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We apologize for having used the term “outlier” in an apparently misleading manner. In this analysis, we asked whether one of the nine haplotype groups influenced FLMb levels in a different manner from the others. We thereby examined whether it had a kind of outlying regulatory role. For this purpose, we performed the analysis by excluding, one by one, each of the nine haplotypes and calculated R2. None of these exclusions substantially changed R2 except when PRO2+AAAAC/INT6+CAA was excluded. When this haplotype was excluded R2 increased from 0.13 to 0.46 indicating that this haplotype substantially and negatively influenced our analysis. Herein, our aim had not been to force the linear model to fit our expectations but to identify a PRO2+/INT6+ haplotype, which possesses a differential effect when tested in transgenic lines when compared to accessions. We have now clarified the legend of Figure 8A, removed the term outlier, and improved the description of the findings in the text. We also removed the correlation analysis of FLMd from Figure 8—figure supplement 1.
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| 100.0 |
“PRO2+ and INT6+ polymorphisms contribute to global variation of FLM levels: The nine PRO2+ and INT6+ haplotypes tested in transgenic experiments were present in 579 (69%) of all 840 accession with available genome sequence information (Figure 8—figure supplement 1A). […] This indicated that PRO2+AAAAC INT6+CAA has an exceptional effect on FLM-ß in the accessions than cannot be predicted based on the results from the transgenics (Figures 4E, 8A and Figure 8—figure supplement 1E).”
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| 100.0 |
3) For the flowering time comparison, the lines were vernalized. However, FLM is known to be repressed by vernalization (as the authors note), so how could it be affecting flowering, and might there be variation among lines in how FLM responds to the vernalization? It seems more likely that other loci that are correlated with FLM are controlling flowering time here. The authors should either measure FLM expression in these vernalized plants and correlate that with flowering, or repeat the experiment without vernalization where FLM and FLC expression in these lines has already been measured. A better way to control for FLC variation would be to statistically account for its effects with multiple regression.
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| 86.5 |
We have extracted from the literature that the role of vernalization on FLM expression is rather unclear. Ratcliffe et al. (2001, Plant Phys.) observed decreased FLM levels after six to eight weeks of vernalization, even though FLM seems not to be as strongly repressed by vernalization as FLC. Scortecci et al. (2001, Plant Journal) did not find an effect on FLM transcript abundance after 30 days of vernalization. However, this observation was revisited in Sung et al. (2006, Genes and Development), who showed that FLM is repressed by vernalization through epigenetic mechanisms. Finally, Lee et al. (2013, Science) did not observe any effect of vernalization on FLM abundance whereby no information on the duration of the vernalization treatment was provided. Even though a repressive effect of vernalization on FLM abundance seems to be the consensus we suppose that the effect of vernalization on FLM abundance needs much more detailed investigation. Especially the duration of vernalization may be a critical factor. We had summarized this literature and findings in our original manuscript. Since we now present an improved analysis based on improved experimental design as described below, we have deleted the respective sections since they were no longer relevant.
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| 99.94 |
Regarding the improved experiment as suggested by the reviewer, we considered it not insightful to conduct an experiment with vernalized plants (and including winter annuals) since the FLC-dependent vernalization pathway is epistatic to the effects of FLM.
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other
| 99.8 |
Instead, we have now performed an experiment with summer annuals not requiring vernalization (all carrying PRO2+/INT6+ haplotypes of the described range). As many summer-annual accessions carry weak FLC alleles with residual low FLC abundance, which still are able to contribute to repression of flowering time {Duncan, 2015 #156;Coustham, 2012 #30;Li, 2014 #111}, we measured FLM and FLC transcript levels and tested their combined and single contribution to flowering time (rosette leaf numbers). Flowering time data was generated in a new experiment. Interestingly, we found a comparable significant contribution of FLMb to flowering time than in the first experiment (R2 = 0.21, before R2= 0.15), with a smaller, however not significant, contribution of the residual FLC levels (R2 = 0.119). We integrated the findings of this new experiment into Figure 8B, changed Figure 8—figure supplement 2, changed the Results, Discussion, and figure legends.
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| 100.0 |
“To examine the correlation between FLM-ß expression and flowering time, we determined flowering time of accessions at 15°C. […] Taken together, we concluded that PRO2+ and INT6+ alleles contribute to variations in FLM-ß levels and that FLM-ß accounts for flowering time in cool ambient spring temperatures in a diverse population of summer-annual Arabidopsis accessions (Figure 9).”
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“The nine PRO2+/INT6+ haplotype combinations included in our transgenic experiments represent 69% of the world-wide PRO2+/INT6+ variation (Figure 8—figure supplement 1A). […] Bearing these possible genetic and environmental interferences in mind, we regard the detected effect of FLM-ß on flowering (21%) in cool (15°C) temperatures as considerable and suggest that it is rather an underestimation.”
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| 100.0 |
“Figure 8—figure supplement 2: Geographic distribution of summer-annual accessions. (A) Geographic distribution of the 27 summer-annual accessions as described in Figure 8B. (B) Correlation of FLC transcript levels with flowering time data (n = 8 – 10) as measured in rosette leaf number of summer-annual accessions. p > 0.05.”
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4) Finally, on the statistical side, the conclusions about "temperature insensitivity" for INT6+ alleles are not backed by statistics – the authors should explicitly test for a change in the temperature sensitivity between FLM-Col0 and INT6+. From the graphs, it appears temperature sensitivity is reduced, but certainly not eliminated (Figure 6).
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| 97.7 |
To address this comment, we have re-evaluated the observation with a new measurement and corrected the calculation and integrated statistical tests. The results and the conclusions were found to be consistent with those presented in the initial manuscript.
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| 99.8 |
In “Natural haplotypes of FLM non-coding sequences fine-tune flowering time in ambient spring temperatures in Arabidopsis" Lutz et al. address the biological relevance of FLM splice variants for variation in flowering time in natural accessions and identify non-coding sequence polymorphisms that contribute to the regulation of basal expression levels and the differential, thermo-responsive generation of the β and δ splice variants. As such, this work provides novel insights into the regulation of flowering in the ambient temperature range and manages to dissect polymorphisms which are relevant for basal FLM expression and temperature-induced effects on the β and δ splice variants.
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The manuscript is overall very well written and the authors provide extensive data which has apparently been subjected to comprehensive and sound statistical analyses. The data is presented in an attractive manner. Main manuscript figures are supported by extensive supplementary material including the presentation of extensive control data, which is greatly appreciated. In some cases the figure descriptions or legends should be extended. Due to the vast amount of data condensed in the figures, the descriptions (at least in the text) should be detailed enough for a wide readership to follow the authors through their argumentation.
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| 99.94 |
1) The authors state that “The presence of intron 1 is sufficient to restore the basal expression of FLM” – while it does restore expression levels to detectable levels, the expression level of the β splice variant at 15°C is considerably lower than the WT, whereas the FLMδ levels are similar the WT at this temperature. This would indicate that intron 1 alone cannot fully account for basal levels of FLM β.
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| 99.94 |
The reviewer is correct in stating that Figure 1 may invite the conclusion that intron 1 does not fully account for basal FLMb levels. However, as we used transgenic lines for this analysis we suggest that no conclusions about the basal expression levels of native FLMb nor FLMd can be drawn from this analysis. Nevertheless, the relative change of transcript levels in response to changing temperature can be detected and compared to the WT construct, which is the main aim of this analysis. To avoid any misunderstandings, we normalized the transcript levels measured at 21°C to “1” and provided an improved description in the legend.
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| 96.3 |
2) The use of pools of independent segregating T2 lines for transcriptional analysis seems unusual. While the controls presented in Figure 2—figure supplement 2 suggest that this can recapitulate the “natural” situation (why not subjected to statistical analysis?), it seems nonetheless risky to deliberately tolerate the presence of non-transgenics in the mix. Could the authors state their reasoning for this unusual procedure and provide the missing statistics here? Also, the phrase “replicate pools compromising…” should probably read “comprising”.
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| 97.4 |
By adopting this pooling strategy, we have followed procedures that were described in publications from leading flowering time labs (e.g. Coustham, 2012, Science; Caroline Dean lab). This is an attractive way to reduce the variability of transgene expression that result from positional effects in different transgenic lines. In view of the ample analysis of transgenic lines presented in our manuscript, this presented the most efficient way for reducing cost and time while still producing a meaningful data set. If we had used T3 generation plants, we would have had to propagate and test the segregation of over 3000 individual plants. We have modified (and thereby improved) the above-described strategy by pooling plants into four pools, which we designate replicate pools. We have also verified the trustworthiness of this pooling system by demonstrating that it is possible to precisely recapitulate the effects observed with the Col-0 and Kil-0 FLM alleles (Figure 2—figure supplement 2C and D). The statistical analysis has now been integrated into the figure as requested by the reviewer.
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| 99.9 |
It should also be noted that we verified that the lines are single insertion lines by scoring their segregation, which was possible due to the use of the FAST vector system, which allows the identification of transgenes by seed fluorescence (Shimada, 2010). Further, we compared the transcript levels to a Col-0 allele reference transgenic construct, which was obtained by the exact same procedure of pooling etc.
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Figure 3—figure supplement 1 provides additional information, but still the color coding of haplotypes which is provided for three different regions (full length, intron 1 or intron 6) is confusing as the composition of the different haplotypes varies depending on the selected gene region (Figure 3—figure supplement 1G). The authors may want to better explain these figures to enable a wide readership to follow their analysis and key points here.
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| 99.9 |
The reviewer is correct in stating that using similar colors for different haplotype regions may be confusing. Here, we used the same color code for each grouping by using the same color for the respective largest group of each the three comparisons. To clarify this, we have now adjusted the shade of these color codes and provided a better description.
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4) Figure 5B and C: Please specify again in the figure caption that this data corresponds to 40 selected accessions. Why was the FLM β level not determined in the vernalized plants? Even though FLC effects seem to be generally negligible, it would have been as easy to do the analysis on the same material.
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| 99.44 |
5) In Figure 5—figure supplement 1D/E: removing the lower quartile (or more!) of flowering time data for the correlation analysis by simple assumption that these represent the non-transformed Nd-1 individuals of the segregating population is questionable. While this may be an attractive assumption, it negates the fact that each line pool may represent its individual set of variances (also present in ND-1 itself). This would at least require a spot check for the presence of transgenes in this pool or the percentage of “clean” Nd-1 individuals. In principle, a simple test by PCR would have provided a solid answer to that. As is, the authors should include these lines in the correlation analysis as the factual genotype is not known.
