Duvelisib attenuates bleomycin-induced pulmonary fibrosis via inhibiting the PI3K/Akt/mTOR signalling pathway

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive interstitial lung disease that seriously threatens the health of patients. The pathogenesis of IPF is still unclear, and there is a lack of effective therapeutic drugs. Myofibroblasts are the main effector cells of IPF, leading to excessive deposition of extracellular matrix (ECM) and promoting the progression of fibrosis. Inhibiting the excessive activation and relieving autophagy blockage of myofibroblasts is the key to treat IPF. PI3K/Akt/mTOR pathway plays a key regulatory role in promoting fibroblast activation and autophagy inhibition in lung fibrosis. Duvelisib is a PI3K inhibitor that can simultaneously inhibit the activities of PI3K-δ and PI3K-γ, and is mainly used for the treatment of relapsed/refractory chronic lymphocytic leukaemia (CLL) and small lymphocytic lymphoma tumour (SLL). In this study, we aimed to examine the effects of Duvelisib on pulmonary fibrosis. We used a mouse model of bleomycin-induced pulmonary fibrosis to evaluate the effects of Duvelisib on pulmonary fibrosis in vivo and further explored the potential pharmacological mechanisms of Duvelisib in lung fibroblasts in vitro. The in vivo experiments showed that Duvelisib significantly alleviated bleomycin-induced collagen deposition and improved pulmonary function. In vitro and in vivo pharmacological experiments showed that Duvelisib dose-dependently suppressed lung fibroblast activation and improved autophagy inhibition by inhibiting the phosphorylation of PI3K, Akt and mTOR. Our results indicate that Duvelisib can alleviate the severity of pulmonary fibrosis and provide potential drugs for the treatment of pulmonary fibrosis.     

SHED aggregate exosomes shuttled miR‐26a promote angiogenesis in pulp regeneration via TGF‐β/SMAD2/3 signalling

ObjectivesPulp regeneration brings big challenges for clinicians, and vascularization is considered as its determining factor. We previously accomplished pulp regeneration with autologous stem cells from deciduous teeth (SHED) aggregates implantation in teenager patients, however, the underlying mechanism needs to be clarified for regenerating pulp in adults. Serving as an important effector of mesenchymal stem cells (MSCs), exosomes have been reported to promote angiogenesis and tissue regeneration effectively. Here, we aimed to investigate the role of SHED aggregate‐derived exosomes (SA‐Exo) in the angiogenesis of pulp regeneration.Materials and MethodsWe extracted exosomes from SHED aggregates and utilized them in the pulp regeneration animal model. The pro‐angiogenetic effects of SA‐Exo on SHED and human umbilical vein endothelial cells (HUVECs) were evaluated. The related mechanisms were further investigated.ResultsWe firstly found that SA‐Exo significantly improved pulp tissue regeneration and angiogenesis in vivo. Next, we found that SA‐Exo promoted SHED endothelial differentiation and enhanced the angiogenic ability of HUVECs, as indicated by the in vitro tube formation assay. Mechanistically, miR‐26a, which is enriched in SA‐Exo, improved angiogenesis both in SHED and HUVECs via regulating TGF‐β/SMAD2/3 signalling.ConclusionsIn summary, these data reveal that SA‐Exo shuttled miR‐26a promotes angiogenesis via TGF‐β/SMAD2/3 signalling contributing to SHED aggregate‐based pulp tissue regeneration. These novel insights into SA‐Exo may facilitate the development of new strategies for pulp regeneration.     

