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The results of Experiments 1 and 2 suggest that elevated rates of lure false recognition with age arise as a result of contributions of both reductions in the availability of object details with age, and impairments in the ability to carry out strategic retrieval processes. However, existing work suggests that these factors may not be affected to a similar degree across older adults (Davidson & Glisky, 2002; Glisky, Rubin, & Davidson, 2001; Migo et al., 2014; Toner et al., 2009). Thus, it may be the case that individual differences in the availability of object details and the ability to execute strategic retrieval processes will impact the susceptibility to false recognition across older adults. Furthermore, if the Forced Choice and Yes/No test formats are differentially sensitive to each of these factors, as suggested by prior work (Guerin et al., 2012; Migo et al., 2009, 2014; Norman, 2010), individual variability in these measures may differentially impact performance in each test format. To investigate these possibilities, we explored the relationship between individual differences in strategic retrieval processes and the availability of object details in relation to older adults’ performance across test formats.
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To capture individual differences in strategic retrieval processes that are necessary to support a recall-to-reject strategy, we included measures of executive function and recall ability, which place common demands on cognitive control processes such as selection, inhibition, and maintenance of stored details. To assess the availability of object details, we used a perceptual task that involves discriminating between objects with overlapping features, thus placing similar demands on the type of object representation needed to support the task, but eliminating any mnemonic demands. We predicted that perceptual discrimination ability would be selectively related to performance in the Forced Choice test, based on the proposal that the Forced Choice test is more sensitive to the availability of object details relative to the Yes/No test. In contrast, we predicted that performance in the Yes/No test would be related to executive function and recall ability, which are critical components of executing a recall-to-reject strategy.
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Forty-two older adults who participated in Experiments 1and 2 returned to the lab to complete a neuropsychological testing battery within 12 months of completing the initial experiment. These participants were randomly selected from the sample of older adults that completed Experiments 1 and 2, with the constraint that individuals were drawn from the full range of performance on the task. The older adults from each experiment did not differ with respect to mean age, years of education, or Shipley Vocabulary Scores (all t < 1). The groups did differ in their scores on the Montreal Cognitive Assessment, however both groups performed well within the normal range (Exp. 1: M = 28.25, SD = 1.12; Exp. 2: M = 27.23, SD = 1.69; t(40) = 2.29, p < .05, d = 0.724). The individuals from each experiment were also matched on object recognition memory performance across test formats (all p > .2) and were combined for all subsequent analyses. A summary of the demographic information can be found in Table 4.
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| 100.0 |
All participants completed a battery of neuropsychological tests assessing memory, executive function, and perception. These included the Logical Memory and Paired Associates subtests from the Wechsler Memory Scale (3rd Edition [WMS-III]; Wechsler, 1997a), the Rey-Osterrieth Complex Figure Test (Osterrieth, 1944), the Verbal Fluency and Trails A & B subtests from D-KEFS (Delis, Kaplan, & Kramer, 2001), and the Digit Span subtest from the Wechsler Adult Intelligence Scale (3rd Edition [WAIS-III]; Wechsler, 1997b). Participants additionally completed a complex visual discrimination task using stimuli developed by Barense and colleagues (2012; see also Newsome et al., 2012 and Ryan et al., 2012). In this task, participants are simultaneously presented with two novel objects and decide if they match or do not match. Critically, when these objects share multiple overlapping features (e.g., high ambiguity trials), convergent evidence from patients with PRC lesions (Barense et al., 2012) and neuroimaging studies in older (Ryan et al., 2012) and younger (Barense et al., 2012) adults indicates that successful performance relies on object-level representations supported by the PRC to resolve feature level ambiguity between exemplars.
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study
| 99.94 |
Scores on each of these subtests were normalized and the z scores averaged to create three different composite scores for each individual: a Representational Quality score, a Recall Performance score, and an Executive Function score. The Representational Quality score consisted of performance on the high ambiguity condition of the visual discrimination task. The Recall Performance score comprised immediate and delayed recall scores from the Logical Memory, Paired Associates, and Rey Complex Figure tests. The Executive Function score comprised Verbal Fluency, Trails B, and Digit Span scores. The group was median split on each composite score to divide participants into high and low scoring groups in each of the three factors. One-way between-participants analyses of variance confirmed that the high and low scoring groups differed significantly on their respective composite scores (Representational Quality Groups: F(1, 41) = 74.69 p < .001; Recall Performance Groups: F(1, 41) = 76.13, p < .001; Executive Function Groups: F(1, 41) = 49.34 p < .001). A full summary of the demographic information and composite scores for each group can be found in Table 5.
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| 100.0 |
We first sought to assess the degree to which the high or low scoring group in each of the three cognitive factors of interest differed in performance across test formats (see Figure 4). To this end, we submitted participants’ d′ scores from the Forced Choice and Yes/No test formats to three mixed ANOVAs with Test Format as a within-subjects factor and Group (high vs. low scoring) as a between-subjects factor. For those participants who completed Experiment 2, we used performance on the 180 item block for their d′ scores to ensure that performance measures were based on comparable experimental conditions across participants.
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We found that those older adults scoring higher in Recall Ability performed significantly better than those individuals in the low scoring group, F(1, 40) = 23.66, p < .001, ηp2 = 0.372, across both test formats (F < 1). This effect remained significant when Executive Function score was taken into account, F(1, 40) = 5.71, p < .05, ηp2 = 0.144. Similarly, we observed a significant difference in memory performance between the High and Low Executive Function groups, F(1, 40) = 11.07, p < .005, ηp2 = 0.217, across test formats (F < 1), and this effect remained when Recall Ability was included as a covariate, F(1, 40) = 4.35, p < .05, ηp2 = 0.100. In contrast, those older adults who scored high and low in Representational Quality did not differ with respect to overall memory performance (F < 1). Instead, we observed a significant Test Format × Group interaction, F(1, 40) = 4.42, p < .05, ηp2 = 0.100, which reflected a selective impact of Representational Quality on Forced Choice performance, t(40) = 2.07, p < .05, d = 0.638, which was not observed in Yes/No performance (t < 1). Critically, this interaction remained significant when Recall Score and Executive Function were included as covariates in the ANOVA, F(1, 40) = 4.12, p < .05, ηp2 = 0.098, indicating an impact of Representational Quality on Forced Choice performance even after the effects of recall and executive function have been accounted for.
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study
| 100.0 |
We next sought to assess whether the benefit to performance gained by the simultaneous presentation of targets and foils in the Forced Choice test was constrained by individual differences in representational quality. To explore this possibility, we computed a difference score quantifying the memory enhancement associated with the Forced Choice test compared with the Yes/No test (see also Westerberg et al., 2013). We then assessed whether the size of this mnemonic benefit varied according to group membership for the three neuropsychological factors. Consistent with predictions, this analysis revealed that those participants in the high Representational Quality group benefited significantly more from the presence of retrieval support than did the low Representational Quality group, t(40) = 2.10, p < .05, d = 0.664, whereas there was no significant difference in this benefit between high and low scoring participants in the Recall Performance or Executive Function groups (ts < 1).
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| 100.0 |
Consistent with our first prediction, older adults with higher scores in representational quality exhibited superior Forced Choice performance, and exhibited larger benefits of the simultaneous presentation of targets and foils in the Forced Choice test, relative to those individuals scoring low in this measure. Critically, this relationship was observed even after accounting for individual differences in recall ability and executive function, consistent with the proposal that the Forced Choice test format enables a direct assessment of the availability of object details. The observation that the size of the Forced Choice benefit was selectively constrained by this factor is also consistent with the proposal that this test format minimizes demands on strategic retrieval processes, resulting in the availability of object details having a larger impact on performance. In contrast, we found that representational quality did not have a direct effect on performance in the Yes/No test, suggesting that the availability of object details is not sufficient to support a recall-to-reject strategy, likely because of the additional demands this test places on the ability to strategically retrieve, maintain, and evaluate stored details.
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study
| 100.0 |
Consistent with this possibility, and our second prediction, older adults who scored higher on measures of both recall ability and executive function performed significantly better in the Yes/No test. This observation replicates previous work identifying a relationship between Yes/No recognition performance with similar foils and a measure of recall ability in older adults (Holden et al., 2013; Migo et al., 2014; Toner et al., 2009), and extends this work by identifying a similar relationship for executive function. These relationships are consistent with the demands this test format places on using a recall-to-reject strategy, which involves cognitive control processes such as selection, inhibition, and postretrieval monitoring that are captured by measures of recall and executive function. Interestingly, those older adults scoring higher on these factors also performed significantly better on the Forced Choice test than those with lower scores. Although we did not predict this relationship, it may reflect the general benefit of memory control processes, which are common to recall and executive function, on memory performance.
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study
| 99.94 |
The current investigation explored the degree to which impairments in the ability to strategically retrieve object details, relative to declines in the availability of these details, contribute to elevated rates of lure false recognition with age. To this end, we assessed older and younger adults’ recognition memory performance in both Yes/No and Forced Choice test formats, on the basis that the simultaneous presentation of targets and foils in the Forced Choice test minimizes demands on strategic retrieval processes, enabling a more direct measure of the availability of object details (Guerin et al., 2012; Migo et al., 2009; Norman, 2010). The results of Experiment 1 revealed that age-related increases in false recognition were evident across test formats, implicating reductions in the availability of object details to increased false recognition among older adults. Experiment 2 assessed whether this factor alone could be driving false recognition across test formats, but found that age differences in Yes/No performance persisted even when performance in the Forced Choice test was matched across groups. Together, these results indicate that both impairments in strategic retrieval processes and reductions in the availability of object details contribute to elevated rates of false recognition with age.
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study
| 100.0 |
The present results complement existing research exploring gist-based false recognition in younger adults by identifying evidence for a substantial contribution of retrieval failure to false recognition of similar lures (Guerin et al., 2012; Migo et al., 2009). Specifically, we found that the incidence of false recognition was considerably reduced in the Forced Choice test format relative to the Yes/No format, indicating that the object details necessary to discriminate between targets and lures are often available in memory, even when they are not retrieved successfully. Importantly, the present observation that older adults benefited to the same degree as younger adults from the simultaneous presentation of targets and foils in the Forced Choice test lends further support to the proposal that this test format improves the accessibility of stored details, and does so across the life span. This enhancement likely arises because performance in the Forced Choice test can more successfully be supported by assessing the relative familiarity between exemplars, whereas Yes/No performance relies critically on a recall-to-reject strategy (Migo et al., 2009; Norman, 2010), a possibility that is supported by the results of the modified Remember-Know judgments included in Experiment 1.