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| 91.0 |
We have provided the full data set without corrections (Figure 5—figure supplement 1D/E) as well as the corrected data set (Figure 5A). For the data presented in Figure 5A, we precisely removed the lower quartile and emphasize that we had confirmed that all lines carried single locus inserinots, using a fluorescence marker. Thus, 25% of the plants should be wild type segregants from the transgenic lines and these should correspond to the early flowering individuals since the genetic background for the transgenic analysis had been the FLM deletion accession Nd-1. In this analysis, the homozygous wild type lines Nd-1 and Col-0 are not included since they do not segregate and the correction procedure of removing the lower quartile could not be applied.
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Regardless of this correction, very similar R2 values were obtained with the corrected (R2 = 0.94) and uncorrected data (R2 = 0.89). We assume that the high correlation between FLMb levels and flowering time is generally very high, regardless of the only small improvements in the correlation analysis when using the corrected or uncorrected flowering time values.
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The new experiment to test this is very helpful, and does provide good evidence that FLMB expression level is important for flowering time variation. My concern is with interpretation: The authors provide several explanations for why the correlation between flowering and FLMB expression in this accession set is lower than in the transgenics. This is trivial because there is much more variation within each haplotype class in the accessions than the transgenics, because they vary at many loci across the genome. I think the real question should be: Is the slope of the relationship between FLMB and flowering time significantly less in the accessions? This would ask if the equivalent change in FLMB expression would cause the same change in flowering. This is a more meaningful metric than change in R2. The correct test would be to include both accessions and transgenics in the same model (flowering_time ~ FLMB * genotype_class), and ask if the interaction is significant.
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| 99.75 |
We performed the suggested test for interaction FLMB:genotype_class and found that the slopes differ (p = 0.0321). We included this analysis in the results (subsection “PRO2+ and INT6+ polymorphisms contribute to global variation of FLM levels”, last paragraph) and Discussion.
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| 100.0 |
This is the aim of the analysis in Figure 8A and Figure 8—figure supplement 1D/E. The main claim is that the expression variation induced by these haplotypes in the transgenics is correlated with the expression variation of accessions carrying the same haplotypes. This conclusion only holds when one of the 9 haplotypes is excluded. The explanation given for excluding this haplotype is that the correlation goes up after removing this. I did a quick simulation study and found that using this algorithm you'd expect that the best correlation found by dropping each of the 9 pairs would be greater than 46% about 21% of the time even if there were actually no correlation at all. So, this is really not very strong evidence that the haplotype effects are similar. Certainly, the statement "We found that the PRO2+/INT6+ effects, as detected in transgenic experiments (R2 = 0.94), partially explain the FLM-ß expression variation in natural Arabidopsis accessions (R2 = 0.46)" is not supported, because this 46% number is only true when you exclude those accessions that don't fit.
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| 99.94 |
As above, I think a more appropriate analysis would be to ask if the sizes of the differences between the haplotypes is the same in the transgenics and the accessions (i.e., is the slope of the graph different from 1?). A more straightforward answer to the question of how much variation in FLMB is explained by these haplotypes is simply to report the R2 for the analysis shown in Figure 8—figure supplement 1D.
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| 99.9 |
We understand the reviewer's concern and deleted the analysis excluding the exceptional haplotype from Figure 8 and reported the R2 of the full dataset shown in Figure 8—figure supplement 1E (R2 = 0.13) as suggested. We changed the respective parts of the text, worded the related conclusions less strongly (subsection “PRO2+ and INT6+ polymorphisms contribute to global variation of FLM levels” and Discussion) and changed Figure 8 accordingly.
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| 99.7 |
This conclusion is important because of the model that FLM controls temperature sensitivity of flowering. New data are presented here relative to the previous version, though where they came from is not clear. The correct test for a change in temperature sensitivity is to ask if the interaction between genotype and temperature is significant (expression ~ Genotype + Temp + Genotype:Temp). The n.s. effect of temperature on expression for INT6+ is not evidence of no temperature effect, only a lack of evidence for a temperature effect. The figure caption states the statistics are done based on a t-test. A t-test can't be used to test for an interaction (except in 6C/D if the 5 transgenic lines per genotype were used as the replicates, n = 5). An ANOVA is needed to conclude that this haplotype affects temperature sensitivity.
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The new data was generated by measuring FLM-β levels with qRT-PCR using the same samples. As suggested by the reviewer, we performed an interaction test between genotype and temperature of the data shown in Figure 6A. We found a significant change in temperature sensitivity (p = 0.012259). We integrated this test in the Results section subsection “The INT6+CAA polymorphism confers temperature-insensitive FLM expression and flowering”) and the figure legend. As the change in temperature sensitivity was now reported by testing the interaction of genotype to temperature, we removed the t-tests from Figure 6A and B.
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1) The authors have clarified that the major concerns about the exclusion of accessions from the correlation of FLM expression and flowering time was largely based on a misunderstanding of the method and intention of the analysis. The changes to the text makes this much more clear now. The exclusion of the “exceptional" haplotypes raises the correlation from 0.13 to 0.46 in a linear regression analysis. Please also provide the corresponding p-values here to make sure that the p-values reflect a robust correlation effect (as is done in the further analysis of FLMß/FLC expression via multiple regression analysis). Please also describe how p-values were obtained for both cases then.
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| 99.56 |
Please see our reply to comment #2 of reviewer #2. According to his comment and reasoning, removing the exceptional haplotype may not be appropriate. Therefore, we decided to simply report the R2 shown in Figure 8—figure supplement 1D in the text. For further details please see our answer to comment #2 of reviewer #2.
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2) I appreciate the explanation regarding the use of the pooling strategy and the inclusion of statistics. However, no information on the performed test is given in the figure caption. was this also a t-test as mentioned in the methods? If so, please state whether a two-sided t-test and correction for multiple testing was implicated. Otherwise perform an ANOVA with suitable post-hoc test.
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A t-test was applied previously. We now performed an ANOVA and Tukey HSD post-hoc test. The interpretation remained unchanged compared to the previously applied t-tests. We changed Figure 2—figure supplement 2C, D and the respective figure legend accordingly.
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| 96.75 |
Esophageal cancer is the 8th most common cancer worldwide with an estimated 456,000 new cases each year.1 Esophageal squamous cell carcinoma (ESCC) is the dominant histological type and accounts for 80% of esophageal cancers.2 With the increased understanding of cancer biology, more and more targeted drugs, such as gefitinib,3 cetuximab4 or imatinib administration5 have been approved for clinical treatment because of their higher efficacy and lower toxicity compared with traditional chemotherapy. However, an effective therapeutic drug targeting ESCC has not yet been developed. ESCC is the 4th leading cause of cancer death in China and 7th in the world because of its ability to develop chemoresistance and tendency to metastasize.6 Even for patients with early stage ESCC, adjuvant therapy cannot prolong their overall survival significantly compared with surgery alone.7
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| 99.9 |
Polyamines are a group of small aliphatic polycations derived from amino acids and are present in all living organisms. The ubiquity of polyamines indicates their indispensable role in key cellular processes, such as cell growth,8 proliferation,9 apoptosis10, and gene expression.11 Aberrant accumulation of polyamines is associated with various pathological consequences, including cancer.11 Ornithine decarboxylase (ODC) is the first rate-limiting enzyme in the polyamine biosynthesis pathway in mammals and its intracellular concentration is tightly controlled. ODC activity is induced in response to cell growth stimuli, and is highly expressed in diseases such as inflammation and cancer. ODC is considered to be a potential oncogene because its over-expression can transform mammalian cell lines,12 indicating that ODC is not only a biomarker for cancer but also a potential target for cancer therapy. Anti-cancer research, including bench work and clinical research, targeting ODC has yielded promising results.13, 14 However, the role of ODC in ESCC development is still unclear. The Wnt signaling pathway is activated in most ESCC cases15 and the Myc loci comprise the most significant regions of amplification.16 Myc has been implicated as a reasonable indicator of the accumulation of various activated and inactivated genes involved in the development of ESCC,17 suggesting Myc expression acts as a driver event of ESCC. As a physiological transcriptional target of c-Myc,18 ODC reportedly plays an important role in ESCC development and progression.
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| 99.7 |
In the present study, we examined the expression pattern of ODC in ESCC cell lines and tissues. Then we investigated the functions of ODC in ESCC development by using shRNA and an irreversible inhibitor of ODC, difluoromethylornithine (DFMO). Our data showed that ODC expression was up-regulated in human ESCC tissues and ESCC progression could be attenuated by suppressing ODC activity, indicating that ODC might be a promising target for ESCC therapy.