The REDUCED LEAFLET Genes Encode Key Components of the trans-Acting Small Interfering RNA Pathway and Regulate Compound Leaf and Flower Development in Lotus japonicus

The endogenous trans-acting small interfering RNA (ta-siRNA) pathway plays a conserved role in adaxial-abaxial patterning of lateral organs in simple-leafed plant species. However, its function in compound-leafed species is largely unknown. Using the compound-leafed species Lotus japonicus, we identified and characterized two independent mutants, reduced leaflet1 (rel1) and rel3, whose most conspicuous defects in compound leaves are abaxialized leaflets and reduction in leaflet number. Concurrent mutations in REL genes also compromise flower development and result in radial symmetric floral organs. Positional cloning revealed that REL1 and REL3 encode the homologs of Arabidopsis (Arabidopsis thaliana) SUPPRESSOR OF GENE SILENCING3 and ARGONAUTE7/ZIPPY, respectively, which are key components of the ta-siRNA pathway. These observations, together with the expression and functional data, demonstrated that the ta-siRNA pathway plays conserved yet distinct roles in the control of compound leaf and flower development in L. japonicus. Moreover, the phenotypic alterations of lateral organs in ta-siRNA-deficient mutants and the regulation of downstream targets by the ta-siRNA pathway in L. japonicus were similar to those in the monocots but different from Arabidopsis, indicating many parallels between L. japonicus and the monocots in the control of lateral organ development by the ta-siRNA pathway.Plant endogenous small RNAs can be categorized into microRNAs (miRNAs) and small interfering RNAs (siRNAs) according to their mechanism of biogenesis (Vaucheret, 2006). trans-Acting siRNAs (ta-siRNAs) are one type of siRNA, and their biogenesis requires several key components, such as SUPPRESSOR OF GENE SILENCING3 (SGS3), RNA-DEPENDENT RNA POLYMERASE6 (RDR6), DICER-LIKE4 (DCL4), ARGONAUTE7 (AGO7)/ZIPPY (ZIP), and dsRNA-BINDING4 (Peragine et al., 2004; Vazquez et al., 2004; Gasciolli et al., 2005; Xie et al., 2005; Yoshikawa et al., 2005; Adenot et al., 2006; Nakazawa et al., 2007). Recent studies revealed that the ta-siRNA pathway is integrated into different processes of plant development, such as vegetative phase transition in Arabidopsis (Arabidopsis thaliana; Hunter et al., 2003; Peragine et al., 2004; Xie et al., 2005; Nakazawa et al., 2007) and shoot apical meristem (SAM) initiation in rice (Oryza sativa; Satoh et al., 1999; Itoh et al., 2000; Nagasaki et al., 2007). Parallel studies of this pathway in simple-leafed species also showed that the ta-siRNA pathway plays critical roles in patterning of leaves and floral organs.In flowering plants, leaves and flowers are produced on the periphery of the apical meristem. These lateral organs are structurally asymmetric with regard to the apical meristem. The adaxial side is adjacent to the meristem, while the abaxial side is away from the meristem. The ta-siRNA pathway was found to play a conserved role in specifying the adaxial identity of lateral organs in both monocots and dicots, but defects in the ta-siRNA pathway caused more severe phenotypes in monocots than in dicot Arabidopsis. In Arabidopsis, no clear leaf polarity defects were detected in the ta-siRNA-defective mutants. However, blocking the ta-siRNA pathway in asymmetric1 (as1) or as2 background, which are regulators of leaf adaxial identity (Lin et al., 2003; Xu et al., 2003), results in enhanced adaxial-abaxial leaf defects (Li et al., 2005; Xu et al., 2006; Garcia et al., 2006). In addition, the as2rdr6 double mutants also display aberrant flowers with sepals failing to enwrap the inner whorl organs and some sepals and petals becoming needle-like structures (Li et al., 2005). In maize (Zea mays), mutations in LEAFBLADELESS1 (LBL1), which encodes the Arabidopsis SGS3 ortholog, give rise to abnormal leaves with partial or complete loss of adaxial cell identity (Timmermans et al., 1998; Nogueira et al., 2007). In severe lbl1 mutants, leaf-like lateral organs of inflorescences and flowers develop as symmetric, thread-like organs, and the immature ear is exposed and arrested in development (Timmermans et al., 1998). In rice, the osdcl4-1 mutants display an abaxialized epidermis in coleoptiles and in the first leaf, and knockdown of OsDCL4 can lead to the awn-like lemma with a radial abaxialized identity and the stamens and carpel not enwrapped by the lemma and pelea (Liu et al., 2007). Transgenic rice plants with