text
string | predicted_class
string | confidence
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|---|---|---|
The Cambodian Demographic Health Surveys conducted in 2010 and 2014 showed similar figures with the reported vaccination coverage by the Health Information Management System of the Ministry of Health of Cambodia as well (Graph 3). It was observed that there was a slightly decreasing trend between each pentavalent vaccine 1, 2, and 3.16
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study
| 99.94 |
The SWOT analysis has been considered as a good tool for a program’s review. The tool was used to review the performance of the NIP at national and subnational levels based on its current policies and strategic areas. The summaries of SWOT analysis related to service delivery, behavioral communication, cold chain management, vaccine preventable disease surveillance, and program management are listed in Table 3.
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other
| 99.9 |
Cambodia is one of the countries with the highest hepatitis B prevalence among the general public in the world. It was observed that the prevalence of hepatitis B in 5-year-old children decreased slightly from 2005 through 2011. This was due to the introduction of the HepB vaccine in 2001; its coverage in 2006 was also low at around 40% for HepB birthdose and 85% for pentavalent (Graph 2 and 3). In an early 2017 survey, it is expected that this prevalence might be below 1% because the birthdose coverage almost doubled in 2010 (71%) compared with 2006.
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study
| 99.94 |
However, the seroconversion has been found low also in some countries. In this particular regard, in some countries with high burden of viral hepatitis B, universal blood screening including hepatitis B antigen, HBe antigen, and HBV deoxyribonucleic acid (DNA) is done, and corresponding actions apart from routine HepB vaccine are composed of hepatitis B immunoglobulin (HBIG), antiretroviral, and cesarean section. Those interventions are provided to increase its effectiveness. Additional measures include the universal testing of pregnant mothers for hepatitis B, hepatitis e antigen, and viral DNA among those who are infected with hepatitis B, where the health infrastructure is capable of performing testing.
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other
| 99.8 |
Outreach activities have played an important role in reaching newborns in high-risk areas. Like other countries, cold chain has been an issue in some countries, especially, in very remote areas and to address this particular issue, the out-of-cold chain approach has been applied in some places of some countries. The policy of the Ministry of Health has made availability of HepB vaccine in very remote communities provided by the community health workers (China).18
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other
| 99.9 |
The NIP shall continue to raise awareness about the importance of the immunization, especially with focus on promoting health facility delivery, and also on the birth dose and pentavalent 1, 2, and 3 administration using current strategy, particularly, for those who live in high risk areas.
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other
| 99.94 |
The NIP should maintain current implementation of the activities according to its policies and strategic plans. Both fixed sites and outreach activities including high-risk communities should be maintained. The capacity building shall be provided, especially, to those who are newly assigned. The cold chain shall be maintained and strengthened. Government’s funding shall be secured and increased when the external funding has decreased. Vaccination targeting selecting adult groups, especially, the health professionals shall be considered.19
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other
| 99.94 |
The NIP has done a remarkable job and the HepB birth-doses and three pentavalents have been very high since 2010. The vaccination coverage was very high, especially, the birthdose. Additional measures shall be considered by revising the current national policy and strategic plan as mentioned in the recommendations.
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other
| 99.94 |
Translation is responsible for the production of proteins that are essential for cellular functions (Gebauer and Hentze 2004; Sonenberg and Hinnebusch 2007; Spirin 2009). One key feature of translation is that cellular mRNAs are translated differently, as a result of the difference in interactions between mRNAs and the translational machinery (Vogel et al. 2010; Gingold and Pilpel 2011). In principle, for a multistep cellular process like translation, regulation can take place at each step and can target numerous proteins or RNAs involved (Merrick and Hershey 1996). Nevertheless, this process is predominantly regulated during initiation (Sonenberg and Hinnebusch 2009; Jackson et al. 2010; Hinnebusch and Lorsch 2012), the step when interactions between 5′ untranslated regions (5′UTRs) of mRNAs and cellular proteins determine how ribosomes will be recruited to the mRNAs to start translation.
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study
| 99.5 |
5′UTRs of mRNAs regulate translation via a diverse set of mechanisms (Sonenberg and Hinnebusch 2009; Jackson et al. 2010; Ingolia et al. 2011; Brar et al. 2012; Hinnebusch and Lorsch 2012; Archer et al. 2016; Hinnebusch et al. 2016). Most notably, the 5′UTR of an mRNA often harbors binding sites for regulatory proteins and translational factors. These RNA-binding proteins (RBPs) are versatile not only in their ways of recognizing partner RNAs but also in interacting with other proteins, which enables them broad yet unique functions in regulating post-transcriptional gene expression (Keene and Tenenbaum 2002; Hogan et al. 2008).
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review
| 98.06 |
Sbp1 is an RNA-binding protein that was initially discovered to coimmunoprecipitate with small nucleolar RNA 10 and 11 (snoRNA10 and snoRNA11) in yeast (Jong and Campbell 1986; Jong et al. 1987; Clark et al. 1990). In addition to its localization in the nucleolus (Jong et al. 1987; Clark et al. 1990), Sbp1 was shown to localize in processing bodies (P bodies) under the stress of glucose starvation in yeast (Segal et al. 2006). Overexpression of Sbp1 results in global translational repression and an increase in the size as well as number of P bodies (Segal et al. 2006). In the same study, Sbp1 was shown to promote decapping of a reporter mRNA with another protein, Dhh1.
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study
| 99.94 |
In line with functional roles of this protein in translation, overexpression of Sbp1 rescues the defect in nonsense suppression caused by SUP35 mutant (Zadorsky et al. 2015). Furthermore, in a study on RNA-binding protein FUS/TLS-dependent cytotoxicity that causes a subset of familial amyotrophic lateral sclerosis (fALS) using yeast as a model system, overexpression of Sbp1 rescues the toxicity caused by FUS/TLS mutations (Ju et al. 2011).
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study
| 99.94 |
Sbp1 has a domain organization with an N-terminal RNA recognition motif (RRM) domain, a central arginine–glycine –glycine (RGG) domain and a second C-terminal RRM domain (Fig. 1A). Because of the diverse roles that RRM and RGG domains play in mediating RNA–protein, protein–protein, and less frequent but important, RNA–RNA interactions (Cuchalová et al. 2010; Rajyaguru and Parker 2012; Daubner et al. 2013; Thandapani et al. 2013), Sbp1 has the potential to target different mRNAs and regulate their translation in a transcript-specific way. Furthermore, unlike the majority of RGG-motif containing proteins which have their RGG repeats in either N or C terminus (data not shown), the location of RGG repeats between the two RRMs suggests potential function of structural modulation of the RNA target by this protein. Despite the importance, very little is known about molecular mechanisms by which Sbp1 regulates transcript-specific mRNA translation.
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| 99.94 |
Sbp1 binds to 5′UTRPab1 specifically and cooperatively. (A) Schematic representation of Sbp1's domain organization. Amino acid residue numbers at the boundaries of individual domains are indicated. (B) An EMSA showing the formation of 5′UTRPab1•Sbp1 complex at 1:1 stoichiometry. 5′UTRPab1 was incubated with increasing concentration of purified Sbp1 (1:0, 1:5, 1:10, 1:15, and 1:20 of 5′UTRPab1:Sbp1 for lanes 1–5, respectively). (C) Competition EMSA showing specific interactions between 5′UTRPab1 and Sbp1. Sbp1 was first incubated with 32P-5′UTRPab1 (lanes 2 and 6). An excess of unlabeled 5′UTRPab1 by five- and 10-fold (lanes 3 and 4) and its complementary RNA, 5′UTRPab1-complement, (lanes 7 and 8) were used to compete with 32P-5′UTRPab1 bound to Sbp1. (D) An EMSA showing weak interactions between the 5′UTRPab1 and individual domains of Sbp1. Sbp1RRM1 (lane 2), Sbp1RGG (lane 4), and Sbp1RRM2 (lane 6) were incubated with 5′UTRPab1. A molar ratio of 1:20 (5′UTRPab1:Sbp1 domain mutants) was used in the experiment. 5′UTRPab1 alone was shown in lanes 1, 3, and 5.
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study
| 100.0 |
Here, we report that Sbp1 inhibits translation of the mRNA that encodes polyadenosine [poly(A)]-binding protein Pab1 in an RNA- and RGG-dependent manner in vitro. We further elucidate the molecular interactions that are important for the translational regulation. Pab1 is an essential regulator of mRNA metabolism in eukaryotes, including splicing (Jenal et al. 2012), 3′ end processing (Jenal et al. 2012), and mRNA export (Bresson and Conrad 2013), localization (Vazquez-Pianzola et al. 2011), translation (Munroe and Jacobson 1990; Tarun and Sachs 1996; Tarun et al. 1997; Wells et al. 1998), and degradation (Bresson and Conrad 2013). Pab1 not only binds to the 3′ poly(A) tail and other adenosine-rich (A-rich) regions of cellular mRNAs (Gilbert et al. 2007), but also directly interacts with the eukaryotic mRNA cap-binding complex via binding with its component, eIF4G (Tarun and Sachs 1996; Tarun et al. 1997; Wells et al. 1998). In addition to stimulating mRNA translation in general, Pab1 inhibits the decapping of mRNAs in the cell (Coller and Parker 2004; Parker 2012). As expected, expression of Pab1 is tightly regulated. In this study, we demonstrate that the two RRM domains of Sbp1 bind specifically to the A-rich region in the 5′UTR of Pab1 (5′UTRPab1) and the RGG domain of Sbp1 directly interacts with Pab1. These molecular interactions explain at least in part how Sbp1 inhibits the translation initiation of Pab1 mRNA.
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To see whether Sbp1 binds to 5′UTRPab1 in the translation initiation pathway of the Pab1 mRNA, we developed a so-called “TRAP” assay. The assay uses molecules known to participate in initiation, including 5′UTRs, translational factors, and 40S ribosomal subunit, as a bait complex for an affinity pull-down procedure followed by mass spectrometry to identify the cellular proteins that interact with these intermediates in initiation. In our experimental design, proteins belonging to the class IV initiation factor family, which includes eukaryotic initiation factors eIF4E, eIF4G, eIF4A, and eIF4B, as well as eIF3 and eIF5, are not used in the bait. These proteins are known to participate in mRNA and 40S activation prior to the start-codon recognition (Jackson et al. 2010), thus their binding to bait complexes provides a valuable positive control for our pull-down results.