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| 100.0 |
Up-regulated ODC expression has been reported in various solid cancers, including skin cancer,19 gastric cancer,20 neuroblastoma,21 and colon cancer.22 In the present study, we explored the expression pattern of ODC in ESCC tissues by immunohistochemistry (IHC). ODC immunostaining was observed in both nucleus and cytoplasm (Fig. 1). Among 110 evaluable ESCC cases, positive staining for ODC was observed in 93.6% of the tissues (103/110). In esophagitis and normal adjacent tissues (NAT), ODC positive staining was 60.0% (9/15) and 52.6% (10/19), respectively (Supplementary Fig. 1). Quantitative analysis showed that the ODC immunostaining integrative optical density (IOD) value was 2255 ± 115 for ESCC, which was significantly higher than the value for esophagitis (1187 ± 236, p < 0.01) or NAT (888 ± 140, p < 0.01; Fig. 1a). The correlations between clinicopathological features and ODC expression in the primary ESCC were determined (Supplementary Table 2). The ODC expression level in ESCC was significantly correlated with lymph node metastasis status (p = 0.02, Fig. 1b) and clinical stage (p = 0.02, Fig. 1c), but was not associated with age, gender, or tumor histological grade.Fig. 1ODC expression is up-regulated in ESCC. a The ODC immunohistochemical IOD value is significantly higher in ESCC compared to esophagitis or NAT. The ODC protein expression level in ESCC is significantly correlated with b status of lymph node metastasis and c clinical stage. Significant differences were determined using the Student’s t test
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ODC expression is up-regulated in ESCC. a The ODC immunohistochemical IOD value is significantly higher in ESCC compared to esophagitis or NAT. The ODC protein expression level in ESCC is significantly correlated with b status of lymph node metastasis and c clinical stage. Significant differences were determined using the Student’s t test
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| 99.94 |
To further investigate the role of ODC in ESCC progression, we introduced shODC into two human ESCC cell lines, KYSE450 and KYSE510, by lentiviral vector and examined the effect of ODC on proliferation and anchorage-independent growth. To select the most effective shRNA, we screened 5 different shRNA sequences targeting ODC. Our data showed that, shODC #2 and #4 worked well in KYSE450 cells, whereas shODC#3 and #4 worked well in KYSE510 cells (Fig. 2a). These shODC sequences were used for experiments. Knocking down ODC expression reduced ODC enzyme activity in both ESCC cell lines by more than 70% (Fig. 2b). Correspondingly, cell proliferation and colony formation in soft agar were also suppressed (Fig. 2c, d), indicating that ODC promotes the progression of ESCC and deserves further investigation.Fig. 2Silencing ODC expression by shRNA suppresses anchorage-dependent and anchorage-independent ESCC cell growth. a ODC expression was analyzed by Western blot in KYSE450 and KYSE510 cells expressing shMock or shODC. b ODC activity was assessed as the release of L-[1-C14] ornithine and results are shown as percentage of the control group (set at 100%). c Anchorage-dependent cell growth was measured by MTS assay. d For anchorage-independent growth, cells were cultured in soft agar for 3 weeks and then colonies were counted using a microscope and the Image-Pro Plus software (v.6.0) program. All data are shown as means ± S.D. of triplicate values from 3 independent experiments
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| 100.0 |
Silencing ODC expression by shRNA suppresses anchorage-dependent and anchorage-independent ESCC cell growth. a ODC expression was analyzed by Western blot in KYSE450 and KYSE510 cells expressing shMock or shODC. b ODC activity was assessed as the release of L-[1-C14] ornithine and results are shown as percentage of the control group (set at 100%). c Anchorage-dependent cell growth was measured by MTS assay. d For anchorage-independent growth, cells were cultured in soft agar for 3 weeks and then colonies were counted using a microscope and the Image-Pro Plus software (v.6.0) program. All data are shown as means ± S.D. of triplicate values from 3 independent experiments
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| 100.0 |
To explain the basis of ODC as a tumor promoter in ESCC progression, we examined apoptosis and cell cycle in shODC-infected ESCC cells. Our results showed that knocking down ODC expression with shODC significantly increased total apoptosis of both KYSE450 and KYSE510 cell lines (Fig. 3a). In addition, compared to shMock, shODC expression also induced G2/M phase arrest and decreased the percentage of cells in S phase in both cell lines (Fig. 3b). We also compared apoptosis- (Fig. 3a) and cell cycle-related (Fig. 3b) markers by Western blot in shMock and shODC-expressing KYSE450 and KYSE510 cells. Our results showed that shODC was generally associated with increases in the expression levels of cleaved caspase 3, cleaved PARP, Bax, p53, p27, p21, E-cadherin, and phosphorylated CDK1 (Tyr15). In contrast, the expression levels of phosphorylated ERK1/2, Bcl-2, PCNA and cyclin B1 were mostly decreased.Fig. 3Silencing of ODC expression by shRNA induces apoptosis and G2/M arrest in ESCC cells. The effects of ODC on a apoptosis and b cell cycle were analyzed by flow cytometry (upper panels). The expression of markers associated with cell cycle and apoptosis were analyzed by Western blot (lower panels). All data are shown as means ± S.D. of triplicate values from 3 independent experiments
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Silencing of ODC expression by shRNA induces apoptosis and G2/M arrest in ESCC cells. The effects of ODC on a apoptosis and b cell cycle were analyzed by flow cytometry (upper panels). The expression of markers associated with cell cycle and apoptosis were analyzed by Western blot (lower panels). All data are shown as means ± S.D. of triplicate values from 3 independent experiments
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| 100.0 |
Based on the in vitro assay results, we investigated the function of ODC in ESCC tumorigenesis in an athymic nude xenograft mouse model. Mice were divided into three groups and inoculated in the right flank with shMock-infected, shODC#2-infected, or shODC#4-infected KYSE450 cells (3 × 106 cells per mouse, 4 mice per group). The body weight of mice increased normally with growth for the duration of the study, indicating no adverse effects from the respective inoculation (Fig. 4a). At 56 days after inoculation, when the tumor volume of the shMock group reached 1000 mm3, all experimental mice were euthanized and tumors extracted for IHC analysis (Supplementary Fig. 2). Our data showed that shMock-infected KYSE450 cells formed markedly larger xenograft tumors in nude mice compared to the shODC-infected groups (p < 0.01, Fig. 4b). IHC results showed that expression of ODC, Ki-67 and PCNA proliferation markers was attenuated. In addition, expression of the anti-apoptosis marker Bcl-2 was also decreased, whereas expression of cleaved caspase 3 was increased (Fig. 4c). These results confirmed that inhibiting ODC expression in ESCC cells suppresses proliferation and induces apoptosis, leading to attenuation of ESCC tumorigenesis.Fig. 4shODC suppresses the tumor-forming ability of ESCC cells. a After inoculation, the body weights of all mice remained stably increased. b shODC significantly suppresses KYSE450 xenograft tumor volume compared with the shMock group. c IHC analysis was performed to determine the expression levels of ODC, PCNA, Ki-67, cleaved caspase 3, and Bcl-2 in ESCC xenograft tumors expressing shMock or shODC. Representative photographs for each antibody and each group are shown. The integrated optical density (IOD) was evaluated using the Image-Pro Plus software (v.6.0) program. All data are shown as mean values ± S.D
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shODC suppresses the tumor-forming ability of ESCC cells. a After inoculation, the body weights of all mice remained stably increased. b shODC significantly suppresses KYSE450 xenograft tumor volume compared with the shMock group. c IHC analysis was performed to determine the expression levels of ODC, PCNA, Ki-67, cleaved caspase 3, and Bcl-2 in ESCC xenograft tumors expressing shMock or shODC. Representative photographs for each antibody and each group are shown. The integrated optical density (IOD) was evaluated using the Image-Pro Plus software (v.6.0) program. All data are shown as mean values ± S.D
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| 100.0 |
Based on the important role of ODC in ESCC progression, ESCC cells were treated with DFMO, a potent substrate analog and specific irreversible inhibitor of ODC.23 The data showed that treatment with DFMO dose-dependently decreased ODC activity in ESCC cells (Fig. 5a). Inhibition of ODC activity was associated with suppression of proliferation and colony formation of ESCC cells (Fig. 5b, c), indicating that DFMO attenuates ESCC progression by targeting ODC.Fig. 5DFMO inhibits ESCC cells in vitro. a The effect of DFMO on ODC activity in KYSE450 and KYSE510 cells was measured as the release of CO2 from L-[1-C14] ornithine and results are shown as percentage of control group (set at 100%). b Anchorage-dependent cell growth was measured by MTS assay. c For measuring anchorage-independent growth, cells were cultured for 3 weeks with different concentrations of DFMO in soft agar and then colonies were counted using a microscope and the Image-Pro Plus software (v.6.0) program. After treatment with DFMO for 72 h, d total apoptosis and e cell cycle were analyzed by flow cytometry. The expression of markers associated with cell cycle and apoptosis was analyzed by Western blot. All data are shown as means ± S.D. of triplicate values from 3 independent experiments
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| 100.0 |
DFMO inhibits ESCC cells in vitro. a The effect of DFMO on ODC activity in KYSE450 and KYSE510 cells was measured as the release of CO2 from L-[1-C14] ornithine and results are shown as percentage of control group (set at 100%). b Anchorage-dependent cell growth was measured by MTS assay. c For measuring anchorage-independent growth, cells were cultured for 3 weeks with different concentrations of DFMO in soft agar and then colonies were counted using a microscope and the Image-Pro Plus software (v.6.0) program. After treatment with DFMO for 72 h, d total apoptosis and e cell cycle were analyzed by flow cytometry. The expression of markers associated with cell cycle and apoptosis was analyzed by Western blot. All data are shown as means ± S.D. of triplicate values from 3 independent experiments
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| 100.0 |
Treatment of ESCC cells with DFMO for 72 h resulted in a dose-dependent increase in total apoptosis (Fig. 5d), G2/M arrest and a decreased S phase cell population (Fig. 5e). Western blot analysis showed that DFMO induced an increased expression of Bax, p53, p21, p27, phosphorylation of CDK1 (Tyr15), cleavage of caspase 3 and PARP and suppressed expression of PCNA, Bcl-2 and cyclin B1.
