Plants are sessile organisms that are permanently restricted to their site of germination. To compensate for their lack of mobility, plants evolved unique mechanisms enabling them to rapidly react to ever changing environmental conditions and flexibly adapt their postembryonic developmental program. A prominent demonstration of this developmental plasticity is their ability to bend organs in order to reach the position most optimal for growth and utilization of light, nutrients, and other resources. Shortly after germination, dicotyledonous seedlings form a bended structure, the so-called apical hook, to protect the delicate shoot meristem and cotyledons from damage when penetrating through the soil. Upon perception of a light stimulus, the apical hook rapidly opens and the photomorphogenic developmental program is activated. After germination, plant organs are able to align their growth with the light source and adopt the most favorable orientation through bending, in a process named phototropism. On the other hand, when roots and shoots are diverted from their upright orientation, they immediately detect a change in the gravity vector and bend to maintain a vertical growth direction. Noteworthy, despite the diversity of external stimuli perceived by different plant organs, all plant tropic movements share a common mechanistic basis: differential cell growth. In our review, we will discuss the molecular principles underlying various tropic responses with the focus on mechanisms mediating the perception of external signals, transduction cascades and downstream responses that regulate differential cell growth and consequently, organ bending. In particular, we highlight common and specific features of regulatory pathways in control of the bending of organs and a role for the plant hormone auxin as a key regulatory component. To compensate for their sessile lifestyle, plants developed unique mechanisms that provide them with an unusual level of developmental plasticity. The adaptive behavior of plants is noticeable throughout their life cycle. Already during early skotomorphogenesis (development in darkness), seedlings of dicotyledonous plants form a hook-like structure at the apical part of the hypocotyl to shield their tender shoot apical meristem and cotyledons from damage while penetrating the soil . This apThe first hints on the molecular basis of the differential growth underlying organ bending date to Darwin’s early studies on coleoptiles. Based on the bending of coleoptiles toward unilateral light, In our review we will discuss the current knowledge on the pathways engaged in the control of tropic responses and apical hook development, the developmental process which is similar to tropic responses largely based on coordinated differential cell growth. Similarities and specificities of mechanisms mediating organ bending in response to environmental stimuli will be highlighted.Table 1.Conversion of light into chemical energy during photosynthesis is essential for sustaining life on earth. Plants belong to the few species capable of such an energy transformation and by storing energy in carbohydrate molecules such as sugars, nutritional resources are generated for other organisms. To adjust their light absorption capacities, plants have evolved different light perception and signaling systems. Among them, phototropism or bending of an organ toward unidirectional blue light, is one of the most important adaptive responses. For the summary of the common and different factors involved in the regulation of phototropism see the Although already in Arabidopsis mutants defective in the response to unilateral light such as non-phototropic1 (nph1), nph2, nph3 and nph4 (NPH1 gene was found to encode a flavoprotein kinase and confirmed to act as a UV-A/blue receptor (NPH1-LIKE1 (NPL1) was shown to contribute to the perception of blue light mainly at high fluence rates (phot1phot2 mutants to blue illumination suggested the involvement of other photoreceptors. The latter proved to be CRYPTOCHROME1 (CRY1) and CRYPTOCHROME2 (CRY2), because the mutation of CRY1 and CRY2 in the phot1phot2 mutant completely abolished the phototropic response  was identified in early screens for mutants defective in phototropism, and despite its essential regulatory function, the mechanism of NPH3 action remained obscure for a long time . Recent  complex  and medi complex  and the  complex . In lighSince Darwin’s experiments, evidence accumulated in support of a crucial role of an asymmetric auxin distribution, depending on functional polar auxin transport, for a proper phototropic response . Yet, whIn contrast, lack of PIN3 activity causes reduced sensitivity to unilateral light, and localization of this transporter at lateral membranes of hypocotyl endodermal cells hinted at PIN3 involvement in the lateral redistribution of auxin in the lower elongation zone of the hypocotyl . Howeverpks1, pks2, and pks4 suggested that PKSs might contribute to the regulation of the phototropic response through modulation of local auxin signaling or lateral auxin transport, alternatively , PKS2, and PKS4. PKSs were identified in a screen for early blue light response genes  and genenatively .2+; 2+ is important for PHOT1-mediated inhibition of hypocotyl growth, and the increase in Ca2+ attributed to PHOT2 function is required for blue light induced phototropism  treatment inhibited a cytosolic Ca2+ increase in response to blue light, while auxin treatment led to a dramatic increase in cytosolic Ca2+  and PIF5  mutant, which lacks endodermal amyloplasts, exhibits hampered shoot gravitropism (SCARECROW (SCR)/SHOOT GRAVITROPISM1 (SGR1) and SHORT-ROOT (SHR)/SGR7, transcription factors of the GRAS gene family, which are indispensable for normal shoot and root endodermis formation, are characterized by a deficient shoot gravitropic response. The deficient shoot gravitropism in the latter mutants supports the function of shoot endodermal cells as statocytes in gravity perception. On the other hand, the unaffected root gravitropism in endodermis defective mutants hints at a difference in the site of gravity perception between shoots and roots, supporting the role of columella cells as primary perception site in the root  mutant, amyloplast sedimentation is dramatically affected due to a low starch content, which correlates with a reduced gravitropic response in roots as well as shoots (pgm mutant upon exposure to a hypergravity stimulus indicates that the amyloplast starch levels might be an important factor in the gravity sensing (starch excess 1 (sex1) mutant phenotype. Due to a defective starch breakdown, amyloplasts are larger in the endodermal cells of light-grown sex1 shoots, while being unaffected in the root columella cells , involved in actin microfilament formation  levels in response to a gravistimulus qualified them as early signaling molecules. How these signaling molecules might coordinate gravitropic bending and possibly the asymmetric redistribution of auxin, the hormonal signal coordinating differential cell growth, will be discussed in the following paragraph.Amyloplast sedimentation in response to gravity is considered to excite a biochemical signal that converts gravity perception into gravitropic curvature. Rapid modulation of the cytoplasmic pH, Caaux1, pin2, pin3 and pin3, pin7 mutants delineated AUX1, PIN2, PIN3 and PIN7 as key transporters coordinating the auxin redistribution upon gravistimulation. Subsequent studies on the polar membrane localization and subcellular dynamics of the auxin transporters, revealed that within a few minutes after gravistimulation, the PIN3 and PIN7localization changed from apolar to polar with maxima at the bottom of root columella cells. This rapid PINs relocation might increase the efficiency of the auxin flow toward the lower side of the gravistimulated root  in these regulations.Although full understanding of the molecular mechanisms controlling gravitropic bending and auxin transport modulation after a gravistimulus is still lacking, there are several indications for the involvement of signaling molecules such as Ca2+ in the shoot within seconds . Furthermore, the dependency of both root and shoot gravitropism on Ca2+ signaling is supported by the defective gravitropism upon administration of Ca2+chelators  mutant, which is characterized by reduced levels of InsP6 , encoding an enzyme involved in phosphatidylinositol metabolism, was found to enhance the root gravitropic response. The reduced sensitivity of the 5PTase13 mutant to the polar auxin transport inhibitor NPA, denoted the involvement of enhanced polar auxin transport and consistently, an altered PIN2 distribution, with an expression extended to the root elongation zone in the 5PTase13 mutant, has been observed . SUPO1 (SAL1/FRY1/HOS2/ALX8/RON1) was shown to code for a protein with inositol polyphosphate 1-phosphatase and 3′(2′),5′-bisphosphate nucleotidase activities  and ARG1-LIKE 2 (ARL2) genes further strengthen the role of actin in the regulation of PIN dynamics. ARG1 interacts with the cytoskeleton, supposedly with actin microfilaments, and arg1 and arg1-like 2 mutants exhibit a reduced shoot and root gravitropism, as well as defects in the asymmetrical auxin redistribution (The function of actin microfilaments in gravitropism is not confined to gravisensing (discussed above). Modulation of the polar membrane localization of auxin transporters after a gravistimulus is largely dependent on the actin cytoskeleton . Stabiliribution . Furtherribution .MASSUGU 2 (MSG2)/IAA19 gene results in a reduced hypocotyl gravitropic response, whereas growth and developmental processes other than differential growth are unaffected (SOLITARY ROOT (SLR)/IAA14, results in agravitropic roots and shoots  side of the hook, while cell elongation is reduced and the arc length is shorter. In contrast, cell elongation is an active process at the convex (outer) side of the hook that permits the arc length to increase. At the basal side, the cell elongation rate is equal between the outer and inner sides and, therefore, without curvature. Thus, coordinated differential cell elongation and cell division mechanisms are essential for proper hook development . Hence, Helianthus annuus L. seedlings treated in the clinostat display a defective apical hook development, hence, supporting the role of gravisensing in hook formation  of apical hook formation remain(s) unknown. Hypothetically, several candidate mechanisms controlling the onset of hook formation are plausible. To fit within a tightly enclosed seed coat, maturing embryos form a typical U-shaped structure with two cotyledons bent toward the embryonic root. Could this embryonic body shape predetermine incipient apical hook formation, after germination? Another factor that might contribute to the control of early apical hook formation is gravity. It has been shown that ormation . On the ormation . Thus, iaxr3 at the concave side of the hook prevents hook formation , short hypocotyl2-2 (shy2-2), massugu (msg2/iaa19) and non-phototropic hypocotyl4 (nph4/msg1/arf7), auxin response factor19 , an ethylene-responsive gene that encodes an N-acetyltransferase. HLS1 was found to inhibit the AUXIN RESPONSE FACTOR2 (ARF2) repressor and, thus, to control the auxin response  gene family are highly expressed in tissues undergoing differential cell expansion , carrot (Daucus carota), and okra . The perception sites for light and gravity differ for the shoot and the root. In addition, the response to these two stimuli rests on different mechanisms. In phototropism, light is perceived through photoreceptors, whereas in gravitropism, gravity is sensed through the sedimentation of starch-containing amyloplasts in statocytes. The triggers and perception site for apical hook formation remain, however, in the dark. To transduce the perception of both light and gravity in organ bending, common, as well as different, biochemical signals and factors are involved. Calcium ions and InsP3 appear as important common biochemical signals in both tropic responses. The increase in cytoplasmic pH in the root cap columella cells in response to gravity is another important biochemical signal, important more specifically for root gravitropism.The ability of plant organs to bend in response to different environmental stimuli represents one of the most crucial adaptive mechanisms compensating for the lack of plant locomotion. Differential growth is the common mechanistic basis underlying all forms of tropic movements in plants. Deciphering the molecular components and mechanisms underlying differential growth in response to various environmental stimuli revealed that multiple common elements from perception to signal transduction, as well as specific elements are involved . Whereas during root gravitropism and apical hook formation auxin accumulates at the inner side of the curvature to restrict cell expansion, shoot gravi- and phototropism and presumably also root phototropism are driven by auxin maxima at the outer side of the curvature, where auxin stimulates cell growth. This apparent discrepancy in auxin accumulation patterns and consequences for local cell growth indicate that auxin might coordinate differential growth through specific mechanisms, which might depend on the developmental context. In addition, auxin is known to regulate cell growth in a concentration-dependent way. At optimal concentrations, auxin stimulates, whereas at supraoptimal concentrations it inhibits cell growth, respectively (Arabidopsis consists of 6 TIR1/AFBs, 29 Aux/IAAs and 23 ARFs homologues are activated in dependence on the auxin concentrations to mediate certain developmental responses. Such a scenario is strongly supported by the recent study of The difference between the perception and the response sites in both gravitropism and phototropism indicates that a signal must be transported between both sites. The polar transport of the plant hormone auxin is indeed indispensable for the differential growth underlying gravitropism, phototropism, as well as apical hook development. In all forms of differential growth, an asymmetrical auxin distribution is found in the bend of the root and the shoot. Despite the firmly established strong correlation between an asymmetric auxin distribution and differential growth, there is a longstanding enigma with respect to auxin accumulation and cell growth patterns (ectively . Hence, The dependence of all forms of differential growth on an asymmetrical auxin distribution indicates that auxin carriers fulfill an indispensable role. PIN3 eﬄux carriers appear to be one of the key components of the polar auxin transport machinery involved in gravitropism, phototropism and apical hook formation . In the Interestingly, several common components of the auxin signaling pathway, such as ARF7/NPH4 and ARF19 as well as IAA14, and IAA19, were found to control the bending of different organs regardless of the nature of the stimulus. This further strengthens the assumption that the developmental context might play a crucial role in defining the specificity of the response. The current knowledge on downstream molecular components and mechanisms which direct differential cell growth during specific tropic responses downstream of auxin is surprisingly scarce. Hence, the identification of factors and detailed understanding of networks coordinating differential growth remain a challenge for future research.The authors declare 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 ketogenic diet (KGD) has been recognized as an effective treatment for individuals with glucose transporter 1 (GLUT1) and pyruvate dehydrogenase (PDH) deficiencies as well as with epilepsy. More recently, its use has been advocated in a number of neurological disorders prompting a newfound interest in its possible therapeutic use in autism spectrum disorders (ASD). One study and one case report indicated that children with ASD treated with a KGD showed decreased seizure frequencies and exhibited behavioral improvements . The KGD could benefit individuals with ASD affected with epileptic episodes as well as those with either PDH or mild respiratory chain (RC) complex deficiencies. Given that the mechanism of action of the KGD is not fully understood, caution should be exercised in ASD cases lacking a careful biochemical and metabolic characterization to avoid deleterious side effects or refractory outcomes. The ketogenic diet (KGD) is a nutritional approach constituted by high-fat content with adequate protein amount for growth but insufficient levels of carbohydrates for metabolic needs , thus fo2 and H2O in the Krebs’ cycle. However, when an imbalance is created between the rate of FA mobilization and the capacity of the Krebs’ cycle to process acetylCoA , the liver converts the excess of acetylCoA into ketone bodies (KB), namely acetoacetate (ACA) and d-β-hydroxybutyrate (BHB). A significant fraction of acetone (~30%), the product of the spontaneous decarboxylation of ACA, is found in urine, sweat, and breath  and/or leptin –9, thus d breath , 11. KB d breath  constitud breath . Most ATd breath . The lowin vitro to in vivo, when exposed to KGD or KGD-mimetics  and glucose transporter 1 (GLUT1) Table . The KGDKetogenic diet has been utilized for >80 years in epilepsy treatment , 40 espeEvidence supporting the use of the KGD for patients with intractable epilepsy and respiratory chain (RC) complex defects has been reported in which the majority of patients responded with decreased seizure frequencies, regardless of the RC complex defect or magnitude of deficit . The admAutism spectrum disorders (ASD) include a complex neurodevelopmental condition characterized by abnormal social interaction, verbal and non-verbal communication, and limited interest in the surrounding environment associated with stereotyped and repetitive behaviors . LimitedBeneficial effects of KGD in children with ASD symptoms have been reported in two independent studies , 55. TheThe other study  reports ad libitum conditions and/or for a relatively short period [see Ref.  from children with high severity scores for ASD has shown impaired PDH activity . The KGDSome patients with ASD have been reported to have defects in fatty acid β-oxidation evidenced as long-chain acyl dehydrogenase deficiency  and highThe KGD might improve mitochondrial function by enhancing mitochondrial biogenesis in murine models , 65. The2 than glucose (2 vs. 5).Children with ASD display an array of mitochondrial dysfunction (MD) of differing severity , 73–75. Recently, KGD-fed rats showed increased brain expression of insulin-like growth factor receptor (ILGFR) and neuronal GLUT3 . The KGDPten) gene haploinsuffciency  have been recently indicated as possible downstream targets of KGD and may be involved in neuroprotective effects associated to the diet . Defectsffciency . Indeed,ffciency  and KGD-ffciency , thus reKetone bodies  inhibit mitochondrial reactive oxygen species (ROS) production in rat neurocortical neurons by increasing NADH oxidation following glutamate (Glu) excitotoxicity . It has Thus, the use of KGD could be beneficial in ASD given that higher rates of mitochondrial ROS production and compromised cellular antioxidant status , 95, 96 The GABA shunt bypasses two steps of the tricarboxylic acid cycle – the α-ketoglutarate (KG) dehydrogenase complex and the succinylCoA synthase – for the conversion of KG into succinate Figure . It invoChanges in GABA neurotransmission by KGD might explain the decrease in seizure frequencies and improved behavior observed in Rett syndrome . StudiesDysfunction in the cholinergic system has been observed when PDH deficits are present  because l-carnitine supplementation have been helpful in selected cases  limitation in protein, carbohydrate, and other nutrients intake can result in a lack of weight gain and growth inhibition , which ced cases . (b) Dysed cases , 128 woued cases , 129 anded cases . These ped cases , should ed cases . (c) KGDed cases . (d) Adved cases . (e) Inied cases . To redued cases . (g) Othed cases , althougMore research is necessary to understand the potential therapeutic use of KGD in ASD as discussed at length for SSADHD . More spAll authors contributed to the design of the work and interpretation of the literature, drafted the work, and gave final approval of the version to be published. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


