Micro-biological degradation and transformation of dissolved organic matter following continuous cropping of tobacco.

In recent years, the problems associated with continuous cropping (CC) that cause soil degradation have become increasingly serious. As a key soil quality property, dissolved organic matter (DOM) affects the circulation of carbon and nutrients and the composition of bacterial communities in soil. However, research on the changes in the molecular composition of DOM after CC is limited. In this study, the soil chemical properties, DOM chemical diversity, bacterial community structure, and their interactions are explored in the soil samples from different CC years (CC1Y, CC3Y, CC5Y, and CC7Y) of tobacco. With increasing CC year of tobacco, most of the soil chemical properties, such as total carbon, total nitrogen and organic matter, decreased significantly, while dissolved organic carbon first decreased and then increased. Likewise, the trends of DOM composition differed with changing duration of CC, such as the tannin compounds decreased from 18.13 to 13.95%, aliphatic/proteins increased from 2.73 to 8.85%. After 7 years of CC, the soil preferentially produced compounds with either high H/C ratios (H/C > 1.5), including carbohydrates, lipids, and aliphatic/proteins, or low O/C ratios (O/C < 0.1), such as unsaturated hydrocarbons. Furthermore, core microorganisms, including Nocardioides, wb1-P19, Aquabacterium, Methylobacter, and Thiobacillus, were identified. Network analysis further indicated that in response to CC, Methylobacter and Thiobacillus were correlated with the microbial degradation and transformation of DOM. These findings will improve our understanding of the interactions between microbial community and DOM in continuous cropping soil.

Matriptase-2 regulates iron homeostasis primarily by setting the basal levels of hepatic hepcidin expression through a nonproteolytic mechanism.

Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease. It plays a key role in iron homeostasis by suppressing the iron-regulatory hormone, hepcidin. Lack of functional MT2 results in an inappropriately high hepcidin and iron-refractory iron-deficiency anemia. Mt2 cleaves multiple components of the hepcidin-induction pathway in vitro. It is inhibited by the membrane-anchored serine protease inhibitor, Hai-2. Earlier in vivo studies show that Mt2 can suppress hepcidin expression independently of its proteolytic activity. In this study, our data indicate that hepatic Mt2 was a limiting factor in suppressing hepcidin. Studies in Tmprss6-/- mice revealed that increases in dietary iron to ∼0.5% were sufficient to overcome the high hepcidin barrier and to correct iron-deficiency anemia. Interestingly, the increased iron in Tmprss6-/- mice was able to further upregulate hepcidin expression to a similar magnitude as in wild-type mice. These results suggest that a lack of Mt2 does not impact the iron induction of hepcidin. Additional studies of wild-type Mt2 and the proteolytic-dead form, fMt2S762A, indicated that the function of Mt2 is to lower the basal levels of hepcidin expression in a manner that primarily relies on its nonproteolytic role. This idea is supported by the studies in mice with the hepatocyte-specific ablation of Hai-2, which showed a marginal impact on iron homeostasis and no significant effects on iron regulation of hepcidin. Together, these observations suggest that the function of Mt2 is to set the basal levels of hepcidin expression and that this process is primarily accomplished through a nonproteolytic mechanism.

Different endodontic treatments induced root development of two nonvital immature teeth in the same patient: A case report.

BACKGROUND: Pulp revascularization is a novel way to treat immature teeth with periapical disease, and the technique has become increasingly well established in recent years. By puncturing the periapical tissue, bleeding is induced, and a blood clot is formed in the root canal. The blood clot acts as a natural bioscaffold onto which mesenchymal stem cells from periapical tissue can be seeded and restore pulp vascularity, thus promoting root development as well as apical closure. Although the effect of pulp revascularization is ideal, there are certain requirements for the apical condition of the teeth. The apical barrier technique and apexification are still indispensable for teeth that cannot achieve ideal blood clot formation. In addition, a meta-analysis of several clinical studies concluded that pulp revascularization has no significant advantages over other treatments. CASE SUMMARY: A 10-year-old girl complained of pain in the right upper and lower posterior teeth for 2 d. Clinical and radiological examinations revealed that both the right maxillary and mandibular second premolars were immature with periapical radiolucency. The right maxillary second premolar was treated by pulp revascularization, while the right mandibular second premolar was treated by conventional apical barrier surgery after revascularization failed. The purpose of this report is to compare the different root maturation processes induced by the pulp revascularization and apical barrier techniques in the same patient in homonymous teeth from different jaws. Twelve months of follow-up showed that the apical foramen of both teeth presented a clear tendency to close; however, the tooth treated with pulp revascularization showed a significant increase in root length as well as root canal wall thickness. CONCLUSION: For the treatment of nonvital immature teeth, pulp revascularization showed a superior therapeutic effect in comparison with the apical barrier technique.