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study
| 100.0 |
Critically, the present results also extend previous work by providing evidence that increases in lure false recognition among older adults cannot be explained solely by retrieval failure, and are in part the result of declines in the availability of object details that can successfully disambiguate targets and foils with overlapping features. This observation is consistent with recent evidence for age-related decline in the ability to discriminate between objects that share overlapping features, even when demands on explicit memory encoding and retrieval are minimized (Burke et al., 2010; Lee et al., 2014; Ryan et al., 2012; Yeung et al., 2013). Together with the present results, this evidence suggests that aging is associated with decline in the quality of online stimulus representations, such that these representations are less able to disambiguate targets and foils that share overlapping features. More specifically, these data are consistent with recent proposals that aging is associated with a reduction in the availability of unique object-level representations, leading to increased reliance on representations of simple features and feature conjunctions that comprise these objects, which remain unaffected (Burke et al., 2010, 2011; 2012; Ryan et al., 2012).
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study
| 99.94 |
This proposal is based on the representational-hierarchical framework, which states that increasingly complex stimulus representations are supported along the posterior-anterior axis of the ventral visual stream, from simple features and feature conjunctions, to the level of a unique object (Bussey & Saksida, 2007; Cowell et al., 2006). According to this view, object-level representations are critical for resolving feature ambiguity between objects sharing overlapping features and are supported by the perirhinal cortex (PRC). When these representations become compromised as a result of damage or dysfunction of PRC, as may occur with increased age (see Burke et al., 2012 and Leal & Yassa, 2015 for reviews), individuals must rely on simple, feature-level representations that are less able to disambiguate targets and foils with overlapping features, resulting in increased false recognition of objects that share common lower level features. Critically, this model makes three specific predictions that are supported by the present data: (a) discrimination between targets and foils with overlapping features should be impaired, even when demands on controlled retrieval processes are minimized, (b) impaired performance arises due to increased vulnerability to feature-level interference, and can be ameliorated by reducing feature-level interference, and (c) common representations support perceptual and mnemonic discriminations of object exemplars sharing overlapping features.
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study
| 89.4 |
In support of these predictions, we identified age-related deficits in Forced Choice performance, as well as evidence that reducing the number of objects viewed by participants can enhance performance in this test format, perhaps by reducing interference from features shared across objects. Furthermore, we identified a relationship between performance in a perceptual discrimination task and Forced Choice performance. This finding provides novel evidence for an association between complex perception and memory ability among older adults, lending support to the proposal that common representations support memory and perception. Finally, although we did not obtain direct measures of PRC structure or function in the present experiment, the current results are nevertheless consistent with the possibility that performance in the perceptual discrimination task and Forced Choice test necessitate object-level representations supported by PRC. In particular, performance in the perceptual discrimination task used here has been linked to PRC recruitment among older adults using fMRI (Ryan et al., 2012), and is impaired in patients with compromised PRC integrity (Barense et al., 2012; Newsome et al., 2012). These findings raise the possibility that our measure of representational quality is sensitive to PRC function.
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study
| 100.0 |
Consistent with this possibility, the relationship between performance in the perceptual discrimination task and Forced Choice performance in the present study bears a striking resemblance to that observed previously among patients with MCI and AD using a direct measure of PRC volume. In particular, PRC volume was selectively related to performance in the Forced Choice test format, as well as the benefit to performance individuals gained in the Forced Choice test relative to the Yes/No test, but not to performance in the Yes/No test format (Westerberg et al., 2013). The correspondence between these findings and the current results is consistent with a contribution of object-level representations supported by PRC to performance in both the perceptual discrimination task and Forced Choice test used here, and lends further support to the possibility that variability in PRC function may contribute to individual differences in Forced Choice performance in the present study.
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study
| 100.0 |
Notably, although the availability of object details that can disambiguate targets and foils is a critical prerequisite for accurate memory performance across test formats, we did not identify a relationship between this factor and Yes/No performance. Instead, we found that performance in this task was related to tests measuring recall and executive function. Importantly, the tasks used in the current battery are thought to be sensitive to hippocampal and prefrontal function, respectively, and may reflect variability in the integrity of these regions in the current sample. This possibility is consistent with the role of the prefrontal cortex in selection, inhibition, maintenance and evaluation of stored details (Badre & Wagner, 2007), and the hippocampus in supporting reinstatement of previous episodes (i.e., pattern completion) and mismatch detection (i.e., pattern separation; Norman & O’Reilly, 2003), which are jointly thought to support a recall-to-reject strategy (Bowman & Dennis, 2016; Gallo, 2004). Importantly, the observation that representational quality was directly related to Forced Choice performance, but not Yes/No performance, suggests that the availability of object-level representations supported by PRC are necessary, but not sufficient, to support performance in the Yes/No test, making it difficult to detect measurable effects of representational quality on Yes/No performance.
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study
| 100.0 |
Consistent with this possibility, previous work using fMRI has found that although PRC is recruited during a Yes/No recognition task with similar lures, only hippocampal activity is related to accurate performance (Reagh & Yassa, 2014). In contrast, PRC activity has been directly related to discrimination performance in a Forced Choice test with similar lures (O’Neil et al., 2015). Similarly, among patients with MTL damage, hippocampal integrity has been related to performance in Yes/No performance, whereas PRC integrity has been associated with Forced Choice performance (Holdstock et al., 2002; Westerberg et al., 2013). Collectively, this empirical evidence indicates that partially dissociable neural mechanisms can support performance across test formats when targets and foils are perceptually similar, consistent with modeling work (Norman, 2010). These direct neural measures complement the present behavioral findings in indicating that the Forced Choice test provides a more direct measure of the quality of object representations and underlying PRC function, relative to the Yes/No test. In doing so, these data further validate the use of the Forced Choice and Yes/No test formats to tease apart contributions of the availability of object details, relative to the ability to retrieve and evaluate these details, to elevated rates of false recognition among older adults.
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study
| 99.94 |
Although we believe these two factors represent the most parsimonious explanation for the present results, we cannot rule out the potential contribution of age-related changes during encoding. Indeed, an important challenge associated with explicit memory tests is the difficulty of separating contributions of processes operating during encoding to the resulting quality of stimulus representations. For example, it is possible that older adults are more likely than younger adults to preferentially focus on semantic as compared with perceptual object information during encoding, thus reducing the availability of perceptual details at test and increasing reliance on semantic gist (Brainerd & Reyna, 2002; Koutstaal et al., 1997, 2003). Although we tried to minimize this possibility by using a perceptually oriented encoding task to encourage comparable processing of images by older and younger adults, it is difficult to completely rule out when concrete, meaningful objects are used as stimuli. Nevertheless, existing evidence argues against the idea that age differences during encoding, and in particular a tendency to predominantly focus on semantic information at the expense of perceptual detail, can account for the pattern of results presented here, as elaborated below.
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study
| 100.0 |
First, existing work has identified age-related deficits in the ability to discriminate between items with overlapping features in the context of recognition memory tests, as well as perceptual discrimination tasks, using both abstract objects (Ryan et al., 2012; Pidgeon & Morcom, 2014) and unfamiliar faces (Bartlett, Leslie, Tubbs, & Fulton, 1989; Crook & Larrabee, 1992; Lee et al., 2014; Megreya & Bindemann, 2015). Such findings suggest that age differences in target-foil discrimination are not specific to stimuli that possess semantic meaning, nor task conditions with explicit ‘encoding’ and ‘retrieval’ phases, but rather any task that involves disambiguating objects with overlapping features. Furthermore, age-related deficits in perceptual discrimination and recognition memory of complex objects that share common features have been observed in aged rats and nonhuman primates (Burke et al., 2010, 2011; see Burke et al., 2012 for review), indicating that these deficits can emerge even when semantic meaning and explicit encoding strategies are unlikely to contribute to performance. Collectively, these findings are consistent with declines in the availability of object-level representations with age, resulting in impaired discrimination of items sharing overlapping features, thus lending further support to this interpretation of the present results.
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study
| 97.44 |
In summary, the results of the current investigation provide evidence for the contribution of two factors to elevated rates of lure false recognition with age: declines in the availability of object details, and impairments in the strategic retrieval and evaluation of these details. Importantly, they also identify two ways in which false recognition can be reduced, including minimizing demands on strategic retrieval processes by increasing environmental support at test, and reducing interference from visual inputs that share common features with to-be-remembered information. The observation that age-related increases in false recognition can be minimized, or even eliminated, in this way may have implications for reducing everyday memory errors among older adults, as well as applications to legal domains such as eyewitness testimony (e.g., sequential vs. simultaneous line-ups; Mickes, Flowe, & Wixted, 2012; Wixted & Mickes, 2014). Finally, the current findings indicate that although false memory errors can increase dramatically and robustly with age, the susceptibility to lure false recognition varies substantially across older adults. Such observations highlight the importance of adopting an individual differences approach to investigations of memory decline in the elderly population.
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study
| 99.94 |
There is a consensus that post-mastectomy radiotherapy (PMRT) is indicated for the breast cancer with locally advanced disease (T3-T4), or four or more positive axillary lymph nodes (LNs) [1, 2]. Additionally, based on the results of a recent meta-analysis , PMRT can be applied to one to three positive axillary LNs.
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review
| 99.9 |
On the other hand, mastectomy is generally considered a sufficient local treatment method for node-negative breast cancer. Nevertheless, a high rate of local recurrence has been reported in a subset of patients with aggressive clinicopathologic factors such as young age, large tumor size, high tumor grade, lymphovascular invasion (LVI), or positive/close resection margin . As the understanding of the molecular biology of breast cancer improves, the biological subtype is receiving attention as a possible prognostic factor with which to distinguish patients with a high versus low risk of local recurrence [5–7]. In the setting of mastectomy, however, the effects of the biological subtype on local recurrence are not consistent [5, 8–10].
|
review
| 99.9 |
The median follow-up period was 5.9 years (range: 0.7-10.4 years). Table 1 summarizes the patient and tumor characteristics. Adjuvant systemic treatment was delivered at the physician's discretion. A total of 182 patients (10.0%) received no systemic treatment. A total of 966 patients (52.8%) received adjuvant chemotherapy; 772 received an anthracycline-based regimen; 126 received a combination regimen comprising cyclophosphamide, methotrexate, and 5-fluorouracil; and only 31 received a taxane-containing regimen. A total of 1,260 patients (68.9%) received endocrine treatment. Among 467 HER2+ patients, only 107 (22.9%) were confirmed to have been treated with trastuzumab (not done, n = 292; unknown, n = 68). Among 255 triple negative (TN) patients, 193 (75.7%) were treated with cytotoxic chemotherapy (not done, n = 62).
