SIRT2 activity is required for the survival of C6 glioma cells

SIRT2 is a tubulin deacetylase, which can play either detrimental or beneficial roles in cell survival under different conditions. While it has been suggested that reduced SIRT2 expression in human gliomas may contribute to development of gliomas, there has been no study that directly determines the effects of decreased SIRT2 activity on the survival of glioma cells. In this study we applied both pharmacological and molecular approaches to determine the roles of SIRT2 in the survival of glioma cells. Our studies, by conducting such assays as flow cytometry-based Annexin V assay and caspase-3 immunostaining, have indicated that decreased SIRT2 activity leads to apoptosis of C6 glioma cells by caspase-3-dependent pathway. Our experiments have further shown that reduced SIRT2 activity produces necrosis of C6 glioma cells. Moreover, our study applying SIRT2 siRNA has also shown that decreased SIRT2 leads to both necrosis and apoptotic changes of C6 glioma cells. Collectively, our study has provided novel evidence indicating that SIRT2 activity plays a key role in maintaining the survival of glioma cells, and that reduced SIRT2 activity can induce both necrosis and caspase-3-dependent apoptosis of C6 glioma cells. These results have also suggested that inhibition of SIRT2 might become a novel therapeutic strategy for gliomas.     

Paclobutrazol and plant-growth promoting bacterial endophyte Pantoea sp. enhance copper tolerance of guinea grass (Panicum maximum) in hydroponic culture

As most gramineous plants, guinea grass (Panicum maximum) comprise cellulosic biomass, which may be used as a feedstock for bioenergy. In order to develop such potential energy plants on copper-polluted lands, the hydroponic experiments with Cu, Paclobutrazol (PP333, a kind of antigibberellin) and plant growth-promoting bacterial endophyte (PGPB) treatments were carried out in a greenhouse. The seedlings of two cultivars of guinea grass, GG1 (P. maximum var. Natsukomaki) and GG2 (P. maximum var. Natsukaze) in 3 weeks old were treated, respectively, with different Cu treatments [0(CK), 100, 200, 300, 400, 500 μM l⁻¹ Cu] for estimating Cu toxicity. The results showed that elevated Cu restrained plant growth and reduced biomass. According to the EC50 value [the Cu concentration when the relative gain in fresh weight ratio was 50% of control] of two tested cultivars, the concentration of Cu for further experiments was decided as 300 μM l⁻¹. Both pretreatments of PP333 (200, 400, 600 mg l⁻¹) and PGPB (Pantoea sp.) significantly alleviated the negative affect caused by stress of 300 μM l⁻¹ Cu. The pretreatment of 400 mg l⁻¹ PP333 promoted both two cultivars in biomass, compared to 300 μM l⁻¹ Cu treat. The inoculation of Pantoea sp. Jp3-3 increased shoot dry weight, compared to Cu treat. The results suggested that the main reason for both PP333 and Pantoea sp. Jp3-3 enhanced Cu tolerance in guinea grass was that their pretreatments significantly decreased Cu absorption and accumulation under excessive Cu stress. The present study has provided a new insight into the exploitation of energy plant in heavy metal polluted condition by the way of plant growth regulation for increasing heavy metal tolerance.     

Kinetic modeling of [18F]VAT,a novel radioligand for positron emission tomography imaging vesicular acetylcholine transporter in non‐human primate brain

Molecular imaging of vesicular acetylcholine transporter (VACh T) in the brain provides an important cholinergic biomarker for the pathophysiology and treatment of dementias including Alzheimer's disease. In this study, kinetics modeling methods were applied and compared for quantifying regional brain uptake of the VACh T‐specific positron emission tomography radiotracer, ((?)‐(1‐(‐8‐(2‐fluoroethoxy)‐3‐hydroxy‐1,2,3,4‐tetrahydronaphthalen‐2‐yl)piperidin‐4‐yl)(4‐fluorophenyl)‐methanone) ([¹⁸F]VAT ) in macaques. Total volume distribution (V _T ) estimates were compared for one‐tissue compartment model (1TCM ), two‐tissue compartment model (2TCM ), Logan graphic analysis (LoganAIF ) and multiple linear analysis (MA 1) with arterial blood input function using data from three macaques. Using the cerebellum‐hemispheres as the reference region with data from seven macaques, three additional models were compared: reference tissue model (RTM ), simplified RTM (SRTM ), and Logan graphic analysis (LoganREF ). Model selection criterion indicated that a) 2TCM and SRTM were the most appropriate kinetics models for [¹⁸F]VAT ; and b) SRTM was strongly correlated with 2TCM (Pearson's coefficients r  > 0.93, p  < 0.05). Test–retest studies demonstrated that [¹⁸F]VAT has good reproducibility and reliability (TRV < 10%, ICC > 0.72). These studies demonstrate [¹⁸F]VAT is a promising VACh T positron emission tomography tracer for quantitative assessment of VACh T levels in the brain of living subjects.