Gut bacterium promotes host fitness in special ecological niche by affecting sugar metabolism in Drosophila suzukii.

As an important fruit pest of global significance, Drosophila suzukii occupies a special ecological niche, with the characteristics of high sugar and low protein contents. This niche differs from those occupied by other fruit-damaging Drosophila species. Gut bacteria substantially impact the physiology and ecology of insects. However, the contribution of gut microbes to the fitness of D. suzukii in their special ecological niche remains unclear. In this study, the effect of Klebsiella oxytoca on the development of D. suzukii was examined at physiological and molecular levels. The results showed that, after the removal of gut microbiota, the survival rate and longevity of axenic D. suzukii decreased significantly. Reintroduction of K. oxytoca to the midgut of D. suzukii advanced the development level of D. suzukii. The differentially expressed genes and metabolites between axenic and K. oxytoca-reintroduced D. suzukii were enriched in the pathways of carbohydrate metabolism. This advancement was achieved through an increased glycolysis rate and the regulation of the transcript level of key genes in the glycolysis/gluconeogenesis pathway. Klebsiella oxytoca is likely to play an important role in increasing host fitness in their high-sugar ecological niche by stimulating the glycolysis/gluconeogenesis pathway. As a protein source, bacteria can also provide direct nutrition for D. suzukii, which depends on the quantity or biomass of K. oxytoca. This result may provide a new target for controlling D. suzukii by inhibiting sugar metabolism through eliminating the effect of K. oxytoca and thus disrupting the balance of gut microbial communities.

Transcriptomic analysis provides insight into defensive strategies in response to continuous cropping in strawberry (Fragaria × ananassa Duch.) plants.

BACKGROUND: Strawberries are an important economic fruit crop world-wide. In strawberry cultivation, continuous cropping (CC) can seriously threaten yield and quality. However, our understanding of the gene expression changes in response to CC and during subsequent defense processes is limited. In this study, we analyzed the impact of CC on the transcriptome of strawberry roots using RNA-Seq technology to elucidate the effect of CC and the subsequent molecular changes. RESULTS: We found that CC significantly affects the growth of strawberry plants. The transcriptome analysis identified 136 differentially expressed genes (DEGs), including 49 up-regulated and 87 down-regulated DEGs. A Gene Ontology (GO) analysis indicated that the up-regulated DEGs were mainly assigned to defense-related GO terms, and most down-regulated DEGs were assigned to nutrient-related GO terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the responsive DEGs were classified in a large number of important biological pathways, such as phenylalanine metabolism, starch and sucrose metabolism, phenylpropanoid biosynthesis, glutathione metabolism and plant-pathogen interaction. We also found that four WRKY transcription factors and three peroxidase genes involved in plant defense pathways were up-regulated in the roots of strawberry plants subjected to CC. CONCLUSION: Several unigenes involved in plant defense processes, such as CNGCs, WRKY transcription factors, PR1, and peroxidase genes with highly variable expression levels between non-CC and CC treatments may be involved in the regulation of CC in strawberry. These results indicate that strawberry roots reallocate development resources to defense mechanisms in response to CC. This study will further deepen our understanding of the fundamental regulatory mechanisms of strawberry resource reallocation in response to CC.

Hepatocyte neogenin is required for hemojuvelin-mediated hepcidin expression and iron homeostasis in mice.