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study
| 99.75 |
Of all 1,828 patients, 52 developed LRR. There were 31 (59.6%) ipsilateral chest wall recurrences, 28 (53.8%) ipsilateral LN recurrences (axillary, 18; internal mammary, 7; supraclavicular, 8; site unspecified, 2), and 7 (13.4%) ipsilateral chest wall and nodal recurrences. The cumulative rates for LRR at 5, 7, and 10 years were 2.8%, 3.8%, and 3.8%, respectively.
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study
| 99.94 |
On univariate analysis, an age of ≤ 40 years (p < 0.001) and T2 stage (p = 0.006) were significantly associated with a high risk of LRR, and the biological subtype was marginally associated with high LRR. With respect to the biologic subtypes, the 7-year cumulative incidence of LRR was 2.2% for luminal A, 7.0% for luminal B, 5.1% for luminal HER2, 4.4% for HER2+, and 5.1% for TN (p = 0.095). The use of trastuzumab in patients with HER2+ did not affect LRR (Table 2). On multivariate analysis (Table 3), an age of ≤ 40 years (HR, 3.3; p < 0.001) and T2 stage (HR, 1.3; p = 0.013) were independently associated with a high risk of LRR.
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study
| 100.0 |
Sixty-one patients developed distant metastasis (DM). Thirteen patients developed DM with synchronous LRR, and two patients developed DM 6 and 11 months after LRR. Forty-six patients had isolated DM. The cumulative rates for DM at 5, 7, and 10 years were 3.2%, 4.1%, and 5.3%, respectively.
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study
| 86.5 |
In total, 98 cases developed AFR; 13 LRR with synchronous DM, 39 LRR without DM, and 46 DM only. Additional two DM was reported after the first recurrence, and the total cases of DM were 61. The cumulative rates for AFR at 5, 7 and 10 years were 5.3%, 6.7% and 7.9%, respectively. On univariate analysis (Table 2), an age of ≤ 40 years (p < 0.001), T2 stage (p < 0.001), a high tumor grade (p = 0.011), and biological subtype (p < 0.001) were significantly associated with a high risk of AFR. With respect to the biologic subtype, the 7-year cumulative incidence of AFR was 4.4% for luminal A, 13.9% for luminal B, 8.2% for luminal HER2, 7.2% for HER2+, and 9.1% for TN (p < 0.001). The use of trastuzumab in patients with HER2 and luminal HER2 subtypes was not associated with AFR (Table 2). To facilitate comparison between the groups in the multivariate analysis, these five subtypes were redefined as binary variables based on the results of the univariate analysis: TN tumors and others (p = 0.006). The use of chemotherapy was significantly associated with a high risk of AFR (p = 0.017), and this was attributed to the development of DM as a first recurrence. On multivariate analysis (Table 3), an age of ≤ 40 years (HR, 2.6; p < 0.001), T2 stage (HR, 1.3; p < 0.001), and the TN biological subtype (HR, 1.6; p = 0.045) were independently associated with a high risk of AFR.
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study
| 99.94 |
For LRR, patient age ( ≤ 40 vs. > 40 years) and tumor size (T1 vs. T2) were used to stratify the risk groups. Of all 1,828 patients, 974 (53.3%) had no risk factors, 766 (41.9%) had one risk factor, and 88 (4.8%) had two risk factors. The 5-year cumulative LRR rates were 1.6% with no risk factors, 3.2% with one risk factor, and 12.4% with two to three risk factors. The latter was defined as the high-risk group for LRR. The 7-year and 10-year cumulative LRR rates were 2.5% with no risk factors, 4.5% with one risk factor, and 12.4% with two risk factors (Figure 1).
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study
| 99.94 |
For ARF, three risk factors were used to stratify the risk groups: patient age, tumor size, and biological subtype. Of 1,656 analyzable patients (172, unknown), 748 (45.2%) had no risk factors and 694 (41.9%), 194 (11.7%), and 20 (1.2%) patents had one, two, and three risk factors, respectively. Because of the small number of patients with three risk factors, patients with two to three risk factors were assigned to the high-risk group (214, 12.9%). The 5-year cumulative ARF rates were 2.8% with no risk factors, 5.9% with one risk factor, and 14.7% with two to three risk factors. The 7-year cumulative ARF rates were 3.9% with no risk factors, 8.4% with one risk factor, and 15.7% with two to three risk factors (Figure 2). The 10-year cumulative ARF rates were 3.9% with no risk factors, 10.6% with one risk factor, and 18.1% with two to three risk factors.
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study
| 100.0 |
The present study showed that the 7-year overall incidence of LRR was 3.8% in patients with pT1-2N0 breast cancer treated with mastectomy but not PMRT. Additionally, the 7-year LRR rate of patients aged less than or equal to 40 years with T2 tumors, who were considered at high risk, was 12.4%. Since no LRR was reported after 7 years, the LRR rate at 7-year and 10-year were identical (Figure 1). These results are in agreement with those of recent studies (Table 4).
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study
| 100.0 |
The indications for PMRT have traditionally been based on the risk of LRR. Although numerous studies have reported the prognostic value of clinicopathologic factors predicting LRR, such as patient age, menopausal status, tumor size, tumor grade, LVI, or resection margin status, variation exists in terms of the significance or magnitude of those factors in the setting of mastectomy without PMRT for pT1-2N0. Among them, young age and large tumor size, which were proven as significant prognostic factors in our analysis, are widely accepted variables associated with a high rate of LRR. With respect to patient age, Sharma et al. , Karlsson et al. , and Yildirim et al. identified an age of ≤ 40 years as an independent predictor of high LRR, and Abi-Raad et al. and Wallgren et al. reported ages of ≤ 50 and ≤ 60 years as cutoffs, respectively. Similarly, Jagsi et al. showed that a premenopausal status was an independent predictor of LRR. Regarding tumor size, T2 tumors (≥ 2 cm) were the most commonly reported cutoff point predicting a high risk of LRR [14–16].
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study
| 99.6 |
Although the EBCTCG adopted AFR (irrespective of LRR or DM) as the primary endpoint for the effect of RT in patients with breast cancer , there are little data regarding AFR in mastectomy without PMRT among patients with pT1-2N0 cancer. We performed an analysis using AFR as another primary endpoint and stratified the risk groups based on three factors proven to be significant in the multivariate analysis: age of ≤ 40 years, T2 tumors, and TN subtype. The 10-year AFR rate of the high-risk groups with two to three adverse factors was 18.1%. There are scarce data with which to directly compare our findings.
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study
| 100.0 |
Notably, the EBCTCG meta-analysis reported that LRR and AFR occurred more frequently in the no-RT group treated with axillary sampling than in the no-RT group treated with axillary dissection; however, the difference disappeared when PMRT was added, irrespective of axillary management. In contrast, we found no difference in the LRR or AFR rate according to axillary management. Moreover, the 10-year LRR and AFR rates were far superior to the outcomes of the EBCTCG in node-negative patients (the 10-year AFR and LRR rates were 7.9% and 3.8%, respectively). Even for the high-risk group defined in our study, the 10-year AFR and LRR rates were 18.1% and 12.4%, respectively. Considering the period of patient enrollment (1964-1986 in the EBCTCG meta-analysis vs. 2005-2011 in our study), it is reasonable that this reduction in recurrence is attributable to improved nodal examination, such as sentinel LN detection or pathologic evaluation, and progress in systemic treatments such as HER2 targeted therapy. Importantly, routine use of systemic treatment, which was given to 90% of our study cohort, could also explain these superior outcomes, in that recent large clinical trials emphasized the impact of systemic treatment in terms of not only DM and survival, but also local control [18–20].
|
study
| 99.94 |
We defined the high-risk group as described above and found a high rate of recurrence in that subset. The remaining question is how many recurrences justify the recommendation for PMRT. Olivotto and Truong suggested that PMRT is indicated when LRR exceeds 25%, but not when it is < 10%, based on the magnitude of absolute LRR reduction and the absolute survival benefit (4:1 ratio according to the EBCTCG) . If the LRR rate is 12.8% (the 10-year rate in our high-risk group), patients’ priorities and preferences should be considered when making decisions regarding PMRT. The EBCTCG suggested a new ratio: for every 1.5 patients in whom AFR is avoided at 10 years, there is an additional survivor at 20 years . This works well for pT1-2N1 patients with mastectomy, but not for pT1-2N0 patients, as previously described . Despite the reduction in AFR by PMRT (34.2% in no-RT vs. 22.1% in RT at 10 years, 2p = 0.0003) in patients who underwent mastectomy and only axillary sampling, breast cancer mortality did not differ (35.8% in no-RT vs. 32.0% in RT at 20 years, 2p > 0.1). Even if PMRT reduced the AFR rate of 18.1% (the 10-year rate in our high-risk group) to a certain degree, it is reasonable that the absolute survival benefit would be small. This small survival benefit cannot be considered as grounds for routine use of PMRT for all high-risk patients with pT1-2N0 cancer. However, considering that most patients in our high-risk group were aged ≤ 40 years, even a small survival benefit may be important. Furthermore, the reduction in LRR or AFR would enable patients to work and live without a disease burden.
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study
| 99.9 |
It is important to recognize the limitations of this study, including those inherent to retrospective studies. First, the follow-up period was not long enough to show all recurrences considering the long natural history of breast cancer. This might weaken the study's statistical power. Second, our study could not directly address the survival benefits in association with reductions in the LRR or AFR by PMRT, because our data regarding patient death included all-cause mortality rather than breast cancer mortality. Despite these limitations, our study is clinically valuable. Although breast-conserving treatment has been generalized for node-negative breast cancer over the past several years, some patients still undergo mastectomy for various reasons, such as the presence of multifocal breast cancer or the patient's preference for breast reconstruction after mastectomy. However, most such reference studies were performed from the 1960s to the 1990s. The present work was a multi-institutional study performed in the 2000s and included a large number of patients with pT1-2N0 breast cancer (n = 1,828) treated with mastectomy without PMRT. The conclusions that can be drawn from our analyses are more relevant to contemporary practice, in that we adopted current diagnostic and therapeutic strategies.
|
study
| 99.94 |
This analysis was performed only in the population who did not receive radiation after mastectomy, prospective study to compare those who received PMRT to those who did not is being planned. Furthermore, the study regarding hypofractionated RT after mastectomy could be considered. Some investigators have suggested that hypofractionated RT is also an alternative option for PMRT , although it has not been thoroughly studied as in post-breast conserving surgery RT [23–25].
|
study
| 99.56 |
In conclusion, our study found that the overall recurrence and LRR rates were substantially low in patients with pT1-2N0 breast cancer treated with mastectomy and systemic therapy without PMRT. This finding shows that mastectomy without PMRT is a sufficient local treatment for pT1-2N0M0 breast cancer. However, there was a patient group at high risk for recurrence: the 7-year LRR rate in patients aged ≤ 40 years with T2 tumors was 12.4%, and the 7-year AFR rate of patients with two to three adverse factors, among those aged ≤ 40 years, T2 tumors and the TN subtype, was 15.7%. PMRT might be considered for these high-risk patients.
