Morphology and Phylogeny of the Soil Ciliate Metopus yantaiensis n. sp. (Ciliophora,Metopida), with Identification of the Intracellular Bacteria

The morphology and infraciliature of a new ciliate, Metopus yantaiensis n. sp., discovered in coastal soil of northern China, were investigated. It is distinguished from its congeners by a combination of the following features: nuclear apparatus situated in the preoral dome; 18–21 somatic ciliary rows, of which three extend onto the preoral dome (dome kineties); three to five distinctly elongated caudal cilia, and 21–29 adoral polykinetids. The 18S rRNA genes of this new species and two congeners, Metopus contortus and Metopus hasei, were sequenced and phylogenetically analyzed. The new species is more closely related to M. hasei and the clevelandellids than to other congeners; both the genus Metopus and the order Metopida are not monophyletic. In addition, the digestion‐resistant bacteria in the cytoplasm of M. yantaiensis were identified, using a 16S rRNA gene clone library, sequencing, and fluorescence in situ hybridization. The detected intracellular bacteria are affiliated with Sphingomonadales, Rhizobiales, Rickettsiales (Alphaproteobacteria), Pseudomonas (Gammaproteobacteria), Rhodocyclales (Betaproteobacteria), Clostridiales (Firmicutes), and Flavobacteriales (Bacteroidetes).     

Single Cell Analysis Linking Ribosomal (r)DNA and rRNA Copy Numbers to Cell Size and Growth Rate Provides Insights into Molecular Protistan Ecology

Ribosomal (r)RNA and rDNA have been golden molecular markers in microbial ecology. However, it remains poorly understood how ribotype copy number (CN)‐based characteristics are linked with diversity, abundance, and activity of protist populations and communities observed at organismal levels. Here, we applied a single‐cell approach to quantify ribotype CNs in two ciliate species reared at different temperatures. We found that in actively growing cells, the per‐cell rDNA and rRNA CNs scaled with cell volume (CV) to 0.44 and 0.58 powers, respectively. The modeled rDNA and rRNA concentrations thus appear to be much higher in smaller than in larger cells. The observed rRNA:rDNA ratio scaled with CV^0.14. The maximum growth rate could be well predicted by a combination of per‐cell ribotype CN and temperature. Our empirical data and modeling on single‐cell ribotype scaling are in agreement with both the metabolic theory of ecology and the growth rate hypothesis, providing a quantitative framework for linking cellular rDNA and rRNA CNs with body size, growth (activity), and biomass stoichiometry. This study also demonstrates that the expression rate of rRNA genes is constrained by cell size, and favors biomass rather than abundance‐based interpretation of quantitative ribotype data in population and community ecology of protists.     

Characterization of Cre recombinase mouse lines enabling cell type-specific targeting of postnatal intervertebral discs

Cre/loxP technology is an important tool for studying cell type-specific gene functions. Cre recombinase mouse lines, including Agc1-CreER^T2, Col2a1-Cre; Col2a1-CreER^T2, Shh-Cre, Shh-CreER^T2, and Osx-Cre, have been proven to be valuable tools to elucidate the biology of long bones, yet the information for their activity in postnatal intervertebral disc (IVD) tissues was very limited. In this study, we used R26-mTmG fluorescent reporter to systematically analyze cell specificity and targeting efficiency of these six mouse lines in IVD tissues at postnatal growing and adult stages. We found that Agc1-CreER^T2 is effective to direct recombination in all components of IVDs, including annulus fibrosus (AF), nucleus pulposus (NP), and cartilaginous endplate (CEP), upon tamoxifen induction at either 2 weeks or 2 months of ages. Moreover, Col2a1-Cre targets most of the cells in IVDs, except for some cells in the outer AF (OAF) and NP. In contrast, the activity of Col2a1-CreER^T2 is mainly limited to the IAF of IVD tissues at either stage of tamoxifen injection. Similarly, Shh-Cre directs recombination specifically in all NP cells, whereas Shh-CreER^T2 is active only in a few NP cells when tamoxifen is administered at either stage. Finally, Osx-Cre targets cells in the CEP, but not in the NP or AF of IVDs tissues at these two stages. Thus, our data demonstrated that all these Cre lines can direct recombination in IVD tissues at postnatal stages with different cell type specificity and/or targeting efficiency, and can, therefore, serve as valuable tools to dissect cell type-specific gene functions in IVD development and homeostasis.     