Neogenin (NEO1) is a ubiquitously expressed multifunctional transmembrane protein. It interacts with hemojuvelin (HJV), a BMP coreceptor that plays a pivotal role in hepatic hepcidin expression. Earlier studies suggest that the function of HJV relies on its interaction with NEO1. However, the role of NEO1 in iron homeostasis remains controversial because of the lack of an appropriate animal model. Here, we generated a hepatocyte-specific Neo1 knockout (Neo1fl/fl;Alb-Cre+) mouse model that circumvented the developmental and lethality issues of the global Neo1 mutant. Results show that ablation of hepatocyte Neo1 decreased hepcidin expression and caused iron overload. This iron overload did not result from altered iron utilization by erythropoiesis. Replacement studies revealed that expression of the Neo1L1046E mutant that does not interact with Hjv, was unable to correct the decreased hepcidin expression and high serum iron in Neo1fl/fl;Alb-Cre+ mice. In Hjv-/- mice, expression of HjvA183R mutant that has reduced interaction with Neo1, also displayed a blunted induction of hepcidin expression. These observations indicate that Neo1-Hjv interaction is essential for hepcidin expression. Further analyses suggest that the Hjv binding triggered the cleavage of the Neo1 cytoplasmic domain by a protease, which resulted in accumulation of truncated Neo1 on the plasma membrane. Additional studies did not support that Neo1 functions by inhibiting Hjv shedding as previously proposed. Together, our data favor a model in which Neo1 interaction with Hjv leads to accumulation of cleaved Neo1 on the plasma membrane, where Neo1 acts as a scaffold to induce the Bmp signaling and hepcidin expression.

The ectodomain of matriptase-2 plays an important nonproteolytic role in suppressing hepcidin expression in mice.

Matriptase-2 (MT2), encoded by TMPRSS6, is a membrane-anchored serine protease that plays a key role in suppressing hepatic hepcidin expression. MT2 is synthesized as a zymogen and undergoes autocleavage for activation. Previous studies suggest that MT2 suppresses hepcidin by cleaving hemojuvelin and other components of the bone morphogenetic protein-signaling pathway. However, the underlying mechanism is still debatable. Here we dissected the contributions of the nonproteolytic and proteolytic activities of Mt2 by taking advantage of Mt2 mutants and Tmprss6-/- mice. Studies of the protease-dead full-length Mt2 (Mt2S762A) and the truncated Mt2 that lacks the catalytic domain (Mt2mask) indicate that the catalytic domain, but not its proteolytic activity, was required for Mt2 to suppress hepcidin expression. This process was likely accomplished by the binding of Mt2 ectodomain to Hjv and Hfe. We found that Mt2 specifically cleaved the key components of the hepcidin-induction pathway, including Hjv, Alk3, ActRIIA, and Hfe, when overexpressed in hepatoma cells. Nevertheless, studies of a murine iron-refractory iron-deficiency anemia-causing mutant (Mt2I286F) in the complement protein subcomponents C1r/C1s, urchin embryonic growth factor, and bone morphogenetic protein 1 domain indicate that Mt2I286F can be activated, but it exhibited a largely compromised ability to suppress hepcidin expression. Coimmunoprecipitation analysis revealed that Mt2I286F, but not Mt2S762A, had reduced interactions with Hjv, ActRIIA, and Hfe. In addition, increased expression of a serine protease inhibitor, the hepatocyte growth factor activator inhibitor-2, in the liver failed to alter hepcidin. Together, these observations support the idea that the substrate interaction with Mt2 plays a determinant role and suggest that the proteolytic activity is not an appropriate target to modulate the function of MT2 for clinical applications.

Extrahepatic deficiency of transferrin receptor 2 is associated with increased erythropoiesis independent of iron overload.

