Temperature, solar radiation and water are major important variables in ecosystem models which are measurable via wireless sensor networks (WSN). Effective data analysis is necessary to extract significant spatial and temporal information. In this work, information regarding the long term variation of seasonal field environment conditions is explored using Hilbert-Huang transform (HHT) based analysis on the wireless sensor network data collection. The data collection network, consisting of 36 wireless nodes, covers an area of 100 square kilometres in Yanqing, the northwest of Beijing CBD, in China and data collection involves environmental parameter observations taken over a period of three months in 2011. The analysis used the empirical mode decomposition (EMD/EEMD) to break a time sequence of data down to a finite set of intrinsic mode functions (IMFs). Both spatial and temporal properties of data explored by HHT analysis are demonstrated. Our research shows potential for better understanding the spatial-temporal relationships among environmental parameters using WSN and HHT. Most popular way to get the HSA results is by using the Hilbert transform . Bedrosix(t), find all the local maxima of the absolute value of the data. Next, connect all these maxima points with a cubic spline curve to form an empiric envelope of the data, described as e1(t).For the given IMF data e1(t) to normalize x(t), that is, y1(t) as the normalized data. Note that the normalized data may still have amplitudes higher than 1 occasionally [x(t) replaced by y1(t) in order to remove any results of this type. Then after n iterations, we get yn(t) are less than or equal to 1, the normalization is finished and turn to Step 3. We need to find its envelope a(t), and carrier cos θ(t), that x(t) = a(t)cosθ(t) = a(t)F(t). The FM part of the x(t), is given bya(t), is defined asUse sionally . Back toAccording to , computeCalculate instantaneous frequency (IF) asThe method above does not need HT and enables us to get exact IF and IA. The Hilbert energy spectrum could also be defined as the squares of the amplitudes. In the subsequent sections, we will use traditional EMD and EEMD to decompose field environment data into IMFs. Then we will use NHT and DQ to process IMFs to calculate IF and IA. The results will be compared and analysed later.2. Total 36 WSN nodes consisting of 108 sensors were deployed in three groups across 3 rural townships. WSN nodes in each group are further divided into several sub-groups as shown in The wireless environment sensor networks (WSN) for data collection in this paper were deployed as a part of the China Next Generation Internet (CNGI) project. This WSN is used to continually monitor crop field environment parameters, including land surface temperature, soil moisture and solar radiation, for the entire growing season. The monitoring field is located in Yanqing County, just outside of the Great Wall in northwest of Beijing CBD and field area is approximately 100 kmWe installed three types of sensors on each of WSN nodes: infrared thermometer (TPT300V/2-B), soil moisture sensor (SWR2) and photonic sensor (SQ-222/SQ-225). The instrumentation was completed in June 2011, with 36 infrared thermometers, 36 soil moisture sensors and 36 photonic sensors. From July to September, more than 230,000 sensor measurements were taken with measurement interval 1 hour and they were stored in a central database.μmol m-2s-1.In http://rcada.ncu.edu.tw/. The iteration stopping criteria I and II used in our algorithm are the same as that described in Section 2.1. In EEMD, the standard deviation of added white noise is set to 0.4, and ensemble time NE is 100. The results of the decomposition of data collected from group 1–2 are shown in All sensor measurements from each WSN nodes were decomposed separately for each of environmental parameters . The HHT on data was carried out using the MATLAB program which was coded based on the source code available at 3 in r(n) reaches its peak in the middle of July, then it slowly decreases between the end of July and September. Since residue r(n) represents “trend” of the original data to be decomposed, this curve displays the temperature trend in Yanqing’s summer correctly. The residue curve of PAR in In Since each IMF represents different intrinsic modes of oscillation, it is possible to calculate the period for each of IMFs. 1 to IMF4 in all three parameters. In particular, the average periods for both moisture and solar radiation values of IMF4 are approximately 24h and for temperature values of IMF3 24h. We highlight those values in bold in Although the three kinds of field environment parameters  are physically dissimilar and are measured in different instruments, the decomposed data show nearly identical periods for the lower IMFs such as IMF8 but not as accurate as that in IMF4. The higher IMFs, which represent the longer periodic oscillations of the original data, have larger differences because there are fewer cycles to average over fluctuations in instantaneous frequencies, as we can find in IMF7,8,9.Moreover, we can see from X (the independent variable) and Y (the dependent variable). If Y tends to increase when X increases, the Spearman correlation coefficient is positive. If Y tends to decrease when X increases, the Spearman correlation coefficient is negative.All the sensor measurements were decomposed into their IMFs. Then we use Spearman’s rank correlation to analyse the relationship among these IMFs. The Spearman correlation coefficient is “nonparametric”, i.e., it can be obtained without prior knowledge of the joint probability distribution for the data to be compared. A high value of the Spearman correlation coefficient implies a stronger relationship between two data series. The sign of the Spearman correlation indicates the direction of association between Temporal correlation of our measurements is discovered by two steps using time series of IMFs. First, we calculate the Spearman’s rank cross-correlation coefficients of each IMFs of parameter pairs , ,  in and between each subgroup with IMFs combination respectively, then we calculate the average cross-correlation coefficients value of each IMFs combination. Average results of parameter pairs , ,  are shown in It is not surprising that the IMFs of temperature are highly correlated with those of solar radiation. These values in From 3-IMF3, IMF4-IMF3, IMF4-IMF4) are smaller than those in the  pairs, which indicates that soil moisture and air temperature are less correlated compared to that between air temperature and PAR. 6 –IMF9) mean that if temperature tends to increase when soil moisture decreases and vice versa. Long-term soil moisture-temperature interactions have only received attention recently in the climate modelling community [Negative correlation values appear in ommunity , 33. Redommunity . The 4th3 and IMF4 are substantially smaller than those in The correlation matrix of soil moisture and PAR  pairs) are shown in The value of correlation matrix  in In this subsection, we investigate how the environmental parameters measured by WSN nodes vary at different geographical locations. First we calculate each IMF’s Spearman’s rank cross-correlation coefficients for each of parameter pairs ,  and  obtained from the corresponding WSN node pair described in 8 and IMF9, little influence of distance on soil moisture’s correlation is observed on such long-time scales.From 3) where all correlation values are above 0.95. Linear regression lines and functions show that the distance has much less influence on land surface temperature than on those of soil moisture. This paper presents a detailed analysis using the HHT based data analysis tools of the environmental data collected with a WSN, deployed in Yanqing, northwest of Beijing CBD in 2011, which provides medium regional-scale measurements of environment parameters for continuous crop field monitoring. The parameters include LST, soil moisture and solar radiation. We introduce the Hilbert-Huang transform as an empirical but adaptive tool to analyse the data set which was collected hourly over a three-month period. HHT based data analysis shows its ability to detect physically meaningful internal components and explain the spatial-temporal relationships among these environmental parameters. It provides new insights of the data with respect to the temporal and spatial variation of agricultural environment. Specifically,This work shows strong localized spectrum information that enables abnormal values to be easily detected using EMD and EEMD techniques. Periodic information, especially the 24 hours cycle, is able to be extracted from data for all observed parameters.Comprehensive correlation analysis is done by calculating the Spearman’s rank cross-correlation coefficients. Temporally, IMF plots illustrate that temperature is highly correlated with solar radiation (close to 0.8) and temperature is also correlated with soil moisture in an approximate diurnal cycle. We confirm from the IMF analysis that the above mentioned correlations are high for spatially widely separated locations. Spatial correlation of soil moisture is significant only when the sensed spots are closely spaced. In addition, IMF plots show a stable correlation between land surface temperature and solar radiation across the measured region.It may be possible to obtain more interesting results if we use HHT to analyse longer environmental data sets when they are available. In addition to the intrinsic relationships among land surface temperature, soil moisture and solar radiation, the HHT based analysis may provide interesting view of the relationship between those mentioned parameters with respect to wind changes and reveal environmental changes as a result of global climate changes. Furthermore, machine learning methods have provided some solutions that achieved excellent performance in a wide variety of fields such as event detection, data-driven decision making and so on ,19. SupeS1 FileThis file includes the photon data of nodes 19 and 23.(RAR)Click here for additional data file.S2 FileThis file includes the soil moisture data of nodes 19 and 23.(RAR)Click here for additional data file.S3 FileThis file includes the temperature data of nodes 19 and 23.(RAR)Click here for additional data file.