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study
| 100.0 |
We cloned 5′UTRPab1 and its sequence complement, 5′UTRPab1-complement, as PCR-generated BamHI–HindIII fragments into the pTRAPv5 vector (Cytostore Inc.) downstream from the two streptavidin-binding S1 aptamers (Srisawat and Engelke 2001). Vectors were linearized with HindIII. Uncapped and aptamer-tagged RNA were transcribed from the DNA templates using T7 polymerase and purified by denaturing polyacrylamide gel electrophoresis or anion-exchange chromatography (Easton et al. 2010). The bait complex that we used in this assay corresponds to the 43S pre-initiation complex (43S PIC) that consists of a ternary complex (eIF2•GTP•Met-tRNAiMet) and a 40S ribosomal subunit (Jackson et al. 2010). Since binding of eIF1 and eIF1A to the 40S ribosome facilitates the recruitment of the ternary complex (Lorsch and Dever 2010), we also included eIF1 and 1A in the bait. Furthermore, we used the nonhydrolyzable GTP analog GDPCP to stabilize the ternary complex in the GTP state.
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First, a stable formation of a 43S PIC on the 5′UTRPab1 was confirmed by an electrophoresis mobility shift assay (EMSA) (Supplemental Fig. S1A). Next, the 5′UTR alone and 5′UTRs with 40S and initiation factors assembled were immobilized onto the streptavidin-coated magnetic beads and were incubated with yeast cell extracts. Following an extensive wash to remove most of the material bound nonspecifically, proteins that bind to the 5′UTR were eluted with D-biotin and analyzed by mass spectrometry. Using this assay, we confirmed that Sbp1 specifically binds to 5′UTRPab1 (Supplemental Fig. S1B). At the same time, we also pulled down Pab1 protein from the cell extract using this assay (Supplemental Fig. S1B, lanes 3,5, and 7). This result is in good agreement with earlier findings that Pab1 binds to the polyadenosine sequences in its own 5′UTR (Sachs et al. 1987).
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| 100.0 |
To test the possibility of a direct interaction between Sbp1 and the 5′UTRPab1, purified Sbp1 (Supplemental Fig. S2A) and in vitro-transcribed 5′UTRPab1 were combined and their interactions were examined by EMSA. As shown in Figure 1B, a complex is formed when Sbp1 and 5′UTRPab1 are combined. Multiple bands are not seen at higher protein concentrations, suggesting that binding of Sbp1 and 5′UTRPab1 is at 1:1 stoichiometry.
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study
| 100.0 |
We further demonstrate that the interactions between 5′UTRPab1 and Sbp1, which leads to the formation of the complex, are specific to each other. In a competition assay, the shifted band is efficiently outcompeted by an excess of unlabeled 5′UTRPab1 (Fig. 1C, lanes 3 and 4) but is not affected by an excess of unlabeled 5′UTRPab1-complement with nucleotide sequence complementary to 5′UTRPab1 (Fig. 1C, lanes 7 and 8). These results suggest that the observed band shifts are the result of formation of a specific, protein–RNA complex.
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| 100.0 |
To see if the domains of Sbp1 bind to the 5′UTRPab1 cooperatively, we studied binding properties of individual domains of Sbp1 to 5′UTRPab1 and compared the results with the wild-type Sbp1. Care was taken to ensure that individual domains—RRM1, RGG, and RRM2 domains of Sbp1 (Sbp1RRM1, Sbp1RGG, and Sbp1RRM2)—were folded properly (Supplemental Fig. S2B). As shown in Figure 1D, while individual domains of Sbp1 bind to the 5′UTRPab1, they interact with the RNA with much weaker affinities compared to the affinity of the full-length Sbp1 with the 5′UTRPab1. The measured apparent dissociation constant (Kd) of full-length Sbp1 to the 5′UTRPab1 is 26.5 ± 1.1 nM with a Hill coefficient of 2.1 ± 0.1 (Supplemental Fig. S3A). In contrast, under the same experimental conditions, the Kd values of its domain mutants, the Sbp1RRM1, Sbp1RRM2, and Sbp1RGG to the 5′UTRPab1 are about 1.1 μM, 0.9 μM, and 2.7 μM, respectively (data not shown). Thus, the binding of full-length Sbp1 to 5′UTRPab1 is over 40 times stronger than that of its individual domains to the same RNA. These results demonstrate convincingly that the domains of Sbp1 bind to the 5′UTRPab1 cooperatively.
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| 100.0 |
To study how the 5′UTRPab1 folds and which region in the RNA binds to Sbp1, we determined the secondary structures of 5′UTRPab1 and 5′UTRPab1–Sbp1 complex using in-line probing (Soukup and Breaker 1999). In-line probing is an RNA structure analysis technique that uses the relative flexibility of RNA backbone conformation to determine secondary structure characteristics of the RNA. As shown in Figure 2, in the absence of Sbp1, the A-rich region in the 5′UTRPab1 adopts a single-stranded conformation (Fig. 2A). Addition of Sbp1 protects the degradation of the RNA in this region (Fig. 2B), suggesting that the protein binds to the A-rich sequence in the 5′UTRPab1 (Supplemental Fig. S4).
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| 100.0 |
Sbp1 binds to the A-rich region in 5′UTRPab1. (A) An in-line probing gel image of 5′UTRPab1. The 5′UTRPab1 in its undigested form (lane 1) was subjected to partial alkaline digestion (lane 2) and RNase T1 treatment in the presence of urea at 37°C (lane 3) and at 55°C (lane 4). Results on in-line probing of 5′UTRPab1 (lane 5) are shown. Double-stranded regions (P1, P2, P3, P4, and P5) and poly(A)-rich region in the RNA are shown. (B) An in-line probing gel image of 5′UTRPab1 in the presence of Sbp1. 5′UTRPab1 RNA partially digested by RNase T1 at 55°C in the presence of urea (lane 1). The cleavage pattern of 5′UTRPab1 after the in-line probing without Sbp1 (lane 2) and with Sbp1 (lane 3) showing protection of the A-rich region by Sbp1. (C) An EMSA showing the formation of 5′UTRPab1•Sbp1•Pab1 complex. Addition of Sbp1 or Pab1 leads to formation of the RNP complexes of 5′UTRPab1•Sbp1 (lanes 2 and 3) or 5′UTRPab1•Pab1 (lane 4), respectively, and results in a slower electrophoretic mobility of the 32P-labeled 5′UTRPab1. In the presence of Sbp1 and Pab1 simultaneously, a band that migrates even slower appears, suggesting the formation of 5′UTRPab1•Sbp1•Pab1 complex (lanes 5 and 6). Lane 1 shows 5′UTRPab1 only.
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| 100.0 |
As mentioned in the “Domains of Sbp1 bind to the 5′UTRPab1 cooperatively” section, the Kd of Sbp1 to the 5′UTRPab1 is 26.5 ± 1.1 nM. Under the same experimental conditions, including ionic strength of binding buffers, the Kd of Pab1 to the 5′UTRPab1 is 31.2 ± 2.5 nM (Supplemental Fig. S3). These results show that Sbp1 and Pab1 bind to the A-rich region in the 5′UTRPab1 with a similar affinity in vitro.
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| 100.0 |
Since Pab1 binds to the poly(A) region in the 5′UTRPab1 (Sachs et al. 1987) and both proteins are pulled down simultaneously on the 5′UTRPab1 with the 43S PIC in our TRAP assay (Supplemental Fig. S1B), these results suggest that Sbp1 and Pab1 likely bind to the 5′UTRPab1 at the same time. Indeed, a higher-order complex of 5′UTRPab1–Sbp1–Pab1 is formed under our experimental conditions, as shown in a native EMSA (Fig. 2C).
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To test whether there is a direct interaction between Sbp1 and Pab1, we performed a glutathione S-transferase (GST) pull-down assay using purified GST-Pab1 and Sbp1 proteins. As shown in Figure 3A (lane 3), Sbp1 is pulled down by Pab1 only in the presence of 5′UTRPab1 RNA. Addition of RNase A abolishes the protein–protein interaction (Fig. 3A, cf. lanes 3 and 4), suggesting that the interaction between Pab1 and Sbp1 is RNA-dependent.
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Sbp1 interacts with Pab1 in an RGG- and RNA-dependent manner. (A–C) GST-pulldown assays showing interactions of GST-tagged Pab1 with Sbp1 (A), Sbp1RGG (B), and Sbp1ΔRGG (C). Sbp1RGG stands for RGG domain of Sbp1; Sbp1ΔRGG stands for mutant with RGG domain replaced by glycine–serine repeats. Purified proteins were incubated in the absence of 5′UTRPab1 (lane 2), with 10× 5′UTRPab1 (lane 3) or with 10× 5′UTRPab1 followed by RNase treatment (lane 4). The proteins pulled down are shown by SDS–PAGE. Lane 1 shows the negative control containing GST only. (D) RNA-binding of Sbp1, not Pab1, is important for the Sbp1–Pab1 interaction. GST-pulldown assays showing Sbp1–Pab1 interaction under different conditions: Sbp1 incubated with Pab1 in the absence of 5′UTRPab1 (lane 2), GST-tagged Pab1 incubated with the Sbp1•5′UTRPab1 complex (lane 3), and Sbp1 incubated with the GST-Pab1•5′UTRPab1 complex (lane 4). In lane 4, 5′UTRPab1 was incubated with GST-Pab1 first, and unbound RNA in excess was removed prior to the addition of Sbp1. Lane 1 shows negative control with GST only. (E) Deletion of the C-terminal domain of Pab1 does not affect Sbp1–Pab1 interaction. GST-pulldown assay was performed for GST-Sbp1 and the C-terminal deleted variant Pab1 (Pab1RRM1-4). Sbp1 and Pab1RRM1-4 interact with each other in the presence of 5′UTRPab1 (cf. lanes 2 and 3), and RNase treatment disrupts the protein interaction (cf. lanes 3 and 4). Proteins were incubated with GST as the negative control (lane 1).
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In contrast, the RGG domain of Sbp1, Sbp1RGG, interacts with Pab1 directly in the absence of 5′UTRPab1 (Fig. 3B, lane 2). Unsurprisingly, neither the presence of an equal stoichiometric amount of 5′UTRPab1 nor an addition of RNase A in the presence of 5′UTRPab1 alters the protein–protein interaction (Fig. 3B, cf. lanes 2, 3, and 4). A replacement of the RGG domain in Sbp1 with 17 glycine–serine repeats (GS repeats) abolishes the interaction between Sbp1 and Pab1 regardless of the presence of 5′UTRPab1 (Fig. 3C, lanes 2, 3, and 4). Furthermore, Pab1 interacts with the Sbp1–5′UTRPab1 complex (Fig. 3D, lane 3), whereas Sbp1 does not interact with the Pab1–5′UTRPab1 complex (Fig. 3D, lane 4). These observations demonstrate that the RNA binding of Sbp1, not Pab1, is important for interactions between Pab1 and Sbp1. As such, our results indicate that exposing the RGG domain of Sbp1 by the RNA binding is required for Sbp1 to bind Pab1.
