circ_SEPT9, a newly identified circular RNA,promotes oral squamous cell carcinoma progression through miR‐1225/PKN2 axis

Circular RNAs (circRNAs) represent a newly discovered class of endogenous non‐coding RNAs which are widely expressed and play important roles in disease progression. However, the function of circRNAs in oral squamous cell carcinoma (OSCC) still remains largely unknown. In this research, we found that circ_SEPT9 was highly expressed in OSCC cell lines and tumour tissues. Results showed that circ_SEPT9 promoted OSCC proliferation and tumour growth. And, circ_SEPT9 also enhanced the migration and invasion of OSCC cells. Mechanically, we found that circ_SEPT9 acted as a sponge for miR‐1225 to rescue PKN2 expression in OSCC cells. Inhibition of circ_SEPT9/miR‐1225/PKN2 pathway could effectively block the proliferation and metastasis of OSCC cells. Our study provides strong evidence that circ_SEPT9/miR‐1225/PKN2 axis is a promising target for OSCC treatment.     

Prevalence of Plasmid-Mediated Quinolone Resistance and Aminoglycoside Resistance Determinants among Carbapeneme Non-Susceptible Enterobacter cloacae

Background

Simultaneous resistance to aminoglycosides and fluoroquinolones in carbapeneme non-susceptible (CNS) isolates will inevitably create problems. The present study was performed to characterize the prevalence of the plasmid-mediated quinolone resistance determinants (QRDs) and aminoglycoside resistance determinants (ARDs) among the CNS Enterobacter cloacae (E. cloacae) isolates in a Chinese teaching hospital, and to acquire their molecular epidemiological characteristics.

Methods

The β-lactamases genes (including class A carbapenemase genes bla_KPC and bla_SME, metallo-β-lactamase genes (MBLs) bla_IMP, bla_VIM and bla_NDM, and extended spectrum β-lactamases (ESBLs),bla_CTX-M, bla_TEM and bla_SHV), QRDs (including qnrA, qnrB, qnrS and aac(6′)-Ib-cr) and ARDs (including aac(6′)-Ib, armA and rmtB) of these 35 isolates were determined by PCR and sequenced bidirectionally. The clonal relatedness was investigated by pulsed-field gel electrophoresis (PFGE).

Results

Of the 35 isolates, 9 (25.7%) harbored a carbapenemase gene; 23 (65.7%) carried ESBLs; 24 (68.6%) were QRD positive; and 27 (77.1%) were ARD positive. Among the 5 bla_IMP-8 positive strains, 4 (80%) contained both ESBL and QRD genes, and all the 5 (100%) harbored ARD genes. Of the 23 ESBLs positive isolates, 6 (26.1%) were carbapenemase positive, 14 (60.9%) were QRD positive, and 18 (78.3%) were ARD positive. PFGE revealed genetic diversity among the 35 isolates, indicating that the high prevalence of CNS E. cloacae isolates was not caused by clonal dissemination.

Conclusion

QRD and ARD genes were highly prevalent among the CNS E. cloacae isolates. Multiple resistant genes were co-expressed in the same isolates. The CNS E. cloacae isolate co-expressing bla_NDM-1, bla_IMP-26, qnrA1 and qnrS1 was first reported.     

氮磷肥配施对冬小麦灌浆期光合参数及产量的影响

在西北绿洲生态条件下, 实验设4个处理, 即165(N₁)和225 kg·hm^-2(N₂)2个氮素(纯氮)水平及105(P₁)和165 kg·hm^-2(P₂)2个磷素(P₂O₅)水平, 研究了氮磷肥配施对冬小麦(Triticum aestivum)品种临抗2号光合特性及产量的影响。结果表明, 低氮(165 kg·hm^-2)处理组的净光合速率(P_n)、气孔导度(G_s)及蒸腾速率(T_r)日变化均呈双峰曲线, 有光合“午休”现象; 高氮(225 kg·hm^-2)处理可减弱甚至使光合“午休”现象消失; 高磷(165 kg·hm^-2)和低磷(105 kg·hm^-2)处理对光合特性的影响差异不显著。N₂P₂具有最高的群体叶面积指数(LAI)、群体光合速率(CAP)、穗粒数、亩穗数、千粒重及产量, 且与N₁P₁和N₁P₂的差异均达显著水平, 与N₂P₁则无显著差异。但N₂P₂水分利用效率(WUE)低于N₂P₁, 显著高于N₁P₁和N₁P₂ (N₁P₁高于N₁P₂, 但无显著差异)。氮肥对光合“午休”的影响大于磷肥, 二者互作效应差异不显著。该实验条件下, 当N、P分别为225和105 kg·hm^-2时有利于提高冬小麦的光合速率及产量。     