Activating mutations in fibroblast growth factor (FGF) receptor 3 and inactivating mutations in the NPR2 guanylyl cyclase both cause severe short stature, but how these two signaling systems interact to regulate bone growth is poorly understood. Here, we show that bone elongation is increased when NPR2 cannot be dephosphorylated and thus produces more cyclic GMP. By developing an in vivo imaging system to measure cyclic GMP production in intact tibia, we show that FGF-induced dephosphorylation of NPR2 decreases its guanylyl cyclase activity in growth plate chondrocytes in living bone. The dephosphorylation requires a PPP-family phosphatase. Thus FGF signaling lowers cyclic GMP production in the growth plate, which counteracts bone elongation. These results define a new component of the signaling network by which activating mutations in the FGF receptor inhibit bone growth. Between birth and puberty, the bones of mammals grow drastically in length. This process is controlled by many proteins, and mutations affecting these proteins can cause bones to either be too long or too short. For example, mutations of a protein called the fibroblast growth factor receptor, or FGF for short, and a protein called NPR2, can cause similar forms of dwarfism – a condition characterized by short stature.The FGF protein controls bone growth, and people with overactive receptors for FGF suffer from a form of dwarfism known as achondroplasia, while people that lack FGF receptors have longer bones. The NPR2 protein, on the other hand, produces a molecule called cGMP, which is necessary for the bones to grow. When NPR2 is blocked, less cGMP is produced, which results in shorter limbs.Previous studies of bone cells grown in the laboratory have shown that these two proteins are linked by a chain of chemical messages. When the FGF receptor is active, phosphate molecules are removed from the NPR2 protein, which reduces the amount of GMP produced. However, until now it was not known whether this mechanism also controls growth in actual bones.Here, Shuhaibar et al. used genetically modified mice in which the phosphate group could not be removed from their NPR2 enzyme. As a result, the bones of these mice were longer than usual. Shuhaibar et al. then developed an imaging technique to examine the region in the bone were growth happens. To see whether FGF reduces the amount of cGMP produced by NPR2 in these areas, cGMP was detected with a fluorescent sensor in order to be tracked.In normal mice, the FGF receptor reduced the rate at which cGMP was produced, but in mice with mutated NPR2, this did not happen. When the cells could not remove the phosphates from NPR2, cGMP levels stayed high and the bones grew longer.These findings reveal new insights into the molecular causes of dwarfism. The next step will be to identify the enzyme responsible for removing phosphate from NPR2. Blocking its activity could help to enhance bone growth. In the future, this could lead to new drug treatments for achondroplasia. Longitudinal growth of limbs and vertebrae depends on division and differentiation of chondrocytes within the cartilage growth plates located at each end of the growing bone , resulting in formation of a scaffold that is subsequently mineralized . These pNpr2 were globally replaced with a sequence encoding NPR2-7E (7E/7ENpr2) demonstrated that dephosphorylation of NPR2 is a physiological mediator of hormonal signaling in ovarian follicles (Activation of the NPR2 guanylyl cyclase requires the extracellular binding of C-type natriuretic peptide (CNP) and also the phosphorylation of multiple intracellular juxtamembrane serines and threonines  Figure . Previouollicles . These fAnother essential regulator of bone elongation is FGF receptor 3 (FGFR3); activating mutations of FGFR3 inhibit bone growth, causing achondroplasia, in which human height is reduced by ~25% . Convers7E/7ENpr2 mice in which NPR2 cannot be inactivated by dephosphorylation, and found that their bones are longer than those of wild type mice. We then developed a live tissue imaging system for monitoring the guanylyl cyclase activity of NPR2 in intact growth plates from wild type and 7E/7ENpr2 mice that express a FRET sensor for cGMP. Using this system, we found that FGF-induced dephosphorylation and inactivation of NPR2 decreases cGMP production in growth plate chondrocytes, thus contributing to FGF-dependent decreases in bone growth.Here we tested this hypothesis by investigating the growth of bones in 7E/7ENpr2 mice, in which NPR2 is modified to mimic a constitutively phosphorylated protein  was added at a saturating concentration (0.5 µg/ml), along with 1 µg/ml heparin . Heparin was included because it enhances FGF receptor activation (Cyclic GMP production was measured using tibias dissected from newborn mice (day 0–1) that globally expressed one or two copies of the cGi500 FRET sensor inserted into the 26 locus . Isolate26 locus . This wativation . ControlAfter the indicated pre-incubations, the distal half of the tibia was cut off, and the proximal piece placed, cut edge up, in a 600 µm deep channel in a plastic slide with access wells for perfusion  . SiliconThe growth plate was imaged using a Pascal confocal system . Imaging was performed at ~34°C. The whole growth plate was imaged with a 10x/0.45 N.A. water immersion objective . For FRETo prepare samples for cGMP ELISA measurements, tibias from newborn mice were treated as described in the legend for To prepare samples for western blotting , epiphys In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Fiona Watt (Senior Editor) and three reviewers, one of whom, Gail Mandel (Reviewer #1), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: Eric Espiner (Reviewer #2).Thank you for submitting your article "Dephosphorylation of the NPR2 guanylyl cyclase contributes to inhibition of bone growth by fibroblast growth factor" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This work illustrates the use of a sensor for cGMP production, in a new intact preparation of mouse bone growth, to show in real time measurements that the NPR2 guanylyl cyclase is part of the FGF pathway for impairing bone growth. Prior studies of NPR2 GC activity have been confined to extracts from cell lines, and while mutations in both proteins cause forms of human dwarfism, whether the two proteins represent parallel or interdependent pathways is not known.Essential revisions:The reviews are explicit for concerns raised by the reviewers, and all three full reviews are attached for your consideration. While we request a point by point response to all of the concerns, in particular, in a revised manuscript, we expect a substantial revision. The major points that are a concern are:1) Although perhaps not the point of the figure, please add data or provide explanation of tibial growth in the absence of CNP.2) Please address whether there are any changes in cGMP levels in WT tibia in the absence of CNP. While we realize that your study measures the production of cGMP, and not basal levels, it might strengthen the real-time measurements if you could provide more information on the basal levels.3) Related to this point, if feasible, an independent experiment supporting the idea that preventing dephosphorylation prevents the FGF effect in the absence of CNP, such as use of a phosphatase inhibitor, would strengthen the study.Reviewer #1:Previous studies have shown that the FGF3 receptor (FGFR3) pathway and, independently, the natriuretic peptide receptor 2, NPR2, are involved in bone growth. Mutations in both proteins cause forms of human dwarfism. However, whether the two proteins represent parallel or interdependent pathways is not known. Here, authors test whether NPR2 is in the FGF3R pathway in the physiological context of mouse bone growth. Using a new and elegant ex vivo preparation of intact tibia from mice expressing a form of NPR2 that cannot be dephosphorylated, authors show that bones are longer in these mice compared to WT mice. Then, by monitoring NPR2 guanylyl cyclase activity, in the intact growth plates, they show that FGF-dependent decreases in bone growth involve FGF-induced dephosphorylation of NPR2, with resulting decreases in cGMP in the growth plate chondrocytes. Thus, part of the mechanism by which FGF signaling inhibits bone elongation is through decreasing NPR2 activity. Of note, the intact system, in combination with the sensor, allow for rapid manipulation of the growth milieu as well as quantitating, in real time, changes in the signaling pathway. Its use here should elevate interest in using it for cGMP measurements in many other physiological contexts in addition to bone growth. Thus, the work will appeal to a wide range of developmental scientists, outside of those having a particular interest in bone growth.I have one major concern that authors should consider addressing in a revised manuscript. While the authors point out that mutations in NPR2 result in a human form of dwarfism, the effects on bone length that they see in the mouse with the NPR2 phospho mimic mutant (~8-14%) are perhaps smaller than one might expect if the mouse model recapitulates the human condition. Authors should clarify in the Discussion their interpretation of the mouse bone length phenotype, observed in this study, with regard to the human phenotype resulting from the NPR2 mutation. They do have a nice discussion relating CNP levels and rescue of FGFR3 achondroplasia, but I could not easily relate their mouse results with the patient NPR2 dwarfism phenotype (the acromesomelic dysplasia Maroteaux type dwarfism) mentioned in the Introduction. Otherwise, with the exception of a couple