Effect of proton pump inhibitor on microbial community, function, and kinetics in anaerobic digestion with ammonia stress

The proton pump is a convincing mechanism for ammonia inhibition in anaerobic digestion, which explained how the ammonia accumulated intercellularly due to diffusion of free ammonia. Proton pump inhibitor (PPI) was dosed for mitigating the accumulation in anaerobic digestion with ammonia stress, with respect to kinetics. Results show PPI inhibited beta-oxidation of fatty acids by targeting ATPase in anaerobic digestion with ammonia stress. Alternatively, PPI stimulated syntrophic acetate oxidization. Random forest located key genera as syntrophic consortia. Methane increased 18.72 +/- 7.39% with 20 mg/L PPI at the first peak, consistent with microbial results. The deterministic Gompertz kinetics and stochastic Gaussian processes contributed 97.63 +/- 8.93% and 2.37 +/- 8.93% in accumulated methane production, respectively. Thus, the use of PPI for anaerobic digestion allowed mitigate ammonia inhibition based on the mechanism of proton pump, facilitate intercellularly ammonia accumulation, stimulate syntrophic consortia, and eliminate uncertainty of process failure, which resulted in efficient methane production under ammonia stress

Overexpression of PpSnRK1α in Tomato Promotes Fruit Ripening by Enhancing RIPENING INHIBITOR Regulation Pathway

As a conserved kinase complex, sucrose non-fermenting-1-related protein kinase 1 (SnRK1) is a major regulator of plant growth and development. In our previous study, overexpression of MhSnRK1 in tomato (Solanum lycopersicum L.) modified fruit maturation: the transgenic fruit ripened earlier than the wild type (WT). However, the mechanism by which fruit maturation is regulated by SnRK1 is not clear; therefore, the test materials used were the transgenic tomato lines (OE-1, OE-3, and OE-4) overexpressing the coding gene of peach [Prunus persica (L.) Batsch] SNF1-related kinase α subunit (PpSnRK1α). The activity of SnRK1 kinase in transgenic tomato lines OE-1, OE-3, and OE-4 was higher than that in the WT at different periods of fruit development; in the pink coloring period the SnRK1 kinase activity increased the most, with 23.5, 28.8, and 21.4% increases, respectively. The content of starch and soluble sugars in red ripe transgenic fruit significantly increased, while the soluble protein and titratable acid content decreased significantly. We also found that the tomatoes overexpressing PpSnRK1α matured approximately 10 days earlier than the WT. Moreover, the yeast-two-hybrid assay showed that PpSnRK1α interacted with the MADS-box transcription factor (TF) SIRIN, which acts as an essential regulator of tomato fruit ripening. The BiFC technology further validated the location of the PpSnRK1α interaction sites within the nucleus. The quantitative real-time PCR analysis showed that RIN expression was up-regulated by PpSnRK1α overexpression; the expression of RIN-targeted TF genes NOR and FUL1 increased during different stages of fruit development. The expression of key genes, ACS2, ACS4, and E8, in ethylene synthesis also changed accordingly, and the ethylene emitted by the red ripe fruit increased by 36.1–43.9% compared with the WT. These results suggest that PpSnRK1α interacts with SIRIN, increasing the expression of RIN, thereby regulating the expression of downstream ripening-related genes, finally promoting fruit ripening

Rescue of failed oocyte activation after ICSI in a mouse model of male factor infertility by recombinant phospholipase Cζ

Artificial oocyte activation to overcome failed fertilization after intracytoplasmic sperm injection (ICSI) in human oocytes typically employs Ca2+ ionophores to produce a single cytosolic Ca2+ increase. In contrast, recombinant phospholipase Czeta (PLCζ) causes Ca2+ oscillations indistinguishable from those occurring during fertilization, but remains untested for its efficacy in a scenario of ICSI fertilization failure. Here, we compare PLCζ with other activation stimuli in a mouse model of failed oocyte activation after ICSI, in which heat-treated sperm are injected into mouse oocytes. We show that increasing periods of 56°C exposure of sperm produces a progressive loss of Ca2+ oscillations after ICSI. The decrease in Ca2+ oscillations produces a reduction in oocyte activation and embryo development to the blastocyst stage. We treated such oocytes that failed to activate after ICSI either with Ca2+ ionophore, or with Sr2+ media which causes Ca2+ oscillations, or we injected them with recombinant human PLCζ. All these treatments rescued oocyte activation, although Sr2+ and PLCζ gave the highest rates of development to blastocyst. When recombinant PLCζ was given to oocytes previously injected with control sperm, they developed normally to the blastocyst stage at rates similar to that after control ICSI. The data suggest that recombinant human PLCζ protein is an efficient means of rescuing oocyte activation after ICSI failure and that it can be effectively used even if the sperm already contains endogenous Ca2+ releasing activity