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study
| 100.0 |
We established 3 ESCC PDX mouse models, referred to as EG20, EG5, and EG37 (Supplementary Table 1) to test the effectiveness of DFMO. None of the patients donating tumors received neo-adjuvant therapy. EG20 tumors were implanted and when the tumors reached an average volume of 250 mm3, mice were divided into 3 groups and treated with vehicle, 2 or 4% (v/v) DFMO in drinking water, respectively. From the 1st day of treatment, body weight and tumor volume were measured every 3 days. Results indicated that over 33 days of treatment, DFMO had no effect on mouse body weight compared with the vehicle-treated group. This indicated that even the highest dose of DFMO (4%) was tolerable and safe (Fig. 6a). Beginning at day 9, the average tumor volume of each DFMO treatment group appeared to be significantly smaller than the average volume of the vehicle-treated group (Fig. 6b). After 33 days of treatment, the tumor volume of the vehicle group reached 1000 mm3 and all experimental mice were euthanized and tumors extracted and weighed. Final results showed that DFMO treatment significantly decreased PDX tumor weight compared to vehicle-treated control mice (p < 0.01, Fig. 6c). No significant difference was observed in either tumor volume or in tumor weight between the two DFMO-treated groups (Fig. 6b, c), indicating that even the lowest dose of DFMO was effective. Similar results were obtained from the other two ESCC PDX models, EG5 and EG37 (Supplementary Fig. 3). Even the low dose of DFMO treatment (2%) could significantly suppress the progression of ESCC PDX tumors. We also examined the expression of PCNA and Ki-67 proliferation markers and cleaved caspase 3 and Bcl-2 apoptosis markers by IHC. Our results showed that DFMO treatment suppressed proliferation and induced apoptosis of ESCC cells (Fig. 6d).Fig. 6DFMO inhibits ESCC progression in a PDX model. A PDX model of mice implanted with human ESCC (EG20) were divided into three groups and treated with vehicle or 2% (v/v) or 4% (v/v) DFMO in drinking water for a total of 33 days. a From the 1st day of treatment, body weight and tumor volume were measured every 3 days. DFMO did not significantly reduce mouse body weight compared with the vehicle group. b From the 9th day of treatment, the average tumor volume of both DFMO-treated groups appeared to be significantly less than the vehicle-treated group. c Compared with vehicle, DFMO treatment significantly decreased the weight of the PDX tumors. d IHC analysis was performed to determine the expression levels of PCNA, Ki-67, cleaved caspase 3, and Bcl-2 in ESCC PDX tumors treated with vehicle or DFMO. Representative photographs for each antibody and each group are shown. The integrated optical density (IOD) was evaluated using the Image-Pro Plus software (v.6.0) program. All data are shown as mean values ± S.D
|
study
| 99.94 |
DFMO inhibits ESCC progression in a PDX model. A PDX model of mice implanted with human ESCC (EG20) were divided into three groups and treated with vehicle or 2% (v/v) or 4% (v/v) DFMO in drinking water for a total of 33 days. a From the 1st day of treatment, body weight and tumor volume were measured every 3 days. DFMO did not significantly reduce mouse body weight compared with the vehicle group. b From the 9th day of treatment, the average tumor volume of both DFMO-treated groups appeared to be significantly less than the vehicle-treated group. c Compared with vehicle, DFMO treatment significantly decreased the weight of the PDX tumors. d IHC analysis was performed to determine the expression levels of PCNA, Ki-67, cleaved caspase 3, and Bcl-2 in ESCC PDX tumors treated with vehicle or DFMO. Representative photographs for each antibody and each group are shown. The integrated optical density (IOD) was evaluated using the Image-Pro Plus software (v.6.0) program. All data are shown as mean values ± S.D
|
study
| 100.0 |
ODC is associated with cell growth, proliferation, and transformation, and is overexpressed in various solid cancers. Moreover, a correlation between polyamine level and tumor stage is well established in colorectal cancers.22 One group previously showed the tumor:normal ratio of ODC mRNA expression in stage III and IV ESCC specimens is significantly higher than in stage I and II ESCC specimens (p = 0.043).24 In the present study, we disclosed the expression pattern of ODC in human ESCC. ODC immunostaining was observed in both the nucleus and cytoplasm (Fig. 1), which agrees with a previous report.25 The expression of ODC was at least twice as high in ESCC compared to esophagitis or NAT (Fig. 1a). Clinically, the expression level of ODC in ESCC was significantly correlated with lymph node metastasis status and TNM stage but not with either age or gender, indicating that ODC might also function as an oncogene in ESCC.
|
study
| 100.0 |
ODC affects numerous processes in carcinogenesis through its regulation of the intracellular polyamine pool. Studies focusing on transgenic mouse models have shown an essential role of polyamines in the early promotion of tumorigenesis. One group26 reported that elevated ODC activity is sufficient to promote skin tumor formation in the carcinogen-exposed skin of K6/ODC transgenic mice, without the addition of tumor-promoting agents. Conversely, suppression of ODC expression is associated with decreased cell proliferation, increased apoptosis and decreased expression of genes affecting tumor invasion and metastasis.27, 28 Our data showed that when ODC expression in ESCC cells was knocked down, both proliferation and anchorage-independent growth of ESCC cells were significantly suppressed (Fig. 2c, d).
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study
| 100.0 |
The relationship between polyamines and apoptosis is still not well defined. Various cell lines respond differently to polyamine depletion. Yuan et al.29 reported that polyamine depletion prevented apoptosis of rat intestinal epithelial cells by decreasing caspase 3 and 9 activities, as well as the translocation of Bax to mitochondria, thus diminishing cytochrome c release. Similarly, Landau et al.30 showed that polyamine depletion in NIH3T3 cells led to cell cycle arrest but not apoptosis. In contrast, most reports demonstrated that polyamine depletion induced apoptosis in tumor cells.31, 32 The mitochondrial pathway is a primary pathway of apoptosis involving Bcl-2 family members, cytochrome c release and activation of caspase 3.33 Wildtype p53 is a tumor suppressor gene that plays a key role in DNA damage repair. But when DNA damage is beyond repair, p53 induces Bax expression, which guides cells into apoptosis.34 The homodimerization of Bcl-2 leads to anti-apoptosis signaling and Bax can heterodimerize with Bcl-2 to induce apoptosis.35 Moreover, Bax has been reported to induce cytochrome c release that in turn activates caspase 3.36 Poly ADP ribose polymerase (PARP) is an important enzyme that plays a key role in DNA repair and apoptosis.37 Once PARP has been cleaved by caspases, apoptosis is induced.38 In the present study, polyamine depletion by shODC enhanced the expression of p53, increasing the expression level of Bax. Then caspase 3 was activated and cleaved PARP, inducing apoptosis (Fig. 3). At the same time, the expression of Bcl-2 was decreased. Our results confirmed that inhibiting polyamine synthesis in ESCC cells resulted in cell death due to apoptosis.
|
study
| 100.0 |
ODC activity displays two peaks during the entire cell cycle process, one associated with the G1/S transition and the other associated with the S/G2 and G2/M phases,39 suggesting that polyamines also play key roles in cell cycle progression. A number of studies reported that polyamine depletion arrests cells at the G1 phase.40–42 Other effects, including arrest at the S or G2/M phase, have been reported in different cell lines,43–45 suggesting that polyamine depletion affects the cell cycle in a cell-type specific manner. Our data showed that knocking down ODC expression arrested ESCC cells at the G2/M phase. Correspondingly, the percentage of cells in the S phase was decreased, with no significant difference at the G1 phase (Fig. 3b). Cell cycle progression is mediated by the family of cyclin-dependent kinases, which comprise a catalytic subunit and requisite positive regulatory subunits known as cyclins. Generally, positive regulation of CDK activity is mediated by the accumulation of cyclins, whereas negative regulation is achieved by phosphorylation of the catalytic subunit or by binding with CDK inhibitors, including p21 and p27.46 Cyclin B1 contributes to the transition of cells from the G2 to M phase and is inactivated by phosphorylation of CDK1.47 In ESCC, cyclin B1 was reported to be an independent prognostic factor in patients,48 indicating that cyclin B1 plays a key role in ESCC cell proliferation. Our results showed that polyamine depletion increased the expression of wildtype p53, which in turn directly down-regulated the transcription of cyclin B1 mRNA and inactivated the CDK1/cyclin B1 complex by phosphorylation of CDK1. Moreover, as a transcription target of p53, p21 also can effectively inhibit the kinase activity of CDK1.49 Altogether, our data suggested that polyamine depletion arrested ESCC cells in the G2/M phase by decreasing the activity of the CDK1/cyclin B1 complex.
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study
| 100.0 |
Based on our in vitro studies, we examined the role of ODC in ESCC progression in a xenograft mouse model. Our results showed that when ODC expression was blocked, ESCC cell proliferation was suppressed and apoptosis induced, causing reduced tumorigenesis in nude mice (Fig. 4). The data demonstrated that ODC plays a key role in ESCC progression, and could be a potential therapeutic target against ESCC.
|
study
| 100.0 |
DFMO, an FDA-approved drug for treatment of Trypanosomiasis, is an enzyme-activated irreversible inhibitor of ODC and acts to deplete polyamine pools. The antitumor effects of DFMO on various solid tumors, including colon cancer,50 skin cancer,14 pancreatic cancer,51 and breast cancer, has been reported in both preclinical and clinical studies.52 However, DFMO is generally cytostatic in mammalian cells, causing a reduction in the rate of proliferation in the absence of cell death.53 Based on our studies using shODC, DFMO’s effect on ESCC was examined. As expected, DFMO treatment not only suppressed proliferation, but also induced apoptosis of ESCC cells by inhibiting ODC activity (Fig. 5).
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study
| 99.94 |
Despite results of research demonstrating impressive antitumor effects in preclinical studies, new therapies often fail to show significant efficacy in clinical trials, indicating that current methods, including immortalized cancer cell lines implanted into xenograft models, are suboptimal in predicting therapeutic efficacy. Major deficiencies of immortalized cancer cell line models are a lack of heterogeneity reflective of the original malignancy and an improper microenvironment.54 The PDX model is considered to be highly relevant to actual human tumor growth because the xenograft maintains the original molecular characteristics and heterogeneity.55 Thus, PDX models could have more predictive power for translating preclinical efficacy into clinical outcomes compared to xenograft models generated from established cell lines or genetically engineered mouse models. To obtain more preclinical data, we established 3 ESCC PDX model cell lines to test the effect of DFMO on ESCC growth. Results showed that DFMO treatment significantly inhibited the progression of ESCC without reducing mouse body weight. Moreover, IHC results reconfirmed that DFMO not only suppressed proliferation, but also induced apoptosis in ESCC cells, leading to the inhibition of ESCC progression. Notably, low doses of DFMO (2%) significantly inhibited the progression of ESCC PDX tumor growth whereas the high dose (4%) did not increase the effect, suggesting that in future clinical trials, increasing the DFMO dose will not enhance its antitumor effects.
|
study
| 99.94 |
In the present study, we reported high ODC expression in ESCC tissues compared with esophagitis or NAT. Our study also demonstrated that polyamine depletion not only suppressed proliferation, but also induced apoptosis of ESCC cells. Overall, the results of this study suggest that ODC is a promising target in ESCC therapy and that DFMO warrants further study in clinical trials to test its effectiveness against ESCC.
|
study
| 99.94 |
A human ESCC tissue microarray (Catalog No. ES244) was purchased from US Biomax, Inc. (Rockville, MD) and DFMO (Catalog No. K100) was from AK Scientific, Inc. (Union City, CA). The primary antibodies against ODC (Catalog No. sc-390366), Bcl-2 (Catalog No. sc-7382), Bax (Catalog No. sc-7480), p21 (Catalog No. sc-6246), p27 (Catalog No. sc-1641), cyclin B1 (Catalog No. sc-245) and β-actin (Catalog No. sc-47778) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The primary antibodies to detect cleaved PARP (Catalog No. 5625), phosphorylated ERKs (T202/Y204; Catalog No. 14474), E-cadherin (Catalog No. 3195), cleaved caspase 3 (Catalog No. 9661), p53 (Catalog No. 2524), phosphorylated CDK1 (Tyr15; Catalog No. 4539) were purchased from Cell Signaling Biotechnology (Beverly, MA) and the Ki-67 (Catalog No. MA5-14520) antibody was from Thermo Scientific (Fremont, CA). The shODC plasmids cloned into the lentiviral expression vector pLKO.1 were obtained from the University of Minnesota (Minneapolis, MN). Human shODC full hairpin sequence is #1. 5′-CCGGGCCATATGGAAGACTAGGATACTCGAGTATCCTAGTCTTCCATATGGCTTTTTG-3′; #2. 5′-CCGGGCCGACGATCTACTATGTGATCTCGAGATCACATAGTAGATCGTCGGCTTTTTG-3′; #3. 5′-CCGGCCTTGTAAACAAGTATCTCAACTCGAGTTGAGATACTTGTTTACAAGGTTTTTG-3′; #4. 5′-CCGGGCGTCTATGGATCATTTAATTCTCGAGAATTAAATGATCCATAGACGCTTTTTG-3′; #5. 5′-CCGGCCTCCAGAGAGGATTATCTATCTCGAGATAGATAATCCTCTCTGGAGGTTTTTG-3′.