     

A thioredoxin NbTRXh2 from Nicotiana benthamiana negatively regulates the movement of Bamboo mosaic virus

An up‐regulated gene derived from Bamboo mosaic virus (BaMV)‐infected Nicotiana benthamiana plants was cloned and characterized in this study. BaMV is a single‐stranded, positive‐sense RNA virus. This gene product, designated as NbTRXh2, was matched with sequences of thioredoxin h proteins, a group of small proteins with a conserved active‐site motif WCXPC conferring disulfide reductase activity. To examine how NbTRXh2 is involved in the infection cycle of BaMV, we used the virus‐induced gene silencing technique to knock down NbTRXh2 expression in N. benthamiana and inoculated the plants with BaMV. We observed that, compared with control plants, BaMV coat protein accumulation increased in knockdown plants at 5 days post‐inoculation (dpi). Furthermore, BaMV coat protein accumulation did not differ significantly between NbTRXh2‐knockdown and control protoplasts at 24 hpi. The BaMV infection foci in NbTRXh2‐knockdown plants were larger than those in control plants. In addition, BaMV coat protein accumulation decreased when NbTRXh2 was transiently expressed in plants. These results suggest that NbTRXh2 plays a role in restricting BaMV accumulation. Moreover, confocal microscopy results showed that NbTRXh2‐OFP (NbTRXh2 fused with orange fluorescent protein) localized at the plasma membrane, similar to AtTRXh9, a homologue in Arabidopsis. The expression of the mutant that did not target the substrates failed to reduce BaMV accumulation. Co‐immunoprecipitation experiments revealed that the viral movement protein TGBp2 could be the target of NbTRXh2. Overall, the functional role of NbTRXh2 in reducing the disulfide bonds of targeting factors, encoded either by the host or virus (TGBp2), is crucial in restricting BaMV movement.     

电针刺激联合局部振动治疗对脊髓损伤并发下肢痉挛患者康复效果及血清BDNF、PDGF的影响

目的:探讨脊髓损伤(SCI)并发下肢痉挛患者经电针刺激联合局部振动治疗后的临床效果。方法:选取本院于2018年1月到2019年6月期间收治的SCI并发下肢痉挛患者90例,根据乱数表法将上述患者分为对照组(n=45)和研究组(n=45),对照组患者给予常规康复系统疗法治疗,研究组在对照组的基础上给予电针刺激联合局部振动治疗,比较两组患者临床疗效、相关量表评分、内收肌角、直腿抬高角以及血清BDNF、PDGF水平。结果:研究组治疗后的临床总有效率为91.11%(41/45),高于对照组的64.44%(29/45)(P0.05)。两组患者治疗后改良Barthel指数、血清BDNF、PDGF水平均升高,且研究组高于对照组(P0.05);改良Ashworth量表、临床痉挛指数评分降低,且研究组低于对照组(P0.05)。两组患者治疗后内收肌角、直腿抬高角均扩大,且研究组大于对照组(P0.05)。结论:电针刺激联合局部振动治疗SCI并发下肢痉挛患者,可有效促进患者康复,改善下肢痉挛,提高生活自理能力,同时还可有效改善血清BDNF、PDGF水平。     

Long noncoding RNA LINC00319 regulates ROMO1 expression and promotes bladder cancer progression via miR-4492/ROMO1 axis

Growing reports indicate that long noncoding RNA (lncRNA) are involved in the regulation of various biological processes of cancer cells. LINC00319 is an ill investigated lncRNA and has been shown to regulate lung cancer, nasopharyngeal carcinoma and ovarian cancer. Nevertheless, its roles in bladder cancer (BCa) remain unclear. In our research, LINC00319 was shown to be an upregulated lncRNA in BCa tissues. LINC00319 expression is negatively correlated with the patient's prognosis. Silencing of LINC00319 suppressed BCa proliferation and invasiveness. In addition, the data indicated LINC00319 was a sponge for miR-4492 and miR-4492 suppressed ROMO1 expression in BCa. Furthermore, our results illustrated miR-4492/ROMO1 axis regulates proliferation, migration, and invasion and LINC00319 exerts oncogenic roles through modulating miR-4492/ROMO1 axis. In sum, this study suggested that LINC00319 acts as oncogenic roles in BCa progression.     