M) maps were acquired from healthy male and female infants and toddlers, 3 to 48 months of age, and used to train a sigmoidal-based maturational model. The validity of the approach was then established by testing the model on 129 different VFM datasets. Results revealed the approach to have high accuracy, with a mean absolute percent error of 13% in males and 14% in females, and high predictive ability, with correlation coefficients between estimated and true ages of 0.945 in males and 0.94 in females. This work represents a new approach toward mapping brain maturity, and may provide a more faithful staging of brain maturation in infants beyond chronological or gestation-corrected age, allowing earlier identification of atypical regional brain development. Hum Brain Mapp 36:1233–1244, 2015. © 2015 The Authors Human Brain Mapping Published by Wiley Periodicals, Inc.The trajectory of the developing brain is characterized by a sequence of complex, nonlinear patterns that occur at systematic stages of maturation. Although significant prior neuroimaging research has shed light on these patterns, the challenge of accurately characterizing brain maturation, and identifying areas of accelerated or delayed development, remains. Altered brain development, particularly during the earliest stages of life, is believed to be associated with many neurological and neuropsychiatric disorders. In this work, we develop a framework to construct voxel-wise estimates of brain age based on magnetic resonance imaging measures sensitive to myelin content. 198 myelin water fraction (VF Brain maturation encompasses a variety of structural and functional processes that respond dynamically and interdependently to learning, environment, and genetic influences. Axonal pruning, dendritic sprouting, synapse generation, and myelination begin at various stages of fetal development and continue post-natally through to adulthood ), also mirrors this histological sequence [Deoni et al., M developmental profiles between 3 months and 5 years of age [Dean et al., M maps from 198  infants, we construct a voxel-wise probabilistic model of VFM maturation. This model can then be inverted to provide brain age estimates across the whole brain from any infant's VFM data. This approach is tested on an additional, independent sample of 129  VFM datasets with mean absolute percent error between estimated age and actual age and reproducibility evaluated. Our results demonstrate the ability to estimate brain maturation within this early age-span, and at the voxel level, for the first time. The approach allows one to objectively examine maturation across the whole brain, potentially allowing the identification and discrimination of brain areas exhibiting accelerated or delayed growth. Such information holds intrinsic value for identifying abnormal development associated with developmental disorders, as well as in establishing structural-functional linkages that may underlie language delay or other learning disorders.Prior qualitative and quantitative MRI studies of healthy infant brain development have revealed a consistent spatiotemporal pattern of evolving white and gray matter contrast that broadly mirrors the histologically established pattern of white matter myelination [Barkovich et al., MRI data used in this study was acquired as part of a much larger, ongoing longitudinal study investigating white matter maturation [Deoni et al., Subjects for this study consisted of 209  healthy volunteers between 76 and 1526 days  of age at recruitment. A total of 327 successful MRI scans were acquired from these 209 subjects: 128 subjects were scanned a single time, 58 subjects were scanned twice, 21 were scanned three times, and 5 were scanned four times. Follow-up MRI scans were acquired at 6 month intervals for children under 2 years of age, and annually for children over 2 years. While males and females were found to significantly differ in birth weight, they did not significantly differ in their gestation-corrected age, gestation duration, or maternal/paternal socioeconomic status  images, two inversion-prepared (IR)-SPGR images, and 16 T1/T2-weighted balanced steady state free precession  images. SPGR and bSSFP images were acquired with incremented flip angles and the bSSFP images were acquired with two phase cycling patterns (0° and 180°).All MRI data were acquired during natural, nonsedated sleep on a Siemens Tim Trio scanner using a 12 channel radio-frequency (RF) head array [Dean et al., 0 and B1 magnetic field inhomogeneity correction) [Deoni M maps were calculated by fitting a three-pool multicomponent relaxation model to the multiangle SPGR and SSFP data [Deoni et al., M map was then nonlinearly aligned to a common, study-specific template in approximate MNI space using the Advanced Normalization Tools software package [Avants et al., M values [Dean et al., M maps to the study specific template.Following acquisition and data preprocessing  Deoni , voxel-wM(age) = α*exp(−exp(β − γ × age) + δ × age) [Gompertz, M datasets  were used. A Gompertz growth curve of the form VFets Table using thM value and the measured value. The algorithm was bounded by a broad age range of 30 to1800 days to ensure the optimization approach would not be biased to converge to a particular age. Calculation of age values was restricted to a central white matter mask that was created by averaging the normalized VFM maps of all subjects and thresholding this average map by 0.05 to avoid regions of gray matter. Additionally, age values were only estimated for voxels that had a VFM value greater than 0.015. This threshold level was specified to guarantee age values were not calculated in nonmyelinated brain regions, for example in younger participants. A global brain age was further estimated by computing the mean of the non-zero voxel-wise age estimates.To invert this model and estimate brain age, a bounded golden search approach Kiefer,  was usedTo evaluate the performance (accuracy and reproducibility) of the method, voxel-wise and global age estimates were calculated for the additional 129 Table datasetsMean global brain age estimates were compared to the true age using the mean absolute error (MAE):In addition to acquiring MRI data from the subjects, each child was administered the Mullen Scales of Early Learning Mullen,  at each M during the investigated age range, representative developmental trajectories with the corresponding fitted Gompertz function for the corpus callosum, frontal white matter, and optic radiations are shown in the top row of M maps used in the training of the Gompertz model. Average VFM values were extracted from the non-zero voxels of the representative regions and plotted with respect to the subjects' gestationally corrected age. Qualitatively, these representative trajectories highlight the observed sigmoidal pattern of development previously reported [Deoni et al., To illustrate the nonlinear development of VFα, controls for the overall scale and shape of the growth curve; β describes the lag period before the initial inflection point, and γ and δ governs the growth rate of the model.Maps corresponding to voxelwise estimates of the four free Gompertz model parameters for the male and female models are also shown in M maps, estimated brain age map, and percent error maps are shown in M maps. While voxels of percent difference are observed to have a wide distribution across the brain, ranging from −98% to 172% and −99% to 232% for males and females, respectively, these were uniformly distributed about zero, as shown by the histograms in Representative matched axial slices through acquired and model-reconstructed VFThe mean global brain age for each subject was calculated from the voxelwise age estimates and was subsequently plotted against the subject's gestation-corrected chronological age (illustrated in T-) scores of male and female subjects are given in Tablez-test , allowing for a standardized basis for comparison [Weiskopf et al., M and provide a better estimation of brain age; however, examination of the histograms of percent differences reveal