Chloroplast Hsp93 Directly Binds to Transit Peptides at an Early Stage of the Preprotein Import Process

Three stromal chaperone ATPases, cpHsc70, Hsp90C, and Hsp93, are present in the chloroplast translocon, but none has been shown to directly bind preproteins in vivo during import, so it remains unclear whether any function as a preprotein-translocating motor and whether they have different functions during the import process. Here, using protein crosslinking followed by ionic detergent solubilization, we show that Hsp93 directly binds to the transit peptides of various preproteins undergoing active import into chloroplasts. Hsp93 also binds to the mature region of a preprotein. A time course study of import, followed by coimmunoprecipitation experiments, confirmed that Hsp93 is present in the same complexes as preproteins at an early stage when preproteins are being processed to the mature size. In contrast, cpHsc70 is present in the same complexes as preproteins at both the early stage and a later stage after the transit peptide has been removed, suggesting that cpHsc70, but not Hsp93, is important in translocating processed mature proteins across the envelope.Most chloroplast proteins are encoded by the nuclear genome as higher M_r preproteins that are fully synthesized in the cytosol before being imported into the chloroplast. The import process is initiated by binding of the N-terminal transit peptide of the preprotein to the translocon at the outer envelope membrane of chloroplasts (TOC) complex, in which Toc159 and Toc34 function as receptors and Toc75 is the outer membrane channel. This step is followed by binding of the transit peptide to the translocon at the inner envelope membrane of chloroplasts (TIC) machinery, the central components of which include the Tic20/Tic56/Tic100/Tic214 channel complex and Tic110. Tic110 functions as the stromal receptor for transit peptides and also as a scaffold for tethering other translocon components (for reviews, see Li and Chiu, 2010; Shi and Theg, 2013; Paila et al., 2015). The actual translocation of the bound preproteins across the envelope is powered by hydrolysis of ATP in the stroma (Pain and Blobel, 1987; Theg et al., 1989), and it is therefore assumed that some stromal ATPase motor proteins bind the preproteins as they emerge from the inner membrane and use the energy of ATP hydrolysis to translocate the preproteins across the envelope into the stroma.Three stromal ATPases have been identified in the translocon complex: cpHsc70 (chloroplast heat shock cognate protein 70 kD), Hsp90C (chloroplast heat shock protein 90), and Hsp93/ClpC (93-kD heat shock protein). Hsp93, the first to be identified, belongs to the Hsp100 subfamily of AAA+ proteins (ATPases associated with various cellular activities) and was detected in coimmunoprecipitation experiments in complexes containing other translocon components and preproteins undergoing import (Akita et al., 1997; Nielsen et al., 1997; Chou et al., 2003; Rosano et al., 2011). In Arabidopsis (Arabidopsis thaliana), Hsp93 exists as two isoforms encoded by the genes HSP93III and HSP93V. Removal of the more abundant Hsp93V results in protein import defects, while double knockout of the two genes causes lethality (Constan et al., 2004; Kovacheva et al., 2007; Chu and Li, 2012; Lee et al., 2015). Purified recombinant Hsp93III can bind to the transit peptide of pea (Pisum sativum) ferredoxin-NADP+ reductase in vitro (Rosano et al., 2011). In addition, the N-terminal domain of Hsp93 is critical both for its in vivo functions and its association with chloroplast membranes and Tic110, suggesting that one of the major functions of Hsp93 requires it to be localized at the envelope with Tic110 (Chu and Li, 2012). However, because many prokaryotic Hsp100 family proteins function as the regulatory components of the Clp proteases (Kress et al., 2009; Nishimura and van Wijk, 2015), and, in Arabidopsis, some Clp proteolytic core components have also been found at the envelope fraction, it has been proposed that Hsp93 is involved in degradation of misfolded or damaged proteins at the envelope (Sjögren et al., 2014). However, whether the Clp proteolytic