PLCζ causes Ca2+ oscillations in mouse eggs by targeting intracellular and not plasma membrane PI(4,5)P2

Sperm-specific phospholipase C ζ (PLCζ) activates embryo development by triggering intracellular Ca2+ oscillations in mammalian eggs indistinguishable from those at fertilization. Somatic PLC isozymes generate inositol 1,4,5-trisphophate–mediated Ca2+ release by hydrolyzing phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the plasma membrane. Here we examine the subcellular source of PI(4,5)P2 targeted by sperm PLCζ in mouse eggs. By monitoring egg plasma membrane PI(4,5)P2 with a green fluorescent protein–tagged PH domain, we show that PLCζ effects minimal loss of PI(4,5)P2 from the oolemma in contrast to control PLCδ1, despite the much higher potency of PLCζ in eliciting Ca2+ oscillations. Specific depletion of this PI(4,5)P2 pool by plasma membrane targeting of an inositol polyphosphate-5-phosphatase (Inp54p) blocked PLCδ1-mediated Ca2+ oscillations but not those stimulated by PLCζ or sperm. Immunolocalization of PI(4,5)P2, PLCζ, and catalytically inactive PLCζ (ciPLCζ) revealed their colocalization to distinct vesicular structures inside the egg cortex. These vesicles displayed decreased PI(4,5)P2 after PLCζ injection. Targeted depletion of vesicular PI(4,5)P2 by expression of ciPLCζ-fused Inp54p inhibited the Ca2+ oscillations triggered by PLCζ or sperm but failed to affect those mediated by PLCδ1. In contrast to somatic PLCs, our data indicate that sperm PLCζ induces Ca2+ mobilization by hydrolyzing internal PI(4,5)P2 stores, suggesting that the mechanism of mammalian fertilization comprises a novel phosphoinositide signaling pathway

Rhythmic actomyosin-driven contractions induced by sperm entry predict mammalian embryo viability.

Fertilization-induced cytoplasmic flows are a conserved feature of eggs in many species. However, until now the importance of cytoplasmic flows for the development of mammalian embryos has been unknown. Here, by combining a rapid imaging of the freshly fertilized mouse egg with advanced image analysis based on particle image velocimetry, we show that fertilization induces rhythmical cytoplasmic movements that coincide with pulsations of the protrusion forming above the sperm head. We find that these movements are caused by contractions of the actomyosin cytoskeleton triggered by Ca(2+) oscillations induced by fertilization. Most importantly, the relationship between the movements and the events of egg activation makes it possible to use the movements alone to predict developmental potential of the zygote. In conclusion, this method offers, thus far, the earliest and fastest, non-invasive way to predict the viability of eggs fertilized in vitro and therefore can potentially improve greatly the prospects for IVF treatment

Regulation of diacylglycerol production and protein kinase C stimulation during sperm- and PLCζ-mediated mouse egg activation

Background information. At fertilization in mammalian eggs, the sperm induces a series of Ca2+ oscillations via the production of inositol 1,4,5-trisphosphate. Increased inositol 1,4,5-trisphosphate production appears to be triggered by a sperm-derived PLCζ (phospholipase C-ζ) that enters the egg after gamete fusion. The specific phosphatidylinositol 4,5-bisphosphate hydrolytic activity of PLCζ implies that DAG (diacylglycerol) production, and hence PKC (protein kinase C) stimulation, also occurs during mammalian egg fertilization. Fertilization-mediated increase in PKC activity has been demonstrated; however, its precise role is unclear

Divergent effect of mammalian PLCζ in generating Ca2+ oscillations in somatic cells compared with eggs

