β, in turn, may be involved in the incision-induced pain hypersensitivity.     

Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport

Human concentrative nucleoside transporter, hCNT3, mediates Na⁺/nucleoside and H⁺/nucleoside co-transport. We describe a new approach to monitor H⁺/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in HEK293 cells expressing mNect.hCNT3 or mNect.hCNT3-F563C, an inactive hCNT3 mutant. Free cytosolic mNect, mNect.hCNT3, and the traditional pH-sensitive dye, BCECF, reported cytosolic pH similarly in pH-clamped HEK293 cells. Cells were incubated at the permissive pH for H⁺-coupled nucleoside transport, pH 5.5, under both Na⁺-free and Na⁺-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mm uridine, providing the first direct evidence for H⁺-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H⁺ flux) in the presence or absence of Na⁺ (1.09 ± 0.11 or 1.18 ± 0.32 mm min⁻¹, respectively). This suggests that in acidic Na⁺-containing conditions, 1 Na⁺ and 1 H⁺ are transported per uridine molecule, while in acidic Na⁺-free conditions, 1 H⁺ alone is transported/uridine. In acid environments, including renal proximal tubule, H⁺/nucleoside co-transport may drive nucleoside accumulation by hCNT3. Fusion of mNect to hCNT3 provided a simple, self-referencing, and effective way to monitor nucleoside transport, suggesting an approach that may have applications in assays of transport activity of other H⁺-coupled transport proteins.Nucleosides are hydrophilic molecules that require transport proteins to mediate their movement across the plasma membrane (1). Human (h)⁷ nucleoside transport (NT) proteins catalyze the vectorial transport of nucleosides, using either concentrative (C) or equilibrative (E) mechanisms (2). hCNTs use either a Na⁺ or H⁺ gradient to accumulate nucleosides against their concentration gradient, whereas hENTs mediate facilitated diffusion of nucleosides down their concentration gradient (3). Nucleoside transporters also transport anti-cancer and anti-viral drugs, and cellular expression of nucleoside transporters is important in cancer therapy as well as in the treatment of cardiovascular, parasitic, and viral diseases (4, 5).Members of the SLC28 family of concentrative nucleoside transporters (CNTs) divide into two phylogenetic subfamilies: hCNT1/2 belonging to one subfamily, and hCNT3 to the other (6–8). Cation substitution and charge/flux ratio studies suggest that hCNT1/2 couple the inward movement of nucleoside to the Na⁺ electrochemical gradient with a 1:1 stoichiometry, whereas hCNT3 can couple nucleoside transport to either the Na⁺ gradient (2 Na⁺:1 nucleoside) or a H⁺ gradient (1 H⁺:1 nucleoside) in the absence of Na⁺ (9, 10). The 2:1 coupling ratio of hCNT3 allows it to develop a trans-membrane nucleoside concentration gradient up to 10-fold higher than that of hCNT1 or hCNT2 (9, 11). At pH 5.5, hCNT3 also transports uridine in the presence of Na⁺ with a 2 cation:1 nucleoside stoichiometry, which raises the possibility that 1 H⁺ and 1 Na⁺ may be transported per nucleoside molecule in these conditions (9–12). Up to this point, however, there has been no direct demonstration that hCNT3 can transport H⁺.Concentrative nucleoside transport has previously been investigated using the Xenopus laevis oocyte expression system and both electrophysiology (two-microelectrode voltage clamp technique) and radioisotope flux measurements (6–9, 12). Electrophysiological experiments are advantageous in that they measure the current induced by addition of substrate in real-time, but they are time-consuming and require specialized equipment and skills. Radioisotope flux assays measure the accumulation of radiolabeled substrate. The need for radiolabeled substrate restricts the range of permeants able to be studied. In addition, radioisotope flux assays are not done in real-time and are labor-intensive, requiring large numbers of oocytes.An attractive alternative approach for the study of hCNT3 would be to measure pH in the immediate vicinity of its intracellular face during H⁺/nucleoside co-transport. These measurements could take advantage of the remarkable progress achieved in the development of genetically encoded fluorophores (13). Indeed, all members of the extended family of homologues and variants of the Aequorea victoria green fluorescent protein (avGFP) exhibit pH-dependent changes in their fluorescent intensity. The spectral changes that occur upon a change in pH can be intensiometric (14), excitation ratiometric (14), emission ratiometric (15), or both excitation and emission ratiometric (16). The apparent pK_a (pK_a′, equal to the pH at which the fluorescence is half-maximal in intensity) for a specific fluorescent protein (FP) is acutely dependent on specific amino acid substitutions in close proximity to the chromophore and can range from less than 3 (17, 18) to greater than 8 (19). Variants with pK_a′ values that are relatively close to intracellular pH values (i.e. ∼7.3 for the mammalian cytosol (20)) are particularly useful as genetically encoded biosensors for dynamic measurement of proton concentrations in living cells.A major development in the area of FP technology has been the identification (21) and subsequent optimization (22, 23) of red fluorescent protein (RFP) homologues of avGFP. The first (monomeric RFP 1 (mRFP1)) (23) and second (the mFruit series) (22) generation-optimized RFPs, derived from tetrameric Discosoma RFP (21), suffer from relatively low brightness relative to other common hues of FP. For example, of the three most red-shifted second generation mFruit variants (mTangerine, mStrawberry, and mCherry) (22), the brightest (mStrawberry) has only 44% of the intrinsic brightness (proportional to the product of extinction coefficient (ϵ) and quantum yield (Φ)) of the popular yellow FP (YFP) Citrine (24) and 76% of the brightness of enhanced avGFP. This limitation has been partially addressed by third generation mRFPs, specifically mApple and TagRFP-T, with fluorescent brightness values on par with, or better than, that of enhanced avGFP (25).Generally speaking, the most red-shifted RFPs derived from Discosoma RFP are relatively pH-insensitive, with the majority of variants having pK_a′ values < 5 (22, 25). A notable exception is the recently reported mApple variant with a pK_a′ of 6.5 (25). The more blue-shifted of the mFruit variants (i.e. mOrange) also have pK_a′ values of 6.5 (22). Several variants of mRFP1 with pK_a′ values >7.5 have been previously reported (26).Here we report the engineering of a pH-sensitive mFruit variant through multiple rounds of directed evolution by random mutagenesis. This RFP, called mNectarine, is appropriate to measure physiological pH changes in mammalian cells, because it has a pK_a′ of 6.9. We have developed a new method to measure H⁺/nucleoside co-transport in mammalian cells, which utilizes hCNT3''s H⁺ coupling characteristics and the pH sensitivity of mNectarine. We fused mNectarine to the cytosolic N terminus of hCNT3 to generate mNect.hCNT3. Fusion of the fluorescent H⁺ sensor to hCNT3 enabled measurement of pH at the intracellular surface of hCNT3, and provided insight into the mechanism of hCNT3 H⁺/uridine co-transport.     