core can form a stable complex with Hsp93 in higher plant chloroplasts remains to be shown.In mitochondria and the endoplasmic reticulum, protein import is driven by the Hsp70 family of proteins. In chloroplasts, accumulating evidence also supports that Hsp70 is important for chloroplast protein import. Purified recombinant Hsp70 can bind in vitro to the transit peptide of the small subunit of RuBP carboxylase preprotein (prRBCS; Ivey et al., 2000). Stromal Hsp70 can be coimmunoprecipitated with preproteins undergoing import and with other translocon components, and mutations resulting in reduced or altered stromal Hsp70 activity cause protein import defects (Shi and Theg, 2010; Su and Li, 2010). Recently, it has been shown, in moss, that increasing the K_m for Hsp70 ATP hydrolysis results in an increased K_m for ATP usage in chloroplast protein import, indicating that stromal Hsp70 is indeed one of the proteins supplying ATP-derived energy to power import (Liu et al., 2014). Finally, stromal Hsp90C has been shown to be part of active translocon complexes in coimmunoprecipitation experiments (Inoue et al., 2013). As further evidence that Hsp90 is important for protein import into chloroplasts, the Hsp90 ATPase activity inhibitor radicicol reversibly inhibits the import of preproteins into chloroplasts (Inoue et al., 2013).Presence of the three ATPases in the translocon was demonstrated by coimmunoprecipitation after solubilization of chloroplast membranes under conditions that preserve the large membrane protein complexes, either by solubilization with nonionic detergents or by treating chloroplasts with crosslinkers that link all proteins in a complex together (Akita et al., 1997; Nielsen et al., 1997; Shi and Theg, 2010; Su and Li, 2010; Inoue et al., 2013). These complexes contain translocon components that directly bind to preproteins, and also other proteins that are associated with these translocon components but have no direct contacts with the preproteins. For example, Nielsen et al. (1997) demonstrated the presence of Hsp93 in the translocon by binding of prRBCS to isolated pea chloroplasts and then solubilization of chloroplast membranes with the nonionic detergent decylmaltoside. Under these conditions, an anti-Hsp93 antibody specifically immunoprecipitated Hsp93 together with Toc159, Toc75, Toc34, Tic110, and prRBCS (Nielsen et al., 1997). The result showed that Hsp93 is in the same complexes with these proteins but did not provide information whether Hsp93 directly binds to them. It is possible that Hsp93 only has direct contacts with, for example, Tic110, which then binds to prRBCS. Direct binding, in particular to the transit peptide region, would provide strong evidence that an ATPase functions as a protein translocating motor, rather than in assisting the assembly of other translocon components or in the folding or degradation of imported proteins. Furthermore, if all three ATPases were found to be involved in preprotein translocation, it would be important to understand how they work together; for example, whether they preferentially bind different preproteins, bind to different regions of a preprotein, or act at different stages of the import process.Here, we examined whether Hsp93 can directly bind to preproteins undergoing import into chloroplasts, and compared the timing of the binding of Hsp93 and cpHsc70 to the preproteins. We used isolated pea chloroplasts, rather than isolated Arabidopsis chloroplasts, because pea chloroplasts exhibit more robust import ability (Fitzpatrick and Keegstra, 2001). Various crosslinkers that react with cysteines were then used to achieve more specific crosslinkings, followed by solubilization with the ionic detergent lithium dodecyl sulfate (LDS) to thoroughly solubilize chloroplast membranes and to disrupt noncovalent protein-protein interactions. Our results show that Hsp93 directly binds to preproteins undergoing import. Import time course experiments further revealed that Hsp93 functions primarily during the early stage of import, whereas cpHsc70 associates with substrates being imported at both the early stage and a later stage after transit peptide removal.     