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study
| 99.94 |
This multi-institutional retrospective study was approved by the Korean Radiation Oncology Group (KROG 14-22) and the Institutional Review Board of each of 10 participating institutions in Korea. After obtaining this approval, we reviewed the medical records of patients with breast cancer treated by mastectomy from 2005 to 2010. The eligibility criteria were (1) a tumor size of ≤ 5 cm (pT1 and pT2), (2) negative LNs (pN0) proven by axillary dissection or sentinel LN biopsy, and (3) no treatment with adjuvant PMRT. The exclusion criteria were (1) male gender, (2) the presence of distant metastasis (DM) at diagnosis, (3) neoadjuvant systemic treatment, (4) a history of radiotherapy (RT) to the chest or neck, (5) a history of malignancies other than papillary/follicular thyroid cancer, and (6) bilateral breast cancer. We identified 1,842 patients according to these eligibility criteria. We then excluded 14 patients who were lost to follow-up < 6 months from the mastectomy date. Finally, 1,828 patients with breast cancer were included in this study.
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study
| 99.94 |
The collected clinicopathological information was presented in Table 1. Positivity of ER, PR, HER2, and Ki-67 was determined by immunohistochemical staining. HER2-positivity was defined as a 3+ immunohistochemical result or a 2+ immunohistochemical result, confirmed by fluorescence in situ hybridization. Using the histologic grade as a surrogate for Ki-67 based on the St. Gallen Expert Consensus , we approximated five breast cancer subtypes based on hormone receptor (ER and PR) status, HER2 status, and histologic grade: luminal A (ER+ or PR+/HER2−/low-intermediate grade), luminal B (ER+ or PR+/HER2−/high grade), HER2+ (ER−/PR−/HER2+), luminal HER2 (ER+ or PR+/HER2+), and triple-negative (TN) (ER−/PR−/HER2−).
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study
| 100.0 |
The primary endpoints were AFR and LRR. AFR was defined as the first tumor recurrence, irrespective of LRR or DM. LRR was defined as any LRR as a first event with or without synchronous DM. Diagnosis of DM within 3 months of an LRR was considered synchronous. LRR occurring after DM was not considered as an LRR event. LRR indicated tumor recurrence in the ipsilateral chest wall; ipsilateral axillary, infraclavicular, internal mammary, or supraclavicular node recurrence; or a combination of these. DM indicated tumor recurrence outside regions identified as LRR sites. The secondary endpoints were DM and overall mortality. The information on date of death was taken from Korea's national database, in which death by breast cancer is not distinguished from death by other causes. Time to any recurrence or death was measured from the date of mastectomy.
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study
| 99.94 |
Cumulative incidence function curves for AFR, LRR, DM, and overall mortality were constructed using the Kaplan-Meier method, and comparisons between groups were performed using log-rank tests. A Cox proportional hazards model was used to estimate hazard ratios (HRs), and to identify correlations between outcomes and risk variables. All statistical analyses were carried out with SPSS version 18.0 (SPSS Inc., Chicago, IL, USA). P values < 0.05 were considered statistically significant.
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study
| 100.0 |
Central serotonin (5-HT) systems have a critical role in the regulation of appetite and body weight. Central serotonin 5-HT2C receptors contribute to the leptin-independent regulation of appetite . Liraglutide, a glucagon-like peptide-1 (GLP-1) receptor agonist used clinically to treat type 2 diabetes and/or obesity, induces feeding suppression in mice 1 h after administration . We previously reported that the acute anorexic effects of liraglutide in mice do not require central 5-HT and functional leptin receptor signaling . A recent report by Anderberg et al., however, suggested that brain-derived 5-HT is critical for maintaining weight loss induced by GLP-1 receptor activation and pharmacologic blockade of central serotonin 5-HT2A receptors using R-96544 attenuates the chronic anorexic and weight loss effects of central injection of exedin-4 (EX4) or intraperitoneal injection of liraglutide in rats .
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study
| 99.94 |
Therefore, in the present study, to determine whether 5-HT is critical for the weight loss induced by GLP-1 receptor activation in mice, we examined the effects of liraglutide on daily food intake and body weight over 4 days in mice treated with or without the tryptophan hydroxylase inhibitor p-chlorophenylalanine (PCPA) for 3 days. In addition, to evaluate the relationship between the acute anorexic effects of the GLP-1 receptor agonist and the expression of hypothalamic 5-HT2A receptors, we examined the effect of liraglutide on the expression of hypothalamic 5-HT2A and 5-HT2C receptors in mice, which are responsive to the administration of liraglutide. We further assessed whether 5-HT2A receptors are involved in the acute anorexic effects of liraglutide by examining the acute effects of liraglutide on food intake in mice pretreated with the high-affinity 5-HT2A agonist (4-bromo-3,6-dimethoxybenzocyclobuten-1-yl) methylamine hydrobromide (TCB-2).
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study
| 100.0 |
In exp 1, 6-week-old C57BL6J mice were intraperitoneally injected with 1% Tween saline or PCPA (500 mg/kg) once a day over 3 days as described previously . Daily body weight changes were determined. In the fourth day, the animals were decapitated and the hypothalamus was removed for RNA extraction, as described previously [2, 4].
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study
| 100.0 |
In exp 2, 6-week-old C57BL6J mice were intraperitoneally injected with 1% Tween saline or PCPA (500 mg/kg) once a day over 3 days. Then, mice were intraperitoneally injected with saline or liraglutide (100 μg/kg) in the light cycle once a day over 4 days. Daily food intake and body weight changes were determined.
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study
| 100.0 |
In exp 3, 6-week-old C57BL6J mice were intraperitoneally injected with saline or liraglutide (100 μg/kg) in the light cycle. One hour later, the animals were decapitated; animals were not fed. The hypothalamus was removed for RNA extraction, as described previously [2, 4].
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study
| 99.94 |
In exp 4, 6-week-old C57BL6J mice were intraperitoneally injected with saline or the high-affinity 5-HT2A agonist, 4-Bromo-3,6-dimethoxybenzocyclobuten-1-yl methylamine hydrobromide (TCB-2) (2.5 mg/kg) in the light cycle. 14 h later and 24 h later, mice were intraperitoneally injected with saline or liraglutide (100 μg/kg) in the light cycle, respectively. Chow pellets were provided 30 min later. The intake of chow pellets was measured for the next 1 h and then 2 h.
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study
| 100.0 |
The dose of PCPA (500 mg/kg) was selected based on evidence that PCPA remarkably decreases brain and serum 5-HT levels in mice [5, 6]. The dose of liraglutide (100 μg/kg) was selected based on evidence that liraglutide acutely induced hypophagia in mice [2, 7]. The dose of TCB-2 (2.5 mg/kg) was selected based on evidence that TCB-2 had no significant effect on food intake .
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study
| 100.0 |
Liraglutide was dissolved in 0.2 ml 0.9% saline. The PCPA was suspended in 0.2 ml 1% Tween saline. The experiment was performed between 9 : 00 and 12 : 00. The animal studies were conducted in accordance with the institutional guidelines for animal experiments at the Tohoku University Graduate School of Medicine.
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other
| 99.9 |
Total RNA was isolated from mouse hypothalamus using the RNeasy Midi kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. cDNA synthesis was performed using a Super Script III First-Strand Synthesis System for RT-PCR Kit (Invitrogen, Rockville, MD) with 1 μg total RNA. cDNA synthesized from total RNA was evaluated in a real-time PCR quantitative system (LightCycler Nano Instrument Roche Diagnostics, Mannheim, Germany). The primers used were listed in Table 1.
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study
| 99.94 |
Data are presented as mean ± SEM (n = 6). Comparisons between the two groups were performed using Student's t-test. Comparisons among more than two groups were performed using analysis of variance with Bonferroni's correction for multiple comparisons. A P value of less than 0.05 was considered statistically significant.
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study
| 99.94 |
Treatment with PCPA (500 mg/kg) for 3 days significantly decreased body weight gain (Figure 1(a)) compared with controls. Treatment with PCPA for 3 days remarkably decreased expression of hypothalamic Tph2 compared with controls (25%) but did not affect the expression of hypothalamic 5-HT2A receptors in mice (Figure 1(b)). These findings demonstrate that despite the inhibition of Tph2, the treatment with PCPA does not affect expression of hypothalamic 5-HT2A receptors in mice.
|
study
| 100.0 |
Changes in daily food intake (Figure 2(a)) and body weight (Figure 2(b)) induced by intraperitoneal administration of saline over 4 days did not differ between mice treated with the PCPA (500 mg/kg) for 3 days and mice without PCPA treatment. Intraperitoneal administration of liraglutide (100 μg/kg) in mice significantly decreased food intake on the first day of treatment and decreased body weight over 4 days compared with saline controls in mice treated with or without PCPA for 3 days. Changes in daily food intake (Figure 2(c)) and body weight (Figure 2(d)) induced by intraperitoneal administration of liraglutide (100 μg/kg) over 4 days did not differ between mice treated with the PCPA (500 mg/kg) for 3 days and mice without PCPA treatment. These findings demonstrate that 5-HT is not required for feeding suppression and body weight loss induced by liraglutide in mice.