Immune-associated biomarkers for early diagnosis of Parkinson’s disease based on hematological lncRNA–mRNA co-expression

Early stage diagnosis of Parkinson’s disease (PD) is challenging without significant motor symptoms. The identification of effective molecular biomarkers as a hematological indication of PD may help improve the diagnostic timelines and accuracy. In the present paper, we analyzed and compared the blood samples of PD and control (CTR) patients to identify the disease-related changes and determine the putative biomarkers for PD diagnosis. Based on the RNA sequencing analysis, differentially expressed genes (DEGs) were identified, and the co-expression network of DEGs was constructed using the weighted gene correlation network analysis (WGCNA). The analysis leads to the identification of 87 genes that were exclusively regulated in the PD group, whereas 66 genes were significantly increased and 21 genes were significantly decreased in contrast with the control group. The results indicate that the core lncRNA–mRNA co-expression network greatly changes the immune response in PD patients. Specifically, the results showed that Prader Willi Angelman Region RNA6 (PWAR6), LINC00861, AC83843.1, IRF family, IFIT family and calcium/calmodulin-dependent protein kinase IV (CaMK4) may play important roles in the immune system of PD. Based on the findings from the present study, future research aims at identifying novel therapeutic strategies for PD.     

Aldosterone enhances high phosphate–induced vascular calcification through inhibition of AMPK‐mediated autophagy

It remains unclear whether the necessity of calcified mellitus induced by high inorganic phosphate (Pi) is required and the roles of autophagy plays in aldosterone (Aldo)‐enhanced vascular calcification (VC) and vascular smooth muscle cell (VSMC) osteogenic differentiation. In the present study, we found that Aldo enhanced VC both in vivo and in vitro only in the presence of high Pi, alongside with increased expression of VSMC osteogenic proteins (BMP2, Runx2 and OCN) and decreased expression of VSMC contractile proteins (α‐SMA, SM22α and smoothelin). However, these effects were blocked by mineralocorticoid receptor inhibitor, spironolactone. In addition, the stimulatory effects of Aldo on VSMC calcification were further accelerated by the autophagy inhibitor, 3‐MA, and were counteracted by the autophagy inducer, rapamycin. Moreover, inhibiting adenosine monophosphate‐activated protein kinase (AMPK) by Compound C attenuated Aldo/MR‐enhanced VC. These results suggested that Aldo facilitates high Pi‐induced VSMC osteogenic phenotypic switch and calcification through MR‐mediated signalling pathways that involve AMPK‐dependent autophagy, which provided new insights into Aldo excess‐associated VC in various settings.     

SALT-OVERLY SENSITIVE5 Mediates Arabidopsis Seed Coat Mucilage Adherence and Organization through Pectins