of things noted below, the paper is well written for a broad audience and the data is very clearly presented.7E/7ENpr2 mice have longer bones due to an intrinsic effect on bone, doesn't seem quite right. I know what they mean, but not sure this is the best way to present it. Might "direct effect of phosphorylation" be better?The second sentence in the Abstract is very confusing with "NPR2 cannot be dephosphorylated" and then "is opposed when NPR2 is dephosphorylated. In Results, the use of the word intrinsic to describe the muscle effects is a bit confusing/awkward. For example, the title in the Results section: Reviewer #2:Having shown that dephosphorylation of nprb by FGFR3 reversibly impairs GC-B activity – but not a phosphomimetic mutant – in rat chondrosarcoma cells , the current study reports that this molecular action of FGF contributes to impaired long bone growth in vivo in mice. The evidence for this is based on i) mitigated inhibition of FGF induced long bone growth in mice with genetic modifications in the CNP receptor preventing dephosphorylation and ii) novel imaging techniques, used ex vivo in intact tissues, showing concordant changes in cGMP within growth plate chondrocytes depending on the phosphorylated state of nprb induced by FGF activity.General Comments. While not surprising in view of their recent report in Cell Signaling, the findings are important new observations that make significant contributions to our understanding of the molecular events underpinning the interplay of two important pathways regulating mammalian long bone growth. The hypotheses are clearly stated and logically pursued in the experimental approach. The methods, genetic models and analyses all appear to be valid, as are the interpretation of the findings.Specific comments.1) In the Introduction, the authors appear to extrapolate findings from NO induced cGMP effects on bone density (JBMR 2017) to similar effects induced by CNP activation of cGMP . However the report of Kondo et al. relates to increased bone turnover-not increased bone density. To date, there is no convincing evidence that CNP promotes bone density per se.2) Introduction, third paragraph. The authors refer to mice lacking FGFR3 having longer bones: they should also cite a similar phenotype in humans with FGFR3 LOF (Makrythanasis Human Mut 2014).3) Reviewer #3:In humans and mice, the short bone phenotype caused by activating mutations in FGFR3 resembles that seen with loss-of-function mutations in NPR2 (or its ligand CNP), and pharmacological doses of hydrolysis-resistant CNP in humans or over-expression of CNP in transgenic mice largely corrects the mutant FGFR3 phenotype. Activation of NPR2 requires both receptor phosphorylation (on seven S/T residues) and binding of CNP; FGF treatment of chondroblastic cells causes NPR2 de-phosphorylation and reversible inhibition of CNP-induced NPR2 activation (as described in an accompanying manuscript by the authors). In the present study, the authors want to test the hypothesis that FGF signaling decreases NPR2 activity by dephosphorylating the receptor in vivo, using existing mice with ubiquitous expression of a cGMP-sensitive FRET sensor and mice carrying a phospho-mimetic mutant NPR2(7E).Consistent with previous data in mice and humans with activating NPR2 mutations, the authors find that the NPR3(7E) mice have longer bones than wild type mice, and tibiae from mutant mice cultured in the presence of CNP grow faster than tibiae from wild type mice. These data suggest either increased basal guanylate cyclase activity of the mutant receptor, or enhanced sensitivity to CNP. Unfortunately, tibial growth in the absence of CNP was not evaluated.The authors demonstrate the feasibility of measuring CNP-induced cGMP generation in intact growth plates in bones isolated from cGMP sensor mice, thereby expanding the number of live tissues in which cGMP generation can be monitored in real time. It appears that the basal CFP/YFP emission ratio in the absence of CNP varies between growth plate preparations e.g.,  and 3, bNo difference in basal cGMP concentrations could be detected between mutant and wild type growth plates, but the assay may not be suitable to detect small changes in basal cGMP concentrations. Therefore, one cannot conclude that NPR2(7E) chondrocytes in vivo have higher cGMP concentrations compared to wild type chondrocytes (as stated at the end of the Results) – but one can speculate that this may be the case given the bone overgrowth phenotype of NPR2(7E) mice. Does treatment with FGF (or phosphatase inhibitors) decrease the CFP/YFP ratio in wild type growth plates in the absence of CNP?Overall, experiments were carefully executed and clearly described, but not all conclusions are supported by the data shown. For some of the conclusions, the biochemical properties of the mutant receptor need to be discussed and contrasted to the wild type receptor . The most novel aspect of the study is the measurement of CNP-induced cGMP concentrations in growth plates, opening the door to assessing the effects of other hormones on cyclic nucleotides. However, the data presented are sparse – there are only two figures and the illustration of the FRET method.  Essential revisions:The reviews are explicit for concerns raised by the reviewers, and all three full reviews are attached for your consideration. While we request a point by point response to all of the concerns, in particular, in a revised manuscript, we expect a substantial revision. The major points that are a concern are:1) Although perhaps not the point of the figure, please add data or provide explanation of tibial growth in the absence of CNP.7E/7ENpr2and wildtype tibia growth in culture to include results of culturing tibias without CNP. The results without CNP are presented in 7E/7ENpr2. We also improved the method of analysis used for 7E/7ENpr2tibias cultured in the presence of CNP showed a 14% higher rate of elongation of the epiphysis (We expanded the data on piphysis .2) Please address whether there are any changes in cGMP levels in WT tibia in the absence of CNP. While we realize that your study measures the production of cGMP, and not basal levels, it might strengthen the real-time measurements if you could provide more information on the basal levels.We addressed the question of basal cGMP by adding ELISA measurements of cGMP in epiphyses of newborn mice new . We used3) Related to this point, if feasible, an independent experiment supporting the idea that preventing dephosphorylation prevents the FGF effect in the absence of CNP, such as use of a phosphatase inhibitor, would strengthen the study.7E/7ENpr2mutation, cantharidin inhibits the decrease in CNP-dependent cGMP production in response to FGF. These experiments provide independent evidence that preventing dephosphorylation prevents the decrease in NPR2 activity, and also indicate that a PPP family phosphatase is responsible for the dephosphorylation. This finding supports the concept of using related phosphatase inhibitors for treatment of achondroplasia.We added experiments to test the effect of the PPP family phosphatase inhibitor cantharidin on the FGF-induced decrease in cGMP production new . Like thReviewer #1:[…] I have one major concern that authors should consider addressing in a revised manuscript. While the authors point out that mutations in NPR2 result in a human form of dwarfism, the effects on bone length that they see in the mouse with the NPR2 phospho mimic mutant (~8-14%) are perhaps smaller than one might expect if the mouse model recapitulates the human condition. Authors should clarify in the Discussion their interpretation of the mouse bone length phenotype, observed in this study, with regard to the human phenotype resulting from the NPR2 mutation. They do have a nice discussion relating CNP levels and rescue of FGFR3 achondroplasia, but I could not easily relate their mouse results with the patient NPR2 dwarfism phenotype (the acromesomelic dysplasia Maroteaux type dwarfism) mentioned in the Introduction. Otherwise, with the exception of a couple of things noted below, the paper is well written for a broad audience and the data is very clearly presented.7E/7ENpr2 genotype. These human mutations result in overly long bones. For example one such patient showed a 14% increase in height (at 15 years of age) compared to the mean , an effect similar in magnitude to what we see with 7E/7ENpr2. In the revised manuscript, we mention this comparison in discussing our results. In contrast, patients with acromesomelic dysplasia type Maroteaux (ADMD) show an ~ 30% decrease in height , rather than an increase . We revised the Introduction to clarify that the mutation we describe is due to an opposite change in NPR2 function compared to that occurring in ADMD. Like the human NPR2 mutation that increases NPR2 activity and bone growth, the 7E/7ENpr2 genotype has a relatively modest effect on bone length. However, mechanistic understanding of this mutation could contribute to identification of strategies to oppose other conditions that result in severe decreases in height.Since the NPR2-7E mutation has not been described in humans, the closest comparison that we can make is to patients with activating mutations in the NPR2 catalytic domain that constitutively activates the enzyme and thus resembles the increased activity seen with the The second sentence in the Abstract is very confusing with "NPR2 cannot be dephosphorylated" and then "is opposed when NPR2 is dephosphorylated.We reworded the Abstract to avoid this confusion.7E/7E mice have longer bones due to an intrinsic effect on bone, doesn't seem quite right. I know what