Transferrin receptor 2 (TFR2) is a transmembrane protein expressed mainly in hepatocytes and in developing erythroid cells and is an important focal point in systemic iron regulation. Loss of TFR2 function results in a rare form of the iron-overload disease hereditary hemochromatosis. Although TFR2 in the liver has been shown to be important for regulating iron homeostasis in the body, TFR2's function in erythroid progenitors remains controversial. In this report, we analyzed TFR2-deficient mice in the presence or absence of iron overload to distinguish between the effects caused by a high iron load and those caused by loss of TFR2 function. Analysis of bone marrow from TFR2-deficient mice revealed a reduction in the early burst-forming unit-erythroid and an expansion of late-stage erythroblasts that was independent of iron overload. Spleens of TFR2-deficient mice displayed an increase in colony-forming unit-erythroid progenitors and in all erythroblast populations regardless of iron overload. This expansion of the erythroid compartment coincided with increased erythroferrone (ERFE) expression and serum erythropoietin (EPO) levels. Rescue of hepatic TFR2 expression normalized hepcidin expression and the total cell count of the bone marrow and spleen, but it had no effect on erythroid progenitor frequency. On the basis of these results, we propose a model of TFR2's function in murine erythropoiesis, indicating that deficiency in this receptor is associated with increased erythroid development and expression of EPO and ERFE in extrahepatic tissues independent of TFR's role in the liver.

The catalytic, stem, and transmembrane portions of matriptase-2 are required for suppressing the expression of the iron-regulatory hormone hepcidin.

Matriptase-2 (MT2) is a type-II transmembrane, trypsin-like serine protease that is predominantly expressed in the liver. It is a key suppressor for the expression of hepatic hepcidin, an iron-regulatory hormone that is induced via the bone morphogenetic protein signaling pathway. A current model predicts that MT2 suppresses hepcidin expression by cleaving multiple components of the induction pathway. MT2 is synthesized as a zymogen that undergoes autocleavage for activation and shedding. However, the biologically active form of MT2 and, importantly, the contributions of different MT2 domains to its function are largely unknown. Here we examined the activities of truncated MT2 that were generated by site-directed mutagenesis or Gibson assembly master mix, and found that the stem region of MT2 determines the specificity and efficacy for substrate cleavage. The transmembrane domain allowed MT2 activation after reaching the plasma membrane, and the cytoplasmic domain facilitated these processes. Further in vivo rescue studies indicated that the entire extracellular and transmembrane domains of MT2 are required to correct the low-hemoglobin, low-serum iron, and high-hepcidin status in MT2-/- mice. Unlike in cell lines, no autocleavage of MT2 was detected in vivo in the liver, implying that MT2 may also function independently of its proteolytic activity. In conjunction with our previous studies implicating the cytoplasmic domain as an intracellular iron sensor, these observations reveal the importance of each MT2 domain for MT2-mediated substrate cleavage and for its biological function.

Matriptase-2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway.

Systemic iron homeostasis is maintained by regulation of iron absorption in the duodenum, iron recycling from erythrocytes, and iron mobilization from the liver and is controlled by the hepatic hormone hepcidin. Hepcidin expression is induced via the bone morphogenetic protein (BMP) signaling pathway that preferentially uses two type I (ALK2 and ALK3) and two type II (ActRIIA and BMPR2) BMP receptors. Hemojuvelin (HJV), HFE, and transferrin receptor-2 (TfR2) facilitate this process presumably by forming a plasma membrane complex with BMP receptors. Matriptase-2 (MT2) is a protease and key suppressor of hepatic hepcidin expression and cleaves HJV. Previous studies have therefore suggested that MT2 exerts its inhibitory effect by inactivating HJV. Here, we report that MT2 suppresses hepcidin expression independently of HJV. In Hjv-/- mice, increased expression of exogenous MT2 in the liver significantly reduced hepcidin expression similarly as observed in wild-type mice. Exogenous MT2 could fully correct abnormally high hepcidin expression and iron deficiency in MT2-/- mice. In contrast to MT2, increased Hjv expression caused no significant changes in wild-type mice, suggesting that Hjv is not a limiting factor for hepcidin expression. Further studies revealed that MT2 cleaves ALK2, ALK3, ActRIIA, Bmpr2, Hfe, and, to a lesser extent, Hjv and Tfr2. MT2-mediated Tfr2 cleavage was also observed in HepG2 cells endogenously expressing MT2 and TfR2. Moreover, iron-loaded transferrin blocked MT2-mediated Tfr2 cleavage, providing further insights into the mechanism of Tfr2's regulation by transferrin. Together, these observations indicate that MT2 suppresses hepcidin expression by cleaving multiple components of the hepcidin induction pathway.

The Tumor Suppressor, P53, Decreases the Metal Transporter, ZIP14.

