Cell Wall-Anchored Nuclease of Streptococcus sanguinis Contributes to Escape from Neutrophil Extracellular Trap-Mediated Bacteriocidal Activity

Streptococcus sanguinis, a member of the commensal mitis group of streptococci, is a primary colonizer of the tooth surface, and has been implicated in infectious complications including bacteremia and infective endocarditis. During disease progression, S. sanguinis may utilize various cell surface molecules to evade the host immune system to survive in blood. In the present study, we discovered a novel cell surface nuclease with a cell-wall anchor domain, termed SWAN (streptococcal wall-anchored nuclease), and investigated its contribution to bacterial resistance against the bacteriocidal activity of neutrophil extracellular traps (NETs). Recombinant SWAN protein (rSWAN) digested multiple forms of DNA including NET DNA and human RNA, which required both Mg²⁺ and Ca²⁺ for optimum activity. Furthermore, DNase activity of S. sanguinis was detected around growing colonies on agar plates containing DNA. In-frame deletion of the swan gene mostly reduced that activity. These findings indicated that SWAN is a major nuclease displayed on the surface, which was further confirmed by immuno-detection of SWAN in the cell wall fraction. The sensitivity of S. sanguinis to NET killing was reduced by swan gene deletion. Moreover, heterologous expression of the swan gene rendered a Lactococcus lactis strain more resistant to NET killing. Our results suggest that the SWAN nuclease on the bacterial surface contributes to survival in the potential situation of S. sanguinis encountering NETs during the course of disease progression.     

Genotyping Analysis for the 46 C/T Polymorphism of Coagulation Factor XII and the Involvement of Factor XII Activity in Patients with Recurrent Pregnancy Loss

Background

Established causes of recurrent pregnancy loss (RPL) include antiphospholipid syndrome, uterine anomalies, parental chromosomal abnormalities, particularly translocations and abnormal embryonic karyotype. A systematic review concluded that coagulation factor XII (FXII) deficiency was associated with RPL. However, it could not be established whether the 46 C/T SNP of FXII or low activity of FXII was a risk factor for RPL, because of the small sample size.

Methods and Findings

We conducted a cross-sectional and cohort study in 279 patients with two or more unexplained consecutive pregnancy losses and 100 fertile women. The association between the lupus anticoagulant (LA) activity and FXII activity was examined. The frequency of the CC, CT and TT genotypes and the FXII activity were also compared between the patients and controls. Subsequent miscarriage rates among the CC, CT, TT genotypes and according to the FXII activity was examined. LA was associated with reduced FXII activity. The CT, but not the TT, genotype was confirmed to be a risk factor for RPL in the cross-sectional study using multivariate logistic regression analysis (OR, 2.8; 95% CI, 1.37–5.85). The plasma FXII activity in the patients was similar to that in the controls. Neither low FXII activity nor the CT genotype predicted the subsequent pregnancy outcome in the cohort study. On the other hand, and intermediate FXII activity level of 85–101% was predictive of subsequent miscarriage.

Conclusions

Low FXII activity was not associated with RPL. The FXII gene was found to be one of the significant susceptibility genes for RPL, similar to the FV Leiden mutation. However, the clinical influence of the CT genotype might be relatively small, because the presence/absence of this genotype did not have any predictive value for the subsequent pregnancy outcome. This was the first study indicating the influence of FXII 46C/T on further pregnancy outcomes.     

Rice Chalky Ring Formation Caused by Temporal Reduction in Starch Biosynthesis during Osmotic Adjustment under Foehn-Induced Dry Wind

Foehn-like extreme hot and dry wind conditions (34°C, >2.5 kPa vapor pressure deficit, and 7 m s⁻¹) strongly affect grain quality in rice (Oryza sativa L.). This is a current concern because of the increasing frequency and intensity of combined heat and water-deficit stress under climate change. Foehn-induced dry wind conditions during the grain-filling stage increase ring-shaped chalkiness as a result of spatiotemporal reduction in starch accumulation in the endosperm, but kernel growth is sometimes maintained by osmotic adjustment. Here, we assess the effects of dry wind on chalky ring formation in environmentally controlled growth chambers. Our results showed that hot and dry wind conditions that lasted for >24 h dramatically increased chalky ring formation. Hot and dry wind conditions temporarily reduced panicle water potential to –0.65 MPa; however, kernel growth was maintained by osmotic adjustment at control levels with increased transport of assimilate to the growing kernels. Dynamic tracer analysis with a nano-electrospray-ionization Orbitrap mass spectrometer and quantitative polymerase chain reaction analysis revealed that starch degradation was negligible in the short-term treatment. Overall expression of starch synthesis-related genes was found to be down-regulated at moderately low water potential. Because the events observed at low water potential preceded the packing of starch granules in cells, we concluded that reduced rates of starch biosynthesis play a central role in the events of cellular metabolism that are altered at osmotic adjustment, which leads to chalky ring formation under short-term hot and dry wind conditions.     