Electrochemical detection of DNA hybridization using a change in flexibility

A novel electrochemical method of detecting DNA hybridization is presented based on the change in flexibility between the single and double stranded DNA. A recognition surface based on gold nanoparticles (GNPs) is firstly modified via mixing self-assembled monolayer of thiolated probe DNA and 1,6-hexanedithiol. The hybridization and electrochemical detection are performed on the surface of probe-modified GNPs and electrode, respectively. Here in our method the charge transfer resistance (R(ct)) signal is enhanced by blocking the surface of electrode with DNA covered GNPs. The GNPs will be able to adsorb on the gold electrode when covered with flexible single stranded DNA (ssDNA). On the contrary, it will be repelled from the electrode, when covered with stiff double stranded DNA (dsDNA). Therefore, different R(ct) signals are observed before and after hybridization. The hybridization events are monitored by electrochemical impedance spectroscopy (EIS) measurement based on the R(ct) signals without any external labels. This method provides an alternative route for expanding the range of detection methods available for DNA hybridization.     

Genome-wide association analyses reveal significant loci and strong candidate genes for growth and fatness traits in two pig populations

Background

Recently, genome-wide association studies (GWAS) have been reported on various pig traits. We performed a GWAS to analyze 22 traits related to growth and fatness on two pig populations: a White Duroc × Erhualian F₂ intercross population and a Chinese Sutai half-sib population.

Results

We identified 14 and 39 loci that displayed significant associations with growth and fatness traits at the genome-wide level and chromosome-wide level, respectively. The strongest association was between a 750 kb region on SSC7 (SSC for Sus scrofa) and backfat thickness at the first rib. This region had pleiotropic effects on both fatness and growth traits in F₂ animals and contained a promising candidate gene HMGA1 (high mobility group AT-hook 1). Unexpectedly, population genetic analysis revealed that the allele at this locus that reduces fatness and increases growth is derived from Chinese indigenous pigs and segregates in multiple Chinese breeds. The second strongest association was between the region around 82.85 Mb on SSC4 and average backfat thickness. PLAG1 (pleiomorphic adenoma gene 1), a gene under strong selection in European domestic pigs, is proximal to the top SNP and stands out as a strong candidate gene. On SSC2, a locus that significantly affects fatness traits mapped to the region around the IGF2 (insulin-like growth factor 2) gene but its non-imprinting inheritance excluded IGF2 as a candidate gene. A significant locus was also detected within a recombination cold spot that spans more than 30 Mb on SSCX, which hampered the identification of plausible candidate genes. Notably, no genome-wide significant locus was shared by the two experimental populations; different loci were observed that had both constant and time-specific effects on growth traits at different stages, which illustrates the complex genetic architecture of these traits.

Conclusions

We confirm several previously reported QTL and provide a list of novel loci for porcine growth and fatness traits in two experimental populations with Chinese Taihu and Western pigs as common founders. We showed that distinct loci exist for these traits in the two populations and identified HMGA1 and PLAG1 as strong candidate genes on SSC7 and SSC4, respectively.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0089-5) contains supplementary material, which is available to authorized users.     

Spatial Structures of the Environment and of Dispersal Impact Species Distribution in Competitive Metacommunities

The correspondence between species distribution and the environment depends on species’ ability to track favorable environmental conditions (via dispersal) and to maintain competitive hierarchy against the constant influx of migrants (mass effect) and demographic stochasticity (ecological drift). Here we report a simulation study of the influence of landscape structure on species distribution. We consider lottery competition for space in a spatially heterogeneous environment, where the landscape is represented as a network of localities connected by dispersal. We quantified the contribution of neutrality and species sorting to their spatial distribution. We found that neutrality increases and the strength of species-sorting decreases with the centrality of a community in the landscape when the average dispersal among communities is low, whereas the opposite was found at elevated dispersal. We also found that the strength of species-sorting increases with environmental heterogeneity. Our results illustrate that spatial structure of the environment and of dispersal must be taken into account for understanding species distribution. We stress the importance of spatial geographic structure on the relative importance of niche vs. neutral processes in controlling community dynamics.