the values to be normally distributed about zero, giving us confidence that the inclusion of data collected at different times from the same subject is justifiable. Nonetheless, executions of longitudinal mixed modeling algorithms are highly desirable and would provide a more complete characterization of developmental patterns.Although scans from the same subject were not used in the training of the developmental growth model, they were used in the testing dataset. These repeated measures may introduce a level of bias into the results presented here, and therefore, greater care would be required to model the observed developmental patterns. Longitudinal analysis techniques, including linear and nonlinear mixed models, provide the means to characterize observed patterns of change from repeated measures and have been demonstrated to be successfully implemented in studies examining brain development [Sadeghi et al. M values, mcDESPOT also provides water-pool specific T1 and T2 estimates, as well as more conventional single-component T1 and T2 values [Deoni et al., T1 and T2 values to decrease exponentially throughout infancy and early childhood, and then to increase gradually throughout adulthood [Deoni et al., Although the presented technique offers a simplistic approach to estimating the brain age, the inclusion of additional information may provide improved accuracy. For instance, alongside VFM maps in healthy infants and toddlers and shown, for the first time, voxel-wise estimates of brain age that correlate strongly with gestation-corrected chronological age and average developmental age equivalent measures in healthy infants and toddlers. The method provides an objective examination of the developing brain, potentially permitting areas of accelerated or delayed maturation to be readily identified. Such information is useful within a research context toward understanding linkages between evolving neurodevelopment and malbehavior, as well as clinical-relevance in premature infants, or infants exposed to alcohol or illicit substances in utero. Although additional work is required to evaluate the utility of the method in these at-risk populations, the high accuracy and robustness of the method demonstrated in healthy infants strongly intimates its feasibility and success.As neurobehavioral and neuropsychiatric disorders are increasingly examined within the context of abnormal development, the ability to accurately gauge regional brain maturity becomes ever more important. Here, we have outlined a framework for age-mapping the brain using mcDESPOT-derived VF


Denosumab is a monoclonal antibody directed against RANKL that has been approved for the treatment of both osteoporosis and bone metastases.1 Although an earlier study had shown a significant increase of 5.5% in bone mineral density (BMD) at the lumbar spine with denosumab in 252 women with non-metastatic breast cancer receiving aromatase inhibitors,2 this trial was not designed to assess fracture risk.The maintenance of bone health has been a clinical challenge in breast cancer patients receiving adjuvant endocrine treatment. Especially in the setting of hormone receptor-positive breast cancer, treatment with aromatase inhibitors, although highly effective as anti-cancer agents, causes rapid and substantial bone loss.et al.T-score of the BMD. There was no difference in adverse events between the placebo and the denosumab group. In fact, most adverse events were considered to be aromatase inhibitor related. No cases of osteonecrosis of the jaw or atypical fractures were reported in either group.3 These findings are important as they clearly demonstrate a substantial clinical benefit with the use of denosumab in the adjuvant treatment of hormone-positive breast cancer, while displaying a favorable safety profile.In a recent study, Gnant 5 Administration of the synthetic progesterone derivate medroxyprogesterone acetate (MPA) and the carcinogen 7,12-dimethylbenz[a]anthracene results in an enhanced carcinogenesis, which is driven by a massive increase of RANKL. Genetic inhibition of RANK markedly reduced the incidence of cancer in this setting.4 Similar results were obtained in another study, where pharmacological inhibition of RANKL attenuated mammary tumor development.5In the past years, emerging preclinical and clinical findings have corroborated a role of the RANKL–RANK system in the pathophysiology of breast cancer exceeding that of being a simple osteoclast differentiation factor . RANKL h7 In a preclinical model of melanoma, neutralization RANKL by its decoy receptor osteoprotegerin markedly reduced bone metastases.6 In addition, mammary cancer metastasis to the lung have been shown to be promoted by the presence of tumor-infiltrating regulatory T cells, which produce high levels of RANKL.8Furthermore, RANKL has been directly linked to the occurrence of bone metastases by increasing the migration of various malignant cells including breast, prostate and melanoma by binding its receptor RANK.9 In breast cancer, RANKL expression was observed in 24/40 samples,10 whereas RANK overexpression was found in 39% of ductal and 53% of lobular breast carcinomas.11Indeed, expression levels of RANKL and RANK are increased in metastatic prostate cancer samples (44% and 49%) compared with primary prostate cancer samples (31% and 38%), respectively.13 Low levels of RANK and high levels of osteoprotegerin were correlated with a longer overall survival in microarray analyses.11 This was confirmed by two different studies that immunohistochemically evaluated 185 and ~600 breast cancer samples for RANK expression and showed a significant association between RANK and poor disease-free survival.13 In addition, RANK has been positively correlated with the development of bone metastases.11Several studies have assessed the prognostic value of RANK expression in breast cancer patients regarding survival and the propensity for bone metastases.The occurrence of bone metastases in the ABCSG-18 trial was too low to assess a benefit of denosumab, which was expected considering the low-recurrence risk in this cohort. However, another trial entitled D-CARE (NCT01077154) is currently underway to specifically address the question whether denosumab treatment can delay the occurrence of bone metastases in patients who are at a higher risk of recurrence than those of the ABCSG-18 trial. The cohort included in this trial has a higher risk of bone recurrence and will provide further insights on the extend of clinical benefit of adjuvant RANKL inhibition in breast cancer patients.In summary, the results from the ABCSG-18 trial show a benefit for patients with hormone-positive breast cancer receiving adjuvant denosumab in terms of fracture reduction while on aromatase inhibitors. These findings are in line with an increasing evidence to suggest that RANKL and RANK may have disease modifying effects in breast cancer by affecting carcinogenesis and the process of bone metastases.


Dietary intake alters specifically human gut microbiota and mucosal immune response. Inflammation is major initiating factor of metabolic associated diseases namely CVD, obesity and diabetes type II.was to provide the scientific background for the implementation of personalised diets for the prevention and treatment of metabolic associated diseases.Sideritis scardica were administered to mice orally for two weeks (15 mg/200µl/day/mouse). Level of IgAs in gut contents and cytokine profiles in supernatants of fragment cultures of gut associated lymphoid tissues was measured by ELISA, and numbers of immune cells quantified by FACS. Human DCs derived from peripheral blood monocytes were exposed to plant extracts at 2 mg/ml for 6 and 24 hours. The expression of CD markers by DCs and levels of secreted cytokines were assessed before and after exposure to the extracts. Clinical trials had been performed in order to detect the early microbial and immunological biomarkers and efficacy of polyphenols reach extracts effect on CVD functions.Classic and modern experimental systems, animal and cells models in preclinical investigation had been used. Methanolic extracts of edible plants: nettle, dill, kale, persimmon, pomegranate, and Edible plants induced a shift in expression of pro-/anti-inflammatory CD1a molecules on a surface of human DCs derived from peripheral blood monocytes. Commensal bacteria act on moDCs differentiation and activation by dose- and strain-specific manner, altering the expression of CD1a, NOD-2, RLH, PPARγ and RARα genes and cytokines production. Plant extracts and commensal microorganisms modulate the humoral and cellular mucosal immune response in GALT tissues on mice models differently regulating IgA secretion by peritoneal cavity B1/B2 cells and compartment of small intestine. Microbiological and immunological biomarkers of metabolic associated diseases as a result of evaluation of all the complexity diagnostic parameters had been found. Selected plants were able affect endothelial function, lipid profile and insulin sensitivity of patients with metabolic and cardiovascular diseases in randomised controlled trial.These results support the concept of potential implementation of personalised diets for the patients with metabolic associated diseases.The current challenge is to perform clinical intervention study according to EFSA demands and to criteria of evidence-based medicine to investigate efficacy of personalised diets developed from components and original recipes of traditional foods.