Loss of p53's proper function accounts for over half of identified human cancers. We identified the metal transporter ZIP14 (Zinc-regulated transporter (ZRT) and Iron-regulated transporter (IRT)-like Protein 14) as a p53-regulated protein. ZIP14 protein levels were upregulated by lack of p53 and downregulated by increased p53 expression. This regulation did not fully depend on the changes in ZIP14's mRNA expression. Co-precipitation studies indicated that p53 interacts with ZIP14 and increases its ubiquitination and degradation. Moreover, knockdown of p53 resulted in higher non-transferrin-bound iron uptake, which was mediated by increased ZIP14 levels. Our study highlights a role for p53 in regulating nutrient metabolism and provides insight into how iron and possibly other metals such as zinc and manganese could be regulated in p53-inactivated tumor cells.

Neogenin Facilitates the Induction of Hepcidin Expression by Hemojuvelin in the Liver.

Hemojuvelin (HJV) regulates iron homeostasis by direct interaction with bone morphogenetic protein (BMP) ligands to induce hepcidin expression through the BMP signaling pathway in the liver. Crystallography studies indicate that HJV can simultaneously bind to both BMP2 and the ubiquitously expressed cell surface receptor neogenin. However, the role of the neogenin-HJV interaction in the function of HJV is unknown. Here we identify a mutation in HJV that specifically lowers its interaction with neogenin. Expression of this mutant Hjv in the liver of Hjv(-/-) mice dramatically attenuated its induction of BMP signaling and hepcidin mRNA, suggesting that interaction with neogenin is critical for the iron regulatory function of HJV. Further studies revealed that neogenin co-immunoprecipitated with ALK3, an essential type-I BMP receptor for hepatic hepcidin expression. Neogenin has also been shown to facilitate the cleavage of HJV by furin in transfected cells. Surprisingly, although cleavage of HJV by furin has been implicated in the regulation of HJV function in cell culture models and furin-cleaved soluble Hjv is detectable in the serum of mice, mutating the furin cleavage site did not alter the stimulation of hepcidin expression by Hjv in mice. In vivo studies validated the important role of HJV-BMP interaction for Hjv stimulation of BMP signaling and hepcidin expression. Together these data support a model in which neogenin acts as a scaffold to facilitate assembly of the HJV·BMP·BMP receptor complex to induce hepcidin expression.

Melatonin attenuates inflammation of acute pulpitis subjected to dental pulp injury.

Acute pulpitis (AP), one of the most common diseases in the endodontics, usually causes severe pain to the patients, which makes the search for therapeutic target of AP essential in clinic. Toll-like receptor 4 (TLR4) signaling is widely involved in the mechanism of pulp inflammation, while melatonin has been reported to have an inhibition for a various kinds of inflammation. We hereby studied whether melatonin can regulate the expression of TLR4/NF-ĸB signaling in the pulp tissue of AP and in human dental pulp cells (HDPCs). Two left dental pulps of the adult rat were drilled open to establish the AP model, and the serum levels of melatonin and pro-inflammatory cytokines, including interleukin 1ß (IL-1ß), interleukin 18 (IL-18) and tumor necrosis factor α (TNF-α), were assessed at 1, 3 and 5 d post injury. At the same time points, the expression of TLR4 signaling in the pulp was explored by quantitative real-time PCR and immunohistochemistry. The AP rats were administered an abdominal injection of melatonin to assess whether melatonin rescued AP and TLR4/NF-ĸB signaling. Dental pulp injury led to an approximately five-day period acute pulp inflammation and necrosis in the pulp and a significant up-regulation of IL-1ß, IL-18 and TNF-α in the serum. ELISA results showed that the level of melatonin in the serum decreased due to AP, while an abdominal injection of melatonin suppressed the increase in serum cytokines and the percentage of necrosis at the 5 d of the injured pulp. Consistent with the inflammation in AP rats, TLR4, NF-ĸB, TNF-α and IL-1ß in the pulp were increased post AP compared with the baseline expression. And melatonin showed an inhibition on TLR4/NF-ĸB signaling as well as IL-1ß and TNF-α production in the pulp of AP rats. Furthermore, melatonin could also regulate the expression of TLR4/NF-ĸB signaling in LPS-stimulated HDPCs. These data suggested that dental pulp injury induced AP and reduced the serum level of melatonin and that supplementation with melatonin may have a protective effect on AP by modulating TLR4/NF-ĸB signaling in the pulp and in pulp cells.