Unconventional Sequence Requirement for Viral Late Gene Core Promoters of Murine Gammaherpesvirus 68

Infection with the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV), is associated with several cancers. During lytic replication of herpesviruses, viral genes are expressed in an ordered cascade. However, the mechanism by which late gene expression is regulated has not been well characterized in gammaherpesviruses. In this study, we have investigated the cis element that mediates late gene expression during de novo lytic infection with murine gammaherpesvirus 68 (MHV-68). A reporter system was established and used to assess the activity of viral late gene promoters upon infection with MHV-68. It was found that the viral origin of lytic replication, orilyt, must be on the reporter plasmid to support activation of the late gene promoter. Furthermore, the DNA sequence required for the activation of late gene promoters was mapped to a core element containing a distinct TATT box and its neighboring sequences. The critical nucleotides of the TATT box region were determined by systematic mutagenesis in the reporter system, and the significance of these nucleotides was confirmed in the context of the viral genome. In addition, EBV and KSHV late gene core promoters could be activated by MHV-68 lytic replication, indicating that the mechanisms controlling late gene expression are conserved among gammaherpesviruses. Therefore, our results on MHV-68 establish a solid foundation for mechanistic studies of late gene regulation.     

Tongxinluo Protects against Hypertensive Kidney Injury in Spontaneously-Hypertensive Rats by Inhibiting Oxidative Stress and Activating Forkhead Box O1 Signaling

Hypertension is an independent risk factor for the progression of chronic renal failure, and oxidative stress plays a critical role in hypertensive renal damage. Forkbox O1(FoxO1) signaling protects cells against oxidative stress and may be a useful target for treating oxidative stress-induced hypertension. Tongxinluo is a traditional Chinese medicine with cardioprotective and renoprotective functions. Therefore, this study aimed to determine the effects of Tongxinluo in hypertensive renal damage in spontaneously hypertensive rats(SHRs)and elucidate the possible involvement of oxidative stress and FoxO1 signaling in its molecular mechanisms. SHRs treated with Tongxinluo for 12 weeks showed a reduction in systolic blood pressure. In addition to increasing creatinine clearance, Tongxinluo decreased urinary albumin excretion, oxidative stress injury markers including malondialdehyde and protein carbonyls, and expression of nicotinamide adenine dinucleotide phosphate oxidase subunits and its activity in SHR kidneys. While decreasing phosphorylation of FoxO1, Tongxinluo also inhibited the phosphorylation of extracellular signal-regulated kinase1/2 and p38 and enhanced manganese superoxide dismutase and catalase activities in SHR kidneys. Furthermore, histology revealed attenuation of glomerulosclerosis and renal podocyte injury, while Tongxinluo decreased the expression of α-smooth muscle actin, extracellular matrixprotein, transforming growth factor β1 and small mothers against decapentaplegic homolog 3,and improved tubulointerstitial fibrosis in SHR kidneys. Finally, Tongxinluo inhibited inflammatory cell infiltration as well as expression of tumor necrosis factor-α and interleukin-6. In conclusion, Tongxinluo protected SHRs against hypertension-induced renal injury by exerting antioxidant, antifibrotic, and anti-inflammatory activities. Moreover, the underlying mechanisms of these effects may involve inhibition of oxidative stress and functional activation of FoxO1 signaling.     