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study
| 100.0 |
Intraperitoneal injection of liraglutide (100 μg/kg) significantly decreased the expression of hypothalamic 5-HT2A receptors compared with saline controls but did not affect the expression of hypothalamic 5-HT2C receptors 1 h after injection (Figure 3). These findings demonstrate that GLP-1 receptors downregulate expression of hypothalamic 5-HT2A receptors in mice.
|
study
| 100.0 |
Although intraperitoneal injection of liraglutide (100 μg/kg) significantly suppressed food intake compared with saline controls for 2 h after injection, the acute anorexic effects of liraglutide were blunted in mice, which were pretreated with TCB-2 (Figures 4(a) and 4(b)). These findings demonstrate that pretreatment with a 5-HT2A agonist partially reverses the acute anorexic effect of liraglutide in mice.
|
study
| 100.0 |
The present study demonstrated that PCPA treatment (500 mg/kg) for 3 days induces body weight loss in mice. These findings support a previous report by other investigators that treatment with PCPA (300 mg/kg) suppresses weight gain by increasing energy expenditure .
|
study
| 100.0 |
Although Tph1-deficient mice fed a normal diet have no significant effect on body weight, Tph1-deficient mice fed a high-fat diet are protected from obesity and insulin resistance by promoting brown adipose tissue-mediated thermogenesis . On the other hand, Tph2-deficient mice fed a normal diet decrease food intake and body weight . Because PCPA inhibits Tph1 and Tph2, both mechanisms could contribute to weight loss.
|
study
| 100.0 |
Although liraglutide reportedly increases brain 5-HT levels in rats and both liraglutide and serotonergic receptor agonists, like lorcaserin, reduced body weight and food intake in rats, the increase in brain 5-HT levels does not always induce reductions of food intake and body weight, especially in mice. Despite increased brain 5-HT levels, ob/ob mice display hyperphagia and obesity . The genetic inhibition of 5-HT synthesis in the brainstem decreases food intake and body weight in ob/ob mice and the wild-type mice . In addition, the pharmacologic inhibition of 5-HT synthesis in the brainstem induced by treatment with a high dose PCPA (500 mg/kg) or trans-2 PCPA decreases body weight and food intake in C57BL6J mice, db/db mice , and high-fat diet-induced obesity . Thus, the decrease in central 5-HT leads to the decrease in body weight and food intake in mice.
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study
| 99.94 |
We previously reported that the pharmacologic inhibition of 5-HT synthesis does not affect the acute anorexic effects of liraglutide in mice . The results of the present study further demonstrated that the pharmacologic inhibition of 5-HT synthesis did not affect the feeding suppression induced by liraglutide for 24 h after injection in mice. Moreover, the inhibition of 5-HT synthesis did not affect both the acute weight loss and maintenance of weight loss induced by liraglutide in mice. Thus, the feeding suppression and weight loss induced by liraglutide could be independent of 5-HT synthesis in mice.
|
study
| 100.0 |
In addition, we demonstrated that liraglutide acutely decreased the gene expression of hypothalamic 5-HT2A receptors in mice, which are responsive to the acute anorexic effects of liraglutide. Despite the chemical inhibition of 5-HT synthesis, the gene expression of hypothalamic 5-HT2A receptors was not changed. Thus, changes in brain 5-HT synthesis and the gene expression of hypothalamic 5-HT2A receptors could be independent, and the decrease in hypothalamic 5-HT2A receptor gene expression induced by liraglutide is unlikely to be compensatory to an increased brain 5-HT levels, although protein levels of the receptor were not measured here and could represent another regulatory mechanism.
|
study
| 100.0 |
Moreover, pretreatment with a 5-HT2A agonist reduced the acute anorexic effects of liraglutide. We previously reported that intraperitoneal injection of sarpogrelate hydrochloride, a blood-brain barrier-penetrating selective 5-HT2A receptor antagonist, acutely suppresses food intake and chronic administration of sarpogrelate hydrochloride decreases daily food intake and body weight in mice . These results suggest that GLP-1 receptors downregulate the expression of 5-HT2A receptors in the hypothalamus and decreased hypothalamic 5-HT2A receptor signaling might be involved in the acute anorexic effects of GLP-1 receptor agonists independently of brain 5-HT.
|
study
| 100.0 |
We cannot rule out that pretreatment with a 5-HT2A agonist might temporarily reduce the downstream signal of a 5-HT2A pathway; thus, the particular timing employed here has a potential to work as an antagonizing treatment to the 5-HT2A signaling pathway. Because the pretreatment might reduce the amount of functional 5-HT2A receptors at the downstream signaling, liraglutide might not be able to induce a full anorexic effect. In addition, the similar effects of 5-HT2A receptor agonist and antagonist on food intake [14–16] might be due to the paradoxical phenomenon that both agonism and antagonism of 5-HT2A receptors induce 5-HT2A receptor's desensitization or downregulation .
|
study
| 100.0 |
These results are in complete contrast to recently reported findings by Anderberg et al. that brainstem-derived 5-HT is critical for maintaining the weight loss induced by GLP-1 receptor activation and that central blockade of 5-HT2A receptors attenuates the weight loss and anorexic effects of GLP-1 receptor agonists in rats . Although the dose of PCPA (500 mg/kg) that we used was higher than that (100 mg/kg) used in their study, the PCPA treatment methods were the same between their study and ours . The different results might be due to species-specific differences between rats and mice or the paradoxical phenomenon of 5-HT2A receptor agonists and antagonists .
|
study
| 99.94 |
Interestingly, effect of liraglutide on food intake in mice is only eliciting an anorexic effect on the first day of treatment. This is not the case for rats, which show a multiday anorexic response that is the primary driver of the weight loss . Despite the anorexic effect of liraglutide disappearing after the first day of treatment in mice, the body weight loss induced by liraglutide persisted for 4 days. These findings suggest that increased energy expenditure is involved in maintaining weight loss induced by liraglutide in mice. Liraglutide reportedly stimulates the central nervous system-mediated brown adipose tissue thermogenesis and adipocyte browning independent of food intake in mice . Because this profile and potential mechanisms of weight loss induced by liraglutide are different between rats in the Anderberg study and mice in the present study, it is not at all surprising that different brain factors are engaged by liraglutide in these two species. In addition, the effect of 5-HT2A agonist on feeding seems to be opposite between mice and rats, at least by comparing the studies. Moreover, our study demonstrated that liraglutide acutely decreased the expression of hypothalamic 5-HT2A receptors and food intake in mice.
|
study
| 99.94 |
These findings suggest that liraglutide reduces appetite and body weight independently of 5-HT synthesis and that GLP-1 receptors downregulate the expression of hypothalamic 5-HT2A receptors in mice. Pretreatment with a 5-HT2A agonist might suppress the acute anorexic effects of liraglutide in mice.
|
study
| 100.0 |
The importance of catalysts with high catalytic activity in achieving “green” or sustainable chemistry has been well documented1. The benefit of high catalytic activity is not limited to reducing the amount of catalyst used. Catalysts with superior activity have the potential to promote unprecedented chemical transformations. As shown in Fig. 1, in the reaction of a nucleophile (Nu) with an electrophile (E), conventional catalysts are used for the synthesis of the 1:1 coupling adduct Nu-E (eq. 1).Figure 1Reaction of a nucleophile (Nu) with an electrophile (E).
|
study
| 99.94 |
If the first coupling product (Nu − E) can be made to react subsequently with further electrophiles through catalysis, 1:2 and/or 1:3 adducts (i.e. Nu-E2, Nu-E3) can be obtained. Although reactions of this type can be seen as over-reactions (eq. 2), such multicomponent coupling reactions are fascinating due to their potential as a direct approach toward highly functionalized advanced materials. List et al. reported an outstanding example: the condensation of acetaldehyde with two molecules of N-Boc imine using 20 mol% proline as a catalyst, which was originally developed for a single condensation reaction2. In order to utilize the double condensation reaction as a rational asymmetric catalytic reaction, new concepts for the design of highly active catalysts are required, and the first coupling product must be smoothly introduced to the second coupling reaction while avoiding the reverse reaction. Here, we report the design and development of a dinuclear palladium catalyst that enables a novel double Mannich reaction.
|
study
| 99.94 |
In this study, malononitrile was selected as a nucleophile with two acidic protons to be directed toward a double Mannich reaction3–6. In the interactions of nitriles with late transition metal salts, malononitrile can bind to two metal atoms7. For example, for the 5th-period elements, the two metal centers should be around 7.5 Å from each side of the malononitrile unit (Fig. 2A). In order to achieve a dinuclear reaction in one asymmetric reaction sphere while retaining the same geometry, a novel phosphoiminoBINOL ligand was designed, as shown in Fig. 2B. The phosphoimino moiety is designed to capture soft metals such as Pd, Rh, Au, Ag, and Cu, and the soft dinuclear complex binds strongly to malononitrile (or an anion of malononitrile generated during the reaction). The phenol functions in the ligand contribute by stabilizing the intermediate through hydrogen bonding. If the phenols incorporate a hard metal such as Li, Mg, or Zn, the resulting metal phenoxide acts as a Brønsted base, enhancing the catalytic activity. The hard Lewis acidity of the metal phenoxide is also characteristic.Figure 2Design of Malononitrile Container. (A) Geometry of malononitrile bound to two metals, (B) Design of the multifunctional dinuclear phosphoiminoBINOL-metal complex for binding malononitrile. (C) Preparation of phosphoiminoBINOL ligands.
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study
| 100.0 |
The synthesis of phosphoiminoBINOLs L1-L4 can be readily achieved through imine formation between 3,3′-formyl BINOL and the corresponding aminophosphine (Fig. 2C). The isopropyl-substituted phosphoiminoBINOL (L1) was stable to handling under air, and showed a high affinity with various soft metal salts. When 2 mol equiv. of Pd(OAc)2 was added to L1, the formation of a dinuclear palladium complex took place, as demonstrated by ESI-MS: an ion peak was detected at m/z = 1117.1705 in CH2Cl2, and this was attributed to [L1(−2H) + Pd2(OAc)]+. Fine crystals were obtained from a CH2Cl2 solution, and the structure of phosphoiminobinaphthoxy (L1)-Pd2(OAc)2 was determined by X-ray crystallographic analysis (Fig. 3).Figure 3X-ray structure of L1-Pd2 (CCDC 1543349).
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study
| 100.0 |
Based on this structure, one of the acetoxy anions of Pd(OAc)2 was replaced with L1 to make a palladium acetoxy phenoxide. The distance between two palladium cations is 6.907 Å, which is suitable for binding malononitrile (or a generated anion of malononitrile), and there is one H2O molecule positioned at the center of the asymmetric sphere. L1-Pd2 reacted smoothly with malononitrile. The 1H NMR spectrum of free malononitrile in CDCl3 shows a single methylene peak at 3.60 ppm. When malononitrile is added to 1 equiv. of L1-Pd2, the peak was shifted to 2.43 ppm 1H NMR (see details in Supplementary Information). ESI-MS analysis of a 1:1 mixture of malononitrile with L1-Pd2 showed a clear new peak at m/z = 1123.1764, attributed to [L1(−2H) + Pd2 + NCCHCN]+.