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Pab1 in yeast is a 71 kDa RNA-binding protein with four RNA recognition motifs (RRM1–4) and a C-terminal protein-binding domain, also known as PABC (Wigington et al. 2014). To determine which regions in the Pab1 interact with Sbp1, we generated domain mutants of Pab1 and tested their bindings with Sbp1. Our results show that the protein construct containing the RRM1–4 domains of Pab1, Pab1RRM1–4, binds to Sbp1 (Fig. 3E). In contrast, no molecular interaction was detected when PABC, or individual RRM domain mutants including Pab1RRM1, Pab1RRM2, Pab1RRM3, and Pab1RRM4 as well as other tandem RRM domains, were tested (Supplemental Fig. S5A–H). These observations, suggest that, together, RRM1–4 domains in Pab1 are responsible for the interaction with Sbp1. Consistent with our previous observations, Sbp1 and the RRM1–4 of Pab1 only interact with each other in the presence of 5′UTRPab1 (Fig. 3E, cf. lanes 2 and 3), and addition of RNase A abolishes the protein–protein interaction (Fig. 3E, cf. lanes 3 and 4). Taken together, these results confirm that molecular interactions between Pab1RRM1-4 and Sbp1 are RNA-dependent: Binding of the RNA is important to expose the RGG repeats of Sbp1, which is required for the interaction with Pab1RRM1–4.
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Thus far, we demonstrated that Sbp1 interacts with Pab1 via its RGG domain in an RNA-dependent manner. It is known that the RRM2 domain of Pab1 binds to eIF4G1 directly (Kessler and Sachs 1998; Safaee et al. 2012). Furthermore, earlier mass spectrometry results show that Sbp1, Pab1, and eIF4G interact with each other (Gavin et al. 2002, 2006). To test formation of a stable 5′UTRPab1–Sbp1–Pab1–eIF4G1 RBP in vitro, we did an IgG-pulldown assay using purified eIF4G1, Pab1, and Sbp1. In this experiment, in order to ensure the correct folding and functionality of the protein, eIF4G1 was chromosomally expressed and affinity-purified in yeast. Furthermore, endogenous RNAs that bind to eIF4G1 and Pab1 were removed (see Materials and Methods) to minimize interference of the bound RNAs to protein–protein interactions. As shown in Figure 4 (cf. lanes 2 and 3), the three proteins bind simultaneously only in the presence of 5′UTRPab1. As expected, formation of the ternary complex (Sbp1–Pab1–eIF4G1) on the 5′UTRPab1 was disrupted by the addition of RNase A (Fig. 4, cf. lanes 3 and 4), suggesting the importance of the RNA in the ternary complex formation.
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Sbp1, Pab1, and eIF4G1 interact with each other and formation of the ternary complex is RNA-dependent. IgG-pulldown assay showing that Sbp1, Pab1, and eIF4G1 interact in the presence of 5′UTRPab1 (cf. lanes 2 and 3), and the molecular interaction is disrupted by RNase treatment (cf. lanes 3 and 4). Lane 1 shows that Sbp1 and eIF4G1 do not interact with each other when Pab1 is absent.
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Methylation at the guanidino group in the arginine side chain, a eukaryotic-specific modification, has been suggested as one of the most extensive protein methylations in mammalian cells (Najbauer et al. 1993; McBride and Silver 2001; Pahlich et al. 2006; Bedford and Clarke 2009; Blanc and Richard 2017). This modification plays important physiological roles from DNA repair, transcriptional regulation, mRNA splicing, to protein translocation and signal transduction. Sbp1 is a known target for the arginine methyltransferase (Hmt1) (Frankel and Clarke 1999), but the physiological function of this modification remains unknown.
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To study roles of the arginine methylation in mediating protein–protein as well as protein–RNA interactions, we first obtained Sbp1 with the methylated RGG domain by coexpressing Sbp1 with its cognate methyltransferase Hmt1 (Supplemental Fig. S2A; Hsieh et al. 2007). We then confirmed methylation of arginines in the RGG domain by mass spectrometry (Supplemental Fig. S6). Next, using the same GST-pulldown of purified proteins and EMSA as described in the previous sections, we demonstrated that RGG methylation (RGGm) compromises the binding of Sbp1 to Pab1 (Fig. 5A, cf. lanes 3 and 4) and to the formation of the ternary complex (Sbp1–Pab1–eIF4G1) on the 5′UTRPab1 (Fig. 5B, cf. lanes 3 and 4). In contrast, methylation of the RGG domain has no influence on the binding of Sbp1 to 5′UTRPab1 (Fig. 5C, cf. lanes 2 and 3).
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Methylation of RGG domain disrupts protein–protein but not protein–RNA interactions. (A) GST-pulldown assay showing RGG methylation in Sbp1 (Sbp1m) disrupts the Sbp1–Pab1 interaction. Sbp1m was incubated with GST-Pab1 without 5′UTRPab1 (lane 2), in the presence of 10× 5′UTRPab1 (lane 3) or RNase A (lane 4). The protein was incubated with GST as a negative control (lane 1). (B) IgG-pulldown assay showing Sbp1m compromises ternary complex formation. Sbp1m and Pab1 were incubated with eIF4G1-TAP without 5′UTRPab1 (lane 2), in the presence of 10× 5′UTRPab1 (lane 3), or an incubation followed by RNase A treatment (lane 4). Sbp1m and eIF4G1 show no interaction in the presence of 5′UTRPab1 (lane 1). (C) EMSA showing that methylation of RGG repeats in Sbp1 does not affect RNA binding. 32P-labeled 5′UTRPab1 was incubated with 10× Sbp1 (lane 2) or 10× methylated Sbp1 (lane 3). Lane 1 is 5′UTRPab1 alone.
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To test the functional importance of these molecular interactions in transcript-specific translation initiation, we carried out in vitro translation assays using luciferase reporter genes (Hofbauer et al. 1982; Hussain and Leibowitz 1986; Wang et al. 2013). We generated a monocistronic reporter construct by cloning the 5′UTR of Pab1 mRNA in-frame with the firefly luciferase open reading frame (ORF) under control of the T7 promoter. Cap-dependent and cap-independent translation, the two pathways for the translation initiation of eukaryotic mRNAs, can be distinguished by adding or omitting the 5′ m7G cap to the mRNAs under study. Furthermore, to inhibit ribosome scanning, a stable hairpin upstream of the uncapped 5′UTRPab1 was used to study the cap-independent translation initiation (Wang et al. 2013) in our investigation. The same strategy was used to generate the monocistronic reporter constructs for the 5′UTR of eIF4E mRNA as a control. Additionally, RNAs with nucleotide sequence complementary to 5′UTRPab1 and 5′UTR4E, 5′UTRPab1-complement and 5′UTR4E-complement, were used as controls to rule out nonspecific interactions. The same amount of RNA was added in each translation assay and all measured activities were normalized to the total protein level in cell extracts. Finally, translation of cricket paralysis virus (CrPV) depends on correct folding of the 5′UTR of the CrPV RNA (5′UTRCrPV, also known as the internal ribosome entry site of CrPV) alone and does not require any additional proteins (Thompson et al. 2001). As such, 5′UTRCrPV was included as an experimental control.
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A capped 5′UTRPab1 or an uncapped 5′UTRPab1 with a stable upstream hairpin was used for measuring the cap-dependent or cap-independent initiation activity, respectively. These RNA constructs were incubated with the yeast extracts from either WT or ΔSbp1 cells with an increasing amount of Sbp1 protein added. As shown in Figure 6A and C (the second column), a decreased translation activity in the presence of an increasing amount of Sbp1 indicated an inhibitory function of this protein in both cap-dependent and cap-independent initiation of the Pab1 mRNA. In contrast, translation of the 5′UTR4E as well as 5′UTRCrPV was almost unchanged as the concentration of Sbp1 increases (Fig. 6D,E).
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Sbp1 inhibits both cap-dependent and cap-independent translation initiation of Pab1 mRNA in an RGG- and RNA-dependent manner. Reporter constructs are shown schematically in the top panel of the figure. In vitro cap-dependent and cap-independent translation activities of 5′UTRPab1 and 5′UTRPab1-Δpoly(A) in the presence of different concentrations of Sbp1 and its variants are shown. (A) Changes of cap-dependent translation initiation activities of 5′UTRPab1 by Sbp1 and its protein variants. In vitro translation assays were performed by incubating the capped 5′UTRPab1 followed by firefly luciferase gene and poly(A) tail [5′UTRPab1-FLuc-poly(A)] in ΔSbp1 cell extract with varying amounts of Sbp1, Sbp1ΔRGG, Sbp1m, Sbp1RGG, and Sbp1mRGG added. Sbp1ΔRGG: a substitution of RGG domain with glycine–serine repeats; Sbp1m: methylated Sbp1; Sbp1RGG: RGG domain of Sbp1; Sbp1mRGG: methylated RGG domain. (B) Changes of cap-dependent translation initiation activities of 5′UTRPab1 at varying concentrations of Sbp1 added to WT cell extracts. (C) Changes of cap-independent translation initiation activities of 5′UTRPab1 by Sbp1 and its protein variants. Experiments were done as in A using a different reporter construct, the uncapped 5′UTRPab1-FLuc-poly(A), which has a hairpin insertion before the 5′UTRPab1 to inhibit ribosome scanning. (D) Changes of cap-dependent translation initiation activities of 5′UTR4E by Sbp1 and its protein variants. Experiments were done as in A, except the capped 5′UTReIF4E-FLuc-poly(A) was used. (E) Changes of cap-independent translation initiation activities of 5′UTRCrPV by Sbp1 and its protein variants. Experiments were done as in A, except the uncapped Hairpin-5′UTRCrPV-FLuc-poly(A) was used. (F) Changes of cap-dependent translation initiation activities of 5′UTRPab1-Δpoly(A) by Sbp1 and its protein variants. 5′UTRPab1-Δpoly(A): 5′UTRPab1 RNA with the internal A-rich region deleted. The integrity of the RNAs was confirmed by either northern blot or ethidium bromide-stained gels under denaturing conditions. Luciferase activities are normalized to the amount of capped or uncapped RNAs and the total protein levels in the study. Reported translation activities are the average of results obtained in three independent experiments. Controls including translation activities of the blank, 5′UTR of eIF4E (5′UTReIF4E) and internal ribosome entry site of cricket paralysis virus (5′UTRCrPV) under the same experimental conditions are shown in D and E.
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Since Sbp1 coimmunoprecipitates with small nucleolar RNA 10 and 11 (Jong and Campbell 1986; Jong et al. 1987; Clark et al. 1990), to rule out possibilities of a change in mRNA translation due to changes of ribosome biogenesis by Sbp1 in the cell, we added purified Sbp1 into the WT cell extract and observed a similar inhibitory function of the translation of Pab1 mRNA by Sbp1 (Fig. 6B).