Most of the research on Ca2+ signaling in plants has been focused on the transport mechanisms for Ca2+ across the plasma- and the vacuolar membranes as well as on the components involved in decoding of cytoplasmic Ca2+ signals, but how intracellular organelles such as mitochondria are involved in the process of Ca2+ signaling is just emerging. The combination of the molecular players and the elicitors of Ca2+ signaling in mitochondria together with newly generated detection systems for measuring organellar Ca2+ concentrations in plants has started to provide fruitful grounds for further discoveries. In the present review we give an updated overview of the currently identified/hypothesized pathways, such as voltage-dependent anion channels, homologs of the mammalian mitochondrial uniporter (MCU), LETM1, a plant glutamate receptor family member, adenine nucleotide/phosphate carriers and the permeability transition pore (PTP), that may contribute to the transport of Ca2+ across the outer and inner mitochondrial membranes in plants. We briefly discuss the relevance of the mitochondrial Ca2+ homeostasis for ensuring optimal bioenergetic performance of this organelle.Plants, being sessile organisms, have evolved the ability to integrate external stimuli into metabolic and developmental signals. A wide variety of signals, including abiotic, biotic, and developmental stimuli, were observed to evoke specific spatio-temporal Ca In animals, when PTP opens only transiently, it possibly mediates Ca2+ release from mitochondria  and function have been identified  mediated by VDACs is unclear, however AtVDAC1 has been shown to interact in two-hybrid yeast system with CBL1, a Ca2+-sensor  selectivity filter sequence (http://aramemnon.uni-koeln.de/). Whether all these isoforms are indeed targeted to mitochondria, whether they form ion channels able to provide a permeation pathway for Ca2+ and whether the various isoforms operate in different tissues and/or at different developmental stages still awaits clarification. Recently obtained experimental evidence indicates that at least one of the isoforms is indeed targeted to mitochondria in Arabidopsis  in vitro -binding domains are located in the cytosol, possibly sensing the cytosolic glutamate/aminoacid concentration  that show high aminoacid sequence similarity to SPG7  and to AFG3L2 . Among those with the highest score, AtFtsH3 and AtFtsH10 are present in mitochondria , the above-described uptake and efflux pathways for calcium might cooperate. For example, Caoduction .In summary, a combination of genetics, fluorescent probe imaging, electrophysiology, bioenergetics and physiology will hopefully provide answers to the numerous open questions related to mitochondrial calcium homeostasis in plants.All authors contributed to the works published by our groups which are described in the minireview. Furthermore, all authors actively participated in writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Total joint replacement surgery is being performed on an increasingly large part of the population. Clinical longevity of implants depends on their osseointegration, which is influenced by the load, the characteristics of the implant and the bone-implant interface, as well as by the quality and quantity of the surrounding bone. Aseptic loosening due to periprosthetic osteolysis is the most frequent known cause of implant failure. Wear of prosthetic materials results in the formation of numerous particles of debris that cause a complex biological response. Dual-energy X-ray Absorptiometry (DXA) is regarded as an accurate method to evaluate Bone Mineral Density (BMD) around hip or knee prostheses. Further data may be provided by a new device, the Bone Microarchitecture Analysis (BMA), which combines bone microarchitecture quantification and ultra high resolution osteo-articular imaging. Pharmacological strategies have been developed to prevent bone mass loss and to extend implant survival. Numerous trials with bisphosphonates show a protective effect on periprosthetic bone mass, up to 72 months after arthroplasty. Strontium ranelate has been demonstrated to increase the osseointegration of titanium implants in treated animals with improvement of bone microarchitecture and bone biomaterial properties. Unfortunately, the introduction of a prosthesis or a dental implant inevitably alters the physiological transmission of loads to the surrounding bone, which starts a remodeling process, resulting in reduction in bone mineral density (BMD)2.Endosseous implantation is one of the most common procedures in orthopedics and dentistry. The ever expanding aging population has also led to an increasing need for total joint replacements6. Mechanical, thermal and chemical intraoperative damage induces necrotic phenomena on the periprosthetic bone, which takes approximately 3 months to repair7. Then the osteocytes, acting as mechanoreceptors, translate the mechanical stimulus into an electrical signal, either activating osteoclasts (OCs) in bone areas no longer subjected to physiological loading or stimulating osteoblastic cell lines where bone is stressed, with consequent hypertrophy10.Aseptic loosening due to bone destruction around the prosthesis has been established as the main cause of implant failure11. After Total Hip Arthroplasty (THA), for example, periprosthetic fractures occur in 0.8% of patients at 5 years, and 3.5% at 10 years12. This is the third most common reason for re-operation14, while implant failure by aseptic loosening could be expected in 3–10% of cases within 15 years12.Despite being a widespread practice, joint arthroplasty almost unavoidably involves a loss of surrounding bone, which can cause periprosthetic fractures resulting in reduced function, subsequent morbidity and increased risk of mortalityA better understanding of the biological basis of peri-implant osteolysis has allowed the development of therapeutic strategies to prevent periprosthetic bone loss, in particular with Bisphosphonates (BPs) and Strontium Ranelate (SrRan).This paper will cover the biological basis of periprosthetic bone resorption, diagnostic techniques and preventive or therapeutic approaches, both from a pharmacological and surgical point of view.15. The basis of this process includes mechanical and biological factors16.Risk factors for periprosthetic bone destruction include osteoporosis, rheumatoid arthritis, revision surgery and stress shielding. These lead to a resorption process in bone areas that are no longer mechanically subjected16. Particles ranging from 0.2 to 10 μm in diameter undergo phagocytosis by macrophages17.Several reports have shown that the cellular responses to biomaterial wear particles play an important roleIn vitro studies of macrophage cultures clearly indicate that smaller particles of polymethylmethacrylate (PMMA) and polyethylene, materials used in implants, (< 20 μm) elicit a significantly increased inflammatory cytokine response, as indicated by increased release of Tumor Necrosis Factor (TNF), IL-1, IL-6, prostaglandin (PG)E2, matrix metalloproteinases, receptor activator of nuclear factor kappa-B ligand (RANKL) and other factors that affect osteoclast differentiation and activity21. Moreover, direct biologic interaction between particles and the cell surface is sufficient to activate osteoclastogenic signaling pathways22, causing bone resorption and periprosthetic BMD loss.23. However, the immediate high postoperative activity of osteoclasts is confirmed by a study on 53 patients followed for 12 months after THA with a cemented femoral stem23. The study showed an early postoperative increase of C-terminal telopeptides of type I collagen (CTX-I) (markers of bone resorption), where the highest level was noted (+21%) 3 weeks after THA, then decreased at 8 weeks after THA (-7% from preoperative levels). This increase was significantly correlated with the bone loss measured by DXA in the calcar region23. These data suggest that a postoperative antiresorptive treatment administered for the period of increased CTX-I levels could prevent periprosthetic bone loss23.Bone turnover markers, due to their high intra-individual variability, have a limited predictive value concerning the extent of periprosthetic bone loss25.Stress shielding is also considered as a potent stimulator of bone resorption. After a total hip arthroplasty, the stem geometry of the implant plays a key role in the load transfer to the femur and consequently in femoral remodeling30. The data on the role of stem design are more consistent24.Conflicting results have emerged regarding the hypothesis that the amount of periprosthetic bone remodeling can be influenced by different factors, including sex, age, weight, Body Mass Index (BMI) and bone mass32. It is also used to assess the effectiveness of these treatments by comparing the medium-term bone density changes between treated and untreated groups. A special piece of software named “metal-removal” enables DXA to analyze periprosthetic bone using seven conventional Regions Of Interest (ROI) called Gruen zones  is considered the most reliable tool to evaluate bone remodeling after THA using implants with different stem designsn zones 33,34.38.Thanks to improvements in software and technology, bone densitometry examinations by DXA may actually allow the detection of periprosthetic bone remodeling that cannot be observed in conventional radiographs as DXA provides an accurate measurement of total and regional periprosthetic BMD after THA38. Analysis of the 7 periprosthetic Gruen zones is the most commonly used protocol to evaluate bone remodeling after the implantation of conventional femoral stems41. As shown in39.DXA scanning is usually performed with the patient in the supine position, the leg placed in a standardized support to ensure a neutral position42. However, in cross-sectional studies, the controlateral unoperated hip has also been analyzed to obtain individual comparative BMD values44.Postoperative measurements are commonly used as baseline values and the measurements at follow-up are expressed as a percentage of the baseline measurements39, its metal-removal software also provides peri-prosthetic measurements for knee arthroplasty49. No DXA protocol is available for ankles, shoulders, elbows, or wrists.Although DXA is regarded as the most accurate method for the detection of small alterations in bone mineral density around hip prostheses47. Bone pixels are isolated and segmented by thresholding, then artificial numbers are assigned to them, while the