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study
| 100.0 |
With this fascinating container for malononitrile (L1-Pd2) in hand, the optimum catalyst for the conventional single Mannich reaction was examined before we attempted the challenge of the double Mannich reaction (Table 1)8–23.Table 1Development of dinuclear phosphoiminoBINOL-metal catalyst for Mannich reaction using malononitrile. EntryLigandAdditiveYield (%)a2a/3aee of 2a (%)1 L1 —9983/1782 L1 LiOAc8684/16103 L1 NaOAc9972/28264 L1 Mg(OAc)29157/43745 L1 Ca(OAc)29474/26306 L1 Zn(OAc)29360/40687 L1 ZnCl28892/878 L1 Zn(OTf)26693/7189 L2 Zn(OAc)24696/41110 L3 Zn(OAc)27285/153711 L4 Zn(OAc)27868/327712 L5 Zn(OAc)27378/22613b) L6 Zn(OAc)27590/101014b) L7 Zn(OAc)26592/8rac15c) L1 Zn(OAc)27073/2791a)Conversion yield (see details for the determination in SI). b)10 mol % of Pd(OAc)2 were used. c)5 mol % of PhosphoiminoBINOL, 10 mol % of Pd(OAc)2, and 5 mol % of Zn(OAc)2 were used. Imine 1a was slowly added over 4 h.
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study
| 100.0 |
Although the Mannich reaction was smoothly catalyzed by the simple use of L1-Pd2, the Mannich product 2a was obtained with only 8% ee. As we expected cooperative effects due to the phenoxy unit of L1-Pd2 (Fig. 2B), the addition of several hard metal salts was examined (Entries 2–8). Several metal acetates were effective in improving asymmetric induction. With assistance from Zn(OAc)2 or Mg(OAc)2, L1-Pd2 smoothly catalyzed the Mannich reaction to give 2a with 68% ee and 74% ee, respectively. The structure-activity relationship of the ligands is also shown in Table 1. The equivalent catalyst using the methyl analog of L1 ((L2)-Pd2) gave 2a with only 11% ee (entry 9). Although the t-Butyl analog L4-Pd2 gave 2a with 77% ee, the catalyst activity was reduced (entry 11). The diastereomeric ligand L5 synthesized from (S)-BINOL, and L6 and L7 for constructing mono-nuclear palladium complex resulted in low levels of asymmetric induction. For L1-Pd2 with a Zn(OAc)2 catalyst system, when the N-Boc imine 1a was added slowly, the Mannich adduct 2a was obtained with 91% ee in 5 mol % catalyst use (entry 15).
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study
| 100.0 |
The results of the L1-Pd2-catalyzed asymmetric Mannich reaction under the optimized conditions are summarized in Fig. 4. Aromatic imines with various substituents were smoothly converted to the Mannich products with high enantioselectivity.Figure 4Dinuclear phosphoiminoBINOL-Pd catalyzed asymmetric Mannich reaction using malononitrile.
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study
| 100.0 |
Using monobenzylated malononitrile, the chiral amine 2 h having adjacent quaternary carbon center was obtained in 97% yield with 93% ee. This suggests the strong catalyst activity of the dinuclear L1-Pd2 complex. We assumed that the highly active dinuclear L1-Pd2 catalyst would facilitate the conversion of the Mannich adducts (2a–d) to the second Mannich reaction for giving the 1,3-diamines 3. Actually, the use of L1-Pd2 with a Zn(OAc)2 catalyst system resulted in the production of a significant amount of the 1:2 adduct 3, as desired. For the synthesis of 2f and 2g, co-production of the same amount of diamines was observed even when 1.5 equiv. of malononitrile was applied to the imine substrate24,25. For the double Mannich reaction, the reaction conditions were modified so that 2.5 equiv. of N-Boc imine were used with respect to the malononitrile. L1-Pd2 with Zn(OAc)2 catalyzed the double Mannich reaction quite smoothly to give the 1,3-diamine 3a in quantitative yield with high diastereoselectivity (dl/meso = 93/7)26–43. The major dl-isomer was obtained in 99% ee. The results of 1,3-diamine synthesis by the double Mannich reaction are summarized in Fig. 5.Figure 5Catalytic asymmetric double Mannich reaction.
|
study
| 100.0 |
For N-Boc-imines derived from both electron-donating and electron-deficient benzaldehydes, chiral 1,3-diamines were obtained in a highly enantioselective manner. A chiral bisfuryl-1,3-diamine(3i) was obtained with 86% ee, and a bisnaphthyl-1,3-diamine(3j) was also obtained successfully with 91% ee. When the second Mannich reaction was examined using rac-2a with N-Boc-imine, 3a was obtained in 73% ee with increasing co-production of the meso-form in dl/meso = 1:1. This result suggests that the second Mannich reaction is catalyzed independently from the first Mannich reaction, and the enantiomeric excess of 3a is improved due to the meso-trick44.
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study
| 100.0 |
The dinuclear L1-Pd2 complex binds to malononitrile at both nitrogen atoms of the nitrile moieties. Although the formation of palladium enolate by simple mixing of malononitrile with L1-Pd2 is suggested by the detection of an ion peak for [L1(−2H) + Pd2 + NCCHCN]+ at m/z = 1123.1764, the addition of Zn(OAc)2 also assists the smooth formation of the palladium enolate. The low asymmetric induction and catalyst activity of the mono-nuclear palladium complex using L6 and L7 suggest the effective role of the dinuclear palladium complex for converting 2a to 3a (Table 1, entries 13, 14) Moreover, because asymmetric induction of the Mannich adducts 2a and 3a is strongly influenced by the selection of hard metal salts, as shown in Table 1, a Zn(OAc)2-driven reactant is incorporated in the asymmetric reaction sphere45–53. When the hard zinc atom is captured by the two hard phenoxy oxygens of L1-Pd2, the zinc atom can act as a Lewis acidic site for activating Boc-imines. During nucleophilic attack by the Pd-enolate malononitrile of the zinc-activated N-Boc imine, the approach shown in model A (Fig. 6) involves serious steric repulsion between a benzene ring from the phosphine in L1 and the N-Boc imine. By flipping the face of the N-Boc imine, as shown in Model B, the steric repulsion can be released, and nucleophilic attack of the Re-face of the N-Boc imine gives the (R)-enriched Mannich product.
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study
| 100.0 |
In conclusion, the dinuclear bis(phosphoimino)binaphthoxy-Pd2(OAc)2 complex described herein facilitated a double Mannich reaction of N-Boc-imine with malononitrile to give chiral 1,3-diamines in a highly enantioselective manner. This demonstrates that over-reaction need not always be useless and undesired: well managed over-reaction can open up novel synthetic processes.
|
study
| 99.94 |
RNA trafficking and localization are important processes that influence cellular physiology at the epigenetic, post-transcriptional, and post-translational levels. Conventional techniques such as qRT-PCR and DNA microarrays are cell lysate-based assays that provide ensemble averages of RNA expression levels. To improve our current understanding of the role of RNAs in health and disease, there is great interest among RNA biologists to visualize individual RNAs in cells. There are currently three popular approaches — fluorescence in situ hybridization (FISH), the MS2 system, and molecular beacons (MBs) — commonly employed by researchers to study RNAs at the single-molecule level in various cellular contexts. Below, we discuss each technique’s usage, advantages, and limitations.
|
review
| 99.9 |
Single-molecule FISH (smFISH) is a powerful technique that uses multiple fluorophores to visualize specific RNA targets at the single-molecule level in cells . As detection of individual fluorophores by conventional fluorescence microscopy is difficult, in order to achieve single-molecule sensitivity, multiple fluorophore-tagged oligonucleotide probes are designed to target different regions of an RNA transcript , , . Hybridization of multiple probes to the same RNA molecule renders the target RNA sufficiently fluorescent upon excitation, allowing each target transcript to be imaged as a bright spot (Figure 1A, Table 1). Currently, smFISH is regarded as the gold standard approach to visualize intracellular distributions of single RNA transcripts in fixed cells and tissues . However, due to the required fixation steps, RNA dynamics data cannot be easily obtained using smFISH. Additionally, smFISH requires permeabilization to allow oligonucleotide probes to enter the cell and hybridize to the target RNAs and washing to remove unbound probes. Therefore, false-negative data can sometimes result from the loss of RNAs.Figure 1Commonly-used techniques for single-molecule RNA imagingA. smFISH labels an endogenous RNA molecule (blue line) in fixed cells using multiple oligonucleotide probes, with each probe designed to hybridize to a different region of the target RNA. B. The MS2 system requires engineering target RNA to harbor multiple MS2-binding sites (blue line) such that binding of GFP-MS2 fusion proteins (indicated in green and red, respectively) to the MS2-binding sites can cause the target RNA to appear as a bright fluorescent spot. C. MBs are stem-loop forming oligonucleotide probes that are labeled with a reporter (red circle) and a quencher (black circle). In the absence of MB target, the reporter is well-quenched. When hybridized to MB target, the fluorophore is separated from the quencher, resulting in restoration of fluorescence. When a target RNA is engineered to harbor multiple MB targets (blue line), hybridization of the MBs to MB targets can illuminate the engineered target RNA as a bright fluorescent spot. smFISH, single-molecule fluorescence in situ hybridization; MB, molecular beacon.Table 1Feature comparison of the currently-available approaches for single-molecule RNA imagingApproachSingle moleculeLive-cell imagingIn vivo stabilityImaging endogenous RNAsProbe size (kDa)Fluorescent selectionHigh signal-to-backgroundRefs.MBYesYesBackbone chemistry dependentYes∼10Organic fluorophoreYes, , MS2YesYesYESNo∼40Fluorescence proteinNo, , , , FISHYesNoN/AYes∼10Organic fluorophoreNo, ,
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review
| 99.9 |
A. smFISH labels an endogenous RNA molecule (blue line) in fixed cells using multiple oligonucleotide probes, with each probe designed to hybridize to a different region of the target RNA. B. The MS2 system requires engineering target RNA to harbor multiple MS2-binding sites (blue line) such that binding of GFP-MS2 fusion proteins (indicated in green and red, respectively) to the MS2-binding sites can cause the target RNA to appear as a bright fluorescent spot. C. MBs are stem-loop forming oligonucleotide probes that are labeled with a reporter (red circle) and a quencher (black circle). In the absence of MB target, the reporter is well-quenched. When hybridized to MB target, the fluorophore is separated from the quencher, resulting in restoration of fluorescence. When a target RNA is engineered to harbor multiple MB targets (blue line), hybridization of the MBs to MB targets can illuminate the engineered target RNA as a bright fluorescent spot. smFISH, single-molecule fluorescence in situ hybridization; MB, molecular beacon.