Sperm PLCζ (phospholipase Cζ) is a distinct phosphoinositide-specific PLC isoform that is proposed to be the physiological trigger of egg activation and embryo development at mammalian fertilization. Recombinant PLCζ has the ability to trigger Ca2+ oscillations when expressed in eggs, but it is not known how PLCζ activity is regulated in sperm or eggs. In the present study, we have transfected CHO (Chinese-hamster ovary) cells with PLCζ fused with either YFP (yellow fluorescent protein) or luciferase and found that PLCζ-transfected cells did not display cytoplasmic Ca2+ oscillations any differently from control cells. PLCζ expression was not associated with changes in CHO cell resting Ca2+ levels, nor with a significantly changed Ca2+ response to extracellular ATP compared with control cells transfected with either YFP alone, a catalytically inactive PLCζ or luciferase alone. Sperm extracts containing PLCζ also failed to cause Ca2+ oscillations in CHO cells. Despite these findings, PLCζ-transfected CHO cell extracts exhibited high recombinant protein expression and PLC activity. Furthermore, either PLCζ-transfected CHO cells or derived cell extracts could specifically cause cytoplasmic Ca2+ oscillations when microinjected into mouse eggs. These data suggest that PLCζ-mediated Ca2+ oscillations may require specific factors that are only present within the egg cytoplasm or be inhibited by factors present only in somatic cell lines

The Action Difference of Lasiodiplodia theobromae on Infecting and Dyeing Poplar Wood in Spatial Growth

Many factors affect the driving force of fungal growth and secretion. To compare the differences of Lasiodiplodia theobromae infected poplar wood, the changes of physical and chemical properties of vertically and horizontally infected poplar wood before and after dyeing were analyzed, and the infection characteristics were studied in this paper. The horizontal infection was more effective than the vertical infection in terms of infection depth, color depth, and microscopic hyphal invasion. The mycelium first intruded into the earlywood tissue and began to secrete a large amount of pigment after twenty days. The crystallinity of mycelium decreased slightly, and the difference in weight loss rate was negligible. The initial contact angle of the dyed specimen on the horizontal infection increased drastically in distilled water, but there was almost no difference between varnish and natural coating. The horizontal infection was more efficient than the vertical infection and had a higher color depth and a better induction effect, which is crucial in future microbial dyeing

Overexpression of <i>PpSnRK1α</i> in Tomato Increased Autophagy Activity under Low Nutrient Stress

Plants suffer from a variety of environmental stresses during their growth and development. The evolutionarily conserved sucrose nonfermenting kinase 1-related protein kinase 1 (SnRK1) plays a central role in the regulation of energy homeostasis in response to stresses. In plant cells, autophagy is a degradation process occurring during development or under stress, such as nutrient starvation. In recent years, SnRK1 signaling has been reported to be an upstream activator of autophagy. However, these studies all focused on the regulatory effect of SnRK1 on TOR signaling and the autophagy-related gene 1 (ATG1) complex. In this study, overexpression of the gene encoding the Prunus persica SnRK1 α subunit (PpSnRK1α) in tomato improved the photosynthetic rates and enhanced the resistance to low nutrient stress (LNS). Overexpression of PpSnRK1α increased autophagy activity and upregulated the expression of seven autophagy-related genes (ATGs). The transcriptional levels of SlSnRK2 family genes were altered significantly by PpSnRK1α, signifying that PpSnRK1α may be involved in the ABA signaling pathway. Further analysis showed that PpSnRK1α not only activated autophagy by inhibiting target of rapamycin (TOR) signaling but also enhanced ABA-induced autophagy. This indicates that PpSnRK1α regulates the photosynthetic rate and induces autophagy, and then responds to low nutrient stress

Preimplantation development of mouse oocytes activated by different levels of human phospholipase C zeta

BACKGROUND A sperm-specific phospholipase C zeta (PLCζ) has been shown to trigger Ca2+ oscillations in mouse and human oocytes and appears to be the sperm factor responsible for activation at fertilization. Previously, complementary RNA (cRNA) injection was used to introduce PLCζ into oocytes, but it was unclear how much PLCζ protein is required for development. Here we have injected cRNA encoding luciferase-tagged human PLCζ (hPLCζ-luc) into mouse oocytes and established the relationship between hPLCζ-luc expression, Ca2+ oscillations and development. METHODS Mouse oocytes were injected with hPLCζ-luc cRNA and a fluorescent Ca2+dye to monitor hPLCζ-luc expression and Ca2+ oscillations, respectively. After inducing diploidy, development in vitro was monitored in hPLCζ-luc cRNA microinjected oocytes and compared with parallel oocytes activated by incubation in Sr2+. RESULTS Repetitive Ca2+ oscillations and oocyte activation were triggered by hPLCζ over a wide range of luciferase expression levels. However, subsequent development of embryos to the blastocyst stage was observed only when expression of hPLCζ-luc was optimized within a specific range. The blastocyst cell number was also affected by the level of hPLCζ expression. CONCLUSIONS Human PLCζ can readily activate mouse oocytes, however, effective development to blastocyst stages is only achieved within a specific window of hPLCζ-luc protein expression levels