A Model of the Intracellular Response of an Olfactory Neuron in Caenorhabditis elegans to Odor Stimulation

We developed a mathematical model of a hypothetical neuronal signal transduction pathway to better understand olfactory perception in Caenorhabditis elegans. This worm has only three pairs of olfactory receptor neurons. Intracellular Ca(2+) decreases in one pair of olfactory neurons in C. elegans, the AWC neurons, following application of an attractive odor and there is a transient increase in intracellular Ca(2+) following removal of odor. The magnitude of this increase is positively correlated with the duration of odor stimulation. Additionally, this Ca(2+) transient is induced by a cGMP second messenger system. We identified likely candidates for the signal transduction molecules functioning in this system based on available gene expression and physiological data from AWCs. Our model incorporated a G-protein-coupled odor receptor, a G-protein, a guanylate cyclase as the G-protein effector, and a single phosphodiesterase. Additionally, a cyclic-nucleotide-gated ion channel and a voltage-gated ion channel that mediated calcium influx were incorporated into the model. We posited that, upon odor stimulation, guanylate cyclase was suppressed by the G-protein and that, upon cessation of the stimulus, the G-protein-induced suppression ceased and cGMP synthesis was restored. A key element of our model was a Ca(2+)-dependent negative feedback loop that ensured that the calcium increases were transient. Two guanylate cyclase-activating proteins acted on the effector guanylate cyclase to negatively regulate cGMP signaling and the resulting calcium influx. Our model was able to closely replicate in silico three important features of the calcium dynamics of AWCs. Specifically, in our simulations, [Ca(2+)] increased rapidly and reached its peak within 10 s after the odor stimulus was removed, peak [Ca(2+)] increased with longer odor exposure, and [Ca(2+)] decreased during a second stimulus that closely followed an initial stimulus. However, application of random background signal ('noise') showed that certain components of the pathway were particularly sensitive to this noise.     

Autophagy regulates inflammation in adipocytes

Autophagy is an essential process for both the maintenance and the survival of cells, with homeostatic low levels of autophagy being critical for intracellular organelles and proteins. In insulin resistant adipocytes, various dysfunctional/damaged molecules, organelles, proteins, and end-products accumulate. However, the role of autophagy (in particular, whether autophagy is activated or not) is poorly understood. In this study we found that in adipose tissue of insulin resistant mice and hypertrophic 3T3-L1 adipocytes autophagy was suppressed. Also in hypertrophic adipocytes, autophagy-related gene expression, such as LAMP1, LAMP2, and Atg5 was reduced, whereas gene expression in the inflammatory-related genes, such as MCP-1, IL-6, and IL-1β was increased. To find out whether suppressed autophagy was linked to inflammation we used the autophagy inhibitor, 3-methyladenine, to inhibit autophagy. Our results suggest that such inhibition leads to an increase in inflammatory gene expression and causes endoplasmic reticulum (ER) stress (which can be attenuated by treatment with the ER stress inhibitor, Tauroursodeoxycholic Acid). Conversely, the levels of inflammatory gene expression were reduced by the activation of autophagy or by the inhibition of ER stress. The results indicate that the suppression of autophagy increases inflammatory responses via ER stress, and also defines a novel role of autophagy as an important regulator of adipocyte inflammation in systemic insulin resistance.     