ectopic expression of SHOOTLESS4 (SHL4), the homolog of Arabidopsis AGO7, exhibit partially adaxialized leaves (Nagasaki et al., 2007; Shi et al., 2007).In addition to the ta-siRNA pathway, other components have also been shown to be involved in the adaxial-abaxial patterning of lateral organs. The Antirrhinum majus PHANTASTICA (PHAN) gene (Waites et al., 1998; Byrne et al., 2000; Xu et al., 2003; Qi et al., 2004), which is the ortholog of Arabidopsis AS1, and CLASS III HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP III) gene family members (McConnell et al., 2001; Emery et al., 2003) contribute to adaxial pattern formation of lateral organs, whereas members of YABBY (YAB; Sawa et al., 1999; Siegfried et al., 1999) and KANADI (Eshed et al., 2001; Kerstetter et al., 2001) gene families, AUXIN RESPONSE FACTOR3 (ARF3) and ARF4 (Pekker et al., 2005), and the miRNAs miR165/166 (Emery et al., 2003; Eshed et al., 2004; Mallory et al., 2004) are required for specifying abaxial identity. How the activities of these adaxial and abaxial determinants are coordinated has been extensively studied. It was found that ARF3 and ARF4 are regulated by the TAS3 ta-siRNA, and this regulation is conserved in both monocots and dicots (Allen et al., 2005; Williams et al., 2005). Recent studies in Arabidopsis suggest that ta-siRNAs act in a non-cell-autonomous manner to spatially restrict ARF activity (Chitwood et al., 2009; Schwab et al., 2009).In contrast to simple leaves with their single lamina, compound leaves are composed of one petiole and several leaflets. It is found that genes required for the adaxial-abaxial patterning of lateral organs in simple-leafed species also play critical roles in compound-leafed species, but these genes play multiple roles in compound leaf development. In tomato (Solanum lycopersicum), down-regulation of PHAN ortholog disturbs the leaf polarity as well as leaflet formation (Kim et al., 2003). Extensive studies of the PHAN expression in diverse compound-leafed species suggest that the function of PHAN in maintaining leaf adaxial identity is associated with leaflet formation in compound leaves and reduced adaxial identity of leaf primordia by down-regulation of PHAN could change pinnate compound leaves into palmate leaves (Kim et al., 2003). In pea (Pisum sativum), the role of PHAN in compound leaf development has also been elucidated by characterization of the phan mutant crispa (cri; Tattersall et al., 2005). However, unlike antisense PHAN transgenic tomato leaves, the cri mutant has the individual leaflet abaxialized, rather than the whole leaf. The number of lateral organs on the cri mutant compound leaves, including leaflets, is not altered, and the leaves remain pinnate. Apart from leaf development, the cri mutation also affects flower development. Although the floral organ identity and organ number are not altered, the laminar floral organ display abaxialized identity (Tattersall et al., 2005).The ta-siRNA pathway plays a critical role in simple-leafed species, but its role in compound-leafed species is not understood. Here, we address this question by analyzing loss-of-function reduced leaflet (rel1) and rel3 mutants in the compound-leafed species Lotus japonicus. Phenotypic characterization shows compound leaves of rel mutants exhibit a conspicuous disturbance in leaflet polarity as well as reduction in leaflet number. Besides the abnormal compound leaves, flower development is also severely affected in rel mutants, showing radial symmetric petals. REL1 and REL3 were identified by map-based cloning and were shown to be homologs of Arabidopsis SGS3 and AGO7, respectively. REL1 and REL3 act in the same genetic pathway and are both required for the biogenesis of TAS3 ta-siRNA. Further investigation reveals that the homolog of the Arabidopsis ARF3 is duplicated in the L. japonicus genome and that the duplicate ARF3 homologs and the ARF4 homolog are all negatively regulated by the ta-siRNA pathway. Furthermore, we found that the expression of LjYAB1, a homolog of Arabidopsis YAB1, was decreased in rel mutants, which may be associated with the reduced lamina.Taken together, our data reveal that the ta-siRNA pathway is integrated into the regulatory networks in the control of lateral organ development in L. japonicus and further emphasize the importance of the ta-siRNA pathway in compound leaf development. Moreover, our results also indicate many parallels between L. japonicus and monocots for the ta-siRNA pathway in the regulation of lateral organs.     