Pre-Dialysis Visits to a Nephrology Department and Major Cardiovascular Events in Patients Undergoing Dialysis

Background and Objectives

Pre-dialysis care by a nephrology out-patient department (OPD) may affect the outcomes of patients who ultimately undergo maintenance dialysis. This study examined the effect of pre-dialysis care by a nephrology OPD on the incidence of one-year major cardiovascular events after initiation of dialysis.

Design, Setting Participants, & Measurements

The study consisted of Taiwanese patients with chronic kidney disease (CKD) who commenced dialysis from 2006 to 2008. The number of nephrology OPD visits during the critical care period (within 6 months of initiation of dialysis) and the early care period (6–36 months before initiation of dialysis) were analyzed. The primary outcome measure was one-year major cardiovascular events.

Results

A total of 1191 CKD patients who initiated dialysis from 2006 to 2008 were included. Binary logistic regression showed that patients with ≧3 visits during the critical care period and those with ≧11 visits during the early care period had fewer composite major cardiovascular events than those with 0 visits. Patients with early referral are less likely to experience composite major cardiovascular events than those with late referral, with aOR 0.574 (95% CI = 0.43–0.77, P<0.001). Patients with both ≧3 visits during critical care period and ≧11 visits during early care period were less likely to experience composite major cardiovascular events (aOR = 0.25, 95% CI = 0.16–0.39, P < 0.001).

Conclusions

Patients with adequate pre-dialysis nephrology OPD visits, not just early referral, may had fewer one-year composite major cardiovascular events after initiation of dialysis. This information may be important to medical care providers and public health policy makers in their efforts to improve the well-being of CKD patients.     

Ability of Clonostachys rosea to Establish and Suppress Sporulation Potential of Botrytis cinerea in Deleafed Stems of Hydroponic Greenhouse Tomatoes

The ability of Clonostachys rosea to establish and persist in deleafed tomato stems and to suppress sporulation potential of Botrytis cinerea was investigated in plots of hydroponic tomatoes in commercial greenhouses. Leaves near lower fruit clusters were removed according to standard practice and deleafed portions of the stems were treated with C. rosea , iprodione or water. Inoculum of B. cinerea was from natural infections. Stem lesions were not produced by the pathogen during the trials. Development of C. rosea and B. cinerea in stems was estimated indirectly by quantifying sporulation on excised stem tissues that were incubated on an agar medium containing paraquat. Incidence and area of sporulation of C. rosea on tissue pieces were high (76-99%) and moderately high (33-79%), respectively, when stems were treated with the agent at 0, 6, 24 or 48 h after deleafing and sampled 11 to 75 days later. In various instances, the agent also sporulated on tissues from water controls and iprodione treatments, apparently after interplot transmission. In most instances, incidence and area of sporulation of B. cinerea on tissue pieces were high (83-100%) and moderate to high (35-76%), respectively, in the water controls, but moderate (31-44%) and moderate to low (5-34%), respectively, for stems treated with C. rosea at 0 to 48 h after deleafing and sampled after 11-75 days. Without exception, C. rosea suppressed B. cinerea as or more effectively than iprodione. Correlations between inoculum density of C. rosea (0-10 6 conidia mL -1 ) and sporulation potential of B. cinerea in deleafed stems were strongly negative in each of three tests ( r = -0.95 to -0.99). Conidial suspensions and a talc formulation of C. rosea were of similar effectiveness against B. cinerea . We conclude that C. rosea persisted and suppressed sporulation potential of B. cinerea in deleafed tomato stems for at least 11 weeks after application.     

HBsAg合成肽段的免疫源性和抗肽血清与天然HBsAg的结合测定

我们将三种乙型肝炎表面抗原的合成肽段在联结载体或不接载体的情况下,分别制备成4种免疫原对兔子进行了免疫。大部分兔子都产生了抗肽抗体。其中抗P_(122-48)(auw)与抗P_(122-148)(adw)都能与天然HBsAg反应,而抗P_(122-148)(adw)的抗HBs活力比文献报道的都高,通过结构分析表明:含有天然蛋白上免疫显性区域的免疫原是合成疫苗的理想选者。