projection profile of metal object is removed47.The study of periprosthetic bone by X-ray techniques can be hindered by artefacts due to the presence of inorganic material in the scan plane. Metal artefacts can be reduced by the use of suitable filters acting during the acquisition, which allow reducing the radiation spectrum and the dynamic range from the beam itself. There are also algorithms to estimate the error due to the presence of artifacts in the acquired images. A method of suppression of high-density artefacts not requiring the introduction of extra bone artefacts was described by Wey and colleagues48. The CT scans were downloaded onto a dedicated software , and automatically evaluated on the basis of an adaptive tracer algorithm that outlined the contour of the outer and inner cortical bone as well as the prostheses, completed by manual correction of metal artefacts48.The effects of femoral adaptive bone remodeling after THA have also been assessed by quantitative computer-tomography assisted osteodensitometry, a method able to differentiate cortical and cancellous bone structures18F-FDG studies by PET/CT scanners49. This method, which allows the anatomical information from CT to be combined with metabolic PET data, is particularly useful in cancer staging, where the improvement in detectability of small lesions located near metal hip implants by this algorithm is very important49.Moreover, an artifact reduction algorithm was developed in50.An innovative device has recently been developed, the BMA, which combines ultrahigh resolution 2D digital X-Ray images and a set of trabecular bone texture analysis parameters, such as 2D fractal analysis (H mean), Co-Occurrence (COOC) and Run Length Encoding (RLE), thus providing a bone microarchitecture quantification independent of bone density measurementin vivo micro-analysis of human bone structure and abnormalities, such as fracture lines that are often uncertain or ignored. All the joints can be examined . The micrometric accuracy in the visualization of joint interspace reflects cartilage thickness, useful for the diagnosis and follow-up of osteoarthritis (OA). The digital X-Ray detector allows examination at a very low dose (effective dose < 2 μSV for a heel exam) due to its excellent Detection Quantum Efficiency (DQE). This performance, added to the quality of a high frequency X-Ray generator, is obtained in less than 1 second exposure time, while the image processing is achieved in less than 2 minutes, facilitating patient workflow and improving productivity50.With a resolution near 100 µm, BMA visualizes the bone structure at the trabecular level, allowing theAlthough not currently widespread, except in few research centers specialized in bone diseases, the employment of BMA may represent a promising device for the study of periprosthetic bone analysis of any joint as well as for arthritis, osteoarthritis and altered bone healing.53 and radiographic studies54, followed by DXA analyses, have been conducted on patients subjected to THA. These studies suggest that the ultra-short implant  can provide immediate postoperative stability and a more physiological load distribution, thus increasing periprosthetic BMD in the medial regions over time24, preserving bone mass and stimulating trabecular bone apposition55. The presence of the lateral flare makes the diaphyseal stem with distal cortical contact almost unnecessary, thus increasing periprosthetic BMD in the medial regions over time24.Since the stem geometry of the implant is believed to play an important role in load transfer to the femur, biomechanical tests24 assessed bone remodeling in patients subjected to two metaphyseal implants, type 1 with a very short stem and type 2 with no diaphyseal stem. Using a 5-ROI protocol of DXA analysis, they found that ultra-short implants can provide immediate postoperative stability and a more natural physiological load distribution in comparison with conventional anatomic implants, thus increasing periprosthetic BMD in the medial regions over time.Albanese and colleagues27. A fundamental feature that enables fixation is the porous surface of the prosthesis56. Cementless THA is increasingly popular. The high rate of osteolysis, aseptic loosening and revision associated with earlier uncemented femoral components has been greatly reduced by using better designed implants incorporating circumferential porous coating56. Moreover, proximal femoral fixation has been shown to prevent stress shielding and a tapered distal tip reduces thigh pain56. The mid-term outcome of a modular, cementless, proximally hydroxyapatite-coated, anatomic femoral stem in THA was reported by Cossetto and Goudar 56. They showed that the modularity of the neck of this femoral component is helpful in both primary and revision settings. In primary procedures, after implantation of the stem, correction can still be made in leg length and offset. In revision procedures, the modularity of the neck facilitates adjustments in leg length, offset and neck version without the need to extract a well fixed femoral component. It also facilitates access to the acetabular component by way of removal of the modular neck and head56. In that study, in case of dislocation, changing the modular neck and head avoided more extensive revision requiring removal of a well-fixed femoral stem. Patients were evaluated pre- and post-operatively , with a clinical evaluation  based on Merle d’Aubigne and Postel scores57 and by anteroposterior and lateral weight-bearing radiographs, in which the femoral component was analyzed according to the 7 zones of Gruen. Contrary to the increased rate of revision in modular hip systems found in the Australian Orthopaedic Association National Joint Replacement Registry, the modular, cementless, proximally hydroxyapatite-coated, anatomic femoral stem provided predictably stable fixation with excellent mid-term outcome58.After stem design, the most important factor known to influence periprosthetic BMD is the fixation of the implant33 conducted a prospective densitometric study by DXA in a group of patients who underwent unilateral bicontact stem implantation. This is a cementless implant made of a titanium forged alloy (Ti6A14V), with a proximal microporous pure titanium plasmapore coating. Despite small signs of stress shielding observed at the tip of the stem, it has shown to provide adequate proximal bone stock preservation33.Similarly, Lerch and colleagues47. A clinically applicable soft foam positioner designed to ensure rotational stability and allow for slight flexion (i.e. 25°) may be safe for clinical use, because this position can be obtained with all normal total knee arthroplasty (TKA) patients both in the early period after surgery and in later follow-ups47.Studying knee arthroplasty is rather difficult when compared to studying THA, due to the position of the patient required for the exam. Full extension of the knee is not possible for most patients in the first days after surgery, therefore while deficits in extension normalize with rehabilitation, individual knee flexion between the baseline and follow-up investigations may be different48 described the changes in bone density over the course of time following a cement-free TKA based on a functional categorization of the measurements in terms of defined ROI by means of DXA. The seven regions were defined as indicated inHowever, a prospective cohort study conducted by Windisch and colleagues48. Statistical analysis revealed that the most significant changes occurred within the first 3 months postoperative with the highest bone density loss found in the region of the proximal medial tibia48.A further aim of that study was to examine the associations between the defined parameters of age, sex, severity of arthrosis, and axis alignment. At 12 months after surgery, a high severity of osteoporosis was associated with low absolute values for periprosthetic bone density. Women demonstrated a lower absolute periprosthetic BMD value than men. The preoperatively determined femur and tibial average Cortical bone Marrow Index (CMI), the varus angle and the BMI showed no significant correlation with the absolute or relative changes of periprosthetic bone density49 has studied the effects of Unicompartmental Knee Arthroplasty (UKA), which has received renewed interest for medial OA within the last decade. UKA has been traditionally used in older, non-obese patients with a sedentary lifestyle. UKA is advantageous as only the severely damaged compartment is replaced and the bone stock is preserved, which is associated with fast recovery times49. Moreover, improvements in surgical technique, implant materials and prosthetic design have made UKAs more durable and reliable49.Another group59.The group measured BMD using DXA and data were collected from multiple ROIs for each patient at several intervals during the first 7 postoperative days. The highest femoral periprosthetic bone loss rate was observed during the first 3 months after UKA. However, BMD changes from 2–7 years were not significant. In particular, there was a significant loss of BMD from distal femoral sites after UKA, while BMD changes were minor in the tibial metaphyseal regions, consistent with a mechanical axis balance between the medial and lateral sides of the tibia. Further, porous tantalum tibial components maintained better periprosthetic BMD compared with cemented tibial implants60. In this regard, it should be noted that differentiation of bone marrow macrophages into mature osteoclasts requires recognition and binding of osteoblasts, fibroblasts, and T cell secreted factor RANKL by its cognate receptor RANK, which is expressed on the surface of osteoclast precursors63. Another osteoblastic factor, namely osteoprotegerin (OPG), acts as a decoy receptor by binding to RANKL and reducing its bioavailability. On the other hand, binding of RANKL to RANK stimulates induction of several intracellular pathways by this receptor, leading to activation of key transcription factors, most notably NF-κB22.In combination with improvements in implant integration, strategies to target the cellular components (osteoblasts and osteoclasts) that contribute to implant failure should be implemented22. In this context, a review by the group of Abu-Amer considers three kinds of approaches22. The first involves targeting OC precursor cells, which are brought to inflammatory sites by circulating cytokines. The second entails targeting precursors that are stimulated by the particle-mediated cellular response