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Importantly, a substitution of the RGG repeats of Sbp1 with GS repeats (column 2 in Fig. 6A,C), or methylation of the RGG domain (column 3 in Fig. 6A,C), or deletion of the internal poly(A) sequence (Fig. 6F, columns 2 and 3), diminishes the inhibitory effect of Sbp1 on the Pab1 mRNA. Taken together, our results show that both the RGG domain and poly(A)-sequence of 5′UTRPab1 are important for Sbp1's function in translation of the Pab1 mRNA.
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Sbp1 is a single-stranded RNA-binding protein that contains two RNA recognition motifs and an arginine–glycine–glycine rich domain. In this study, we investigated molecular interactions important for the transcript-specific translational regulation of Sbp1 on an essential mRNA that encodes the poly(A)-binding protein Pab1 in vitro. Our results demonstrate that the RNA-binding property and RGG domain of Sbp1 are important for its function.
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We show that the two RRMs of Sbp1 bind to the poly(A) region of 5′UTRPab1 specifically, and this protein–RNA interaction is important for the inhibition of translation initiation of Pab1 mRNA. Four observations support this conclusion. First, direct binding of Sbp1 to 5′UTRPab1 was shown by a TRAP assay where Sbp1 was pulled down from cell lysates using 5′UTRPab1 alone and 5′UTRPab1 assembled with a preformed 43S PIC (Supplemental Fig. S1B). Consistent with this result, a recent genome-wide, cross-linking study shows that Sbp1 preferentially binds to the 5′UTRs of mRNAs (Mitchell et al. 2013). Second, 5′UTRPab1, but not its sequence complement, 5′UTRPab1-complement, competes for the binding of Sbp1 (Fig. 1). Third, binding analyses based on EMSAs reveal that the individual domains of Sbp1, while folded properly, have much weaker affinities for the 5′UTRPab1 than the full-length protein and, therefore, suggests that the domains in the protein act cooperatively for RNA binding.
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Fourth, results from our in-line probing experiments on 5′UTRPab1 alone (Fig. 2A) and the 5′UTRPab1–Sbp1 complex (Fig. 2B) show that the A-rich region in 5′UTRPab1 is protected upon the binding of Sbp1, indicating that this region in 5′UTRPab1 is likely to be the major binding site of Sbp1 (Supplemental Fig. 4). The measured Kd of Sbp1 and 5′UTRPab1 is 26.5 ± 1.1 nM. Templated poly(A)-rich tracts have been found in UTRs of many cellular mRNAs in eukaryotes including yeast and humans (Gilbert et al. 2007; Wigington et al. 2014). It is conceivable that Sbp1 likely binds to the A-rich region in these mRNAs in a similar way.
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It has been shown that the first two RRM domains of the Pab1 bind to the polyadenosine sequence in the 5′UTRPab1 with nanomolar affinity (Sachs et al. 1987; Kessler and Sachs 1998), which contributes to the mechanism of an autoregulated translation of Pab1 mRNA in vitro and in vivo (de Melo Neto et al. 1995; Bag and Wu 1996; Hornstein et al. 1999). The apparent Kd of the 5′UTRPab1 and Pab1 is 31.2 ± 2.5 nM (Supplemental Fig. S3), which is slightly higher compared to that obtained on a 5′UTRPab1–Sbp1 complex (26.5 ± 1.1 nM) under the same experimental conditions.
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As shown in Figure 6A, C, and F, an increasing amount of Sbp1 leads to a 20%–60% decrease in the cap-dependent translation initiation activity of 5′UTRPab1. Similar changes in the cap-independent activity of 5′UTRPab1 were also observed. In contrast, the cap-dependent translation initiation activities of 5′UTRPab1-Δpoly(A) remain nearly unchanged when the concentration of Sbp1 increases.
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In addition to RNA binding, the RGG domain of Sbp1 is important for the function of Sbp1. Substitution of the RGG repeats with GS repeats, or methylation of arginines in the RGG repeats, abolishes the inhibitory function of Sbp1 on the Pab1 mRNA in both cap-dependent and cap-independent initiation pathways (Fig. 6A,C).
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To rule out the possibility that RNA or a tertiary factor present in cell extracts mediates the protein–protein interaction, we carried out the pulldown assay using purified proteins such as Sbp1 and Pab1 (Supplemental Fig. S2A). Heparin column and stringent multiple washing procedures were used to eliminate residual RNAs associated with these RNA-binding proteins (see Materials and Methods). Under our experimental conditions, Sbp1 directly binds to initiation factors Pab1 via its central RGG domain. Importantly, the RGG domain of Sbp1, not the RRM domains, is required for the protein–protein interactions, as demonstrated by results obtained from the pulldown assay using domain mutants of Sbp1 (Fig. 3B,C). We further showed that four RRM domains in Pab1 (RRM1-4) are required for the binding with Sbp1. Consistent with our results, Sbp1 was identified to associate with Pab1 in a recent study (Richardson et al. 2012).
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Pab1 and eIF4G were known to interact with each other (Tarun and Sachs 1996; Tarun et al. 1997; Kessler and Sachs 1998). Furthermore, Sbp1 was shown to interact with eIF4G1, eIF4G2, and Pab1 in the cell in a proteomic investigation using open-reading frame tagging, affinity purification, and mass spectrometry (Gavin et al. 2002, 2006). It is conceivable that higher-order complexes can be formed on RNA by these proteins. Indeed, interactions between Sbp1, Pab1, and eIF4G1 on 5′UTRPab1 were observed (Fig. 4).
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Importantly, addition of RNase disrupts the Sbp1–Pab1 as well as Sbp1–Pab1–eIF4G1 interactions on 5′UTRPab1 (Figs. 3A, 4), but leaves Sbp1RGG–Pab1 and Pab1–eIF4G1 interactions unchanged (Figs. 3B, 4). These results confirm that the exposure and conformation of the RGG domain in Sbp1 by RNA binding is important for the protein–protein interaction, which explains why the interaction between Sbp1–Pab1 is RNA-dependent. Furthermore, we noticed that the RGG repeats in Sbp1 are located in between the two RRM domains, much unlike many other RGG-motif proteins which contain the RGG repeats in the terminal regions (data not shown). This domain organization of Sbp1 would likely remodel the RNA structure that it binds by positioning of the central RGG repeats for protein interactions in an RNA-dependent manner.
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It is noteworthy that the interaction between Sbp1 and eIF4G1 is mediated by Pab1 on 5′UTRPab1. Using purified RNA-free proteins, we observed no direct interactions between eIF4G1 and Sbp1, and between eIF4G1 and the RGG domain of Sbp1 (Sbp1RGG) (Supplemental Fig. S7). Previously, Sbp1 was shown to interact with translation initiation factor eIF4G1 via its RGG domain by a GST-pulldown assay using overexpressed eIF4G1 in E. coli cell extracts (Rajyaguru et al. 2012). We reasoned that the observed interaction between Sbp1 and eIF4G1 under the described experimental condition is likely mediated by endogenous RNAs or a ternary protein.
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Post-translational modifications of the RGG domain of Sbp1 were known to modulate its molecular interactions in the cell (Hsieh et al. 2007). In this study, we demonstrated that arginine methylation in the RGG repeats of Sbp1 compromised the binding of Sbp1 to Pab1. In contrast, the same post-translational modification has little effect on the binding of Sbp1 to the RNA (5′UTRPab1) in vitro. Results from our in vitro translation assay show that the methylation of the RGG domain abolishes the translation repression activity of Sbp1 on 5′UTRPab1.
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Arginine methylation has been known to be important in changing protein–protein interactions as well as in controlling subcellular localization of several proteins (Shen et al. 1998; Sinha et al. 2010). Furthermore, the expression of Hmt1 is cell cycle-dependent and the protein is active under different growth conditions in yeast (Messier et al. 2013), suggesting Sbp1 may play a role in the 5′UTR activation during cell cycle progression. Further investigations are needed to understand how the interactions between Sbp1 and Pab1, as well as the formation of a higher-order complex with eIF4G1 involved, are regulated by arginine methylation and the functional consequences of this regulation in translation of targeted mRNAs in the cell.
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Based on the results, our mechanistic hypothesis for the Sbp1-specific translation regulation is the following (Fig. 7): The two RRM domains of Sbp1 bind to 5′UTRs cooperatively, and the center RGG domain recruits other proteins. These interactions lead to formation of an RNP assembly on the UTR, which modulates the translation of targeted mRNAs. In the case of 5′UTRPab1, the RGG domain interacts with other proteins including Pab1, and the resulting RNP complex inhibits the translation initiation of Pab1 mRNA. Because Pab1 is a general translation factor, the observed down-regulation of this protein by Sbp1 may explain at least in part why Sbp1 inhibits the global translation of mRNAs in the cell.
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A proposed mechanism of Sbp1-specific translation regulation. The two RRM domains of Sbp1 bind to 5′UTRs cooperatively, and the center RGG domain recruits proteins such as initiation factor Pab1. These interactions remodel the 5′UTR assembly, leading to changes in the recruitment of the 43S PIC to the target mRNAs and the downstream translational events.
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Consistent with this hypothesis on the importance of RNA binding, the distribution patterns of Sbp1 and Sbp1m (arginine methylated Sbp1) in polysome profiles of 5′UTRPab1 differ substantially, as shown in Figure 8. In this experiment, in vitro translations of 5′UTRPab1 in-frame with the firefly luciferase reporter were carried out in the ΔSbp1 cell extracts in the presence of Sbp1 or methylated Sbp1m. Polysome profiles on the transcript were studied and presence of the protein in different fractions was detected by western blot (see Materials and Methods). As shown in Figure 8, Sbp1 mainly cosediments with RNP and 40S fractions. A small portion of the protein also cosediments with the 80S and polysome fractions. These results are consistent with the observation that tagged Sbp1 was pulled down with 80S and polysomes (Zhang et al. 2014; Wang et al. 2016).
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Methylation of the RGG repeats disrupts ribosome association of Sbp1. (A) Distribution of His-tagged Sbp1 proteins in the polysome profile of 5′UTRPab1-FLuc-poly(A). (B) Distribution of His-tagged methylated Sbp1m proteins in the polysome profile of 5′UTRPab1-FLuc-poly(A). Capped 5′UTRPab1 with luciferase reporter, 5′UTRPab1-FLuc-poly(A), was incubated with ΔSbp1 cell extract in the presence of either 3 µM Sbp1 (A) or Sbp1m (B). 10%–15% sucrose gradient was used for polysome profiling, and distribution of the protein was monitored by western blot. Peaks of polysome, 80S, 60S, 40S, and mRNA are indicated.
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In contrast, Sbp1m only cosediments with the RNP fraction and does not show interactions with the ribosome. This result suggests that the methylation state of the RGG repeats in Sbp1 abolishes its interaction with the ribosome. In addition, because of changes in the protein–protein interaction by methylation, the nature of RNPs formed with Sbp1 and Sbp1m on 5′UTRPab1 is likely to be different.