Interactions between cell wall polymers are critical for establishing cell wall integrity and cell-cell adhesion. Here, we exploit the Arabidopsis (Arabidopsis thaliana) seed coat mucilage system to examine cell wall polymer interactions. On hydration, seeds release an adherent mucilage layer strongly attached to the seed in addition to a nonadherent layer that can be removed by gentle agitation. Rhamnogalacturonan I (RG I) is the primary component of adherent mucilage, with homogalacturonan, cellulose, and xyloglucan constituting minor components. Adherent mucilage contains rays composed of cellulose and pectin that extend above the center of each epidermal cell. CELLULOSE SYNTHASE5 (CESA5) and the arabinogalactan protein SALT-OVERLY SENSITIVE5 (SOS5) are required for mucilage adherence through unknown mechanisms. SOS5 has been suggested to mediate adherence by influencing cellulose biosynthesis. We, therefore, investigated the relationship between SOS5 and CESA5. cesa5-1 seeds show reduced cellulose, RG I, and ray size in adherent mucilage. In contrast, sos5-2 seeds have wild-type levels of cellulose but completely lack adherent RG I and rays. Thus, relative to each other, cesa5-1 has a greater effect on cellulose, whereas sos5-2 mainly affects pectin. The double mutant cesa5-1 sos5-2 has a much more severe loss of mucilage adherence, suggesting that SOS5 and CESA5 function independently. Double-mutant analyses with mutations in MUCILAGE MODIFIED2 and FLYING SAUCER1 that reduce mucilage release through pectin modification suggest that only SOS5 influences pectin-mediated adherence. Together, these findings suggest that SOS5 mediates adherence through pectins and does so independently of but in concert with cellulose synthesized by CESA5.Cellulosic cell walls are a defining feature of land plants. Primary cell walls are composed of three major classes of polysaccharides: cellulose, hemicelluloses, and pectins. In addition, approximately 10% of the primary cell wall is composed of protein (Burton et al., 2010). Cell walls provide mechanical support for the cell, and cell wall carbohydrates in the middle lamellae mediate cell-cell adhesion (Caffall and Mohnen, 2009). Current models of cell wall structure depict a cellulose-hemicellulose network embedded in an independent pectin gel (for review, see Albersheim et al., 2011). These components are believed to interact through both covalent and noncovalent bonds to provide structure and strength to the cell wall, although the relative importance of pectin and its interactions with the hemicellulose-cellulose network remain unclear (for review, see Cosgrove, 2005).Another gap in our understanding of cell wall structure and assembly is the role of arabinogalactan proteins (AGPs). AGPs are a family of evolutionarily conserved secreted proteins highly glycosylated with type II arabinogalactans, and they can be localized to the plasma membrane by a C-terminal glycophosphatidylinositol (GPI) lipid anchor (for review, see Schultz et al., 2000; Showalter, 2001; Johnson et al., 2003; Seifert and Roberts, 2007; Ellis et al., 2010). AGPs can be extensively modified in the cell wall; many glycosyl hydrolases can affect AGP function by cleaving their glycosyl side chains (Sekimata et al., 1989; Cheung et al., 1995; Wu et al., 1995; Kotake et al., 2005). The GPI anchor can also be cleaved, releasing the AGPs from the membrane into the cell wall (Schultz et al., 2000). Although their exact roles are still unclear, AGPs have been proposed to interact with cell wall polysaccharides, initiate intracellular signaling cascades, and influence a wide variety of biological processes (for review, see Seifert and Roberts, 2007; Ellis et al., 2010; Tan et al., 2013).Many fasciclin-like AGPs (FLAs), which contain at least one fasciclin domain (FAS) associated with protein-protein interactions, have been suggested to influence cellulose biosynthesis or organization (Seifert and Roberts, 2007; Li et al., 2010; MacMillan et al., 2010). FLA3 RNA interference lines have reduced intine cell wall biosynthesis and loss of Calcofluor white (a fluorescent dye specific for glycan molecules) staining in aborted pollen grains (Li et al., 2010). A fla11 fla12 double mutant was shown to have reduced cellulose deposition, altered cellulose microfibril angle, and reduced cell wall integrity (MacMillan et al., 2010). The fla11 fla12 double mutant also had reductions in arabinans, galactans, and rhamnose (MacMillan et al., 2010). FLA4/SALT-OVERLY SENSITIVE5 (SOS5) was identified in a screen for salt sensitivity in roots. The SOS5 gene encodes an FLA protein with a GPI anchor, two AGP-like domains, and two FAS domains (Shi et al., 2003). Plants homozygous for the loss-of-function conditional allele sos5-1 have thinner root cell walls that appear less organized (Shi et al., 2003). The presence of the two FAS domains has led to the suggestion that SOS5 may interact with other proteins, forming a network that strengthens the cell wall (Shi et al., 2003). SOS5 is involved in regulation of cell wall rheology through a pathway involving two Leu-rich