to differentiate into OCs. The third approach involves targeting activation mechanisms of mature osteoclasts22.It is known that NF-κB activation, when induced by factors such as TNF and PMMA particles, exacerbates osteoclastogenesis and inflammatory responses65, but the successful preclinical findings, i.e. the ability to prevent and reverse wear debris-induced osteolysis, have not been confirmed by clinical trials. At present, the monoclonal antibody anti-RANKL is available, known as ‘denosumab’, which shows significant effectiveness in the inhibition of bone resorption due to osteoporosis66. Transduction of a dominant-negative form of the NF-κB inhibitory protein, IκB, by retaining NF-κB in the cytoplasm, has been revealed to be able to block osteoclast formation and activity69. Another viable approach is to block activation of the upstream IKK complex, which is responsible for phosphorylation of IκB and subsequent activation of NF-κB, by introducing a small peptide that hinders assembly of the IKK complex70. Notably, administration of the dominant negative IκB protein or the IKK inhibitory small peptide to arthritic mice blocks bone erosion and particle-induced osteolysis of calvaria in mice71.An example of the first strategy is the application of RANKL decoy molecules such as OPG and the soluble fusion protein RANK-Fc71. Selective inhibitors of the c-src tyrosine kinase have shown great promise in reducing osteoclast activity73.Further targets of therapy are NF-κB mediated genes. Recent studies have revealed that proinflammatory cytokines such as TNF act directly on some of these genes and their products, in particular c-src and NF-κB, to accelerate osteoclast formation and cause a strong osteoclastic response74, potent anti-resorptive drugs widely used in the treatment of osteoporosis, which inhibit osteoclast function and induce their apoptosis. In animal models subjected to implantation77, oral BP showed reduced radiographic periprosthetic radiolucency, as inhibiting debris-induced osteolysis, although the levels of PGE2 and IL-1 remained elevated in tissue cultures from these implants. These studies have served as a basis for clinical trials using alendronate, one of the most commonly used BPs, in patients with radiographically evident osteolytic lesions. In other studies, bone loss around implants was prevented and treated by alendronate81.Another promising approach involves the use of bisphosphonates (BP)83. Moreover,in vivo trials showed a direct action of some BPs in stimulating the osteoblastic proliferation, which might play an essential role in increasing periprosthetic bone ingrowth84. The mechanism by which BPs are supposed to act on the osteoblasts is by up-regulating the expression of genes coding the synthesis of some morphogenetic proteins, including BMP-285.More recently, human clinical trials have revealed the efficacy of BPs in reducing particle-induced osteolysis over the first year of life of cemented and cementless hip and knee replacement prostheses, with better and more durable results when treatment was started early after surgery and continued for over 6 months2. Thirty-five women over 60 years old, not necessarily suffering from osteoporosis, were subjected to THA. They were examined by DXA at the 15th day after surgery (T0) and at 6 and 12 months, either at the spine, contralateral femur or periprosthetic femur, both totally and at the 7 Gruen regions. Of these patients, 19 patients received 3 mg ibandronate intravenously within 5 days after surgery and then passed to oral administration with a monthly dose of 150 mg, plus calcium carbonate (1 g) and cholecalciferol (880 IU) supplementation. The other 16 patients formed the control group and were treated only with calcium carbonate (1 g) and cholecalciferol (880 IU) supplementation. As a result, a reduction in the BMD was observed over the first 6 months from T0 in both groups; smaller reductions were observed in the treated group (-7.7% compared to the control group). In contrast, at 12 months, a marked trend reversal was observed, with a statistically significant BMD percentage recovery compared to the baseline value at T0 of about 1.74% of the global BMD in the treatment group. This was more evident in region R1 (+3.81%) and in the lateral metaphyseal region (R2) (+4.12%). On the other hand, no global BMD recovery was observed in the control group, which had virtually stabilized compared to values at 6 months2.Muratore and colleagues assessed the effect of ibandronate, another type of BP that can peculiarly be administered either orally or intravenously, with extended dosing intervals, thanks to its high affinity for the bone mineral component and its consequent long-term persistence in the skeletal tissue, which therefore ensures excellent adherence to therapyConsidering that periprosthetic remodeling occurs within the first 6–12 months after surgery, this study therefore concluded that ibandronate reduces periprosthetic resorption, in particular in the medial metaphyseal region , the one at greater risk with respect to the life of the prosthesis.86. The more potent nitrogen-containing bisphosphonates  are not metabolized in this way but inhibit key enzymes of the mevalonate/cholesterol biosynthetic pathway, such as farnesyl pyrophosphate synthase, compromising the function of essential intracellular messengers, thus causing osteoclast inactivation and apoptosis87.Bisphosphonates, which are chemically stable analogues of inorganic pyrophosphate, can be classified into at least two groups with different molecular modes of action. The simpler non-nitrogen-containing bisphosphonates  can be metabolically incorporated into nonhydrolysable analogues of adenosine triphosphate, which interfere with adenosine triphosphate-dependent intracellular pathways11 of 14 randomized controlled trials employing BPs after joint arthroplasty found that the protective effect of these drugs, probably modified by BP generation and the prosthesis location, could persist in a middle-term follow-up after surgery and for 18 to 70 months after drug discontinuation. The efficacy was more potent for amino-BPs, than for the first generation of BPs11.A meta-analysis11.However, since the trials did not address the clinically relevant outcomes, it is imperative to perform a randomized clinical trial with an adequate number of patients and sound methodology in order to establish the definitive role of BPs in joint arthroplasty, and make recommendations for their optimal administration. It is also necessary to better understand the mechanisms of their actions and potential side effects90, alterations in normal collagen cross-linking and matrix heterogeneity93, reduced vascularity95 and decreased cortical bone toughness98, as well as a small number of subtrochanteric or diaphyseal femoral fractures102. The exact mechanism of these ‘atypical’ fractures is unknown. One theory is that they occur in the subtrochanteric region of the femur because it is subject to high bending forces106. The latter would cause the formation of micro-cracks, normally repaired through bone remodeling. Bisphosphonates’ suppression of bone turnover results in a failure to repair these micro-cracks102.Moreover, prolonged use of BPs has recently been associated with severe suppression of bone turnover107. It has been shown that 200 IU of salmon calcitonin administered nasally decreases osteoporotic fractures109. A Finnish clinical trial107 randomized 60 patients who underwent THA using cemented Exeter prostheses into a treatment group  and placebo group  for 6 months. They were followed with DXA, bone turnover markers and dynamic histomorphometry on bone biopsies taken from the femoral neck at the time of discharge, after 6 and 12 months. Calcitonin was not shown to promote any additional value on calcium substitution in preventing aseptic osteolysis107.Calcitonin is a 32-amino acid polypeptide hormone (produced predominantly in C-cells of the thyroid gland) which mainly acts by inhibiting osteoclast function1. The beneficial effects of SrRan have previously been reported in various animal models, where it has been shown to prevent bone loss by maintaining bone formation at a high level and inhibiting bone resorption114. Thesein vivo results were consistent within vitro data which show that SrRan reduces bone resorption by osteoclasts and increases bone formation by osteoblasts118. In particular, SrRan has been shown to enhance preosteoblastic cell replication and osteoblastic differentiation and to decrease abilities of osteoblasts to induce osteoclastogenesis, both through the calcium-sensing receptor and an increase in the OPG/RANKL ratio118.Another antiosteoporotic treatment, Strontium Ranelate (SrRan), was shown to be of considerable interest in investigations to improve implant osseointegration119. Furthermore, treatment with SrRan is not associated either with osteonecrosis of the jaw or with low energy atypical fractures of the femur1.It has also been demonstrated that SrRan is able to improve bone biomechanical and structural properties1 showed that SrRan significantly improves mechanical fixation of titanium implants inserted into the tibias of female rats, with both a positive effect on bone microarchitecture and on bone biomaterial properties in the vicinity of the implant. SrRan increased pull-out strength compared to controls (+34%), with a significant improvement of bone microarchitecture around the implant, a more plate-shape structure and an increase in bone-to-implant contact (+19%)1.A group from the Bone Division of Geneva120 was designed to evaluate the effect of systemic SrRan treatment on fixation of hydroxyapatite (HA)-coated titanium screws in ovariectomized (OVX) rats. The OVX rats were randomly divided into the following groups: OVX (without treatment), OVX+SRL (treated with a low SR (i.e. SrRan) dose of 500 mg/kg/day) and OVX+SRH (with a high SR dose of 1000 mg/kg/day). Micro-CT and biomechanical push-out tests were performed twelve weeks after treatment, in order to evaluate bone blocks with implants. The two groups treated with SR showed an increase of bone volume ratio, osseointegration and maximal force, compared to OVX animals, suggesting that SrRan treatment can improve HA-coated screw fixation dose-dependently in OVX rats and facilitate the stability of the implant in the osteoporotic bone120.Another studyThese results may support the potential benefits of SrRan in enhancing osseointegration in orthopaedic and dental surgery.Aseptic loosening, due to periprosthetic osteolysis, is the most common cause of implant failure.Among the other clinical and biomechanical criteria, bone status should be considered before proceeding with arthroplasty, in order to select the most adequate implant model as well as to evaluate the necessity of an anti-osteoporosis therapy.Dual-energy X-ray Absorptiometry allows evaluation of bone density around hip or knee prosthesis, and further data may be provided by the new Bone Microarchitecture Analysis device.The comprehension of the principal mechanisms of periprosthetic bone loss has led to the development of pharmacologic strategies aiming at the enhancement of bone mass recovery after surgery and consequently to the prolongation of implant survival.BPs, potent anti-resorptive drugs widely used in the treatment of osteoporosis and other disorders of bone metabolism, were shown to be particularly effective in reducing periprosthetic bone resorption in the first year after hip and knee arthroplasty, both cemented and cementless.SrRan, due to its antiresorbing and bone-forming activity, also promises to facilitate the stability of dental and joint implants in both healthy and osteoporotic bone. This is a nice review that more addresses bone loss directly following TJR as opposed to the consequences of wear driven osteolysis that can initiate many years after surgery. Nevertheless, the discussions on treatment approaches are useful. Adding that denosumab is now entering clinical trials for peri-implant osteolysis  might be warranted.