|
other
| 84.1 |
Human ESCC cell lines (KYSE450, KYSE510) and the human embryonic kidney cell line (HEK293T) were purchased from American Type Culture Collection (ATCC; Manassas, VA). Each vial of frozen cells was thawed and maintained in culture for a maximum of 8 weeks. All cells were cytogenetically tested and authenticated before freezing. All cell culture conditions were performed following ATCC’s instructions.
|
other
| 99.7 |
The lentiviral packaging vectors (pMD2.0G and psPAX) were purchased from Addgene Inc. (Cambridge, MA). To prepare shODC lentiviral particles, the lentiviral vector and packaging vectors were transfected into HEK293T cells using iMFectin Poly DNA Transfection Reagent (GenDEPOT, Barker, TX) following the manufacturer’s suggested protocols. The transfection medium was changed at 8 h after transfection and then cells were cultured for 36 h. The lentiviral particles were harvested by filtration using a 0.45 µm sodium acetate syringe filter and then combined with 8 μg/ml of polybrane (Millipore, Billerica, MA) and infected overnight into 60% confluent KYSE450 and KYSE510 cells. The cell culture medium was replaced with fresh complete growth medium and after 24 h, cells were selected with 2 μg/ml of puromycine for an additional 24 h. The selected cells were used for experiments.
|
study
| 99.94 |
ODC activity was measured as the release of CO2 from L-[1-C14] ornithine as previously described.56 Briefly, cell lysates were incubated at 37 °C for 60 min after addition of 0.5 µCi L-[1-C14] ornithine hydrochloride (Perkin Elmer, Waltham, MA), 40 mM sodium phosphate buffer (pH 7.8) containing 0.64 mM pyridoxal phosphate, 0.8 µM EDTA and 8 mM dithiothreitol. The reaction was terminated by addition of 10% trichloroacetic acid. The released 14-CO2 was soaked in scintillation solution (Research Products International Corp., Mount Prospect, IL) and radioactivity was measured by scintillation counter (LS6500, Beckman Coulter, Fullerton, CA) and the results are expressed as percentage of the control, which was set at 100%.
|
study
| 99.94 |
Cells (1 × 103 per well) were seeded into 96-well plates. After overnight incubation, cells were treated with different concentrations of DFMO (0, 0.25, 0.5, 1, and 1.5 mM), and incubated for 24, 48, or 72 h. Then 20 μl Cell Titer 96 Aqueous One Solution (Promega Corporation, Madison, WI) were added and cells incubated for another 1 h. Absorbance was read at 492 nm.
|
study
| 99.94 |
Cells (8 × 103 per well) suspended in complete growth medium (Eagle’s basal medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics) were added to 0.3% agar with different doses of DFMO in a top layer over a base layer of 0.6% agar with the same doses of DFMO. The cultures were maintained at 37 °C in a 5% CO2 incubator for 3 weeks and then colonies were counted under a microscope using the Image-Pro Plus software (v.6.0) program (Media Cybernetics, Inc. Rockville, MD).
|
study
| 99.94 |
For analysis of apoptosis, cells (2 × 105 cells per well) were seeded into 6-well plates and cultured for 24 h, and then exposed to DFMO (0, 0.25, 0.5, 1, or 1.5 mM) for 72 h. Cells were trypsinized and washed twice with cold phosphate-buffered saline (PBS) solution, and then resuspended with PBS and incubated for 5 min at room temperature with annexin V-fluorescein isothiocyanate (FITC) plus propidium iodide. Cells were analyzed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA). For cell cycle analysis, cells were treated with DFMO, harvested and washed twice with PBS, and fixed with cold 70% ethanol overnight at −20 °C. Stained cells were detected and quantified by flow cytometry.
|
study
| 99.94 |
Protein concentrations of ShODC or DFMO-treated ESCC cell lysates were determined using a protein assay kit (Bio-Rad Laboratories, Inc. Hercules, CA). Total proteins (20 to 100 μg) were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore, Billerica, MA). After blocking in 5% non-fat milk, the membranes were probed with specific primary antibodies overnight at 4 °C, then washed 3 times with TBS-Tween 20 followed by incubation at room temperature 1 h with a horseradish peroxidase (HRP)-conjugated secondary antibody. The protein bands were visualized with a chemiluminescence reagent (GE Healthcare Biosciences, Pittsburgh, PA).
|
study
| 99.94 |
NU/NU nude female mice (6 weeks old; Charles River, Chicago, IL) were randomly divided into three groups of four each and inoculated in the right flank with KYSE450-shMock, KYSE450-shODC #2 or KYSE450-shODC #4 lentiviral-infected cells (3 × 106 cells permouse). Mice were maintained under “specific pathogen-free” conditions based on the guidelines established by the University of Minnesota Institutional Animal Care and Use Committee. Tumor volume and body weight were measured once a week. Tumor volume was calculated from measurements of three diameters of the individual tumor base using the following formula: tumor volume (mm3) = (length × width × height × 0.52). Mice were monitored until tumors reached 1000 mm3 total volume, at which time mice were euthanized and tumors extracted.
|
study
| 99.94 |
Human ESCC tumor fragments were obtained from three male ESCC patients who underwent esophagectomy at the First Affiliated Hospital of Zhengzhou University (Zhengzhou, Henan, China) without any neo-adjuvant therapy (Supplementary Table 1). In China, ESCC is much more common in males than in females. According to the epidemiological investigation of ESCC incidence in 2003, ESCC incidence in males is 19.68/100,000 compared to 9.85/100,000 in females. Thus for these initial studies, we used tumor fragments from male patients only, but will examine tumors from female patients in future studies. A frozen section was stained with H&E and evaluated to confirm the diagnosis. A fresh ESCC tissue fragment was collected and transferred at 4 °C in FBS-free RPMI-1640 medium with antibiotics. Within 2 h of surgical resection, the tumor tissue was trimmed, cut into 3-5 mm pieces and implanted subcutaneously in anesthetized 6–8 week old female C.B-17 severe combined immunodeficient mice (Vital River Laboratories Co., Ltd., Beijing, China). Once mass formation reached about 1500 mm3, mice of this first generation of xenografts (named G1) were sacrificed and the tumors were passaged and expanded for two more generations (named G2 and G3). When G3 tumors reached an average volume of 250 mm3, mice were divided into three groups (seven mice per group) randomly and treated with vehicle or 2% (v/v) or 4% (v/v) DFMO in drinking water, respectively. The concentration of DFMO (2% v/v in drinking water) was founded on a previous report.13 Based on average mouse daily water intake (4 ml per day), 80 mg (4 g/kg body weight) DFMO would be consumed by a mouse per day. At the same time, we also set a higher concentration group (4%) for comparison. Tumor volume and body weight were measured every three days. Tumor volume was calculated from measurements of three diameters of the individual tumors using the following formula: tumor volume (mm3) = (length × width × height × 0.52). Mice were monitored until tumors reached 1000 mm3 total volume, at which time mice were euthanized and tumors extracted. Seven animals per group were recruited to achieve statistical significance. Mice were randomly grouped and treated without investigator blinding.
|
study
| 100.0 |
Tissue sections were deparaffinized in xylene and microwaved in 10 mM citrate buffer (pH 6.0) to unmask the epitopes. Endogenous peroxidase activity was blocked by incubation for 10 min with 0.03% hydrogen peroxide in methanol. Immunohistochemical staining was performed using the indirect avidin biotin-enhanced HRP method according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA). After developing with 3,3′-diaminobenzidine, all sections were counterstained with hematoxylin and observed by microscope (200×). Quantitative analysis of IHC staining was performed using the Image-Pro Plus software (v.6.0) program (Media Cybernetics, Inc. Rockville, MD).
|
study
| 91.6 |
All quantitative results are expressed as mean values ± S.D. of triplicate values from three independent experiments. Significant differences were compared using the Student’s t test (single tailed). A p value of <0.05 was considered to be statistically significant.
|
study
| 99.44 |
The insertion of the quadriceps femoris into the patella is traditionally described as a common tendon with a tri-laminar arrangement (Andrikoula et al. 2006; Iriuchishima et al. 2012; Sonin et al. 1995; Warwick & Williams 1973; Yablon et al. 2014), with the most superficial fibers originating from the rectus femoris, the deepest layer from the vastus intermedius and the intermediate layer from the vastus lateralis and vastus medialis. Other studies have suggested that the quadriceps tendon anatomy is more variable with a two- to four-layered or even more complex organisation, often with unequal contributions from its tendinous constituents (Waligora et al. 2009; Zeiss et al. 1992). Considering the consistent components of the quadriceps muscle group, the published variability of the tendon composition seems surprising.
|
study
| 87.0 |
In recent anatomical studies, an intervening muscle, between the vastus lateralis and vastus intermedius – named the tensor vastus intermedius - has been identified (Grob et al.; Rajasekaran & Hall 2016). Depending on the relation to the adjacent vastus lateralis and vastus intermedius muscles, different morphological types of tensor vastus intermedius were identified. The aponeurosis of the tensor vastus intermedius consistently fused into the middle layer of the quadriceps tendon and inserted at the superior medial border of the patella. As this muscle was previously attributed to other parts of the quadriceps muscle group, its role in the organization of the quadriceps tendon was unknown (Grob et al.).