they mean, but not sure this is the best way to present it. Might "direct effect of phosphorylation" be better?In Results, the use of the word intrinsic to describe the muscle effects is a bit confusing/awkward. For example, the title in the Results section: Npr2We revised the wording as suggested.NPR2 is expressed in the growth plate even before birth , so it might seem surprising that no difference in bone length is seen at birth. One possible explanation is that the NPR2 agonist, CNP, might not be expressed until after birth. Possibly growth becomes NPR2 dependent in parallel with the development of CNP expression within the first few weeks after birth. To our knowledge, the developmental time course of CNP expression in the growth plate has not been examined.This information was added to the figure legend now .Reviewer #2:[…] Specific comments.1) In the Introduction, the authors appear to extrapolate findings from NO induced cGMP effects on bone density (JBMR 2017) to similar effects induced by CNP activation of cGMP . However the report of Kondo et al. relates to increased bone turnover-not increased bone density. To date, there is no convincing evidence that CNP promotes bone density per se.We omitted the sentence and references about bone density.2) Introduction, third paragraph. The authors refer to mice lacking FGFR3 having longer bones: they should also cite a similar phenotype in humans with FGFR3 LOF (Makrythanasis Human Mut 2014).Thank you for pointing out this reference, which we added to the Introduction.3) We added this information to the legend of the current Reviewer #3:[…] Consistent with previous data in mice and humans with activating NPR2 mutations, the authors find that the NPR3(7E) mice have longer bones than wild type mice, and tibiae from mutant mice cultured in the presence of CNP grow faster than tibiae from wild type mice. These data suggest either increased basal guanylate cyclase activity of the mutant receptor, or enhanced sensitivity to CNP. Unfortunately, tibial growth in the absence of CNP was not evaluated.7E/7ENpr2.We added data addressing this question . This fiThe authors demonstrate the feasibility of measuring CNP-induced cGMP generation in intact growth plates in bones isolated from cGMP sensor mice, thereby expanding the number of live tissues in which cGMP generation can be monitored in real time. It appears that the basal CFP/YFP emission ratio in the absence of CNP varies between growth plate preparations e.g.,  and 3, bThank you for pointing out that in the example shown in CNP treatment results in a robust FRET change that is larger in mutant compared to wild type growth plates, and pre-treatment with FGF reduces cGMP accumulation in wild type, but not mutant growth plates. The authors conclude that "FGF signaling reduces cGMP production by dephosphorylating NPR2 in the growth plate." However, there is no data presented (or cited) to correlate NPR2 phosphorylation with GC activity.In the revised manuscript, we added a sentence to the Introduction to refer to previous papers that correlate NPR2 phosphorylation and activity. We also added a sentence to the Results to clarify that we have not yet succeeded in establishing methods to measure the phosphorylation state of NPR2 from growth plate tissue, and thus have not been able to determine whether the dephosphorylation seen in the chondrocyte cell line  also occurs in vivo.The 7E substitutions may or may not alter receptor conformation and function the same way phosphorylation does (this should be discussed in light of data shown in the accompanying manuscript).m for GTP is only slightly different for NPR2-7E and wildtype proteins, and that the EC50 for CNP is the same.To address the question of whether our results could be explained by an effect of the 7E mutation on an aspect of NPR2 function unrelated to phosphorylation state, we added experiments with a phosphatase inhibitor  that proNo difference in basal cGMP concentrations could be detected between mutant and wild type growth plates, but the assay may not be suitable to detect small changes in basal cGMP concentrations. Therefore, one cannot conclude that NPR2(7E) chondrocytes in vivo have higher cGMP concentrations compared to wild type chondrocytes (as stated at the end of the Results) – but one can speculate that this may be the case given the bone overgrowth phenotype of NPR2(7E) mice.7E/7ENpr2mice compared to wild type, and clarified that our measurements provide information about rates of cGMP production for the 2 genotypes, but not about cGMP concentrations in vivo.We omitted the statement that chondrocyte cGMP concentrations are higher in Does treatment with FGF (or phosphatase inhibitors) decrease the CFP/YFP ratio in wild type growth plates in the absence of CNP?Pre-incubation with FGF does not decrease the CFP/YFP ratio measured in the absence of CNP , 6A, or Overall, experiments were carefully executed and clearly described, but not all conclusions are supported by the data shown. For some of the conclusions, the biochemical properties of the mutant receptor need to be discussed and contrasted to the wild type receptor .m, EC50, and protein expression levels of NPR2-7E are similar to the values obtained for the wild type receptor.This information was added to the text. In relation to this issue, we also added a western blot showing that the level of NPR2 protein expression, normalized to total protein, is the same for wild type and mutant NPR2 . These aThe most novel aspect of the study is the measurement of CNP-induced cGMP concentrations in growth plates, opening the door to assessing the effects of other hormones on cyclic nucleotides. However, the data presented are sparse – there are only two figures and the illustration of the FRET method.The revised manuscript includes new data, 3 additional figures, and an additional supplementary figure.We prefer to keep the separate graphs since we think that they effectively present the experimental results.The explanatory schemata of NPR2 phosphorylation/ activity are redundant between An improved version of


Endoscopic techniques have been developed to treat patients with mild obesity and ameliorate comorbidities, but endoscopic skills are needed, beside the costs of the devices. Bariatric surgery is currently the most effective method to ameliorate co-morbidities as consequence of morbidly obese patients with BMI over 35 kg/mTo report a new technique for internal gastric plication using an intragastric single port device in an experimental swine model.  Twenty experiments using fresh pig cadaver stomachs in a laparoscopic trainer were performed. The procedure was performed as follow in ten pigs: 1) volume measure; 2) insufflation of the stomach with CO2; 3) extroversion of the stomach through the simulator and installation of the single port device (Gelpoint Applied Mini) through a gastrotomy close to the pylorus; 4) performance of four intragastric handsewn 4-point sutures with Prolene 2-0, from the gastric fundus to the antrum; 5) after the performance, the residual volume was measured. Sleeve gastrectomy was also performed in further ten pigs and pre- and post-procedure gastric volume were measured.  The internal gastric plication technique was performed successfully in the ten swine experiments. The mean procedure time was 27±4 min. It produced a reduction of gastric volume of a mean of 51%, and sleeve gastrectomy, a mean of 90% in this swine model.  The internal gastric plication technique using an intragastric single port device required few skills to perform, had low operative time and achieved good reduction (51%) of gastric volume in an in vitro experimental model. Between November and December 2015, 20 procedures were performed in the experimental surgery at our institution. The experimental model consisted of ex-vivo fresh pig cadaver stomachs installed in a laparoscopic single port trainer  -3. The procedure was performed as follows in ten pigs: 1) for measuring the initial pre-procedural gastric volume, each stomach was filled with water to a pressure of 50 mmHG (0.06 Bar); 2) the stomach wall was then extroverted and fixed to the laparoscopic trainer single port orifice, and opened a 2 cm anterior incision close to the pylorus; 3) the single port device  was inserted intragastrically through the 2 cm gastrotomy  A-B; 4) In further ten pigs, sleeve gastrectomy was performed by stapling the antrum and greater curvature over a 36F bougie. Post-procedural gastric volume was also measured. As a result of the internal plication, the technique produced macroscopic alterations in the form and capacity of the plicated stomach . PlastinA total of 10 internal gastric plication procedures and 10 sleeve gastrectomy procedures were performed in an ex-vivo swine cadaver stomach model. Mean total procedure time for the plications was 27±4 min per stomach. The mean preoperative volume of the pig stomachs was 884 ml (510ml-1260ml). The mean volume after the plications was 341±169 ml. The mean volume after the sleeve gastrectomy was 63±23ml. The mean percentage of the volume reduction for IGS was 51±25% . The meaPlastinate models were produced filling the lumen of procedural alterated specimens with colored cyanoacrylate to evaluate internal luminal anatomy for IGP and SG. The models showed the altered anatomy after the plication and the disposition of the remaining cavities .Bariatric surgery is the most effective and sustainable treatment for obesityNew less invasive methods have been developed to promote an effective therapy without the potential problems of penetrating or resecting the gastric wall. To reduce the capacity of the stomach