古田山常绿阔叶林凋落量时间动态及冰雪灾害的影响

2006年10月至2009年12月期间,我们通过对古田山24 ha常绿阔叶林动态样地169个种子雨收集器的凋落物进行烘干、分类、称量和数据分析,研究了凋落量的组成特征和时间动态,以及受2008年2月特大冰雪灾害的影响.2007年和2009年凋落量分别为532.05 g/m2和375.17 g/m2,年际变化显著,这与2008年的冰雪灾害有关.2007年古田山常绿阔叶林各组分在凋落量中所占比例依次为:叶(78.99%)>枝(14.69%)>皮(3-33%)>其他(2.99%);叶凋落量中各组分所占比例依次为:常绿阔叶树种叶(78.70%)>落叶阔叶树种叶(12.37%)>针叶树种叶(8.92%),其中甜槠(Castanopsis eyrei)、木荷(Schima superba)、马尾松(Pinus massoniana)和短柄袍(Quercus serrata vat.brevipetiolata)4个优势种年凋落量合计占叶凋落总量的71.36%,它们的动态直接影响着凋落总量的变化格局.凋落量高峰发生在春季(4月)和秋冬季(10月末至12月初),其中总凋落量、叶凋落量动态呈双峰型,枝条凋落量和树皮凋落量动态为不规则峰型,其他凋落量动态为单峰型.冰雪灾害后总凋落量、叶凋落量、枝条凋落量显著减少(P<0.05),其中甜槠、杨梅叶蚊母树(Distylium myricoides)叶凋落最显著减少(P<0.05)、木荷叶凋落量减少达到边缘显著水平(P<0.1),这也反映了冰亏灾害期间森林群落的受损情况.其他凋落量(主要成分为虫粪)在4B份增加极其显著(P<0.01),说明灾后植物的枝叶出现了补偿性生长.     

Progressive surface B cell antigen receptor down-regulation accompanies efficient development of antinuclear antigen B cells to mature, follicular phenotype

Previous studies have suggested that B cell Ag receptor (BCR) down-regulation by potentially pathological autoreactive B cells is associated with pathways leading to developmental arrest and receptor editing, or anergy. In this study we compare the primary development of B cells in two strains of mice expressing transgenic BCRs that differ by a single amino acid substitution that substantially increases reactivity for nuclear autoantigens such as DNA. Surprisingly, we find that both BCRs promote efficient development to mature follicular phenotype, but the strongly autoreactive BCR fails to promote marginal zone B cell development. The follicular B cells expressing the strongly autoreactive BCR do not appear to be anergic, as they robustly respond to polyclonal stimuli in vitro, are not short-lived, and can participate in germinal center reactions. Strikingly however, substantial and progressive down-modulation of surface IgM and IgD takes place throughout their primary development in the BM and periphery. We propose that BCR-autoantigen interactions regulate this pathway, resulting in reduced cellular avidity for autoantigens. This process of "learned ignorance" could allow autoreactive B cells access to the foreign Ag-driven memory B cell response, during which their self-reactivity would be attenuated by somatic hypermutation and selection in the germinal center.     