repeat receptor-like kinases, FEI1 and FEI2 (Xu et al., 2008). SOS5 and FEI2 are also required for normal seed coat mucilage adherence and hypothesized to do so by influencing cellulose biosynthesis (Harpaz-Saad et al., 2011, 2012).Arabidopsis (Arabidopsis thaliana) seed coat mucilage is a powerful model for studying cell wall biosynthesis and polysaccharide interactions (Arsovski et al., 2010; Haughn and Western, 2012). Seed coat epidermal cells sequentially produce two distinct types of secondary cell walls with unique morphologies and properties (Western et al., 2000; Windsor et al., 2000). Between approximately 5 and 9 d approximate time of fertilization (DPA), seed coat epidermal cells synthesize mucilage and deposit it in the apoplast, creating a donut-shaped mucilage pocket that surrounds a central cytoplasmic column (Western et al., 2000, 2004; Haughn and Chaudhury, 2005). From 9 to 13 DPA, the cytoplasmic column is gradually replaced by a cellulose-rich, volcano-shaped secondary cell wall called the columella (Beeckman et al., 2000; Western et al., 2000; Windsor et al., 2000; Stork et al., 2010; Mendu et al., 2011).Seed mucilage is composed primarily of relatively unbranched rhamnogalacturonan I (RG I) with minor amounts of homogalacturonan (HG), cellulose, and hemicelluloses (for review, see Haughn and Western, 2012). When mucilage is hydrated, it expands rapidly from the apoplastic pocket, forming a halo that surrounds the seed. Mucilage separates into two fractions: a loose nonadherent fraction and an inner adherent fraction that can only be released by vigorous shaking, strong bases, or glycosidases (for review, see North et al., 2014). Galactans and arabinans are also present in mucilage, and their regulation by glycosidases is required for correct mucilage hydration (Dean et al., 2007; Macquet et al., 2007b; Arsovski et al., 2009). For example, β-XYLOSIDASE1 encodes a bifunctional β-d-xylosidase/α-l-arabinofuranosidase required for arabinan modification in mucilage, and β-xylosidase1 mutant seeds have a delayed mucilage release phenotype (Arsovski et al., 2009). MUCILAGE MODIFIED2 (MUM2) encodes a β-d-galactosidase, and mum2 seeds fail to release mucilage when hydrated in water (Dean et al., 2007; Macquet et al., 2007b). MUM2 is believed to modify RG I galactan side chains but may also affect the galactan component of other mucilage components (Dean et al., 2007; Macquet et al., 2007b). Galactans are capable of binding to cellulose in vitro and could affect mucilage hydration through pectin-cellulose interactions (Zykwinska et al., 2005, 2007a, 2007b; Dick-Pérez et al., 2011; Wang et al., 2012), although carbohydrate linkage analysis suggests that the galactan side chains are very short.Several studies indicate that seed mucilage extrusion and expansion are also influenced by methylesterification of HG. For example, both SUBTILISIN-LIKE SER PROTEASE1.7 and PECTIN METHYLESTERASE INHIBITOR6 are required for proper methyl esterification of mucilage (Rautengarten et al., 2008; Saez-Aguayo et al., 2013). Mutations in another gene, FLYING SAUCER1 (FLY1; a transmembrane E3 ubiquitin ligase), reduce the degree of pectin methylesterification in mucilage and cause increased mucilage adherence and defective mucilage extrusion (Voiniciuc et al., 2013). fly1 seeds have disc-like structures at the edge of the mucilage halo, which are outer primary cell wall fragments that detach from the columella during extrusion and are difficult to separate from the adherent mucilage (Voiniciuc et al., 2013).Recently, CELLULOSE SYNTHASE5 (CESA5) and SOS5 were proposed to facilitate cellulose-mediated mucilage adherence (Harpaz-Saad et al., 2011; Mendu et al., 2011; Sullivan et al., 2011). A simple hypothesis for the role of CESA5 in mucilage adherence is that it synthesizes cellulose, which interacts with the mucilage pectin to mediate adherence. Loss of CESA5 function results in a reduction of mucilage cellulose biosynthesis and a less adherent mucilage cell wall matrix (Mendu et al., 2011; Sullivan et al., 2011). The role of SOS5 in mucilage adherence is more difficult to explain. SOS5 null mutations cause a loss-of-adherence phenotype similar to cesa5-1 seeds, suggesting that SOS5 may regulate mucilage adherence by influencing CESA5 function (Harpaz-Saad et al., 2011). However, the mechanism through which SOS5 could influence CESA5 and/or cellulose biosynthesis is not clear.To better understand the role of SOS5 in mucilage adherence and its relationship to CESA5, we thoroughly investigated the seed coat epidermal cell phenotypes of the cesa5-1 and sos5-2 single mutants as well as those of the cesa5-1 sos5-2 double mutant. We also investigated how cellulose, SOS5, and pectin interact to mediate mucilage adherence by constructing double mutants with either cesa5-1 or sos5-2 together with either mum2-1 or fly1. Our results suggest that SOS5 mediates mucilage adherence independently of CESA5. Furthermore, compared with CESA5, SOS5 has a greater influence on mucilage pectin structure, suggesting that SOS5 mediates mucilage adherence through pectins, not cellulose.