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This review nicely covers the current work in enhancing bone integration with modern implants, however this field will be truly exciting over the next 5-10 years as we will see a vast improvement in device design with biologic materials with rapid prototyping and tissue engineering.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. This is a comprehensive evaluation of periprosthetic bone loss in mostly total hip arthroplasty that is helpful for orthopaedic surgeons. Currently, most orthopaedic surgeons only evaluate bone loss with plain radiographs. Having the knowledge to use DXA and BMA may be helpful in selective cases, although ordering advanced imaging on all patients may not be clinically necessary. Additionally, being able to combat bone loss with therapeutic treatments such as bisphosphonates and strontium ranelate may prove useful in a select patient population for a specified duration of time. Overall, this is a helpful article with cutting edge technology that may be useful for imaging techniques to better evaluate bone loss, and holds potential for evaluating future treatments to prevent and restore bone loss.Title/Abstract: Both are appropriateImaging techniques for the evaluation of periprosthetic BMD loss:- This should include a section on x-ray imaging and CT imaging with metal subtraction.- What is the accessibility of BMA? What type of facilities have this? Are there studies comparing this technology with other imaging techniques with implants?Therapeutic strategies for enhancing bone mass recovery after arthroplasty:- Good sections on surgical and pharmacological considerationsPharmacological strategies to prevent aseptic loosening,” the authors mentioned the use of OPG and the soluble fusion protein RANK-Fc. However, there are no studies presented evaluating this in the setting of implants and only in the osteoporosis model. This may not be an appropriate to mention in this article .- Under “- It would be helpful to provide the mechanism of action of Strontium Ranelate.Conclusions: There should be a sentence on imaging, as mentioned in the article.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.  Imaging techniques for the evaluation etc., before the last sentence, we would add: "Although not currently widespread, except in few research centers specialized in bone diseases, the employment of BMA..."As regards BMA technique, it is not currently widespread for a clinical use, except in few research centers specialized in bone diseases, like ours at Orthopedic Trauma Center in Florence, Italy. As its entrance is very recent, and its potential applications are very wide, it is not yet available a protocol to compare this technique with other ones for peri-prosthetic bone study. So, at the end of the paragraphAbout metal subtraction option in X-ray and CT imaging, as it represents a real possibility to correct artifacts, we are preparing a brief paragraph to add: thank you for the suggestion!but the successful preclinical findings, i.e. the ability to prevent and reverse wear debris-induced osteolysis, have not been confirmed by clinical trials".OPG and RANK-Fc were the first molecular strategy, so we would cite them, adding the sentence "In particular, SrRan has been shown to enhance preosteoblastic cell replication and osteoblastic differentiation and to decrease abilities of osteoblasts to induce osteoclastogenesis, both through  the calcium-sensing receptor and an increase in the OPG/RANKL ratio :1291–1300)."Regarding Strontium Ranelate, we would add: "Dual-energy X-ray Absorptiometry allow to evaluate bone density around hip or knee prosthesis, and further data may be provided by the new device called Bone Microarchitecture Analysis." can be added after the second paragraph.In the conclusions, the sentence "We will provide a new version of the paper with all these corrections as soon as possible.The authors would like to thank the referee for his response. I would like to congratulate Loredana Cavalli and Maria Luisa Brandi on this really quite enjoyable review. For me, I found it quite comprehensive but there were some minor omissions such as the role of electrical charge on bone integration and role of tissue engineering and rapid prototyping on prosthetic design in the future. I would also have liked to have more information from the lessons learnt from failed implants in terms of the pathobiology at the interface. Otherwise it was a good review of the literature and worthy of publication.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. The authors would like to thank the referee for his response.


DNA methylation primarily occurs within human cells as a 5-methylcytosine (5mC) modification of the cytosine bases in CpG dinucleotides. 5mC has proven to be an important epigenetic mark that is involved in the control of gene transcription for processes such as development and differentiation. However, recent studies have identified an alternative modification, 5-hydroxymethylcytosine (5hmC), which is formed by oxidation of 5mC by ten-eleven translocation (TET) enzymes. The overall levels of 5hmC in the mammalian genome are approximately 10% of 5mC levels, although higher levels have been detected in tissues of the central nervous system (CNS). The functions of 5hmC are not yet fully known, but evidence suggests that 5hmC may be both an intermediate product during the removal of 5mC by passive or active demethylation processes and also an epigenetic modification in its own right, regulating chromatin or transcriptional factors involved in processes such as neurodevelopment or environmental stress response. This review highlights our current understanding of the role that 5hmC plays in neurodegenerative diseases, including Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), fragile X-associated tremor/ataxia syndrome (FXTAS), Friedreich ataxia (FRDA), Huntington's disease (HD), and Parkinson's disease (PD). FMR1) gene  enzymatic conversion of cytosine residues in CpG dinucleotides to 5-methylcytosine (5mC) , formed due to oxidation of 5mC by ten-eleven translocation (TET) enzymes, a family of 2-oxoglutarate- and Fe(II)-dependent dioxygenases, consisting of three proteins, TET1, TET2, and TET3  is the most common neurodegenerative disorder, characterized by progressive decline of cognitive functions, neuronal cell loss, and two hallmarks of pathology, extracellular amyloid beta plaques and intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein Tanzi, . The cauAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective loss of motor neurons within the brain and spinal cord  is a late-onset neurodegenerative disease caused by CGG repeat expansion mutations within the 5' untranslated region (UTR) of the FXN gene, leading to decreased expression of the essential mitochondrial protein frataxin  is a rare autosomal recessive neurodegenerative disorder caused by GAA repeat expansion mutation within intron 1 of the HTT gene, leading to abnormal polyglutamine formation within the amino-terminus of the HTT protein  of HD patients compared with age-matched controls  is an autosomal dominant progressive neurodegenerative disease, characterized by chorea, dystonia, and cognitive decline, with the main site of pathology being the striatum. HD is caused by CAG repeat expansion mutation within exon 1 of the SNCA , PARK2 (parkin), PTEN induced Putative Kinase 1 (PINK1), PARK7 (DJ-1), Leucine Rich Repeat Kinase 2 (LRRK2) and ATP13A2  is the second most common neurodegenerative disease after AD, characterized by the progressive loss of substantia nigra dopaminergic neurons, resulting in muscle rigidity, bradykinesia, tremor, and instability. The causes of PD are unknown, but mutations in several genes have now been identified in rare cases of inherited PD, including the genes To date, the investigations of 5hmC in relation to neurodegenerative diseases can be considered to be within their early days. Of the few studies that have been performed, no common features have emerged regarding 5hmC alterations and neurodegenerative diseases; both global increases and decreases of 5hmC levels have been identified in different diseases, and in the case of AD, within the same disease. It is likely that poorly reproducible techniques of immunohistochemical quantification have contributed to this lack of clarity. One impetus behind the recent studies of 5hmC is the potential to identify changes in 5hmC as a potential biomarker of neurodegenerative disease. Alterations of 5hmC levels may be useful, irrespective of whether they are considered to be a cause or a consequence of the disease process. However, initial studies of sporadic ALS have revealed no correlation between alterations of 5hmC, or indeed in 5mC, in spinal cord and blood, suggesting that these would not be suitable as biomarkers of this neurodegenerative disease (Figueroa-Romero et al., All authors contributed to draft the manuscript and all authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.