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Our study demonstrates that 5′UTR remodeling by RBP is important for the transcript-specific translation regulation. The RNA binding is critical for exposing Sbp1's central RGG repeats, which in turn is important for the molecular interactions between Sbp1, Pab1, and eIF4G1. In contrast, deletion of the RRM domain, whereby leaving the RGG domain alone, regardless of its methylation state, inhibits the translation of all the examined RNAs including Pab1, eIF4E, and CrPV in both cap-dependent and cap-independent pathways (Fig. 6, columns 5 and 6).
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While our data strongly suggest a synergistic model described, we cannot, however, rule out a competitive binding of Sbp1 and Pab1 on the same RNA. Since both proteins can bind to the polyadenosine or A-rich sequences, they could potentially compete for the same target sequences. In this scenario, RNA remodeling is also likely to be accompanied when one protein replaces the other. Pab1 contains four tandem RRMs in which the first two RRM domains mediate the polyadenosine RNA binding (Sachs et al. 1987; Kühn and Pieler 1996; Deo et al. 1999; Safaee et al. 2012). Furthermore, a stretch of the RNA containing 12 consecutive adenosines or longer is required for a high affinity binding to Pab1 (Sachs et al. 1987). On the other hand, in the case of Sbp1, we showed that the protein mainly binds to the A-rich sequence in the RNA cooperatively using multiple domains, especially the two RRM domains flanking the central RGG repeats. Thus, the distinctive RRM domain architecture in Pab1 and Sbp1 will most likely lead to different conformations when these two proteins interact with the same RNA and, as a result, remodel the RNA structure differently. A related question is whether Sbp1 prefers binding to specific transcripts containing poly(A) or perhaps even the same transcript in different stages of mRNA biogenesis, considering that Sbp1 also binds to mRNA sequences other than poly(A) (Mitchell et al. 2013; Wang et al. 2016). Clearly, further studies involving genomic and structural investigations are required to answer these questions.
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Recently, decapping activator Dhh1 has been shown to promote decapping by slowing ribosome movement on mRNA (Sweet et al. 2012). Since Sbp1 was suggested to stimulate decapping activity of mRNAs with Dhh1 (Segal et al. 2006) and we also observed that Sbp1 interacts with polysomes (Fig. 8), a fascinating question to ask next is whether, similar to Dhh1, Sbp1 also targets an elongating ribosome to control the translation of mRNA. Further investigations will be needed to provide a complete picture of Sbp1-specific translational regulation.
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The YS602 strain from Saccharomyces cerevisiae (a generous gift from Professor Maurille J. Fournier from UMass Amherst) was used to generate Sbp1-depleted strain (ΔSbp1::kan) by homologus recombination using standard methods, as previously described (Baudin et al. 1993; Wach et al. 1994). Monocistronic luciferase reporters were generated by inserting the firefly luciferase gene between BamHI and KpnI restriction sites of the pUC18 vector (Clontech). A poly(A)62 was introduced between KpnI and EcoRI restriction sites downstream from the luciferase gene. 5′UTRs of Pab1 and eIF4E, including a 36-nt-long endogenous sequence in the open reading frame to maintain the AUG context, were cloned into XbaI and BamHI restriction sites of pUC18, in-frame and upstream of the luciferase gene. RNAs with sequence complementary to the 5′UTRPab1 and 5′UTReIF4E, termed 5′UTRPab1-complement and 5′UTReIF4E-complement, were used as an RNA length and sequence control. The 5′UTRPab1-complement and 5′UTReIF4E-complement were generated in the same way as the 5′UTRPab1. The 5′UTRCrPV sequence was amplified from a pEJ1014 vector (a generous gift from Professor Eric Jan from the University of British Columbia) (Ren et al. 2012) and also cloned in the same way as the 5′UTRPab1.
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A T7 promoter sequence or the same sequence followed by a stable hairpin was cloned into the HindIII and XbaI restriction sites upstream of the 5′UTRs for the cap-dependent or cap-independent translation assays, respectively. The hairpin sequence (5′-CTGCAGCCACCACGGCCCAAGCTTGGGGG CCGTGGTGGCTGCAGGAGAGAGATTCC-3′) was used to inhibit ribosome scanning (Wang et al. 2013).
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Genes encoding translation initiation factor eIF1, eIF1A, Pab1, Sbp1 and individual domains of Sbp1 and Pab1 were amplified from the genomic DNA of S. cerevisiae and cloned into the pETNKI-GST-LIC-Amp or pETNKI-His6-LIC-Amp vectors (Luna-Vargas et al. 2011). The type I arginine methyltransferase (HMT1) gene from S. cerevisiae and the eIF2 β and γ subunits were cloned into the pETNKI-His6-LIC-Kan vector. eIF2α was cloned into the pET22b vector. All the pETNKI vectors are generous gifts from Dr. Patrick Celie at the Netherlands Cancer Institute Protein Facility.
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His-tagged or GST-tagged eIF1, eIF1A, Sbp1, and Pab1 and their mutants containing individual domains were overexpressed in E. coli BL21 (DE3) with 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 18°C. The proteins were purified by affinity chromatography using either Ni-NTA or glutathione agarose, which is followed by ion-exchange chromatography and HiTrap Heparin HP columns (GE Healthcare). Circular dichroism (CD) was performed on the wild-type Sbp1 and its individual RRM domains to confirm proper folding of the RRM domain mutants. eIF2 was expressed in yeast and purified as previously described (Acker et al. 2007). A TAP tag with a calmodulin binding peptide and two IgG binding domains of protein A (Dharmacon) was inserted at the end of the eIF4G1 gene in its chromosome locus. The C-terminal TAP-tagged eIF4G1 was expressed in yeast cells and purified with affinity chromatography using buffer containing 20 mM Hepes-KOH, pH 7.9, 150 mM KCl, 1 mM Mg(OAc)2, 0.1% NP-40, 1 mM DTT followed by a HiTrap Heparin HP column (GE Healthcare) using buffer containing 20 mM Tris–HCl, pH 7.5, 150 mM KCl, 0.1% NP-40. RNase A and high salt wash with 1 M KCl were used to remove endogenous RNAs bound to eIF4G1.
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RNAs were synthesized by run-off transcription from linearized DNA plasmid templates and purified by either gel electrophoresis under denaturing conditions or by anion-exchange chromatography (Easton et al. 2010). The purity and integrity of the RNA was monitored by an acrylamide gel under denaturing conditions or by northern blot.
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All steps were performed in a buffer of 50 mM Hepes pH 7.3, 50 mM KCl, 10 mM Mg(C2H3O2)2, 2 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM benzamide. Of note, 40 U RNase inhibitor (NEB) were added per 1 mg/mL of total protein concentration. Cell extracts from S. cerevisiae were precleared with 60 µL of paramagnetic streptavidin-conjugated magnetic beads (Dynabeads M-280; Invitrogen) for 1 h at 4°C to decrease the amount of protein bound nonspecifically to the beads. S1-tagged 5′UTRPab1 RNAs alone and the 5′UTRPab1-43S PIC were incubated with the beads for 2 h at 4°C in the buffer consisting of 50 mM Hepes, pH 7.3, 100 mM KCl, 2 mM MgCl2, and 2 mM DTT. The formation of the 43S PIC was carried out as described previously (Acker et al. 2007). To facilitate ribosome-loading, 5′UTRPab1-construct1 was used in which the RNA sequence downstream from the start codon AUG was replaced by AAAAAAAAA.
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Subsequently, RNA-bound streptavidin beads were incubated with cell extracts for 2 h at 4°C. Proteins bound to the 5′UTR assembly were eluted by D-biotin (Invitrogen), examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and identified and quantified by liquid chromatography–tandem mass spectrometry (LC–MS/MS).
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Cells were harvested at OD600 = 0.85–0.9 and washed five times with translation buffer comprised of 22 mM Hepes-KOH, pH 7.4, 120 mM potassium acetate, 1.5 mM magnesium acetate, 0.75 mM ATP, 0.1 mM GTP, 25 mM creatine phosphate (Invitrogen), 0.04 mM of each of the twenty amino acids, 1.7 mM 1,4-dithiothreitol (DTT), 5 µg creatine kinase (Invitrogen), and 10 U RNasin Plus (Promega). Cell extracts were treated with micrococcal nuclease and prepared as previously described (Iizuka et al. 1994). An m7G cap was added at the 5′ end of the RNA using the Vaccinia capping enzyme (NEB). In vitro translation reactions were carried out at 25°C for 40 min in translation buffer. m7G-capped or uncapped RNAs were added to the extracts in the presence of Sbp1 and its domain variants at varying concentrations, and luciferase activities were measured using the ONE-Glo Luciferase Assay kit (Promega). The measured reporter activity was normalized to the concentration of total proteins in the extract obtained by Bradford assay. All reported activities are an average of at least three independent luciferase assays.
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32P-labeled 5′UTRPab1 and proteins bound—including eIF4G1, Pab1, Sbp1, and its domain variants—were mixed at different stoichiometric ratios. Binding of the RNA to the protein was examined by electrophoresis in an 8% (bis-acrylamide 29:1) polyacrylamide under native conditions in THEM buffer (34 mM Tris Base, 57 mM Hepes, 0.1 mM EDTA, 2.5 mM MgCl2). Bound and unbound RNAs were quantified using ImageQuant software (Molecular Dynamics). The dissociation constant (Kd) and the Hill coefficient (n) was obtained by fitting data to the following equation: [RNA]bound([RNA]bound+[RNA]free)=[P]totaln(Kdn+[P]totaln), where [P]total is the total concentration of the protein, [P]total ≈ [P]free under the conditions used, and [RNA]bound and [RNA]free are concentrations of RNA in the protein-bound and free forms, respectively.
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GST-tagged Pab1 or Sbp1, immobilized on a glutathione–sepharose resin, was incubated with the target proteins or their mutants containing individual domains with or without 5′UTRPab1 at 4°C for 30 min in the binding buffer (50 mM Tris–HCl, pH 7.5, 120 mM KCl, 2 mM DTT, 2 mM MgCl2, 0.5% Triton-X-100). The resin was then washed four times with cold binding buffer and subsequently boiled in 1× SDS-loading buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 10% Glycerol, 1% β-mercaptoethanol, 0.02% bromophenol blue) to elute bound proteins. RNase treatment was carried out after the formation of RNP by incubating RNase A with the RNP at 25°C for 30 min. The proteins were resolved by SDS–PAGE and detected by either Coomassie Brilliant Blue or SYPRO Ruby staining.