Low intracellular iron increases the stability of matriptase-2.

Matriptase-2 (MT2) is a type II transmembrane serine protease that is predominantly expressed in hepatocytes. It suppresses the expression of hepatic hepcidin, an iron regulatory hormone, by cleaving membrane hemojuvelin into an inactive form. Hemojuvelin is a bone morphogenetic protein (BMP) co-receptor. Here, we report that MT2 is up-regulated under iron deprivation. In HepG2 cells stably expressing the coding sequence of the MT2 gene, TMPRSS6, incubation with apo-transferrin or the membrane-impermeable iron chelator, deferoxamine mesylate salt, was able to increase MT2 levels. This increase did not result from the inhibition of MT2 shedding from the cells. Rather, studies using a membrane-permeable iron chelator, salicylaldehyde isonicotinoyl hydrazone, revealed that depletion of cellular iron was able to decrease the degradation of MT2 independently of internalization. We found that lack of the putative endocytosis motif in its cytoplasmic domain largely abolished the sensitivity of MT2 to iron depletion. Neither acute nor chronic iron deficiency was able to alter the association of Tmprss6 mRNA with polyribosomes in the liver of rats indicating a lack of translational regulation by low iron levels. Studies in mice showed that Tmprss6 mRNA was not regulated by iron nor the BMP-mediated signaling with no evident correlation with either Bmp6 mRNA or Id1 mRNA, a target of BMP signaling. These results suggest that regulation of MT2 occurs at the level of protein degradation rather than by changes in the rate of internalization and translational or transcriptional mechanisms and that the cytoplasmic domain of MT2 is necessary for its regulation.

[Effects of different host plants on the cold-resistant substances in overwintering larvae of Carposina sasakii Matsumura (Lepidoptera: Carposinidae)].

To evaluate the influence of different host plants including apple, wild jujube, jujube, pear and hawthorn on the cold-tolerance substances in overwintering larvae of the peach fruit moth Carposina sasakii Matsumura, we measured the larvae super-cooling capacity, the water content (W), total fat content (TFC), total protein content (TPC) and total glycogen content (TGC) in the body. Results showed that the mean super-cooling point (SCPs) and freezing point (FPs) of overwintering larvae from the 5 host plant fruits differed significantly, ranging from -15.53 to -8.50 degrees C and -11.31 to -4.04 degrees C, respectively. The overwintering larvae fed on hawthorn owned the highest SCP, FP, TGC and the lowest W, while those fed on apple had the lowest SCP, FP, TFC and TGC but the highest W and TPC. The fresh mass (FM) of the overwintering larvae fed on pear was the highest, while those fed on jujube was very low. Those fed on jujube accumulated the highest TFC but the lowest TPC.

An iron-regulated and glycosylation-dependent proteasomal degradation pathway for the plasma membrane metal transporter ZIP14.

Protein degradation is instrumental in regulating cellular function. Plasma membrane proteins targeted for degradation are internalized and sorted to multivesicular bodies, which fuse with lysosomes, where they are degraded. ZIP14 is a newly identified iron transporter with multitransmembrane domains. In an attempt to dissect the molecular mechanisms by which iron regulates ZIP14 levels, we found that ZIP14 is endocytosed, extracted from membranes, deglycosylated, and degraded by proteasomes. This pathway did not depend on the retrograde trafficking to the endoplasmic reticulum and thus did not involve the well-defined endoplasmic reticulum-associated protein degradation pathway. Iron inhibited membrane extraction of internalized ZIP14, resulting in higher steady-state levels of ZIP14. Asparagine-linked (N-linked) glycosylation of ZIP14, particularly the glycosylation at N102, was required for efficient membrane extraction of ZIP14 and therefore is necessary for its iron sensitivity. These findings highlight the importance of proteasomes in the degradation of endocytosed plasma membrane proteins.