|
study
| 99.94 |
A thorough understanding of the architecture of the knee extensor mechanism is of clinical importance. The quadriceps tendon is involved in many orthopaedic procedures around the knee joint including surgical approaches (Apostolopoulos et al. 2010; Koninckx et al. 2014; Rossi et al. 2014), tendon injuries (Rehman & Kovacs 2015) or harvesting as a tendon graft (Geib et al. 2009; Kim et al. 2009; Lund et al. 2014; Noyes & Albright 2006). Patellar problems are also common after total knee arthroplasty (Russell et al. 2014). A better understanding of the quadriceps tendon anatomy is therefore fundamental for an improvement in surgical techniques and for the radiological interpretation of a traumatized extensor apparatus of the knee joint (Yablon et al. 2014; Zeiss et al. 1992).
|
review
| 99.9 |
The newly described tensor vastus intermedius contributes to the extensor apparatus of the knee joint (Grob et al.; Rajasekaran & Hall 2016). It can be hypothesized that the tensor vastus intermedius as a fifth component of the quadriceps muscle group might represent a specific section in the in the quadriceps tendon. It has been shown, that the aponeurotic tendon fuses into the quadriceps tendon and inserts at the superior medial border of the patella (Grob et al.; Rajasekaran & Hall 2016). The purpose of the present study was to further investigate the multi-layered structure of the quadriceps tendon with special emphasis on on all components of the extensor apparatus.
|
study
| 100.0 |
Ten cadaveric lower limbs from 7 specimens, three paired and four unpaired (5 men and 2 women; mean age at death 78 years) were investigated using macro dissection techniques. The cadaver parts were obtained from the institutional body donation program (http://www.anatomy.uzh.ch/de/koerperspende.html) following the ethical guidelines “On the use of cadavers and parts of cadavers in medical research and for pre-, postgrad and continued education and research with human subjects” by the Academy of Medical Sciences (SAMS). All lower limbs were embalmed in a formalin-based solution. The thighs were examined on the basis of a standardized dissection protocol. Each lower limb was placed supine on the dissection table. The hip joint was approached from the anterior aspect and the tensor fasciae latae muscle mobilized laterally. The femoral nerve and artery were localized via a second ilio-inguinal approach, and traced distally. With the aid of these neurovascular structures, the muscle bellies of the rectus femoris, the vastus lateralis, the tensor vastus intermedius, vastus intermedius, and vastus medialis were identified. For better visualization the rectus femoris and sartorius were transsected in the mid portion and reflected. Each muscle with its aponeurosis was traced from proximal to distal until they merged into the quadriceps tendon. Connections between the different aponeurotic layers of each muscle were studied from origin to insertion (Fig. 1), with special emphasis on corresponding muscle fibers from the medial and lateral elements. The fusing points of each layer were marked. Their distance to the patella and the distances between the fusing points were measured.Fig. 1Overview of the quadriceps muscle group including the newly discovered fifth component, the tensor vastus intermedius. Anterior view to the left thigh is shown. The red stickers mark the medial and lateral femoral condyles and center of the neck of the femur respectively. For better visualization the sartorius and rectus femoris muscle are transected and reflected. The components of the extensor apparatus are arranged like the layers of a husk of a corn (on the left top). Superficial lateral and medial fibers in the proximal aspect of the thigh are piled in deeper inner layers at the level of the quadriceps tendon. The vastus medialis is released from its insertion into the vastus intermedius, rectus femoris and patella
|
study
| 99.94 |
Overview of the quadriceps muscle group including the newly discovered fifth component, the tensor vastus intermedius. Anterior view to the left thigh is shown. The red stickers mark the medial and lateral femoral condyles and center of the neck of the femur respectively. For better visualization the sartorius and rectus femoris muscle are transected and reflected. The components of the extensor apparatus are arranged like the layers of a husk of a corn (on the left top). Superficial lateral and medial fibers in the proximal aspect of the thigh are piled in deeper inner layers at the level of the quadriceps tendon. The vastus medialis is released from its insertion into the vastus intermedius, rectus femoris and patella
|
review
| 99.9 |
All portions of the extensor apparatus fused over a region ranging from 13 to 90 mm (mean 44 mm, SD +/− 21) proximal to the superior pole of the patella medial to the mid-line of the quadriceps tendon. The different components were structured in onion-like layers or similar to a husk of a corn. Superficial lateral and medial fibers in the proximal aspect of the thigh were piled in deeper inner layers at the level of the quadriceps tendon (Fig. 1). The thickness of the quadriceps tendon increased steadily as more aponeurotic layers of the extensor apparatus joined both medially and laterally. At the patella insertion the quadriceps tendon reached its maximal thickness of 79 mm (range 65 to 95 mm, SD +/− 0.9). Deep to the quadriceps tendon between the tendon and the femur, the muscle bundles of the articularis genus extended to the suprapatellar bursa, and the synovial membrane of the knee joint. The fibers of the articularis genus did not contribute to the architecture of the quadriceps tendon, but merely fused with the dorsal side of the aponeurosis of the vastus intermedius.
|
study
| 100.0 |
The tendon of the vastus intermedius showed a complex multi-layered structure. It converged towards the patella and divided into a lateral and medial part. The lateral part of the vastus intermedius aponeurosis formed the deepest layer of the quadriceps tendon. These lateral fibers were oriented towards the medial femoral condyle (Fig. 2). The medial part of the vastus intermedius aponeurosis separated into a superficial and deep medial layer with an orientation towards the lateral femoral condyle (Figs. 2 and 3a). The fibers of the deep medial vastus intermedius aponeurosis were located above the lateral part of the vastus intermedius aponeurosis. Five to 10 mm medial to the mid-line of the quadriceps tendon, the superficial and deep layer of the medial vastus intermedius aponeurosis fused with the aponeurosis of the tensor vastus intermedius and vastus lateralis respectively. Together they formed the two-layered intermediate layer of the quadriceps tendon. Therefore, the vastus intermedius contributed first to the deep layer of the quadriceps tendon through its lateral part of the vastus intermedius aponeurosis and second to the two-layerd intermediate layer of the quadriceps tendon by its superficial and deep medial VI aponeurosis (Fig. 3). Jointly the different fibers continued towards the patella. The fusion point of the fibers for the deep intermediate layer was on average 56 mm (range, 30 to 90 mm, SD +/− 21) proximal to the patella (Fig. 3b). In the distal aspect the superficial medial layer of the vastus intermedius aponeurosis turned into a tendinous gliding layer of the vastus medialis. The latter, in turn, extended to the medial proximal margin of the patella, and in the deeper aspect to the tendon of the rectus femoris. The superficial medial layer of the vastus intermedius aponeurosis that fused with the aponeurosis of the vastus lateralis (superficial intermediate layer) met 33 mm (range, 13 to 53 mm, SD +/− 14) above the patella (Fig. 3b). The fibers of the vastus lateralis were often composed of bundles of individual thin fiber strands (Fig. 4). The meeting point of the superficial intermediate layer was always distal (23 mm, range, 12 to 41 mm, SD +/− 0.9) to the meeting point of the deep intermediate layer (Fig. 3b).Fig. 2Orientation of the multilayered structure of the extensor apparatus of the knee joint. The distal aspect of a right thigh is shown. Red stickers mark the medial and lateral femoral condyles above the knee joint space. The vastus medialis is released from its insertion into the vastus intermedius and rectus femoris (reflected laterally) freeing the view to the complex multi-layered aponeurosis of the vastus intermedius. The lateral part of the vastus intermedius aponeurosis form the deepest layer of the quadriceps tendon. The medial part of the vastus intermedius aponeurosis separates into a superficial and deep medial layer with an orientation towards the lateral femoral condyle (lateral red stick). The fibers of the medial vastus intermedius aponeurosis are located above the lateral part of the vastus intermedius aponeurosis. Generally, lateral fibers are oriented towards the medial femoral condyle (medial red stick). The blue dots mark the fusing points of the intermediate layer of the quadriceps tendon and the superior base of the patella. For better visualization the fusing points are underlined with black paper. The white dotted lines indicate the fiber direction towards the condyles Fig. 3Architecture of the multilayered quadriceps tendon. The distal aspect of a right thigh a with corresponding distal section of the quadriceps tendon b is shown. a The architecture of the quadriceps tendon consisting of the rectus femoris (R), vastus lateralis (VL), tensor vastus intermedius (TVI), vastus intermedius (VI) is shown. The medial part of the VI aponeurosis separates into a superficial and deep medial layer. Sartorius (S), patella (P). The insertions of the vastus medialis (VM) into the patella and capsule of the knee joint (blue arrow), rectus femoris (red arrow) and vastus intermedius (green arrow) is marked. Anterior insertion of the vastus medialis in the the vastus intermedius (VM ant), posterior insertion of the vastus medialis into the vastus intermedius (VM post). b The proximal two blue dots (F1 and F2) mark the fusing points of the intermediate layer of the quadriceps tendon. All portions of the extensor apparatus fuse over a region ranging from 13 to 90 mm proximal to the superior pole of the patella (distal blue dot). Lateral portions of the vastus intermedius (lateral VI) form the deepest layer of the quadriceps tendon. The superficial and deep layer of the medial vastus intermedius aponeurosis (black dotted arrows) fuse 56 mm (range, 30 to 90 mm) and 33 cm (range, 13 to 53 mm) proximal to the patella with the aponeurosis of the tensor vastus intermedius (TVI) and vastus lateralis (VL) respectively. Together they built the two-layered intermediate layer of the quadriceps tendon. The tendon of the rectus femoris (R) forms the superficial layer of the quadriceps tendon. For better visualization the fusing point are underlined with black paper. The vastus medialis is released from its insertion into the vastus intermedius and rectus femoris. Depending on the level of virtual transection one finds two, three or four layers (white arrows with numbers). An oblique transection could lead to the impression of a complex multi-layered arrangement of the quadriceps tendon Fig. 4Distal aspect of the extensor apparatus of the knee joint above the patella. The rectus femoris muscle is reflected medially displaying the anterior insertion of the vastus medialis into the vastus intermedius (see also Fig. 3a). The vastus medialis further inserts into the rectus femoris and patella. The medial deep and medial superficial layer of the vastus intermedius aponeurosis are covered by the inserting muscle fibers of the vastus medialis and therefore not visible. The aponeurosis of the vastus lateralis is separated into two fiber bundles. The fibers of the vastus lateralis and the tensor vastus intermedius are oriented towards the medial femoral condyle. Together with parts of rectus tendon the vastus medialis occupies the supero-medial half of the upper semi-circle of the patella (green dotted line). The strong muscle belly of the vastus lateralis inserts at the supero-lateral semi-circle of the patella (red dotted line)
|
study
| 100.0 |