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Methylation of the arginine in the RGG domain of Sbp1 was done essentially as previously described (Hsieh et al. 2007) with the following modifications. Briefly, plasmids containing Sbp1 and type I arginine methyltransferase Hmt1 were cotransformed and coexpressed in E. coli BL21 (DE3) cells. Methylated Sbp1 was purified by affinity chromatography followed by ion-exchange chromatography using HiTrap Heparin HP column (GE Healthcare). Methylation of the arginines in the RGG domain was confirmed by LC–MS/MS.
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Western blot was done as previously described (Coller and Parker 2005; Sweet et al. 2012; Zeng and Jin 2016). Briefly, fractions from polysome profiling were resolved by SDS–PAGE; and proteins in unfixed gels were transferred to nitrocellulose membranes (GenScript) for 30 min at 100 V in a Mini Trans-Blot apparatus (Bio-Rad). Protein bands were detected with anti-GST (Invitrogen) or anti-His antibodies (Sigma-Aldrich). Northern blots were done as previously described (Coller and Parker 2005; Sweet et al. 2012). RNA samples were separated on a 5% urea acrylamide gel, transferred to nylon membranes, and probed overnight with a luciferase gene-specific 32P-labeled DNA probe. Blots were exposed to PhosphorImager screens, scanned by a Storm 840 scanner, and quantified with ImageQuant software.
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Transistor-type organic memory devices (TOMDs) have attracted keen interest because of their potentials toward flexible or bendable plastic memory modules12345. TOMDs are effective in terms of module (array) configuration because they have both memory and driving (switching) elements in single device, whereas additional transistor circuits are necessary for driving array modules in the case of resistor-type organic memory devices (ROMDs)678. To date, various organic materials and structures, such as ferroelectric polymers, charge-trapping polymers, polymeric layers with metal nanoparticles and polymer energy well structures, have been employed for TOMDs, but they could not deliver both low-voltage operation and high stability at the same time91011121314151617181920. Very recently, encouraging data retention characteristics at room temperature have been reported for TOMDs with poly(vinyl alcohol) memory gate-insulating layers2122.
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However, TOMDs with the data retention stability at high temperatures have never been reported so far, even though organic materials (films) are generally understood physically softer and weaker than inorganic materials (films) leading to relatively inferior stability at high temperatures232425. The weak high temperature characteristics of typical organic devices can be attributed to the fact that organic films in devices are made with organic molecules by weak intermolecular (interchain for polymers) interactions such as van der Waals forces, slightly strong hydrogen bonding, etc.26272829. In the case of flash memory devices commercialized with inorganic materials, the guaranteed retention stability (program-erase cycle) reaches 1,000 times and 10,000 times for the triple-level cells (TLCs) and the multi-level cells (MLCs), respectively303132. In addition, such inorganic flash memory devices can be operated at elevated temperatures up to 70 °C333435. Therefore, it is necessary for TOMDs to secure the data retention stability at high temperatures in regard to the positive consideration of commercialization.
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In order to achieve the high temperature stability of TOMDs, the core memory layer needs to be thermally stable without loss of memory functions at elevated temperatures. In this regard, organic/inorganic hybrid materials can be one of the effective approaches when it comes to the complementary role of organic and inorganic components in the hybrid materials36373839. In particular, employing polymers as the organic component can be further advantage to the hybrid materials in terms of better stability for flexible TOMDs due to the toughening role of polymers rather than small molecules404142.
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In this work, we synthesized novel hybrid nanostructures, featuring polymer nanodot-embedded alkyl silicon oxide (ASiO) networks, by two-step (sol-gel and thermal cross-linking) reactions between poly(4-vinylphenol) (PVP) and triethoxyvinylsilane (or vinyltriethoxysilane - VTES). The resulting cross-linked ASiO hybrids embedded with PVP nanodots (nanoparticles) (X-ASiO-PVPNP) were employed as a thermally stable memory layer for the TOMDs with the poly(3-hexylthiophene) (P3HT) channel layers. The hybrid TOMDs could be operated at low voltages (1~5 V) and exhibited outstanding operation stability at high temperatures (150 °C) due to the pronounced hysteresis by the PVP nanodots and the high thermal stability by the cross-linked VTES-derived ASiO network structures.
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As shown in Fig. 1a, VTES was first hydrolyzed to vinyltetrahydroxysilane (VTHS) in the presence of water and acetic acid, followed by addition of PVP. As soon as VTES is converted to VTHS, sol-gel reactions are considered to begin among VTHS molecules as well as between VTHS and PVP (Si-OH groups in VTHS and C-OH groups in PVP), leading to the precursor sol solutions (VTES- PVP). The precursor solutions were coated on quartz substrates or indium-tin oxide (ITO)-coated glass substrates leading to the VTES-PVP precursor films, which were finally converted to the cross-linked hybrid (X-ASiO-PVPNP) films via thermal curing reaction between double bonds in the silicon atoms. The optical measurement disclosed that the optical absorption spectra (absorption edge) of hybrid (X-ASiO-PVPNP) films were gradually red-shifted as the PVP content increased (see Fig. 1b). However, the optical transparency of films was well maintained even though the film color was changed to slightly yellowish (see the inset photographs in Fig. 1b and Fig. S1). Here we note that the precursor films before thermal cross-linking reactions could be as thick as more than 200 μm by drop-casting from their precursor solutions (see Fig. 1c). The formation of Si-O-C bonds, which are caused by the reaction between the hydroxyl (C-OH) groups in the PVP chains and the silanol (Si-OH) groups in the VTHS domains during sol-gel reactions, was proven by the X-ray photoelectron spectroscopy (XPS) and Fourier Transform-Infrared (FT-IR) spectroscopy measurements (see Fig. 2). As shown in Fig. 1d, the cross-linked hybrid (X-ASiO-PVPNP) films were well coated on the patterned ITO-glass substrates so as to make a gate-insulating memory layer that is placed beneath the P3HT channel layer in the hybrid TOMDs (see the optical microscope images for the channel area in Fig. 1e).
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study
| 100.0 |
In order to verify whether the hydroxyl (C-OH) groups in the PVP chains were certainly reacted with the silanol (Si-OH) groups in the VTHS domains during sol-gel reactions, X-ray photoelectron spectroscopy (XPS) was employed to measure the evolution of Si-O-CPhenyl bonds. As shown in Fig. 2a, the C1s peaks (shoulders) at ca. 286.5 eV and 288.4 eV were emerged as the PVP content increased, which can be ascribed to the aromatic C-OH because the XPS intensity at this energy range was ignorable for the pristine ASiO films without PVP. Similarly, the O1s shoulders at around 535 eV (aromatic C-O) became pronounced with the PVP content though no XPS signal was measured at this region for the pristine ASiO films. These results confirm the presence of PVP in the hybrid films. Finally, the Si2p peaks revealed the formation of Si-O-CPhenyl bonds because the shoulders between 105 eV and 106 eV were gradually increased with the PVP content compared to no signal for the pristine PVP films (note that the Si-O-CEthyl bonds were almost completely removed during hydrolysis reaction). The evolution of Si-O-Si and Si-O-CPhenyl bonds was measured at the wavenumber range of 900~1200 cm−1 from the Fourier Transform-Infrared (FT-IR) spectra (Fig. 2d)43, which also delivered the gradual increase in the aromatic C-C and C = C peaks with respect to the PVP content44.
|
study
| 100.0 |
The nanostructures of the cross-linked hybrid (X-ASiO-PVPNP) films were first investigated by employing synchrotron radiation grazing incidence X-ray diffraction (GIXD) techniques. As shown in the 2D GIXD images (Fig. 3a), very weak (almost unrecognizable) Debye diffraction ring was measured for the pristine ASiO films (PVP content = 0 wt.%), while no particular diffractions were measured for the pristine PVP films. Interestingly, an intense Debye ring was measured for the cross-linked hybrid (X-ASiO-PVPNP) films with 5 mol.% PVP. The similar strong Debye ring was also measured for the hybrid films with 10 mol.% PVP. Based on the scattering vectors for the Debye rings, the d-spacing (d) values calculated were in the range of 0.62~1.57 nm. The detailed investigation with the 1D GIXD profiles (Fig. 3b) disclosed that the intense Debye rings have a maximum peak at around qxy = 0.66 Å−1 (2θ = 6.75°), leading to d = 1 nm, in the directions of both out-of-plane (OOP) and in-plane (IP). Considering the GIXD results, it is shortly concluded that the addition of PVP had a strong influence on the reorganization (recrystallization) of VTES molecules.
|
study
| 100.0 |
Both high-resolution and scanning transmission electron microscopy (HRTEM and STEM) measurements were performed to further understand the nanostructure of the cross-linked hybrid films. As shown in Fig. 3c, the HRTEM measurements revealed the existence of very small nanodots that are randomly distributed with various sizes (1~3 nm) in the cross-linked hybrid (X-ASiO-PVPNP) films (see also Fig. S2a,b). Further HRTEM measurement with high magnification showed that the small nanodots consist of a particular lattice nanostructure with an inter-lattice spacing of ca. 0.15~0.2 nm, indicative of highly ordered states (see the bottom images in Fig. 3c). Interestingly, as shown in Fig. 3d, the nanodots were identified as the PVP domains by the STEM measurement through the composition analysis (see the focused area in Fig. S2c). Because the nanodots exhibited dark spots in the HRTEM images and bright spots in the STEM images, they are confirmed a well-ordered phase that is sufficient to make proper electron diffractions45. Taking into account the TEM analysis results, it is supposed that the nanodots consist of well-ordered PVP chains as illustrated in Fig. 3d (right). This well-ordered state could be made via nanoscale-phase separation processes during sol-gel and thermal cross-linking reactions because of the increased viscosity leading to the different surface energy between the PVP phase and the ASiO precursor sol phase.
|
study
| 100.0 |
Next, the cross-linked hybrid (X-ASiO-PVPNP) films were examined as a gate-insulating layer for the transistor structure as illustrated in Fig. 1d. As shown in the output curves (Fig. 4a), all devices exhibited typical p-type transistor characteristics and could be operated at low voltages (−1~−5 V in absolute value). Interestingly, a hysteresis was measured in the output curves for the devices with the X-ASiO-PVPNP layers, which became more pronounced as the content of PVP increased. In addition, the drain current (ID) at the same voltage condition was higher for the devices with the X-ASiO-PVPNP layers (10 mol.% PVP) than those with the pristine X-ASiO layers or the pristine PVP layers. Note that the devices with the pristine PVP layers showed high leakage currents even at zero gate voltage (VG = 0 V). As observed from the transfer curves in Fig. 4b, the current hysteresis between forward and backward sweeps in the devices with the pristine X-ASiO layers was quite small and not improved even by increasing the drain voltage (VD) from −1 V to −5 V. However, the hysteresis became certainly pronounced when 5 mol.% PVP was added. Further addition of PVP (10 mol.%) led to more improved hysteresis in the transfer curves (see Fig. S3 for the gradual hysteresis change with the drain voltage). In addition, the on/off ratio at VD = −5 V was noticeably improved from 1.2 × 103 (0 mol.%) to 1.1 × 104 (5 mol.%) and 4.4 × 105 (10 mol.%) by addition of PVP. The PVP addition contributed to the improved hole mobility from 2.89 × 10−3 (0 mol.%) to 2.28 × 10−2 (5 mol.%) and 1.3 × 10−1 (10 mol.%) (see Fig. S4).