Plant-pollinator interactions in tropical monsoon forests in Southeast Asia

Forests with different flora and vegetation types harbor different assemblages of flower visitors, and plant-pollinator interactions vary among forests. In monsoon-dominated East and Southeast Asia, there is a characteristic gradient in climate along latitude, creating a broad spectrum of forest types with potentially diverse pollinator communities. To detect a geographical pattern of plant-pollinator interactions, we investigated flowering phenology and pollinator assemblages in the least-studied forest type, i.e., tropical monsoon forest, in the Vientiane plain in Laos. Throughout the 5-year study, we observed 171 plant species blooming and detected flower visitors on 145 species. Flowering occurred throughout the year, although the number of flowering plant species peaked at the end of dry season. The dominant canopy trees, including Dipterocarpaceae, bloomed annually, in contrast to the supra-annual general flowering that occurs in Southeast Asian tropical rain forests. Among the 134 native plant species, 68 were pollinated by hymenopterans and others by lepidopterans, beetles, flies, or diverse insects. Among the observed bees, Xylocopa, megachilids, and honeybees mainly contributed to the pollination of canopy trees, whereas long-tongued Amegilla bees pollinated diverse perennials with long corolla tubes. This is the first community-level study of plant-pollinator interactions in an Asian tropical monsoon forest ecosystem.     

Tenascin C interacts with ecto-5'-nucleotidase (eN) and regulates adenosine generation in cancer cells

Tenascin C is expressed in invasive human solid tumors; however its specific role in cancer biology remains obscure. Previously, we have found that ecto-5'-nucleotidase (eN) is a marker of ER (-) breast carcinoma and elevated expression correlates with invasive mesenchymal cell phenotype. To investigate for the potential relationship between eN and protein components of the extracellular matrix (ECM) we measured adenosine generation from AMP in cells incubated with soluble ECM proteins. We found that tenascin C was the only ECM component that strongly inhibited ecto-5'-nucleotidase (eN) activity in situ and adenosine generation from AMP (75% inhibition, p < 0.01). The inhibition was comparable to that induced by concanavalin A, a well-defined and strong inhibitor of eN. Resin immobilized tenascin C, but not collagen, and only weakly fibronectin, specifically and quantitatively bound cell-extracted eN. We further developed breast cancer cell line with reduced eN expression and tested changes in cell adhesion on different ECM. Breast cancer cells expressing reduced eN attached 56% weaker (p < 0.05) to immobilized tenascin C. This difference was not detected with other ECM proteins. Finally, control breast cancer cells migrated slower on tenascin C when compared with clone with reduced eN expression. These data suggest that eN is a novel and specific receptor for tenascin C and that the interaction between these proteins may influence cell adhesion and migration and also lead to decreased generation of local adenosine.     

Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice

Four enzymes, namely, the maize C(4)-specific phosphoenolpyruvate carboxylase (PEPC), the maize C(4)-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C(3)-specific NADP-malic enzyme (ME), were overproduced in the mesophyll cells of rice plants independently or in combination. Overproduction individually of PPDK, MDH or ME did not affect the rate of photosynthetic CO(2) assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO(2) assimilation in PEPC overproduction lines remained unaffected by overproduction of PPDK, ME or a combination of both, however it was significantly restored by the combined overproduction of PPDK, ME, and MDH to reach levels comparable to or slightly higher than that of non-transgenic rice. The extent of the restoration of CO(2) assimilation, however, was more marked at higher CO(2) concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO(2) inside the chloroplast. Transgenic rice plants overproducing the four enzymes showed slight stunting. Comparison of transformants overproducing different combinations of enzymes indicated that overproduction of PEPC together with ME was responsible for stunting, and that overproduction of MDH had some mitigating effects. Possible mechanisms underlying these phenotypic effects, as well as possibilities and limitations of introducing the C(4)-like photosynthetic pathway into C(3) plants, are discussed.     

Contribution of symbiotic mycangial fungi to larval nutrition of a leaf-rolling weevil

Some phytophagous insects have been known to inoculate certain fungi on plant substrates. In many cases of such insect–fungi relationships it has been considered that fungi contribute to insects by decomposing lignin or polysaccharides, and that the insects feed on the decomposition products or fungi themselves. Females of the leaf-rolling weevil in the genus Euops (Attelabidae) store spores of symbiotic fungi in the mycangia and inoculate them on leaf rolls. To determine the effect of mycangial fungi on larval nutrition in E. lespedezae, the nutritional value was compared between leaves with and without mycangial fungi. Two Penicillium species were isolated from the mycangia. These mycangial fungi showed little effect on the decomposition of lignin and polysaccharides, and showed little effect on enhancement of soluble sugars within leaves. Thus, the mutualism between Euops and its mycangial fungi contrasts with the mainly nutritional mutualisms between wood-infesting insects (termites, bark/ambrosia beetles, and wood wasps) and lignin/polysaccharide-decomposing fungi.