The innervation and adaptive significance of extensively distributed neuromasts in <Emphasis Type="Italic">Glossogobius olivaceus</Emphasis> (Perciformes: Gobiidae)

Components of the lateral line system and their innervation were examined in Glossogobius olivaceus (Gobiidae), with almost all of the trunk scales bearing a row of superficial neuromasts, the latter comprising some 2,900 of the total (ca. 4,800) neuromasts on the body. The relationship between orientation and innervation of the superficial neuromasts on the head showed the buccal and mandibular rami to be clearly separated. On the trunk, the lateral ramus detached a number of branches, typically comprising dorsal, lateral and ventral ramules, to innervate neuromasts. Extensively distributed neuromasts were considered as an adaptation to a nocturnal habit, compensating for reduced vision.     

Hypoxia-inducible factor-2α regulates Fas-mediated chondrocyte apoptosis during osteoarthritic cartilage destruction

Apoptosis of articular chondrocytes is associated with the pathogenesis of osteoarthritis (OA). Recently, we demonstrated that hypoxia-inducible factor (HIF)-2α, encoded by Epas1, causes OA cartilage destruction by regulating the expression of various matrix-degrading enzymes. Here, we investigated the involvement of HIF-2α in chondrocyte apoptosis and OA cartilage destruction. HIF-2α levels in human and mouse OA chondrocytes were markedly elevated in association with increased apoptosis of articular chondrocytes. Overexpression or knockdown of HIF-2α alone did not cause chondrocyte apoptosis. However, HIF-2α expression markedly increased chondrocyte apoptosis in the presence of an agonistic anti-Fas (CD95) antibody. HIF-2α enhanced Fas expression and potentiated downstream signaling pathways, increasing the activity of initiator and executioner caspases. Overexpression of HIF-2α in mouse cartilage tissue, either by intra-articular injection of Epas1 adenovirus (Ad-Epas1) or in the context of chondrocyte-specific Epas1 transgenic mice, increased chondrocyte apoptosis and cartilage destruction. In contrast, chondrocyte-specific knockout of Epas1 in mice suppressed DMM (destabilization of the medial meniscus)-induced chondrocyte apoptosis and inhibited OA cartilage destruction. Moreover, Fas-deficient mice exhibited diminished chondrocyte apoptosis and OA cartilage destruction in response to Ad-Epas1 injection or DMM surgery. Taken together, our results demonstrate that HIF-2α potentiates Fas-mediated chondrocyte apoptosis, which is associated with OA cartilage destruction.     

Polymorphisms of the 5′ regulatory region of the porcine <Emphasis Type="Italic">PPARGC1A</Emphasis> gene and the effects on muscle fiber characteristics and meat quality

The purpose of this study was to determine the structure of the porcine PPARGC1A 5′ upstream region, and to find suitable molecular markers for improved meat quality and good lean meat production. Ten DNA polymorphisms, including 7 SNPs, 2 microsatellites, and 1 insertion or deletion were newly found in the 5′ upstream region of PPARGC1A. Three SNPs that had restriction enzyme site were evaluated for associations with muscle fiber characteristics and production traits. Two hundred fifty-two pigs (Yorkshire and Landrace) were used in this analysis. The c.-2894G>A genotypes was significantly associated with muscle fiber characteristics, including the number of fiber type I and IIb composition (P < 0.05), mean cross-sectional area of fibers (P < 0.01), and fiber number per unit area (P < 0.05). The animals with the GG genotype had a higher percentage of type I fibers and a lower percentage of type IIb fibers with better meat quality [higher pH value (P < 0.05) and lower drip loss (P < 0.05)] and lean meat production [larger loin eye area (P < 0.05)]. Moreover, the mRNA expression levels of PPARGC1A among genotypes were significantly different with the highest level of GG genotype. The c.-2885G>T and c.-1402A>T sites showed similar results that had significant effects on the mean cross-sectional area (CSA; P < 0.05), fiber number per unit area (P < 0.05) and loin eye area (P < 0.01). Therefore, we suggest that the c.-2894G>A polymorphism in the 5′ upstream region of the porcine PPARGC1A gene can be used as a meaningful molecular marker for simultaneous improvement of lean meat production and quality traits.     

Novel reporter systems for facile evaluation of I-SceI-mediated genome editing

Two major limitations to achieve efficient homing endonuclease-stimulated gene correction using retroviral vectors are low frequency of gene targeting and random integration of the targeting vectors. To overcome these issues, we developed a reporter system for quick and facile testing of novel strategies to promote the selection of cells that undergo targeted gene repair and to minimize the persistence of random integrations and non-homologous end-joining events. In this system, the gene target has an I-SceI site upstream of an EGFP reporter; and the repair template includes a non-functional EGFP gene, the positive selection transgene MGMTP140K tagged with mCherry, and the inducible Caspase-9 suicide gene. Using this dual fluorescent reporter system it is possible to detect properly targeted integration. Furthermore, this reporter system provides an efficient approach to enrich for gene correction events and to deplete events produced by random integration. We have also developed a second reporter system containing MGMTP140K in the integrated target locus, which allows for selection of primary cells with the integrated gene target after transplantation. This system is particularly useful for testing repair strategies in primary hematopoietic stem cells. Thus, our reporter systems should allow for more efficient gene correction with less unwanted off target effects.     