and allowing mechanical restriction to food intake is a known effective mechanism to promote weight loss for morbidly obese patients2, and 714 had completed at least ten years of follow-up. They showed 47% of excess weight loss at 15 years. Angrisani et al compared the laparoscopic adjustable gastric banding with the RYGB with 51 patients in a period of five years2. The mean BMI at 12 months after surgery was 35 for the RYGB and 39 for the LAGB, decreasing to 29 for the RYGB and 36 for the LAGB after three years and staying the same for the RYGB at 29 and still decreasing for the LAGB at 35 after five years. Excess weight loss was significantly higher in the RYGB group after five years (67% RYGB and 48% LAGB). Himpens et alO'Brien et al2; the mean EWL was 20% after one month (n=779), 67% after 12 months (n=491), 70% after 24 months (n=356), 66% after three years (n=251), 62% after four years (n=176) and 55% after five years (n=134) following surgery. Severe complications occurred in 1% of the patients, including gastric necrosis and enteric fistulae. They had to perform operative revisions because of regain in 32 cases and failure in six cases. These findings show in the literature similar effects in EWL of LGCP to the SG,Talebpour et al shared their 12 years experience in 800 patients submitted to LGCP-Endoscopic alternatives were developed to fill the gap between bariatric procedures with high effectiveness but with more complications, and the conservative therapy, less harmful but less efficient in obtaining sustainable weight loss. The endoscopically placed gastric balloon seems to be a safe procedure for losing weight, but a metanalysis showed that it is only a short-term effective treatment, but it is not yet capable to maintain a weight loss over a long period of time2. The procedure time was 172 to 245 min and they placed 26 sutures2 to 30.9±5,1 kg/m2 at one year follow up. The indication of this therapy is currently applied to low-BMI patients,,nd stage strategy is recently described,The novel endoscopic suturing device available in the market, Apollo Overstich(r), has been currently used to perform a gastric volume reduction in the style of the SG without resection of the stomachThe physiology of our proposed IGP technique is potentially similar to the endoscopic gastric plication and not similar to the laparoscopic gastric plication, where the greater curvature is denervated and devascularized, and the plicated stomach wall is compressed to the lumen. In IGP, there is rather a delaying in gastric emptying caused by the shortening of the stomach and the impaired motility due to the sutures. In fact, in a recent study evaluating postoperative scintigraphic findings on patients submitted to endoscopic sleeve gastroplasty, Abu Dayyeh reported a delayed gastric emptying, recognizing a complete different mechanism of action of this therapy, if compared with SG, where on the contrary a rapid gastric emptying is expectedThe results of our study show that the IGP-IGS technique is feasible and produces, ex-vivo, a significant reduction in the gastric volume by restricting its area of distension. It is possible to reduce the volume of the stomach without resecting or bypassing it. It is also possible to perform a surgical gastric plication without devascularization, by acting intraluminally. The entry wound in the antral region does not produces relevant morbidity in our clinical experience with IGS for resection of intragastric tumors. In studies, LGCP seems to be as effective as the sleeve gastrectomy regarding EWL and ameliorating comorbidities. The IGP-IGS can potentially be as effective as that with less operative time, as there is no need to entry the rest of the peritoneal cavity nor to liberate the gastric vessels or vessel ligation. Leaks, gastric necrosis or bleeding are improbable, as the technique does not induce gastric ischemia. A shorter learning curve is expected, it is a procedure with only one incision and only one technical step. It also is reversible by just cutting the suture lines, endoscopically, within the first weeks after the operation. As the endoscopic similar technique - endoscopic sleeve gastroplasty - it can be followed by surgical malabsorptive methods as a second stage operation in cases with insufficient weight loss without any change to previous surgery.Survival studies have to be addressed to evaluate the feasibility and safety of the technique. The consequences of this study cannot be applied yet in the clinical practice, as experimental survival studies are needed to prove the feasibility and safety of IGP. Its evaluation as a valuable method for therapy of morbid obesity, in achieving sustainable weight loss, is the next step of our research.


Metastasis is the primary cause of mortality and morbidity in cancer patients. The bone marrow is a common destination for many malignant cancers, including breast carcinoma (BC), prostate carcinoma, multiple myeloma, lung carcinoma, uterine cancer, thyroid cancer, bladder cancer, and neuroblastoma. The molecular mechanism by which metastatic cancer are able to recognize, infiltrate, and colonize bone are still unclear. Chemokines are small soluble proteins which under normal physiological conditions mediate chemotactic trafficking of leukocytes to specific tissues in the body. In the context of metastasis, the best characterized role for the chemokine system is in the regulation of primary tumor growth, survival, invasion, and homing to specific secondary sites. However, there is ample evidence that metastatic tumors exploit chemokines to modulate the metastatic niche within bone which ultimately results in osteolytic bone disease. In this review, we examine the role of chemokines in metastatic tumor growth within bone. In particular, the chemokines CCL2, CCL3, IL-8/CXCL8, and CXCL12 are consistently involved in promoting osteoclastogenesis and tumor growth. We will also evaluate the suitability of chemokines as targets for chemotherapy with the use of neutralizing antibodies and chemokine receptor-specific antagonists. Cancer is the second leading cause of death in the developed world. Metastatic spread of tumor cells to vital organs results in mortality and morbidity , 2. The Clinicians and researchers have noted for over a century that metastasis is not a random process and that specific cancers tend to predominantly metastasize to certain organs. The “seed vs soil” hypothesis first put forth by the physician Stephen Paget to explain organotropism suggested that local microenvironment of target organs (“soil”) provided an appropriate environment for tumor (“seed”) colonization and growth . The molChemokines are a family of low molecular weight proteins which function in directing leukocyte cell chemotaxis to various tissues in both steady-state homeostasis and inflammatory conditions . There aChemokines are recognized by a family of receptors that belong to the superclass of seven transmembrane spanning G-protein coupled receptors (GPCRs). Nomenclature for the receptors is based on the subfamily of ligands they bind: CCR receptors activated by CCL ligands, designated CCR1–10; CXCR receptors bind to CXCL ligands, designated CXCR1–8; XCR is the sole receptor for XCL1 and XCL2 ligands; and the CX3CR1 receptor and CX3CL1 ligand form an exclusive pair. There is extensive promiscuity between the CC ligands and their receptors. There is some degree of promiscuity between the CXC ligands and their receptors, but it is not as extensive as that observed with CC ligands  triggers a series of biochemical events characteristic of GPCR signaling in general with activation of heterotrimeric G proteins (Gαβγ). Most chemokine receptors have been shown to couple to multiple Gα proteins, each of which can engage and modulate the activity of effector proteins. These include Gi, that effects adenylyl cyclase results in the inhibition of the second messenger molecule cAMP; and Gq that effects phospholipase C ultimately resulting in the release of calcium and activation of calcium-dependent enzymes. Some chemokine receptors have been shown to activate GG-protein coupled receptors have proven to be excellent pharmacological targets and amenable to high-throughput screening techniques . Chemokiin vivo. Blockade of CXCR4 with neutralizing antibodies prevented the BC cell line MDA-MB-231 from metastasizing to the liver and lung as these organs express the CXCR4 ligand, CXCL12 (SDF-1). Since this paper was published, there have been many studies which examined the role of chemokines in the various stages of metastasis , OCLs, mesenchymal stem cells, bone marrow endothelial cells, hematopoietic cells, and adipocytes , 28. OBsIndicators of OCL maturation include positive tartrate-resistant acid phosphatase (TRAP) staining and the presence of proteases, such as cathepsin K and matrix metalloproteinase 9.Tumor cells within the bone generally promote osteolysis in the process of creating a favorable microenvironment as the bone matrix is rich in growth factors . Tumors after colonization of bone secrete pro-OCL maturation factors, such as parathyroid hormone-related protein, IL-11, and TNFα which stimulate OBs to increase RANKL and decrease OPG production . OCL matIn this review, we focus on the role of chemokines in directly influencing components of the bone microenvironment which in turn enable osteolysis and tumor growth. The focus will be on BC, PC, and MM, cancer types for which bone is a prominent metastatic target. Particular attention will be paid to animal models where injection of tumor cells directly into bone allows for measuring the effect of chemokine blockade specifically on tumor