Hydroxysafflor yellow A (HYSA) inhibited the proliferation and differentiation of 3T3-L1 preadipocytes

Hydroxysafflor yellow A (HSYA), a main component of safflor yellow, has been demonstrated to prevent steroid-induced avascular necrosis of femoral head by inhibiting primary bone marrow-derived mesenchymal stromal cells adipogenic differentiation induced by steroid. In this study, we investigate the effect of HSYA on the proliferation and adipogenesis of mouse 3T3-L1 preadipocytes. The effects of HSYA on proliferation and differentiation of 3T3-L1 cells and its possible mechanism were studied by 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide spectrophotometry, Oil Red O staining, intracellular triglyceride assays, real-time quantitative RT-PCR, transient transfection and dual luciferase reporter gene methods. HSYA inhibited the proliferation of 3T3-L1 preadipocytes and cell viability greatly decreased in a dose and time dependent manner. HSYA (1 mg/l) notably reduced the amount of intracellular lipid and triglyceride content in adipocytes by 21.3 % (2.13 ± 0.36 vs 2.71 ± 0.40, P < 0.01) and 22.6 % (1.33 ± 0.07 vs 1.72 ± 0.07, P < 0.01) on days 8 following the differentiation, respectively. HSYA (1 mg/l) significantly increased hormone-sensitive lipase (HSL) mRNA expression and promoter activities by 2.4- and 1.55-fold, respectively (P < 0.01), in differentiated 3T3-L1 adipocytes. HSYA inhibits the proliferation and adipogenesis of 3T3-L1 preadipocytes. The inhibitory action of HYSA on adipogenesis may be due to the promotion of lipolytic-specific enzyme HSL expression by increasing HSL promoter activity.     

NKG2D ligand RAE1ε induces generation and enhances the inhibitor function of myeloid‐derived suppressor cells in mice

Expression of surface NKG2D ligands on tumour cells, which activates nature killer (NK) cells and CD8⁺ T cells, is crucial in antitumour immunity. Some types of tumours have evolved mechanisms to suppress NKG2D‐mediated immune cell activation, such as tumour‐derived soluble NKG2D ligands or sustained NKG2D ligands produced by tumours down‐regulate the expression of NKG2D on NK cells and CD8⁺ T cells. Here, we report that surface NKG2D ligand RAE1ε on tumour cells induces CD11b⁺Gr‐1⁺ myeloid‐derived suppressor cell (MDSC) via NKG2D in vitro and in vivo. MDSCs induced by RAE1ε display a robust induction of IL‐10 and arginase, and these MDSCs show greater suppressive activity by inhibiting antigen‐non‐specific CD8⁺ T‐cell proliferation. Consistently, upon adoptive transfer, MDSCs induced by RAE1ε significantly promote CT26 tumour growth in IL‐10‐ and arginase‐dependent manners. RAE1ε moves cytokine balance towards Th2 but not Th1 in vivo. Furthermore, RAE1ε enhances inhibitory function of CT26‐derived MDSCs and promotes IL‐4 rather than IFN‐γ production from CT26‐derived MDSCs through NKG2D in vitro. Our study has demonstrated a novel mechanism for NKG2D ligand⁺ tumour cells escaping from immunosurveillance by facilitating the proliferation and the inhibitory function of MDSCs.     

1, 3-丙二醇发酵过程中盐浓度的胁迫作用

通过对克雷伯氏菌(Klebsiella pneumoniae)甘油发酵生产1, 3-丙二醇(1, 3-PD)过程的研究发现, 盐浓度对 1, 3-PD发酵有胁迫作用。盐浓度较低时, 菌体生长和产物生成均维持较高速率; 盐浓度较高时会导致菌体生长减慢, 1, 3-PD最终浓度, 甘油到1, 3-丙二醇的转化率降低, 同时1, 3-丙二醇氧化还原酶受到抑制。在5 m3罐中控制合适的盐浓度可以提高1, 3-PD的发酵水平, 使1, 3-PD的最终浓度达到64 g/L, 转化率61%, 生产强度2.1 g/(L·h)。