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study
| 91.9 |
The MS2 system takes advantage of the ability of the bacteriophage MS2 coat protein (MCP) to bind to an aptamer sequence (known as the MS2 aptamer) with high specificity and affinity (Figure 1B, Table 1) , , , . To enable single-RNA imaging, target RNA is genetically modified to harbor multiple tandem repeats of the MS2 aptamer. When co-expressed with an MCP-GFP fusion protein, each target RNA can then be tagged by multiple GFPs through MS2-MCP interactions, thus appearing as a bright fluorescent spot. To date, the MS2 system has been the most popular approach for imaging single engineered RNA transcripts in living cells, owing to its biostability and ease of delivery , , , , . However, this approach cannot be used for imaging endogenous RNAs, and its use of fluorescent proteins limits fluorophore brightness that is necessary for high-quality imaging. Furthermore, MCP-GFP fusion proteins weigh nearly 40 kDa. Thus binding of multiple large probes to an RNA may potentially interfere with its normal activities and functions .
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study
| 99.25 |
MBs are antisense stem-loop forming oligonucleotide probes labeled with a fluorophore at one end and a quencher at the other end (Figure 1C, Table 1). In the “closed” or “off” configuration, the complementary sequences flanking the loop domain anneal to form a stable stem, placing the quencher in close proximity with the reporter fluorophore, quenching its fluorescence. In the “open” or “on” configuration, target hybridization with the loop domain disrupts the stem, bringing the quencher away from the fluorophore to restore its fluorescence . With careful selection of fluorophore-quencher pair, MB fluorescence can increase 20–100 fold upon hybridization to target RNA . To date, MBs have been the most widely utilized tool for imaging endogenous RNA levels based on ensemble measurements , , , , , , , , , , , , , , , , , . To achieve single-molecule sensitivity, target RNA is engineered with tandem repeats of an MB target sequence, so that multiple MBs can hybridize to a target RNA, illuminating the RNA as a bright spot , , . Despite these advantages, one major limitation for the use of MBs is their biostability. The chemistry of the MB oligonucleotide backbone influences susceptibility to nuclease degradation or nonspecific protein binding, which could cause false-positive signals (FPSs) , .
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review
| 99.75 |
In addition to the techniques described above, other techniques, including RNA-targeting CRISPR associated protein 9 (RCas9) , RNA-mimics of GFP-based systems , and sequence-specific Pumilio-based probes , have been developed for visualization of subcellular localization and trafficking of specific RNA molecules based on ensemble fluorescence measurement. Further work is required to explore their potential for imaging RNA transcripts in living cells at the single-molecule level.
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review
| 99.75 |
Both the MS2 and MB systems are capable of imaging single RNA transcripts in living cells. Nonetheless, the MS2 system has been used more widely, despite the fact that MBs offer several advantages including smaller probe size, versatility in fluorophore/quencher selection, improved signal-to-background due to quenching, and the ability to image endogenous RNAs (Table 1). One major obstacle that hampers the widespread use of MBs is their tendency to be sequestered into the nucleus where they can generate FPSs as a result of nonspecific protein binding and/or nuclease degradation , , , , , .
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review
| 99.5 |
To reduce nonspecific signals, MBs have been conjugated to macromolecules that are either too big to traverse the nuclear pores, such as quantum dots and pegylated NeutrAvidins , or are quickly exported to the cytoplasm, such as tRNAs and small interfering RNA (siRNA)-like molecules . Alternatively, MBs have been synthesized with degradation-resistant oligonucleotides containing locked nucleic acids (LNA) or modified internucleotide linkages (such as phosphorothioate, PS) , , . By incorporating PS linkages throughout the loop domain of a 2′-O-methyl (2Me) MB backbone, we have recently developed an MB architecture called the 2Me/PSLOOP MB, based on the latter approach . The 2Me/PSLOOP MB exhibits a marginal level of FPSs in various cell types and can be used for imaging single RNA transcripts in living cells . Here we highlight the work undertaken to develop 2Me/PSLOOP MBs and explore their capabilities for single-molecule RNA imaging. We envision that the use of 2Me/PSLOOP MBs to study RNAs in living cells can further our knowledge of the role of RNAs in health and disease.
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review
| 88.25 |
Conventional MBs, including those that are synthesized with backbones composed of DNA or 2Me RNA linked with phosphodiester bonds (DNA or 2Me MBs), can be highly sensitive to nuclease degradation. To confer nuclease resistance, a non-bridging oxygen of the phosphate may be replaced with a sulfur atom to form a chemically-modified internucleotide linkage known as the PS bond. Yeh et al. reported the first use of MBs that incorporate PS linkages throughout the probe backbone (2Me/PSFULL MBs) and showed that these MBs enable detection of Coxsackie viral RNA replication for up to 12 h . Supporting this finding, we showed that 2Me/PSFULL MBs have longer intracellular stability and bioactivity than 2Me MBs . Despite these attributes, however, we found that 2Me/PSFULL MBs still cause a detectable FPS . Several hours after entry, 2Me/PSFULL MBs could exhibit a punctate staining pattern that can be easily misinterpreted as RNA granules or even single RNA transcripts . Puncta were primarily detected in the nucleus as expected, since highly-PS-modified oligodeoxyribonucleotides (ODNs) are widely reported to bind nonspecifically to the nuclear matrix , , , , , .
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| 100.0 |
We hypothesized that partially-PS-modified probes may exhibit an optimal balance of nuclease resistance while avoiding excess nonspecific binding. To test this, MBs were synthesized with different numbers and distributions of PS linkages. In a variety of cell types including HEK293, HeLa, Jurkat, and primary BJ cells, we observed a general trend of MB performance relative to the degree of PS modification (Figure 2). For example, 2Me MBs and 2Me/PSSTEM MBs, which have a fully PS-modified stem, were both highly susceptible to nonspecific opening . Incorporating PS in the loop domain significantly improved MB stability, as 2Me/PS10-LOOP MBs exhibited lower FPSs than 2Me/PSSTEM MBs, despite having the same total number (10) of PS modifications. Consistent with this observation, 2Me/PSLOOP MBs, which have a fully-PS-modified loop domain and a phosphodiester stem, exhibited even lower FPSs than 2Me/PS10-LOOP MBs . Only 1%−3% of the 2Me/PSLOOP MBs opened nonspecifically within 10 h after delivery into several different cell types . FPSs generated by 2Me/PSLOOP MBs were lower than those generated by 2Me/PSFULL MBs, suggesting that when the loop domain is highly PS-modified, stem domain modification is disadvantageous, as the additional PS groups can induce nonspecific binding while offering no additional increase in nuclease resistance. Overall, these findings demonstrate the feasibility of reducing the number of PS modifications in the MB backbone to reduce nonspecific binding while maintaining nuclease resistance.Figure 2Nonspecific opening of non-PS-modified and PS-modified MBs in living cells over timeFollowing microporation of 5 µM MBs that have no endogenous RNA targets into HeLa, HEK293, Jurkat, or primary BJ cells, the extent of MB opening was quantified over the course of 10 h . The MBs tested include 2Me (●), 2Me/PSSTEM (○), 2Me/PS10-LOOP (▴), 2Me/PSALT (△), 2Me/PSLOOP (■), and 2Me/PSFULL (□). Data are presented as mean ± S.E. from at least 30 cells. B. Representative images of MBs in HeLa cells, acquired at 10 h post microporation. The inset shows an expanded segment of the image. Arrows point to bright spots indicative of nonspecific binding in the nucleus (Scale bar, 10 µm). PS, phosphorothioate; MB, molecular beacon; 2Me, 2′-O-methyl. The graphs and images are reproduced with permission from Elsevier .
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study
| 100.0 |
Following microporation of 5 µM MBs that have no endogenous RNA targets into HeLa, HEK293, Jurkat, or primary BJ cells, the extent of MB opening was quantified over the course of 10 h . The MBs tested include 2Me (●), 2Me/PSSTEM (○), 2Me/PS10-LOOP (▴), 2Me/PSALT (△), 2Me/PSLOOP (■), and 2Me/PSFULL (□). Data are presented as mean ± S.E. from at least 30 cells. B. Representative images of MBs in HeLa cells, acquired at 10 h post microporation. The inset shows an expanded segment of the image. Arrows point to bright spots indicative of nonspecific binding in the nucleus (Scale bar, 10 µm). PS, phosphorothioate; MB, molecular beacon; 2Me, 2′-O-methyl. The graphs and images are reproduced with permission from Elsevier .
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study
| 100.0 |
Currently, debate over the primary causes of MB FPSs in living cells remains unresolved. Our findings that MBs with different PS modifications exhibit large differences in the degree of nonspecific opening can help explain why MBs open nonspecifically in cells. For example, single-stranded endonucleases appear to be the primary cause of MB nonspecific opening, as levels of FPSs are inversely correlated with the extent of PS modifications in the single-stranded loop domain . Exonucleases appear to have little impact on MB degradation, as PS modifications in the stem have little effect on MB stability . Presumably, the fluorophore and the quencher sterically block access by 5′- and 3′-exonucleases. The tendency of highly-PS-modified MBs to aggregate and emit FPSs is consistent with previous studies showing that PS-modified ODNs are prone to nonspecific binding to cellular proteins , , , , , . Accordingly, the higher nonspecific signals emitted by 2Me/PSFULL than 2Me/PSLOOP MBs suggest that any nuclease resistance gained by PS modifications in the stem domain is offset by increased nonspecific binding due to greater number of PS modifications.
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study
| 100.0 |
Our finding that 2Me/PSLOOP MBs exhibit a marginal level of FPS raises the possibility of using MBs to image the dynamics and localization of single RNA transcripts in living cells with high accuracy. To determine whether 2Me/PSLOOP MBs can accurately detect single RNA transcripts, we developed a plasmid construct that encodes a transcript carrying an EGFP coding sequence followed by 32 tandem repeats of an MB target sequence (pEGFP-N1-32x) . As the MB target sequence and EGFP coding sequence are transcribed as one RNA molecule, we hypothesized that if MBs could hybridize to the engineered transcript, the target RNA should appear as a bright fluorescent spot reflecting MB-target hybridization. Furthermore, the MB fluorescent spot should colocalize with smFISH spots visualized using a set of probes targeting unique regions on the EGFP sequence. We found that following microporation of 2Me/PSLOOP MBs and smFISH processing, bright MB and smFISH spots could be detected in the nucleus and the cytoplasm (Figure 3A). Analysis of colocalization between MB and smFISH signals in three dimensions showed nearly 90% colocalization of the MB and smFISH signals, indicating that 2Me/PSLOOP MBs can detect engineered transcripts with high accuracy (Figure 3B). By contrast, in cells microporated with 2Me/PSFULL MBs and processed by smFISH, only 60% of the MB signals colocalized with smFISH signals. Thus, consistent with the analysis showing that 2Me/PSLOOP MBs generate lower FPS compared to 2Me/PSFULL MBs, 2Me/PSLOOP MBs can image single RNA transcripts more accurately than 2Me/PSFULL MBs in living cells.Figure 3Single-molecule RNA transcript detection using 2Me/PSLOOP MBs and smFISHHeLa cells stably expressing pEGFP-N1-32x were fixed and permeabilized 8 h or 24 h after microporation with 5 µM 2Me/PSLOOP MBs. smFISH was then performed with a pool of probes designed to target EGFP to assess MB detection accuracy for single RNA transcripts. A. Representative maximum intensity projection images of 2Me/PSLOOP MBs (Atto647NN-labeled) and EGFP smFISH probes (TAMRA-labeled) at 8 h or 24 h after microporation (Scale bar, 10 µm). B. The percentage of MB signals that colocalized with smFISH signals (left) and the percentage of smFISH signals that colocalized with MB signals (right) was analyzed on a cell-by-cell basis using a custom MATLAB program. Data are presented as mean ± SD from at least 10 cells. Significant difference from the 2Me/PSFULL MBs is indicated with asterisks (P < 0.05). The images are reproduced with permission from Elsevier .