growth as opposed to earlier steps in the metastatic cascade where chemokines are known to play a role.via a neutralizing antibody was shown to inhibit metastasis to bone of a strongly metastatic MDA-MB-231 subline  is the primary ligand for the CCR2 receptor which is normally expressed on monocyte/macrophages . This si subline . OB-deri subline , 40. Sev subline , 41, 42. subline .in vitro and in vivo. Human PC3 cell conditioned media induces bone marrow cultures and RAW 264.7 cells to form OCLs in vitro and this effect is partially blocked by anti-CCL2 neutralizing antibodies  is the principal chemokine ligand associated with MM growth in bone –62. MM iin vitro . The in el of MM . In thisel of MM . The priel of MM , 65. CCRel of MM , 67. Theel of MM . Small mturation , 70. A sInterleukin-8 (IL-8/CXCL8) is a member of the CXCL class of chemokines which bind to the receptors CXCR1 and 2 , 73. IL-in vitro  enhances colonization of bone . In addiLung carcinoma metastasis of bone also involves CXCR4/CXCL12 signaling, however, the relative importance of pathway in survival, homing, and osteolysis is not clear , 106. Rein vitro but limited in its ability to form tumors in bone marrow in vivo, clearly highlighting the role of the microenvironment for promoting tumor expansion. A similar phenotype to knockdown of CCL4 was observed in CCR5 loss of function experiments. When 4T1.3 cells were injected into the bone of either CCR5 knockout mice or mice treated with a CCR5 antagonist, they failed to grow efficiently. The ongoing efforts to develop CCR5-specific antagonists are, therefore, expected to have efficacy against metastatic tumors that utilize CCL3 and CCL4 to condition the bone microenvironment.Other cell types within the bone marrow stroma can contribute to tumor growth. The murine cell line 4T1 model is used to study BC in the context of an immunocompetent animal. A subline (named 4T1.3) was selected for bone metastasis by repeated cycles of isolation of mammary fat pad injection and isolation from bone marrow . DNA micin vivo remains to be studied.Renal cell carcinoma (RCC) is a relatively rare tumor of the kidney which often metastasizes to bone. Kominsky et al. showed that metastatic RCC expressed higher levels of CCL15 than patient matched primary tumors . The stuin vitro in a CXCL1 and CXCL2-dependent fashion. Consistent with this role for adipocytes, a high fat diet was observed to enhance PC-mediated bone osteolysis  is also associated with bone metastasis. CX3CL1 is predominantly expressed in tissue macrophages and certain lymphoid subsets and it exists as a membrane bound precursor which is ultimately processed into the mature soluble ligand. Blockade of proteolytic CX3CL1 cleavage and release has been suggested as an alternative therapeutic avenue as a means of disrupting the chemokine gradient that attracts CX3CR1-bearing tumor cells to the bone . It was radium-223 dichloride (Xofigo®) a calcium-mimetic agent that specifically targets bone lesions, has been shown to have an impact on survival rates for this disease (Current therapies for treating bone metastasis attenuate osteoclast activity. These include bisphosphonates which are taken up by OCLs and interfere with their ability to lyse bone and anti-RANKL antibodies (such as denosumab) which prevent OCL maturation. Interestingly, certain bisphosphonates (such as zoledronic acid) may impart some of their benefit by acting on chemokine signaling , 119. Di disease . The curIn vivo blockade of both of these pathways may prove to be very effective on treating bone-metastatic cancers.Although many different chemokine systems are involved in these processes depending on the tumor type studied, a broad consensus has emerged. There is clear evidence that carcinomas, such as BC, PC, lung, and OSCC often utilize both CCL2 and IL-8 to condition the bone microenvironment and promote OBD , 75, 76.The role of CCL3 and its receptor CCR1 in bone metastasis have been best characterized in the context of MM and blockade of this receptor has promising effects in pre-clinical animal cancer models. Lending weight to this therapeutic avenue is the fact that CCL15, another CCR1 agonist, is associated with osteoclastogenesis and bone metastasis . This paThe role of CXCL12/CXCR4 in cancer cell trafficking to bone has been explored extensively; however, there is a potential function for this pathway in modulating OCLs and bone resorption. Blockade of CXCR4 in most tumor models prevents OCL maturation and osteolysis. However, it is has been reported that mice reconstituted with CXCR4 null hematopoetic cells display more OCLs and osteolysis  It was nin vitro and ex vivo 3D culture systems are currently being developed to augment the study of tumor cells within the bone microenvironment  is hampered by the death of human cell lines which are capable of exhibiting consistent and reliable metastatic behavior in mouse models. Even the cell lines that do provide metastasis in mouse models often fail to display the full spectrum of malignant features. Most pre-clinical studies have been carried out using only a limited number of cell lines or genetic mouse models of cancer. Novel ironment , 125. HoThe use of neutralizing antibodies against soluble ligands and small molecule pharmacological inhibitors that target the relevant chemokine receptors have yielded encouraging results in a number of pre-clinical models Table . For thoin vivo pre-clinical model studies, targeting a single receptor fails to completely inhibit bone metastasis and the effect on prolonging animal survival is modest. It is possible in these cases that other chemokine receptors are involved in the residual migration, invasion, and growth within the bone. For example, the expression of several chemokines by BC cells  have been shown in various models to promote growth within bone. Blockade of one of these results in upregulation and/or usage of one of the others to carry out the pro-metastatic function. Advanced cancers are notorious for being able to develop chemoresistance and switching from one chemokine system to another would seem to be a likely strategy adopted by malignant tumors. To target multiple chemokine ligand/receptor pairs would require combinations of anti-chemokine agents or use of pharmacological inhibitors which have a broader target range. Either strategy may be advantageous in this scenario. Given the diversity of chemokines used by a given tumor type to metastasize to bone, it may be necessary to profile individual patients for chemokine ligand/receptor expression to determine which agent is likely to be most effective for a given patient. The chemokine expression profile would also have to be monitored over treatment time as it is the possible tumor cells which may acquire resistance by switching ligand expression.Given that multiple chemokine ligand/receptor pairs may be driving bone metastasis, a combinatorial approach targeting multiple chemokine pathways simultaneously, may be required for effectively preventing bone metastasis. In most of the The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The primary aim of this retrospective pilot study was to evaluate the clinical outcome of overdentures on four non-splinted maxillary implants compared to the mandible using locator attachments and secondly to assess patient's opinion of the treatment.The treatment protocol used here is summarised as a single-stage surgical approach followed by immediate loading (same day in 12 of 17 patients) of a removable prosthesis in the maxilla and mandible. Most of the implants were installed into fresh extraction sockets. Clinical outcomes were evaluated in 68 southern implants, straight (non co-axis) or angulated (co-axis) in 17 patients. Patients were examined by independent examiners at an average follow-up of 14.5 months after implant placement.P=0.01) was found between bone level, from implant head to bone contact in the maxilla  and the mandible . The marginal bone-to-implant head distance with the angulated co-axis implants was 1.9 mm  compared to non co-axis, mean 1.2 mm  (P=0.01). The OHIP-14 overall mean was 3.3 (out of a maximum of 56).Outcomes measured were implant survival, bone loss, bleeding on probing, probing pocket depths and plaque score in addition to quality of life measured with OHIP-14 questionnaires. An overall implant survival of 100% was achieved. The mean marginal bone level (mm) over the entire cohort of 66 measured implants was . A significant difference (The implant survival was 100% and the patients benefited from the overdenture treatment on four non-connected implants. The extremely low OHIP-14 indicated a very high level of patient satisfaction following treatment. The results of this study merit further long-term investigation to fully investigate the success of immediately loading implants in the maxilla as well as cost-benefit. Also immediate loading is predictable with single implants or when multiple implants are splinted with a fixed reconstruction in maxilla and mandible.2 Machined surface implants yield high failure rates and often four–six implants are suggested to support a splinted reconstruction. Sadowsky and Zitzmann2 recently carried out a systematic review of 23 publications relating to 20 cohort studies looking at maxillary implant overdentures. They concluded that four–six implants are widely applied in successful cohort studies and found no distinct evidence that implant splinting with a bar is superior to single attachments in terms of implant survival, in general splinted and solitary anchorage systems are both advocated. Furthermore, technical issues with attachments or soft tissue