Orientation of the multilayered structure of the extensor apparatus of the knee joint. The distal aspect of a right thigh is shown. Red stickers mark the medial and lateral femoral condyles above the knee joint space. The vastus medialis is released from its insertion into the vastus intermedius and rectus femoris (reflected laterally) freeing the view to the complex multi-layered aponeurosis of the vastus intermedius. The lateral part of the vastus intermedius aponeurosis form the deepest layer of the quadriceps tendon. The medial part of the vastus intermedius aponeurosis separates into a superficial and deep medial layer with an orientation towards the lateral femoral condyle (lateral red stick). The fibers of the medial vastus intermedius aponeurosis are located above the lateral part of the vastus intermedius aponeurosis. Generally, lateral fibers are oriented towards the medial femoral condyle (medial red stick). The blue dots mark the fusing points of the intermediate layer of the quadriceps tendon and the superior base of the patella. For better visualization the fusing points are underlined with black paper. The white dotted lines indicate the fiber direction towards the condyles
|
other
| 95.0 |
Architecture of the multilayered quadriceps tendon. The distal aspect of a right thigh a with corresponding distal section of the quadriceps tendon b is shown. a The architecture of the quadriceps tendon consisting of the rectus femoris (R), vastus lateralis (VL), tensor vastus intermedius (TVI), vastus intermedius (VI) is shown. The medial part of the VI aponeurosis separates into a superficial and deep medial layer. Sartorius (S), patella (P). The insertions of the vastus medialis (VM) into the patella and capsule of the knee joint (blue arrow), rectus femoris (red arrow) and vastus intermedius (green arrow) is marked. Anterior insertion of the vastus medialis in the the vastus intermedius (VM ant), posterior insertion of the vastus medialis into the vastus intermedius (VM post). b The proximal two blue dots (F1 and F2) mark the fusing points of the intermediate layer of the quadriceps tendon. All portions of the extensor apparatus fuse over a region ranging from 13 to 90 mm proximal to the superior pole of the patella (distal blue dot). Lateral portions of the vastus intermedius (lateral VI) form the deepest layer of the quadriceps tendon. The superficial and deep layer of the medial vastus intermedius aponeurosis (black dotted arrows) fuse 56 mm (range, 30 to 90 mm) and 33 cm (range, 13 to 53 mm) proximal to the patella with the aponeurosis of the tensor vastus intermedius (TVI) and vastus lateralis (VL) respectively. Together they built the two-layered intermediate layer of the quadriceps tendon. The tendon of the rectus femoris (R) forms the superficial layer of the quadriceps tendon. For better visualization the fusing point are underlined with black paper. The vastus medialis is released from its insertion into the vastus intermedius and rectus femoris. Depending on the level of virtual transection one finds two, three or four layers (white arrows with numbers). An oblique transection could lead to the impression of a complex multi-layered arrangement of the quadriceps tendon
|
clinical case
| 89.7 |
Distal aspect of the extensor apparatus of the knee joint above the patella. The rectus femoris muscle is reflected medially displaying the anterior insertion of the vastus medialis into the vastus intermedius (see also Fig. 3a). The vastus medialis further inserts into the rectus femoris and patella. The medial deep and medial superficial layer of the vastus intermedius aponeurosis are covered by the inserting muscle fibers of the vastus medialis and therefore not visible. The aponeurosis of the vastus lateralis is separated into two fiber bundles. The fibers of the vastus lateralis and the tensor vastus intermedius are oriented towards the medial femoral condyle. Together with parts of rectus tendon the vastus medialis occupies the supero-medial half of the upper semi-circle of the patella (green dotted line). The strong muscle belly of the vastus lateralis inserts at the supero-lateral semi-circle of the patella (red dotted line)
|
study
| 99.7 |
The superficial layer of the quadriceps tendon was formed by the tendon of the rectus femoris. Proximal to the meeting points of the two-layered intermediate layer, the aponeurosis of the rectus femoris was located directly on the lateral part of the vastus intermedius aponeurosis (= deep layer of the quadriceps tendon). In other words, 56 mm (range, 30 to 90 mm) proximal to the superior border of the patella, an intermediate layer was missing (Table 1). Therefore, at this site the quadriceps tendon was seen to be composed of two layers only, separated by thin partitions of fat. In contrast, distal to the meeting points of the two-layered intermediate layer, the quadriceps tendon was composed of four layers (Fig. 3b). The various layers of the quadriceps tendon were joined to each other through light, divisible crosswise fibers. The latter had also inlets of fatty tissue to a differing extent. Distally the superficial medial vastus intermedius aponeurosis and the deep gliding aponeurosis of the vastus medialis fused with the tendon of the rectus femoris and the patella. Together with parts of rectus femoris tendon the vastus medialis occupied the supero-medial half of the upper semi-circle of the patella (Fig. 4). Remainder of the aponeurosis of the rectus femoris continued superficial to the patella to join the patellar ligament.Table 1Fusion points of the two-layered intermediate layer of the quadriceps tendonCase Nr.sideDeep intermediate layer (mm)Superficial intermediate layer (mm)Distances between the fusion points (mm)1right3018122left4530153left7253194right9049415left8146356right5936237right6641258right4625219left34191510right341321mean55.73322.7max905341min301312Table 1 indicates the individual data (n = 10) of the fusion points (distances to the patella in millimeters) of the two-layered intermediate layer of the quadriceps tendon. The medial deep layer of the vastus intermedius aponeurosis fused with the aponeurosis of the tensor vastus intermedius (deep intermediate layer). The medial superficial layer of the vastus intermedius aponeurosis fused with the aponeurosis of the vastus lateralis (superficial intermediate layer). The meeting point of the superficial intermediate layer was always distal (23 mm, range 12 to 41 mm, SD +/− 0.9) to the meeting point of the deep intermediate layer. All elements of the quadriceps tendon fused over a region proximal to the patella ranging from 13 to 90 mm (Fig. 3b)
|
study
| 100.0 |
Table 1 indicates the individual data (n = 10) of the fusion points (distances to the patella in millimeters) of the two-layered intermediate layer of the quadriceps tendon. The medial deep layer of the vastus intermedius aponeurosis fused with the aponeurosis of the tensor vastus intermedius (deep intermediate layer). The medial superficial layer of the vastus intermedius aponeurosis fused with the aponeurosis of the vastus lateralis (superficial intermediate layer). The meeting point of the superficial intermediate layer was always distal (23 mm, range 12 to 41 mm, SD +/− 0.9) to the meeting point of the deep intermediate layer. All elements of the quadriceps tendon fused over a region proximal to the patella ranging from 13 to 90 mm (Fig. 3b)
|
study
| 100.0 |
The strong muscle belly of the vastus lateralis inserted at the supero-lateral semi-circle of the patella (Fig. 4). In a fully extended knee joint, fibers of the lateral components of the extensor apparatus were oriented towards the medial superior border of the patella and subsequently towards the medial femoral condyle. Fibers of the medial components of the extensor apparatus were oriented towards the lateral superior border of the patella and subsequently towards the lateral femoral condyle (Figs. 2, 3 and 4).
|
clinical case
| 52.84 |
In four cases an independent and strong aponeurosis of the tensor vastus intermedius was found (n = 4 independent type) (Grob et al.). In one case the aponeurosis of the tensor vastus intermedius was rather weak and greater portions of the lateral vastus intermedius aponeurosis divided into two layers. An identical pattern was found in three cases where the aponeurosis of the tensor vastus intermedius emerged from the lateral part of the vastus intermedius (n = 4 vastus intermedius type) (Grob et al.). In these situations, the anterior part of the lateral vastus intermedius aponeurosis fused with the deep medial vastus intermedius aponeurosis forming the deep intermediate layer of the quadriceps tendon. In two other cases the aponeurosis of the tensor vastus intermedius arose from the vastus lateralis (n = 2 vastus lateralis type) (Grob et al.). Thus the vastus lateralis contributed to both layers of the intermediate layer of the quadriceps tendon (Fig. 5).Fig. 5Schematic drawing of the three-layered architecture of the quadriceps tendon. The superficial layer (I) of the quadriceps tendon is formed by the tendon of the rectus femoris (R). The intermediate layer (II) is further sub-divided. The tendon of the vastus intermedius (VI) itself shows a complex multi-layered structure consisting of the lateral part of the vastus intermedius aponeurosis (lateral VI) and the medial deep and medial superficial layers of the vastus intermedius aponeurosis. The medial superficial and the medial deep layer of the vastus intermedius aponeurosis fuse with the aponeurosis of the tensor vastus intermedius (TVI) and vastus lateralis (VL), respectively. The lateral part of the vastus intermedius aponeurosis forms the deepest layer (III) of the quadriceps tendon. In some cases, the aponeurosis of the tensor vastus intermedius emerges either from the lateral part of the vastus inermedius (bended blue arrow) or vastus lateralis (bended orange arrow) (Grob et al.). The vastus medialis (VM) is not directly involved in the architecture of the quadriceps tendon. It inserts into the aponeurosis of the vastus intermedius and tendon of the rectus femoris on its anterior and posterior side (indicated by the red dots and lines)
|
study
| 99.94 |
Schematic drawing of the three-layered architecture of the quadriceps tendon. The superficial layer (I) of the quadriceps tendon is formed by the tendon of the rectus femoris (R). The intermediate layer (II) is further sub-divided. The tendon of the vastus intermedius (VI) itself shows a complex multi-layered structure consisting of the lateral part of the vastus intermedius aponeurosis (lateral VI) and the medial deep and medial superficial layers of the vastus intermedius aponeurosis. The medial superficial and the medial deep layer of the vastus intermedius aponeurosis fuse with the aponeurosis of the tensor vastus intermedius (TVI) and vastus lateralis (VL), respectively. The lateral part of the vastus intermedius aponeurosis forms the deepest layer (III) of the quadriceps tendon. In some cases, the aponeurosis of the tensor vastus intermedius emerges either from the lateral part of the vastus inermedius (bended blue arrow) or vastus lateralis (bended orange arrow) (Grob et al.). The vastus medialis (VM) is not directly involved in the architecture of the quadriceps tendon. It inserts into the aponeurosis of the vastus intermedius and tendon of the rectus femoris on its anterior and posterior side (indicated by the red dots and lines)
|
other
| 99.4 |
Published data about the structure of the quadriceps tendon are diverse. While some authors observed three layers (Andrikoula et al. 2006; Iriuchishima et al. 2012; Sonin et al. 1995; Warwick & Williams 1973; Yablon et al. 2014), others report two, three or more layers (Waligora et al. 2009; Zeiss et al. 1992). Anatomy textbooks often give no special attention to the structure of the quadriceps tendon and state briefly that the four muscular elements of the quadriceps muscle group fuse in the quadriceps tendon (Moore et al. 2014; Netter 2011; Pabst 2008; Platzer 2013; Schünke et al. 2011). In the present dissections a consistent tri-laminar structure of the quadriceps tendon was found. However, the intermediate layer could be further sub-divided. Besides this description of the laminar organization the present findings provide information about the fiber orientation and the insertion of the different components of the extensor apparatus of the knee joint into the patella.