Complete Genome Sequence of Staphylococcus aureus Bacteriophage GH15

GH15 is a polyvalent phage that shows activity against a wide range of Staphylococcus aureus strains. In this work, the complete genome sequence of GH15 was determined. With a genome size of 139,806 bp (double-stranded DNA), GH15 is the largest staphylococcal phage sequenced to date. The complete genome encodes 214 open reading frames (ORFs) and 4 tRNAs. The closest relatives are the class III staphylococcal myobacteriophages, including K, A5W, ISP, Sb-1, and G1. Interestingly, although corresponding gene sequences demonstrate very high similarity, all the introns and inteins present in the phages listed above are absent in GH15. As such, GH15 can be considered phylogenetically unique among the staphylococcal myobacteriophages, indicating the diversity of this family.     

Prediction of lysine ubiquitination with mRMR feature selection and analysis

Ubiquitination, one of the most important post-translational modifications of proteins, occurs when ubiquitin (a small 76-amino acid protein) is attached to lysine on a target protein. It often commits the labeled protein to degradation and plays important roles in regulating many cellular processes implicated in a variety of diseases. Since ubiquitination is rapid and reversible, it is time-consuming and labor-intensive to identify ubiquitination sites using conventional experimental approaches. To efficiently discover lysine-ubiquitination sites, a sequence-based predictor of ubiquitination site was developed based on nearest neighbor algorithm. We used the maximum relevance and minimum redundancy principle to identify the key features and the incremental feature selection procedure to optimize the prediction engine. PSSM conservation scores, amino acid factors and disorder scores of the surrounding sequence formed the optimized 456 features. The Mathew’s correlation coefficient (MCC) of our ubiquitination site predictor achieved 0.142 by jackknife cross-validation test on a large benchmark dataset. In independent test, the MCC of our method was 0.139, higher than the existing ubiquitination site predictor UbiPred and UbPred. The MCCs of UbiPred and UbPred on the same test set were 0.135 and 0.117, respectively. Our analysis shows that the conservation of amino acids at and around lysine plays an important role in ubiquitination site prediction. What’s more, disorder and ubiquitination have a strong relevance. These findings might provide useful insights for studying the mechanisms of ubiquitination and modulating the ubiquitination pathway, potentially leading to potential therapeutic strategies in the future.     

The importance of mitochondrial metabolic activity and mitochondrial DNA replication during oocyte maturation in vitro on oocyte quality and subsequent embryo developmental competence

Mitochondrial metabolic capacity and DNA replication have both been shown to affect oocyte quality, but it is unclear which one is more critical. In this study, immature oocytes were treated with FCCP or ddC to independently inhibit the respective mitochondrial metabolic capacity or DNA replication of oocytes during in vitro maturation. To differentiate their roles, we evaluated various parameters related to oocyte maturation (germinal vesicle break down and nuclear maturation), quality (spindle formation, chromosome alignment, and mitochondrial distribution pattern), fertilization capability, and subsequent embryo developmental competence (blastocyst formation and cell number of blastocyst). Inhibition of mitochondrial metabolic capacity with FCCP resulted in a reduced percent of oocytes with nuclear maturation; normal spindle formation and chromosome alignment; evenly distributed mitochondria; and an ability to form blastocysts. Inhibition of mtDNA replication with ddC has no detectable effect on oocyte maturation and mitochondrial distribution, although high-dose ddC increased the percent of oocytes showing abnormal spindle formation and chromosome alignment. ddC did, however, reduce blastocyst formation significantly. Neither FCCP nor ddC exposure had an effect on the rate of fertilization. These findings suggest that the effects associated with lower mitochondrial DNA copy number do not coincide with the effects seen with reduced mitochondrial metabolic activity in oocytes. Inhibiting mitochondrial metabolic activity during oocyte maturation has a negative impact on oocyte maturation and subsequent embryo developmental competence. A reduction in mitochondrial DNA copy number, on the other hand, mainly affects embryonic development potential, but has little effect on oocyte maturation and in vitro fertilization.