|
study
| 100.0 |
Based on the hysteresis characteristics, memory operation tests were performed for the devices with the X-ASiO-PVPNP layers. The writing-once-reading-many (WORM) test showed that both 5 mol.% and 10 mol.% PVP devices properly made proper operations of reading (VG = −1 V and VD = −3 V) after writing (VG = −5 V and VD = −3 V) (see Fig. 5a). However, the 10 mol.% PVP devices showed better WORM characteristics with stable signals. In the case of writing-reading-erasing-reading (WRER) cycle test (Fig. 5b), the 10 mol.% devices exhibited more stable memory functions with clearer drain current difference between reading-1 (R1) after writing (W) and reading-2 (R2) after erasing (E). In contrast, the 5 mol.% devices showed less stable WRER characteristics in the presence of small drain current difference. The superior memory performance of the 10 mol.% PVP devices is also evidenced from the repeated WORM and WRER operations (see Fig. S5). Hence the 10 mol.% PVP devices can be called one of TOMDs with proper memory functions.
|
study
| 100.0 |
Taking into account the nanostructures measured in Fig. 3, the charge traps in the PVPNP parts of the X-ASiO-PVPNP layers can be proposed as a core factor for memory operation for the present hybrid TOMDs. As illustrated in Fig. 5c, the PVPNP parts are charged simultaneously when the X-ASiO-PVPNP layers undergo polarization upon applying bias between the gate and source electrodes (VG = −5 V). However, the charges made in the PVPNP parts cannot easily go away but trapped inside the PVPNP parts by the heterogeneous dielectric interfaces between PVPNP and X-ASiO because of different nature of materials. So the charged PVPNP parts play a critical role in delivering the hysteresis characteristics of the hybrid TOMDs.
|
study
| 100.0 |
Considering the outstanding WORM and WRER characteristics, the TOMDs with the X-ASiO-PVPNP layers (PVP = 10 mol.%) were chosen for the data retention test, which is usually performed by repeating the WRER operation over several thousand cycles464748. Prior to the retention test, the TOMDs were encapsulated with the cross-linked poly(dimethylsiloxane) (PDMS) layers (see Fig. 6a and the method section for the detailed process). As shown in Fig. 6b, water drops were well spread over the channel area in the case of the bare devices without any encapsulation, which implies good interactions between Al electrodes and water molecules. However, the devices encapsulated with the cross-linked PDMS layers showed a restricted spreading of water drop. Next, both devices were immersed into water for 24 h in order to examine the performance of encapsulation. As displayed in Fig. 6c, the Al electrodes in the channel area were safe without any defects in the case of the encapsulated devices, whereas the Al electrode parts were seriously damaged for the bare devices (without encapsulation).
|
study
| 100.0 |
Hence the 10 mol.% PVP devices, which were encapsulated with the cross-linked PDMS layers, were used for the measurement of retention characteristics and high temperature operations. As shown in Fig. 6d (left), the encapsulated devices exhibited very stable retention characteristics up to 5000 WRER cycles. The deviation of reading-1 (R1) current was only 4.3% after 5000 cycles, while that of reading-2 (R2) after erasing was 15.5% compared to the initial value. Next, the encapsulated devices were loaded on a hot stage in order to examine the retention characteristics at 150 °C. As shown in Fig. 6d (right), the drain current deviation after 4750 cycles at 150 °C was 1.5% and 44% for reading-1 (R1) and reading-2 (R2), respectively. Here we note that the drain current level at 150 °C was slightly changed from that at room temperature, which can be attributable to the thermal effect on charge transport (mobility) as well as the different contact environment between the Al electrodes and the probes in the measurement system. Here it is also worthy to note that the P3HT channel layers are also strong enough to withstand the thermal shock at 150 °C because the present P3HT polymers with high regioregularity (96%) possess relatively high glass transition temperatures, close to but lower than the melting points (210~240 °C), due to the very small portion of amorphous (regiorandom) parts (4%)495051. In contrast to the outstanding retention characteristics of the encapsulated devices at such high temperature (150 °C), a catastrophic current fluctuation was measured for the bare devices at 150 °C and even at room temperature (see Fig. S6). It is also worthy to note that the pristine PVP devices (PVP = 100 mol.%) exhibited much poorer hysteresis at 150 °C than room temperature, indicative of an impossible state for memory operation (see Fig. S7). Hence this result implies that the present encapsulated devices with the X-ASiO-PVPNP layers (PVP = 10 mol.%), hybrid TOMDs, can be used as a durable memory device for high temperature applications such as security camera built in car, control systems for fire-fighters and steel mill industries, rescue robots for nuclear power stations, memory systems for space shuttles, etc.
|
study
| 100.0 |
In conclusion, novel polymer nanodot-embedded alkyl silicon oxide hybrids, X-ASiO-PVPNP, were successfully prepared via sol-gel and chemical cross-linking reactions. The surface of PVP nanodots (PVPNP = 1~3 nm from HRTEM) with a particular lattice nanostructure (inter-lattice spacing = 0.15~0.2 nm) was found to make covalent bonds with the cross-linked alkyl silicon oxide (X-ASiO), which might be a driving force to generate such a small PVPNP leading to high thermal stability. The hybrid transistor-type organic memory devices (TOMDs) with the X-ASiO-PVPNP layers exhibited stable operation at 150 °C during >4750 WRER cycles, which is the first record for TOMDs tested at a high temperature. Therefore, the present X-ASiO-PVPNP hybrid materials are expected to be a landmarking milestone for achieving durable organic memory devices with excellent operation stability at high temperatures. In addition, the synthesis protocols of X-ASiO-PVPNP materials can be useful to invent super-strong flexible transparent substrates and thermo-resistive functional coatings for flexible electronic devices because the embedded (hybridized) polymer nanodots are expected to play a critical role in compensating mechanical stresses together with high thermal stability by the X-ASiO domains.
|
study
| 100.0 |
The hybrid solutions were prepared via sol-gel reactions of vinyl triethoxysilane (VTES) and poly(4-vinylphenol) (PVP, average molecular weight = 25 kDa), which were purchased from Sigma-Aldrich Co. (St Louis, Mo, USA), in the presence of deionized (DI) water and acetic acid (Sigma-Aldrich). The composition of PVP to VTES was 0, 5, and 10 mol. %, while the molar ratio of DI water and acetic acid was fixed at 1:1:6. The mixture solutions, which contain VTES, PVP, DI water and acetic acid, were subject to vigorous stirring initially, followed by sol-gel reactions at room temperature for 24 h.
|
study
| 100.0 |
Indium-tin oxide (ITO)-coated glass substrates (sheet resistance = 10 Ω/cm2) were subject to a photolithography process to make the ITO patterns with a 1 × 12 mm stripe for gate (G) electrodes. After cleaning the patterned ITO-glass susbtrates in an ultrasonic bath with acetone and isopropyl alcohol (30 min), the clenaed ITO-glass substrates were treated with UV-ozone (28 mW/cm2 for 20 min) by utilizing UV-ozone cleaner (UVO cleaner, Ahtech LTS Co., Ltd) in order to remove any organic contaminants remained. Next, the VTES-PVP precursor films were spin-coated on the ITO-glass substrates, followed by thermal curing processes at 250 °C for 6 h leading to chemically cross-linked hybrid (X-ASiO-PVPNP) films (thickness = 700 nm). On top of the X-ASiO-PVPNP layers, the P3HT channel layers (thickness = 60 nm) were spin-coated at 1500 rpm for 30 s by using the P3HT solutions, which were prepared by dissolving the P3HT polymer (weight-average molecular weight = 70 kDa, polydispersity index = 1.8, regioregularity = 96%, Rieke Metals) in toluene at a solid concentration of 13 mg/ml. The spin-coated P3HT channel layers were soft-baked at 70 °C for 15 min and transferred to a vacuum chamber installed inside a nitrogen-filled glove box system. Finally, the source (S) and drain (D) electrodes were formed by successively depositing nickel (Ni, thickness = 15 nm) and aluminum (Al, thickness = 60 nm) electrodes on the P3HT layers through a shadow mask. The channel length and width of devices were 70 μm and 2 mm, respectively (see Fig. 1d). The fabricated devices were encapsulated by the cross-linked poly(dimethyl siloxane) (PDMS) layers (Sylgard 184, 10 g clip-pack, Sigma-Aldrich Co., St Louis, Mo, USA, thickness = 450 nm), which were cured at room temperature. We note that the X-ASiO-PVPNP films were prepared on quartz substrates for the measurement of optical absorption spectra but indium-tin oxide (ITO)-coated glass substrates were used for other measurements including XPS spectra, FT-IR spectra and GIXD images.
|
study
| 100.0 |
The thickness of films was measured using a surface profiler (Alpha Step 200, Tencor). An ultraviolet-visible absorption spectrometer (Optizen 2120UV, Mecasys) was used for the measurement of optical absorption spectra of films. The cross-sections of hybrid memory transistors were examined with a field-emission scanning electron microscope (FESEM, S-4800, Hitachi), while an optical microscope (SV-55, Sometech) was used to inspect the surface of hybrid films and the top part of devices. The memory transistor charateristics of devices were measured using a semiconductor parameter analyzers (Keithley 4200, Keithley 2636B, Keithley Instruments Inc.), which are connected to a sample holder system with heating units inside a glove-box probe station (PS-CPSN2, Modu-Systems). The XPS spectra of hybrid films were measured with a X-Ray photoelectron spectrometer (ESCALAB 250, Thermo Scientific, Inc.), while a Fourier transform-infrared spectrometer (FT-IR, 5700 Continum, Thermo Scientific, Inc.) with the attenuated total reflection (ATR) mode was used for the measurement of functional groups in the hybrid film samples. The nanostructure of hybrid films was measured using a synchrotron radiation grazing incidence angle X-ray diffraction system (GIXD, wavelength = 1.212969 Å, Pohang Accelerator Laboratory).