irritations under bars have been described and difficulty of cleaning has been reported with bar anchorages. The success of non-axially loaded implants as a technique to overcome the problem of bone resorption particularly in the posterior maxilla has been described in some detail.3A recent systematic review indicated that the number of studies providing information on overdentures in the maxilla is rather limited.1 only two prospective clinical studies were available. Eccellente et al.4 used the Ankylos Syncone system  in 45 subjects with four non-splinted implants. A prefabricated conical crown was adapted to the relined existing denture. The conical crown concept resulted in stable complete denture retention, a reduced denture base and facilitated oral hygiene. The overall implant survival rate was 97.8% during an average observation period of 26 months. Pieri et al.5 attached maxillary overdentures in 22 consecutive patients on four–five implants connected by a bar and this yielded a 97.1% survival after one year. The most common prosthetic complication was frequent relining of the denture in the initial weeks in 27% of the patients. The patient’s subjective appreciation of function and satisfaction increased significantly in comfort, functional and aesthetic parameters. The patients found it difficult to maintain the high level of oral hygiene required. Although it is suggested that treatment outcome may be good, one should realise that current evidence to recommend this treatment on a routine basis is insufficient and comparative studies to provide surgical or prosthetic guidelines related to patient selection are unavailable.There is evidence in the dental literature showing successful immediate loading of implant overdentures in the mandible, but there is a paucity of evidence regarding the maxilla. This is mainly related to jeopardised bone condition making immediate loading more problematic in the maxilla, especially in elderly patients. In a systematic review on immediate loading in various treatment indications,The primary aim of this retrospective pilot study was to evaluate the clinical outcome of overdentures on four non-splinted maxillary implants compared to the mandible using locator attachments  and second to assess patient’s opinion of the treatment.From 2011 to 2014, 21 either dentate or edentulous patients were treated at Vermilion – The Smile Experts Ltd, Edinburgh with implants in the maxilla and mandible to provide stability for an overdenture. Smokers, diabetics and patients with history of periodontitis were not excluded and there was no restriction based on the opposing dentition. Patients were personally invited by the dental surgeon (DO) by phone or mail to participate in a clinical examination and 17 patients agreed to be checked by independent examiners from Ghent University, Belgium. NHS ethical approval was not required under the terms of the Governance Arrangements for the Research Ethics Committee (South East Scotland Ethics) and the advice was based on the study being limited to utilisation of data previously collected in the course of normal care (reference number NR/1408AB14).Surgery was carried out by one oral surgeon (DO) by initially raising of flap followed by implant placement following the manufacturer’s recommendations . ImplantAfter implant installation, immediate placement of locator abutments was carried out with immediate loading of the interim prosthesis (same day) whenever implant insertion torque exceeded 25 Ncm . After sClinical evaluation was carried out at a mean follow-up of 14.5 months after implant placement. Bleeding on probing and plaque was measured on four sites  using a dichotomous score  leading to an implant score between 0 and 4. Probing depth was registered on the four sites with a periodontal probe. Clinical implant stability was examined clinically and retention of the prosthesis was checked by rocking the prosthesis and clinical inspection of damaged matrices or fractures.Peri-implant bone levels were measured on radiographs taken at a mean follow-up of 14.5 months after implant placement with the Nomad Pro* handheld X-ray system for intraoral radiographic imaging . Digital peri-apical radiographs were taken for each individual implant using the parallel long-cone technique in order to visualise the implant threads and marginal bone-to-implant contact level using the implant–abutment interface as reference point . A guidi6–8 The index measures the people’s perception of the social impact of oral disorders on their well-being on a 5-point scale ranging from very negative (score 4) to very positive (score 0). The average score per question as well as the total score of 14 items was calculatedThe patients filled out the Oral Health Impact Profile (OHIP-14) questionnaire at a mean follow-up of 14.5 months after implant placement to evaluate the impact of the overdenture treatment on oral health-related quality of life by focusing on eating and speaking comfort as well as physical, psychological and social discomfort.The mesial and distal bone was measured around each implant, and a Wilcoxon signed-rank test was applied to test the differences between the mesial and distal bone levels. Because these were not statistically significantly different, the mesial and distal values were averaged to obtain one value per implant.U-tests were used to analyse differences in marginal bone level changes based on location (mandible versus maxilla), angulation (angulated implants versus straight implants and smoking behaviour (smokers versus non-smokers). All statistical analyses were performed using SPSS 22.0  with a pre-set significance level of P⩽0.05.Mann–Whitney Data were included from 21 consecutively treated patients, 17 patients agreed to participate . The mean follow-up time was 14.5 months . In total, 68 implants were examined and clinically inspected. Twenty-six implants in nine patients were placed in the mandible and 42 in 11 patients were placed in the maxilla; 45 were straight and 21 co-axis. Three patients indicated they were smokers and 14 were non-smokers. All clinically examined implants were immobile without signs of pain and all supporting a functional overdenture. This trial showed good implant survival without any failures. The average probing depth was 2.5 mm  with only three patients showing probing depths greater than 3 mm. The mean plaque index was 20%  and the mean bleeding index was 0.21 .et al.9 described an acceptable bone loss of 1.5 mm during the first 12 months and a further maximum of 0.2 mm yearly. A total of six implants in two patients in this study lay outside this acceptable limit. The first of these patients prior to implant placement had aggressive periodontal disease, was a smoker, a bruxist and had an atrophic maxilla lacking height and width. The other patient’s implants were placed in the edentulous mandible; she had a periodontal disease history and was also a past smoker.Analysed data included bone loss related to 66 implants. Albrektsson P=0.01) was found between bone level, from implant head to bone contact in the maxilla  and the mandible . The marginal bone to implant head distance with the angulated 12° or 24°, co-axis implants were 1.9 mm  as compared to the non co-axis, mean 1.2 mm  (P=0.01). The bone loss was measured on a total of 13 implants measured in smokers  and 53 implants from non-smokers . Although the difference in bone loss was not significantly different between the groups (P=0.15), there is also trend towards more expressed bone loss in the smoking group.A significant difference  . These rPatients completed the OHIP-14 questionnaire only at the time of the final clinical examination. An extremely low overall mean of 3.6  from a possible maximum of 56 OHIP-14 points indicating a high quality of life after treatment .1 This clinical outcome on implant survival is in line with other reports in the literature.10 There has become an increasing amount of evidence that the modern implant design do not yield a high failure rate when implants are placed into fresh sockets and then immediately loaded.1 There have been some comprehensive systematic reviews of early or immediate loading of mandibular overdentures compared to delayed loading.12 These analyses concluded there was no significant difference between the two protocols with a loading period of up to 2 years. Another systematic review based on 25 studies reported bone loss of 0–0.2 mm in early loading studies and 0.7 mm in immediate loading studies.1 This difference has been explained by the time the baseline radiographs are taken. In immediately loaded implants, the baseline is taken at the time of surgery and not after a period of prolonged healing.This is a retrospective study describing an overdenture treatment protocol that can be summarised as a single-stage surgical approach followed by immediate loading (same day in 12 of the 17 patients) of a removable prosthesis. Most of the implants were installed into fresh extraction sockets. Our aim here was to reduce the amount of surgery and the restoration placement without compromising the success of the implant. An overall implant survival of 100% was achieved, which is in line with similar reports on immediate loading in fully edentulous maxillae and mandibles.et al.,5 is 1.5 mm during the first year and then a stabilizing of crestal bone loss to only 0.2 mm yearly. Our data show this apart from one patient whose bone loss in all four implants was over 2.5 mm and up to 5.5 mm. This patient before placement of implants had aggressive periodontal disease, was smoker, a bruxist and had an atrophic maxilla. There were no significant