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study
| 100.0 |
HeLa cells stably expressing pEGFP-N1-32x were fixed and permeabilized 8 h or 24 h after microporation with 5 µM 2Me/PSLOOP MBs. smFISH was then performed with a pool of probes designed to target EGFP to assess MB detection accuracy for single RNA transcripts. A. Representative maximum intensity projection images of 2Me/PSLOOP MBs (Atto647NN-labeled) and EGFP smFISH probes (TAMRA-labeled) at 8 h or 24 h after microporation (Scale bar, 10 µm). B. The percentage of MB signals that colocalized with smFISH signals (left) and the percentage of smFISH signals that colocalized with MB signals (right) was analyzed on a cell-by-cell basis using a custom MATLAB program. Data are presented as mean ± SD from at least 10 cells. Significant difference from the 2Me/PSFULL MBs is indicated with asterisks (P < 0.05). The images are reproduced with permission from Elsevier .
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study
| 100.0 |
Given their ability to detect single engineered RNA transcripts with high accuracy in live cells, 2Me/PSLOOP MBs may be a promising tool to study the trafficking and localization of single RNA transcripts in real time. Figure 4A shows diffusion coefficients of single pEGFP-N1-32x RNAs in cells as measured by MB imaging. The high variance of diffusion coefficients among transcripts in both the nucleus and cytoplasm indicates a heterogeneous nature of RNA dynamics . On average, RNAs in the nucleus move nearly 4 times slower than transcripts in the cytoplasm, consistent with previous findings showing that the nucleoplasm is more viscous than the cytoplasm . Similar results have been obtained in cells transfected with pEGFP-C1-32x RNAs (Figure 4B), in which the 32 MB target repeats are located in the 3′-UTR. These findings suggest that an RNA transcript can be targeted by 2Me/PSLOOP MBs at either 5′- or 3′-UTR. Furthermore, binding of the 2Me/PSLOOP MBs to 32 tandem repeats does not cause interference with EGFP translation as seen when 64 repeats are used (Figure 4C), suggesting that MBs can be used to image target RNA containing up to 32 tandem repeats without perturbing physiological functions of target RNAs. There is no change in cell viability or cell spreading detected in cells microporated with varying concentrations of MBs (Figure 4D–E), suggesting that 2Me/PSLOOP MBs do not affect cellular growth or physiology. Overall, these findings suggest that 2Me/PSLOOP MBs can be a noninvasive platform for imaging single RNA transcripts in living cells.Figure 4Noninvasive imaging and measurement of RNA dynamics at the single-molecule levelSingle-particle tracking analysis reveals diffusion coefficients of single pEGFP-N1-32x (A) or pEGFP-C1-32x (B) mRNAs in the nucleus and the cytoplasm of HeLa cells. Insets show average diffusion coefficients (mean ± SE) in the nucleus and the cytoplasm. Significant difference is indicated with asterisks (P < 0.05). (C) The effect of 2Me/PSLOOP MBs on EGFP protein expression. Total EGFP protein level was assessed using Western blotting 24 h after microporation of 2Me/PSLOOP anti-repeat MBs at concentrations of 0, 1, or 5 µM into HeLa cells stably expressing pEGFP-N1-32x or pEGFP-N1-64x RNAs. Protein expression is normalized to that in cells microporated with 0 µM MB. Total GAPDH protein level was measured as a loading control. Significant difference from 0 µM MBs is indicated with asterisks (P < 0.05). Average spreading (D) and proliferation (E) 24 h after microporation with different concentrations of 2Me/PSLOOP MBs. All data are presented as mean ± SD of at least three independent experiments. The images are reproduced with permission from Elsevier .
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study
| 100.0 |
Single-particle tracking analysis reveals diffusion coefficients of single pEGFP-N1-32x (A) or pEGFP-C1-32x (B) mRNAs in the nucleus and the cytoplasm of HeLa cells. Insets show average diffusion coefficients (mean ± SE) in the nucleus and the cytoplasm. Significant difference is indicated with asterisks (P < 0.05). (C) The effect of 2Me/PSLOOP MBs on EGFP protein expression. Total EGFP protein level was assessed using Western blotting 24 h after microporation of 2Me/PSLOOP anti-repeat MBs at concentrations of 0, 1, or 5 µM into HeLa cells stably expressing pEGFP-N1-32x or pEGFP-N1-64x RNAs. Protein expression is normalized to that in cells microporated with 0 µM MB. Total GAPDH protein level was measured as a loading control. Significant difference from 0 µM MBs is indicated with asterisks (P < 0.05). Average spreading (D) and proliferation (E) 24 h after microporation with different concentrations of 2Me/PSLOOP MBs. All data are presented as mean ± SD of at least three independent experiments. The images are reproduced with permission from Elsevier .
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study
| 100.0 |
Conventional MBs have been used to image RNAs in various cellular contexts, but their propensity for nonspecific opening in living cells limits their widespread applications in studies where more sensitive detection is necessary, such as imaging RNA localization and dynamics at the single-molecule level , , . We have recently developed a new MB architecture, known as the 2Me/PSLOOP MB, that elicits a marginal level of FPSs in cells as compared with conventional MBs . We show that 2Me/PSLOOP MBs could accurately image single mRNA transcripts harboring 32 tandem repeats of an MB target sequence using conventional fluorescence microscopy. Currently, RNA dynamics at the single-molecule level has been studied primarily based on engineered RNA molecules that harbor large insertions of MB target or MS2 aptamer sites that potentially interfere with the activities of target RNAs. With further possible approaches for optimizing signal-to-background, such as fluorophore/quencher selection, and the use of more sophisticated imaging techniques, we anticipate that 2Me/PSLOOP MBs can be a promising platform for live-cell, single-molecule imaging of minimally-engineered RNA molecules, or even endogenous RNA molecules, providing researchers with opportunities to study RNAs with unprecedented spatial and temporal resolutions.
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| 99.94 |
Integrins contribute to essential components of tumor progression such as survival, proliferation, and cell motility1. Specifically, integrin α6β4 is a known driver of tumor cell invasion2, which in turn promotes metastasis3. In cancer cells, integrin α6β4 signaling is activated upon binding to laminin extracellular matrix proteins and in cooperation with growth factor receptors such as EGFR, RON, and c-MET4–6. Activation of integrin α6β4 results in stimulation of downstream signaling pathways including PI3K, MAPK, Src family kinases, Rho family small GTPases, and the Nuclear Factor of Activated T-cells (NFAT)7–9 that contribute to invasion, angiogenesis, anoikis-resistance, cell survival, and proliferation10. Integrin α6β4 enhances these properties in part through transcriptional upregulation of pro-tumorigenic genes including S100A4 in breast cancer11, 12 and the EGFR ligands AREG and EREG in pancreatic carcinomas13.
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study
| 99.9 |
The importance of AREG and EREG in tumor progression, therapeutic resistance, and as a potential prognostic and predictive biomarker has been well established in multiple cancer types14, 15. Cleavage of pro-AREG and pro-EREG by the MMPs results in protein release and autocrine signaling to activate EGFR13. AREG and EREG are unique in their ability to cause EGFR recycling back to the plasma membrane for reactivation16, 17. EGFR signaling by AREG and EREG is enhanced in pancreatic carcinomas and contributes to the aggressive nature of the disease18, 19. We have shown that AREG and EREG are required for HGF-mediated migration and invasion in response to signaling from integrin α6β4, further demonstrating their importance to invasive properties of cancer cells13. We and others find that AREG13 and EREG13, 20 gene expression is controlled by DNA methylation. However, the mechanisms guiding the demethylation of these promoters have not been elucidated.
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study
| 100.0 |
Transcriptionally silenced genes have a repressive epigenetic state that compacts chromatin. Repressive epigenetic marks include non-acetylated histones, lysine methylation at H3K27 and H3K4 and cytosine methylation at CpGs21. Active DNA demethylation is tightly regulated and requires a series of enzymatic reactions that proceed through the BER pathway. This mechanism of epigenetic alteration is likely responsible for upregulation of pro-tumorigenic genes, as it has been identified for dynamic, context dependent modification of DNA22, 23.
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study
| 100.0 |
The ten-eleven translocation methylcytosine dioxygenase (TET1) is the first crucial step in DNA demethylation as this protein recognizes specific 5-mCs to be targeted for removal by DNA repair and conversion from 5-mC to 5-hydroxymethyl cytosine (5-hmC)23. 5-hmC can be further oxidized by TET proteins to 5-carboxycytosine (5-caC) and 5-formylcytosine (5-fmC); however, these derivatives are found less often in the genome, and their complete function is still being characterized24.
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| 99.9 |
5-mC products are identified by growth arrest and DNA damage inducible alpha (GADD45A). GADD45A is responsible for recruitment of other repair factors to CpG sites for removal of methyl groups, and has been implicated as a necessary step in DNA demethylation by providing a link between epigenetics and DNA repair25, 26. GADD45A recruits Activation Induced Cytidine Deaminase (AID) and Apolipoprotein B mRNA Editing Enzyme, Catalytic polypeptide-like (APOBEC) proteins26, which deaminates 5-hmC to 5-hmU, generating a G-U DNA mismatch. This mismatch is removed by thymine DNA glycosylase (TDG) or methyl-binding protein 4 (MBD4). This cleavage activates the normal functions of the BER pathway including cleavage of the DNA backbone by AP-endonuclease and repair back to a non-methylated cytosine by XRCC-1, PARP-1, DNA ligase, and DNA polymerase27.
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| 100.0 |
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