Foliar aphid feeding recruits rhizosphere bacteria and primes plant immunity against pathogenic and non-pathogenic bacteria in pepper

Background and Aims

Plants modulate defence signalling networks in response to different biotic stresses. The present study evaluated the effect of a phloem-sucking aphid on plant defence mechanisms in pepper (Capsicum annuum) during subsequent pathogen attacks on leaves and rhizosphere bacteria on roots.

Methods

Plants were pretreated with aphids and/or the chemical trigger benzothiadiazol (BTH) 7 d before being challenged with two pathogenic bacteria, Xanthomonas axonopodis pv. vesicatoria (Xav) as a compatible pathogen and X. axonopodis pv. glycines (Xag) as an incompatible (non-host) pathogen.

Key Results

Disease severity was noticeably lower in aphid- and BTH + aphid-treated plants than in controls. Although treatment with BTH or aphids alone did not affect the hypersensitive response (HR) against Xag strain 8ra, the combination treatment had a synergistic effect on the HR. The aphid population was reduced by BTH pretreatment and by combination treatment with BTH and bacterial pathogens in a synergistic manner. Analysis of the expression of the defence-related genes Capsicum annum pathogenesis-related gene 9 (CaPR9), chitinase 2 (CaCHI2), SAR8·2 and Lipoxygenase1 (CaLOX1) revealed that aphid infestation resulted in the priming of the systemic defence responses against compatible and incompatible pathogens. Conversely, pre-challenge with the compatible pathogen Xav on pepper leaves significantly reduced aphid numbers. Aphid infestation increased the population of the beneficial Bacillus subtilis GB03 but reduced that of the pathogenic Ralstonia solanacearum SL1931. The expression of defence-related genes in the root and leaf after aphid feeding indicated that the above-ground aphid infestation elicited salicylic acid and jasmonic acid signalling throughout the whole plant.

Conclusions

The findings of this study show that aphid feeding elicits plant resistance responses and attracts beneficial bacterial populations to help the plant cope with subsequent pathogen attacks.     

Osmotic stress regulates mammalian target of rapamycin (mTOR) complex 1 via c-Jun N-terminal Kinase (JNK)-mediated Raptor protein phosphorylation

mTOR complex 1 (mTORC1) is a multiprotein complex that integrates diverse signals including growth factors, nutrients, and stress to control cell growth. Raptor is an essential component of mTORC1 that functions to recruit specific substrates. Recently, Raptor was suggested to be a key target of regulation of mTORC1. Here, we show that Raptor is phosphorylated by JNK upon osmotic stress. We identified that osmotic stress induces the phosphorylation of Raptor at Ser-696, Thr-706, and Ser-863 using liquid chromatography-tandem mass spectrometry. We found that JNK is responsible for the phosphorylation. The inhibition of JNK abolishes the phosphorylation of Raptor induced by osmotic stress in cells. Furthermore, JNK physically associates with Raptor and phosphorylates Raptor in vitro, implying that JNK is responsible for the phosphorylation of Raptor. Finally, we found that osmotic stress activates mTORC1 kinase activity in a JNK-dependent manner. Our findings suggest that the molecular link between JNK and Raptor is a potential mechanism by which stress regulates the mTORC1 signaling pathway.     

Verification of the interdomain contact site in the inactive monomer, and the domain-swapped fold in the active dimer of Hsp33 in solution

Upon dimerization by oxidation, Hsp33 functions as a molecular chaperone in prokaryotes. Previously published structures of both the inactive and active species are of doubtful relevance to the solution conformations since the inactive (reduced) crystal structure was dimeric, while the active (oxidized) species was crystallized with a truncation of its regulation domain. The interdomain contact site of the inactive monomer, identified in this work, is consistent with that previously observed in the reduced dimer crystal. In contrast, fluorescence quenching of the active dimer contradicted the results expected from the domain-swapped fold observed in the truncated dimer crystal. The results of this study provide important new information concerning controversial issues in the activation process of Hsp33.     

Growth of ammonia-oxidizing archaea and bacteria in cattle manure compost under various temperatures and ammonia concentrations

A recent study showed that ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) coexist in the process of cattle manure composting. To investigate their physiological characteristics, liquid cultures seeded with fermenting cattle manure compost were incubated at various temperatures (37°C, 46°C, or 60°C) and ammonium concentrations (0.5, 1, 4, or 10?mM NH (4) (+) -N). The growth rates of the AOB and AOA were monitored using real-time polymerase chain reaction analysis targeting the bacterial and archaeal ammonia monooxygenase subunit A genes. AOB grew at 37°C and 4 or 10?mM NH (4) (+) -N, whereas AOA grew at 46°C and 10?mM NH (4) (+) -N. Incubation with allylthiourea indicated that the AOB and AOA grew by oxidizing ammonia. Denaturing gradient gel electrophoresis and subsequent sequencing analyses revealed that a bacterium related to Nitrosomonas halophila and an archaeon related to Candidatus Nitrososphaera gargensis were the predominant AOB and AOA, respectively, in the seed compost and in cultures after incubation. This is the first report to demonstrate that the predominant AOA in cattle manure compost can grow and can probably oxidize ammonia under moderately thermophilic conditions.