|
study
| 100.0 |
In late autumn 2008, a 49-year old woman died. She suffered from type 1 diabetes, familial mediterranean fever, peripheral neuropathy, and foot ulcers. In February 2008, she was admitted to the Department of Infectious Diseases, University Hospital in Linköping for amputation of her left foot and received piperacillin-tazobactam intravenously and a blood transfusion post-operatively (Fig. 1). Despite this, her clinical status deteriorated critically within one week and she developed acute liver and renal insufficiency. The suggested diagnosis was hemolytic uremic syndrome and the result of an allergic reaction to piperacillin-tazobactam. She was treated with plasmapheresis, corticosteroids, imipenem, and metronidazole. The culture from the foot ulcer prior to amputation revealed growth of Enterococcus fecalis (E. fecalis) together with several gram-negative bacterial strains. She recovered partially, but the renal function was permanently destroyed and she was treated with hemodialysis and released on March 11th 2008. On March 24th, she was readmitted to the Department of Infectious Diseases for fever, shivering, and diarrhea. The blood cultures revealed growth of E. fecalis and Staphylococcus aureus. She was transferred to the Department of Cardiology, University Hospital in Linköping for mitral valve endocarditis and underwent surgery for biological valve prosthesis. The antibiotic treatment was composed of meropenem, vancomycin, and clindamycin and continued for 8 weeks. The heart valve culture revealed E. fecalis. She was dismissed on June 5th 2008 with moxifloxacin. She developed vomiting after 5 days at home and was readmitted on June 23rd. Blood cultures revealed growth of E. fecalis again and she was treated with vancomycin. Echocardiography showed a paravalvular leak that was treated at the Department of Cardiology. On July 28th 2008, while still on the ward, she experienced sudden temporary blindness. A computed tomography (CT) scan did not show new pathologic signs that might explain the symptom. Doktacillin was complementary to vancomycin in the treatment. However, she developed hemolytic anemia and antibiotic therapy was interrupted on August 8th. She suffered from anxiety, depression, severe heart failure, pain, and loss of vision, but she had no fever. Blood tests revealed a high sedimentation rate, anemia, high white blood cell count, and a high level of C-reactive protein. Blood cultures from eight different occasions yielded no growth. Two short episodes of antibiotic therapy with tigecycline and imipenem were interrupted due to negative blood cultures. The symptoms were judged as Mediterranean fever and she was transferred to the Department of Nephrology with high dose of corticosteroids on October 1st. She died October 13th due to septic shock and E. fecalis grew in all blood cultures that were taken before she died.Fig. 1Chronic ulcer in a patient with persistent Enterococcus fecalis infection and organ dysfunction in 2006
|
clinical case
| 100.0 |
Persistent infection in biofilms has been the subject of clinical studies since 1981. Biofilms are a collection of microbes that adhere to surfaces by producing a matrix that shields them from environmental elements. It has been speculated that bacteria colonizing chronic wounds are part of the highly persistent biofilms . Molecular analyses of chronic wound specimens revealed diverse polymicrobial communities but it has been very difficult to identify specific bacteria of the entire individual, especially strictly anaerobic bacteria, by culture methods . Traditional culturing methods may be extremely biased as a diagnostic tool as they select for easily cultured organisms e.g. S. aureus, but not bacteria difficult to culture such as anaerobes . The formation of bacterial biofilms could lead to chronic inflammation. Detachment of the biofilm enables bacteria to enter into the blood stream, causing bacteremia and vascular embolism . The establishment of a non-cultural method for analysis of infections may help to identify the key bacteria that cause pathogenic biofilms [5–7]. Thus, it is most important to identify the underlying causative infectious agent in the symbiotic community of biofilms in order to antagonize the development of a suitable environment for opportunistic pathogens and thereby improve the wound healing process.
|
review
| 99.8 |
E. fecalis is a successful pathogen and is able to adapt to the hostile environment to grow within the host and has been shown to facilitate the survival of other bacteria within the biofilms . Therapy directed to eliminate adherent gram-positive bacteria, such as E. fecalis, may effectively destroy the biofilm by disturbing the growth of rapid growing gram-negative bacteria [9–11].
|
study
| 95.9 |
As part of serial studies to identify the role of the key bacteria in several associated diseases, this study aims to:Develop anti-bacterial human antibodies in vitro from patients who have been treated for systemic infection.Immobilize the produced antibodies in a SPR system and use the response to injected heat-killed pathogens for assessment of specificity.Analyze the ulcer secretions from patients and healthy volunteers by the SPR system and compare the results to data from culture methods.To use these antibodies as an alternative tool for detecting bacteria in biofilms.
|
study
| 100.0 |
The blood samples were collected at convalescence from 10 patients (described in the following section) who met the study leader at Department of Infectious Diseases and attended a follow-up visit within 4–8 weeks after onset of systemic infection. The informed consent was obtained and the samples were handled unidentified.
|
study
| 99.94 |
The ulcer secretion was collected from subjects (n = 24, described in the following section). This material has been presented previously . The study was approved by the local ethics committee at Linköping University Hospital. All participants provided written consent (03–322, M133-09).
|
other
| 96.4 |
Blood samples were gathered in EDTA tubes (Venoject K2K, Terumo Europe NV, Leuven, Belgium) from patients who had systemic infections caused by E. fecalis (male, 70 years old, septicemia), S. aureus (male, 26 years old, severe periodontitis, ulcers and septicemia), Pseudomonas aeruginosa (p. aeruginosa) (female, 35 years old, decubital ulcer), Staphylococcus epidermidis (S. epidermidis) (female, 76 years old, prosthesis infection), Clostridium perfringens (female, 75 years old, decubital abscess), Bacterioides fragilis (male, 61 years old, perianal abscess and septicemia), Prevotella oralis (male, 60 years old, severe periodontitis and bacteremia), Escherichia Coli (E. coli) (female, 38 years old, pyelonephritis) and Streptococcus sp. (78 years old male, septicemia) at convalescence.
|
study
| 99.94 |
Ulcer secretion was gathered from ten patients (8 women, 44–89 years of age, median 76 years old) with chronic leg ulcers that had been stable for at least 6 months. The etiologies of the ulcers were venous insufficiency or a combined venous and mild arterial insufficiency determined by clinical judgment and physiological measurements, i.e., toe and ankle pressure measurements. Cultures from the ulcers revealed growth of several species of bacteria; S. aureus, Enterobacter cloacae, P. aeruginosa, E. fecalis, and Proteus mirabilis. Skin biopsies were taken from the left arm of nine healthy volunteers (all women, 40–60 years of age, median: 54 years) and were used as controls. The ulcer secretion was collected within 24 h after injury and the cultures from these healthy controls were negative. Ulcer secretions were also collected from 5 patients within 2 weeks of operation for breast cancer without any signs of metastasis (all women, 31–85 years of age, median 64 years). Cultures revealed growth of S. aureus in two cases and were negative in the rest of these patients.
|
study
| 99.94 |
During a 24-h period, equal amounts of ulcer secretion were collected by absorption using 1 cm2 of absorbent material (Mepilex, Mölnly Health Care AB, Box 13080, SE 40252, Göteborg, Sweden) placed under the dressing and then transferred into a flask (scintillation vial 20 mL, Sarstedt AB, SE- 26151 Landskrona, Sweden) containing 5 ml physiological sodium chloride (NaCl). The sample was vortexed (Vortex-Genie, Scientific Industries, Inc., New York, USA) and centrifuged at 3000 × g for 10 min. The supernatant was transferred into tubes (Nunc Cryo Tube, Nunc Brand Products, Denmark) and stored at −70 °C before analysis.
|
study
| 99.94 |
Whole blood was diluted in 0.9 % NaCl solution in a 1:1 ratio and lymphocytes were isolated by density gradient centrifugation using lymphoprep solution (Axis-shield PoC, Oslo, Norway) upon centrifugation at 1800 × g at 20 °C for 20 min. A clear distinct layer of mononuclear cells containing lymphocytes was carefully pipetted from the tube and cultured in L-15 medium (ATCC, Borås, Sweden), supplemented with 10 % fetal bovine serum (FBS; Sigma Aldrich, Stockholm, Sweden), at 37 °C. The lymphocytes were then stimulated with heat-killed E. fecalis (CCUG, Gothenberg University, Sweden) at an optical density (OD) of 1 and incubated at 37 °C for 3 weeks (Additional file 1). After 3 weeks, the medium was collected and centrifuged at 3000 × g for 10 min to pellet the cells. The supernatant was filtered using 0.45 μm sterile filters (Thermofisher Scientific, Stockholm, Sweden) and centrifuged again using 100 KDa Amicon Microcon Centrifugal Filters (Millipore, Molsheim, France) at 3000 × g for 60 min. Antibodies with molecular weight over 100KDa were recovered from the filters by pipetting and were stored at −70 °C until further use.
|
study
| 100.0 |
A bacterial co-culture of E. fecalis, S. aureus and E. coli was prepared by diluting the heat-killed bacteria in 1:100 ratio in PBS (pH 7.4, Apoteket, Linköping, Sweden), and in vitro specific anti-E. fecalis antibodies were diluted 1:200 in PBS. The bacteria were incubated overnight with anti- E. fecalis antibodies at 4 °C. Samples were then incubated with nano gold-anti-human IgG antibodies (Nanoprobes, New York, USA) diluted 1:10 in PBS for 2 h. The specimen was mounted on the formvar coated copper grids and counter stained with 2 % Osmium tetroxide (Sigma-Aldrich, Sweden) and were analyzed using TEM1230 Gantan Electron microscope (Linköping University, Sweden).
|
study
| 100.0 |
The SPR measurements and ligand immobilization procedures were conducted at 760 nm in a fully automatic Biacore 2000 instrument (GE-Healthcare GmbH, Uppsala, Sweden) equipped with four flow cells, and the temperature was 25 °C in all experiments. The running buffer was HBS-EP (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005 % surfactant P20, pH 7.4) (GE-Healthcare GmbH). Ligands were coupled to carboxymethylated dextran CM5 chips (GE Healthcare GmbH) by conventional carbodiimide chemistry using 200 mM N-ethyl-N-(3 diethylaminopropyl) carbodiimide (EDC) and 50 mM N-hydroxysuccinimide (NHS). The activation time was 7 min, followed by a 7 min ligand injection. Deactivation of the remaining active esters was performed by a 7 min injection of ethanolamine/hydrochloride, pH 8.5. A flow rate of 5 μl/min was used during the immobilization and measurement procedures. Antibodies developed against bacteria were diluted 1:10 in 10 mM acetate buffer, pH 4.5, below the isoelectric point of the protein, thus enhancing the electrostatic interactions between the dextran matrix and the ligands. Anti-guinea pig IgG and anti-human IgG (Sigma Aldrich) and heat-killed ATCC bacterial strains (CCUG, Gothenburg, Sweden) were used as controls. The contact time was 7 min, which resulted in immobilization levels between 15000 and 40000 response units (RU). Ulcer secretions were diluted 1:1 in PBS (Apoteket AB, Umeå, Sweden). Injection of equal mixture of 1 M NaCl and 10 mM glycine, pH 2, followed by one injection of borate pH 8.5, were used for regeneration (1 min).
|
study
| 100.0 |
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