differences between the maxillary and mandibular bone levels. Also the co-axis (angulated) implants did not seem to result in differences in peri-implant bone level compared to straight implants. Other studies have also shown no difference in bone loss between straight or tilted implants.13–15In this study, the mean marginal bone level (mm) over the entire cohort of 66 measured implants was ). The acceptable bone loss according to the success criteria, described by internationally well acclaimed Albrektsson et al.17 focussed on implants in the mandible and measured patient mediated and economic factors following immediate loading of implants with mandibular overdentures. In the current study, we measured the patient satisfaction with treatment using the OHIP-14. The overall mean of 3.3 (out of a maximum of 56) was extremely low indicating a very high level of patient satisfaction following treatment. This study contained no baseline measurement for the OHIP-14 questionnaire, this could be attributed to the immediate loading protocol known to yield high satisfaction compared to delayed loading.18Very few studies have evaluated patient satisfaction with implant treatment. These two papers by Attard 1 The patients included in this study follow regular maintenance of their implants and prostheses with adjustments made regularly, this may overcome some of the biological and technical issues that may occur when cases are not followed up regularly.It is not known what the implications of this procedure over the long term are going to be. Dental implants are known to function well, but an ongoing study of this cohort over time will show us if this procedure has an impact on the success of implants and the prosthesis in the long term.We realise we have a limited number of cases here because this is a working daily practice. However, the study can continue with honest reporting on all issues over time to get a gauge on the success of this procedure particularly in the maxilla. There was one notable aspect in regard to the OHIP-14 results in every patient group, the scores are very low, indicating a good quality of life. Because we had over 80% (17/21) patients willing to come back and participate in the study, there may be a bias in the patient satisfaction (OHIP-14) results.Another limitation of this retrospective study was no baseline measurements taken for OHIP-14 or radiographs for bone measurement. The only radiographs available were taken as routine radiographs and were not accurate or clear enough to carry out bone measurements. In prospective studies, the authors will be taking baseline radiographs from the day when the trans-mucosal part pierces the mucosal tissue and annually thereafter.The implant survival was 100% and the patients benefited from the overdenture treatment on four non-connected implants. There is limited long-term data on procedural recommendations for the minimum number of implants required, length and the success of non-splinted immediately loaded prosthesis in the maxilla. The results of this study merit further long-term investigation to fully investigate the success of immediately loading implants in the maxilla as well as cost-benefit.


Although the role of cancer-activated stroma in malignant progression has been well investigated, the influence of an activated stroma in therapy response is not well understood. Using retrospective pilot cohorts, we previously observed that MRI detected stromal contrast enhancement was associated with proximity to the tumor and was predictive for relapse-free survival in patients with breast cancer receiving neoadjuvant chemotherapy. Here, to evaluate the association of stromal contrast enhancement to therapy, we applied an advanced tissue mapping technique to evaluate stromal enhancement patterns within 71 patients enrolled in the I-SPY 1 neoadjuvant breast cancer trial. We correlated MR stromal measurements with stromal protein levels involved in tumor progression processes. We found that stromal percent enhancement values decrease with distance from the tumor edge with the estimated mean change ranging  The imaging component of this trial was conducted as a companion study to the American College of Radiology Imaging Network (ACRIN) 6657 and Cancer and Leukemia Group B (CALGB) 150007. Participation in both ACRIN 6657 and CALGB 150007 was required to enroll in the I-SPY 1 TRIAL. All patients gave a signed consent to participate. Eligible patients had histopathologically confirmed breast cancers without evidence of distant metastatic disease. The NAC included an initial four cycles of anthracycline-cyclophosphamide (AC) after which patients either underwent surgery or received four cycles of taxane-based treatment prior to surgery.2.2,The I-SPY 1 TRIAL patients underwent contrast-enhanced MRI at visits before the start of chemotherapy (T1), after one cycle of AC (T2), between AC and taxane-based (T3) regimen, and at the completion of chemotherapy prior to surgery (T4). Images were acquired as previously described.Despite the long scan duration, the first postcontrast time sample occurred at 2.5 min using the standard 2.3For breast MRI, contrast enhancement kinetics, as captured by the signal intensity–time curves are used to distinguish among malignant  and those who did not. A linear mixed effect model was utilized to assess the PE and SER radial trend in the range of 5 to 40 mm from the tumor edge.33.1As shown in the consort diagram , there wOur survival endpoint was RFS, both at 3 years and as a censored variable. Of the 71 patients with MRI stromal evaluation, 17 patients had recurred after 3 years and 51 had not (three were missing due to insufficient follow up). Univariable Cox proportional hazards analysis showed that clinical stage was significantly associated with RFS  at the tumor periphery within the nonrecurrent and recurrent groups. In general, the PE signal was lower in the later time point, with the only exception being between T1 and T2 in the nonrecurrent group. The difference was significant only between T2 and T3 in the recurrent group. Here the estimated (pseudo-) median difference was 3.2.2Using the same mixed effects model as for PE, we observed an increasing trend, rather than a decrease, in mean SER as a function of distance from the tumor edge at the pretreatment time point (T1). Mean SER values increased significantly at T1 only. Here the estimated mean increase per mm was 0.004  . In MRIsSimilar to PE, we added the recurrence status as a group variable to the mixed effects model with distance to assess differences in SER enhancement patterns in recurrent and nonrecurrent patients. We focused on T1, since that was the only significant time point. The estimated mean increase (95% CI) in the nonrecurrent group was 0.0046  the small sample size in which data for both MR and protein measurements were available; (2) differences in MR enhancement attributed to protein/phosphoprotein levels that were not measured in this study; and (3) the average numeric PE and SER descriptors may be limited in describing the regional variation of stromal behaviors. Recent advances in radiomics-based analysis have demonstrated the power of transforming imaging data into multidimensional mineable radiologic featuresIn addition to measuring radial dependence of PE, we also evaluated the average PE of the entire stroma or global stromal PE at each time point. In the present cohort, global stromal PE decreased with the increasing exposure tochemotherapy from T3 to T4. These findings suggest that the average stromal vascular permeability decreased with chemotherapy, suggesting there are global effects on stromal vasculature that are of unknown significance.We also measured SER values, which in contrast to PE, reflect washout kinetics of the gadolinium contrast. In our previous studies, we found an association with higher global SER values at posttreatment time points with improved RFS. We did not find this association in the present cohort. However, we observed a small increasing trend in mean SER with distance from the tumor edge at T1. We observed similar associations in our pilot cohort.We recognize that the current study population was limited to a cohort of patients enrolled in the I-SPY 1 neoadjuvant trial. Due to relatively small numbers and events, limited conclusions could be drawn from this cohort. However, the findings in this study are consistent with observations made in previous cohorts, namely the radial dependence of PE and SER in the stroma. A new finding is the persistence of the radial dependence of PE despite the treatment with chemotherapy, and the decrease in global PE with the increasing exposure to chemotherapy from T3 to T4.,,Enhancement threshold plays a significant role in defining tumor regions and subsequent stromal analyses. In the past, we found that functional tumor volume defined by PE threshold set at 70% has the strongest prediction of RFSWe recognize that the current study relied on measurements of average numeric global PE and SER descriptors that may over simplify the behaviors of the tumor microenvironment. However, the current proximity mapping technique uniquely accounts for tissue vascular permeability in all voxels by their location relative to the closest tumor voxel. Ongoing efforts to refine the proximity analysis and integrate methods with radiomic-based analysis may improve the predictive performance for survival. While PE and SER will remain the primary imaging parameters for stromal analysis in DCE-MRI, the proximity mapping methodology can also be extended to diffusion-weighted imaging. Other descriptors such as apparent diffusion coefficient