|
study
| 99.44 |
Similar to previous reports (Andrikoula et al. 2006; Iriuchishima et al. 2012; Sonin et al. 1995; Waligora et al. 2009; Warwick & Williams 1973; Yablon et al. 2014; Zeiss et al. 1992) the present study found variations in the structure of the quadriceps tendon. However, the variability was restricted to the fusion point rather than the number or structure of the individual layers (Fig. 3).
|
study
| 100.0 |
In four cases an independent aponeurosis of the tensor vastus intermedius could be traced. In five cases the aponeurosis of the tensor vastus intermedius either arose from the vastus intermedius or the vastus lateralis. In one case an independent but weak aponeurosis of the tensor vastus intermedius was observed. However, these variations did not change the general architecture of the quadriceps tendon.
|
study
| 99.9 |
In contrast to textbooks of anatomy (Moore et al. 2014; Netter 2011; Pabst 2008; Platzer 2013; Schünke et al. 2011) we found a two-layered medial aponeurosis of the vastus intermedius. Thus, it contributed to the deepest as well as to the intermediate layer of the quadriceps tendon. The vastus medialis, as an important dynamic stabiliser against laterally directed forces, inserted in all layers of the vastus intermedius aponeurosis (Figs. 2, 3a and 5). Hence, not only the vastus medialis, but also the vastus intermedius represents a dynamic restraint to lateral tracking of the patella. In contrast to some publications (Andrikoula et al. 2006; Iriuchishima et al. 2012; Sonin et al. 1995; Warwick & Williams 1973; Yablon et al. 2014) the vastus medialis is not directly involved in the architecture of the quadriceps tendon. It inserts into the aponeurosis of the vastus intermedius and tendon of the rectus femoris on its anterior and posterior side.
|
study
| 99.94 |
We postulate six elements of the multi-layered quadriceps tendon based on the current dissection (Fig. 5): Lateral aponeurosis of the vastus intermedius, deep and superficial medial aponeurosis of the vastus intermedius, vastus lateralis, tensor vastus intermedius and rectus femoris. All these elements – with differences in fiber direction - join each other a certain distance proximal to the patella (Fig. 3). Despite the complex structure of the quadriceps tendon and individual differences its anatomic arrangement is well structured.
|
study
| 90.44 |
The situation becomes complex and confusing when the quadriceps tendon is viewed at different cross sections. There is a high variability regarding the fusing point of the superficial and deep intermediate layer (between 13 and 90 mm proximal to the superior base of the patella). This and the oblique orientation of the two-layered intermediate layer appear to be the major reasons for the published diversity of the architecture of the quadriceps tendon (Waligora et al. 2009; Zeiss et al. 1992). Depending on the level of transection or MRI cut one finds two, three or four layers (Fig. 3b). Additionally, depending on the direction of the plane the corresponding layers can be complete or incomplete. An oblique transection or MRI cut could easily lead to the impression of a complex multi-layered arrangement of the quadriceps tendon. Furthermore, the aponeurosis of the vastus lateralis, can separate into two or three fiber bundles (Fig. 4) causing additional confusion. Zeiss et al. studied the MRI appearance of 52 knees with normal tendons. They described that the interpretation of the architecture of the quadriceps tendon is especially difficult in its intermediate layer. They found that the number of laminations was variable, with either two (30 %), three (56 %) or four layers (6 %). In 8 % of the knees, the laminations were barely visible (Zeiss et al. 1992).
|
study
| 61.72 |
In contrast to previous investigations, the present study traced all components of the extensor apparatus from the origin to insertion (Figs. 1 and 3). This enabled us to outline the different layers over the whole expansion of the muscle components. The architecture of the quadriceps tendon based on cross- and longitudinal transections (Waligora et al. 2009; Zeiss et al. 1992) is limited and makes an interpretation difficult or even impossible.
|
study
| 100.0 |
The medial and lateral muscle fibers of the extensor apparatus lie opposite each other and join 5 to 10 mm medial to the mid-line of the tendon. This arrangement and its orientation towards the medial and lateral femoral condyles support the view that medial and lateral forces of the quadriceps muscle group balance each other. The vastus lateralis, the tensor vastus intermedius and the lateral part of the vastus intermedius counterbalance the medial parts of the vastus intermedius (superficial and deep layer) and the inserting vastus medialis. The rectus femoris also predominantly inserts into the medial aspect of the superior border of the patella. The vastus intermedius and rectus femoris provide an extensive area for the attachment of the vastus medialis (Fig. 3a).
|
study
| 99.94 |
A quadriceps tendon graft may be used to reconstruct the anterior cruciate ligament (Crall & Gilmer 2015; Geib et al. 2009; Lee et al. 2016; Lee et al. 2007; Marshall et al. 1979; Slone et al. 2015), the posterior cruciate ligament (Chen et al.; Chen et al. 2004; Wu et al. 2007), the medial patellofemoral ligament (Lenschow et al. 2015; Steiner et al. 2006), the lateral collateral ligament (Chen et al. 2001) and the Achilles tendon (Arriaza et al. 2016). This autograft shares biological and mechanical properties with other grafts such as the patellar ligament or hamstrings, sometimes with superiority (Han et al. 2008). Harvesting the quadriceps tendon (with or without patellar bone) might have an impact on the function of the extensor apparatus of the knee joint as a whole. The removal of a tendon graft probably alters the delicate interplay between different layers of the extensor apparatus. Chen et al. reported that 9 % of subjects exhibited donor site pain after quadriceps graft harvesting, and the risk of occult partial rupture of the remaining quadriceps tendon may exist. Late quadriceps tendon rupture at the donor site following harvesting of a quadriceps tendon graft has been reported (Pandey et al. 2015). Loss of quadriceps muscle strength of 20 % after harvesting the quadriceps tendon graft for anterior cruciate ligament reconstruction and prolonged weakness of knee extension strength, predominantly in women, have also been reported (Chen et al. 2006; Yasuda et al.). However, others report low donor-site morbidity when using a quadriceps tendon graft compared to a bone tendon bone graft of the patellar ligament (Gorschewsky et al. 2007; Han et al. 2008; Lund et al. 2014). The harvesting technique may also impact the outcome. For example, if the quadriceps tendon is harvested at the fusing points (Fig. 3b) it is questionable that such a graft is suitable as a firm graft. A harvest of the quadriceps tendon medial to the fusing points of the intermediate layers violates the insertion of the vastus medialis with potential consequences on the terminal phase of extension and patellar stability (Lieb & Perry 1971; Pocock 1963; Toumi et al. 2007). On the other hand a lateral harvest of the quadriceps tendon compromises the insertion of the vastus lateralis and the tensor vastus intermedius. Based on the present anatomic findings it can be assumed that a harvest of a tendon graft lateral of the fusing points of the two-layered intermediate layer would be of better quality than a medial graft removal (Fig. 3a). Questions arise whether a partial- or full-thickness graft should be harvested and how closure of tendon defects should be performed. Latter questions also arise with regards to parapatellar approaches to the knee joint.
|
review
| 99.9 |
In conclusion, the findings of the present study revealed a complex but constant architecture of a three-layered quadriceps tendon which is formed by six elements. These are 1. lateral aponeurosis of the vastus intermedius, 2. deep and 3. superficial medial aponeurosis of the vastus intermedius, 4. vastus lateralis, 5. tensor vastus intermedius and 6. rectus femoris. These elements of the extensor apparatus join each other proximal to the patella in a complex onion-like architecture. Its two-layered intermediate layer shows variable fusions points. The vastus medialis contributes to the quadriceps tendon with its medial insertion into all layers of the quadriceps tendon. Further studies are needed to translate the anatomical findings into clinical relevance in patellofemoral pathology or knee surgery.
|
study
| 100.0 |
Our study has few limitations. Inter individual differences between specimens’ height were not considered in the present study. An other important limitation is that the quadriceps tendon was investigated in embalmed cadaveric specimens from elderly donors. Age-related muscle atrophy may well have distorted some results. In addition, embalmed tissue has been reported to shrink by 2.2 to 12 % (Cutts 1988; Friederich & Brand 1990). This could have affected absolute values for variables such as measured fusing points of each layer, their distance to the patella and the distances between the fusing points of the quadriceps tendon and therefore are not likely to be representative of normal healthy adults. Nevertheless, the fundamental architecture of the quadriceps muscle group is likely to have been preserved. Considering the complexity of the quadriceps tendon further investigation of its morphology in healthy young individuals is warranted.
|
study
| 100.0 |
Activators of 5′-AMP-activated protein kinase (AMPK) are widely used for treatment of metabolic disorders, type 2 diabetes, heart disease, anti-proliferative therapy and sports medicine. AMPK, a heterotrimeric serine/threonine protein kinase, is activated in response to reduced intracellular ATP to AMP ratio and controls numerous metabolic pathways. Activation of this kinase improves glucose uptake, fatty acid oxidation and insulin sensitivity in skeletal muscles, carbohydrate and lipid metabolism in liver and adipocytes, and release of insulin by pancreatic β cells (Long and Zierath, 2006).
|
review
| 99.8 |
A variety of medications work through AMPK and its downstream pathways. For instance, the biguanide drug metformin, in use since the 1950s, is a very effective agent for the treatment of type 2 diabetes, insulin resistant states, pre-diabetes, and cancer (Rojas and Gomes, 2013). Metformin acts primarily in the liver by reducing glucose output and, secondarily, by augmenting skeletal myocyte glucose uptake. Many mechanisms are involved in the beneficial effects of metformin on cellular metabolism; the activation of an upstream kinase, liver kinase B1 (LKB1), increased cellular AMP levels, and consequent AMPK activation are considered to be key mechanisms for the action of this drug (Zhou et al., 2001; Grahame Hardie, 2014).
|
review
| 99.9 |
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