Although acupuncture is gaining popularity for the treatment of nonspecific pain, the immediate analgesic effect of acupuncture has never been reviewed. We conducted a systematic review and meta-analysis of randomized controlled trials (RCTs) on disease-related pain to critically evaluate the immediate effect of acupuncture for pain relief. The PubMed and Cochrane Central Register of Controlled Trials databases as well as three Chinese databases including the China National Knowledge Infrastructure (CNKI), Wanfang, and VIP platforms were searched through November 2016. The outcome was the extent of pain relief from baseline within 30 min of the first acupuncture treatment. We evaluated all RCTs comparing acupuncture with other interventions for disease-related pain. Real acupuncture showed statistically significantly greater pain relief effect compared to sham acupuncture  and analgesic injection . No serious adverse events were documented. Acupuncture was associated with a greater immediate pain relief effect compared to sham acupuncture and analgesic injections. Further RCTs with stricter design and methodologies are warranted to evaluate the immediate pain relief effect of acupuncture for more disease-related pain. Pain is a major health problem with serious social and economic consequences. The annual cost of pain management in the USA in 2010 was $560–635 billion, which was a conservative estimate because it excluded the cost of management of pain affecting institutionalized individuals , 5. GiveAcupuncture, which is a mainstay in the healthcare practices of traditional Chinese medicine, is commonly used for the treatment of pain. There is substantial evidence for acupuncture being effective in the treatment of acute –8 and chVarious factors, such as acupuncture manipulation , 20, acuhttps://doi.org/10.1155/2017/3837194) systematic review was conducted with the following aims: (1) to compare the immediate analgesic effect and safety of acupuncture in the treatment of disease-related pain with those of sham acupuncture and other active treatments; (2) to identify specific factors associated with positive results; and (3) to identify areas for future treatment and research.To date, there is no published systematic review or meta-analysis of the immediate analgesic effect of acupuncture. Therefore, we conducted a systematic review and meta-analysis to evaluate the immediate effect of acupuncture for various disease-related pain in order to summarize the available evidence, evaluate the quality of that evidence, and offer suggestions for future research and treatment. This PRISMA-compliant . We searched through the following databases to retrieve records from the earliest publications to those published till November 15, 2016: PubMed/MEDLINE, the Cochrane Central Register of Controlled Trials, and three Chinese databases including the China National Knowledge Infrastructure (CNKI), Wanfang, and VIP platforms. Acupuncture-related terms including acupuncture, electroacupuncture, and needle and pain-related terms including pain, ache, and analgesia were used as the key search terms in the English databases. The following key terms were used in the Chinese digital databases: zhenjiu, zhenci, dianzhen, zhen, and tong . Our search was restricted to trials published in English and Chinese. The reference lists of all of the retrieved trials and reviews were screened; relevant conference proceedings and abstracts as well as on-going and unpublished studies were also manually searched. Two reviewers independently evaluated each of the reports for eligibility. Disagreements were resolved by discussion.In the present study, we applied the review methods advocated by the updated Cochrane Handbook for Systematic Reviews of Interventions . The proStudies that met the following criteria were included in the present review: (1) randomized controlled clinical trials; (2) trials comparing acupuncture with sham acupuncture, no treatment, or effective western medications ; (3) studies including participants suffering from nonspecific pain; that is, there were no restrictions on the type, cause, or duration of pain; and (4) studies measuring self-reported pain relief using scales such as the visual analogue scale (VAS), numeric rating scale (NRS), or verbal rating scale (VRS). The outcome for this review was pain relief immediately following the first treatment  from the baseline level.We exclude RCTs comparing different types of acupuncture among each other, or those using transcutaneous electrical nerve stimulation (TENS) as a treatment, or those of perioperative pain management, or those using pressure/palpation pain as the unique outcome.Two authors (AX and KC) independently extracted the study characteristics and outcome data from the included studies. Disagreements between the authors were resolved by discussion, and in case of continued disagreement, a third reviewer (SL) was consulted. Since the outcome for our review was the improvement in pain immediately after the end of the first treatment, in case of RCTs reporting the outcomes at multiple time points after treatment, we used the data at the time point closest to the end of the treatment. In cases where only the final and baseline scores were available, we calculated the mean change of the score by subtracting the mean final value from the mean baseline value and computed the change-from-baseline standard deviation using a correlation coefficient . In caseFor each of the included studies, we assessed the risk of bias using the Cochrane Collaboration's risk of bias tool , which eIn the assessment of the blinding of the participants and assessors, we assigned sham-controlled trials a judgment of “unclear” unless we were certain that the sham control was convincing enough in fully blinding the participants to the treatment being evaluated. We considered sham-controlled trials as having a low risk of bias for blinding if the RCT either (i) evaluated the credibility of the sham treatment and found it to be indistinguishable from true acupuncture or (ii) used a penetrating sham needle or a previously validated sham needle . Two ofTwo acupuncturists (SL and XS) with a combined clinical experience of nearly 40 years in treating the pain syndrome with acupuncture, and who had previously worked on RCTs of acupuncture, assessed the adequacy of the acupuncture administered in the trials. Four aspects of each acupuncture intervention were assessed for adequacy: the choice of acupuncture points, needling technique, duration of treatment, and experience of the acupuncturist . The totI2 statistic [I2 statistic; an I2 statistic value of 50% or more was considered as indicating substantial heterogeneity. All continuous data reported for all of the studies were represented in forest plots. We did not carry out meta-analysis when there were less than two studies in a comparison according to the definition of meta-analysis [We only pooled the data from the trials that used similar controls . For the pooled data, the summary test statistics were calculated with the RevMan software, version 5.1 , using ttatistic , which itatistic . The staWe analyzed the penetrating and nonpenetrating sham acupuncture-controlled trials separately; however, in cases where there were no large or significant differences in the pooled effect between these two subgroups of trials, we pooled the data of all such trials available.Two of the authors (AX and KC) independently graded the overall quality of the evidence for each outcome using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) classification .The major outcomes of the review were the standardized mean differences (SMDs) in the pain outcomes between acupuncture and each of the control groups. We used the SMDs as the principal measure of effect size because although the RCTs had assessed the same outcomes, they had performed the measurements using different scales .P values, pooled estimates, and I2 values of each of the two relevant subgroups for the subgroup comparisons of both characteristics.We performed the subgroup analysis of two clinical characteristics that might influence the immediate analgesic effect of acupuncture on pain: (1) the type of sham, penetrating or nonpenetrating; (2) the duration of pain, acute (≤3 months) or chronic (>3 months) . We perfConsidering that clinical pain included in present study was induced by various diseases, we also conducted the sensitivity analysis using the leave-one-out approach. The study by Zhang et al.  was exclWe finally evaluated a total of 13 studies including a total of 1,077 participants with a mean age of 32 years . For all of the eligible RCTs, the participants were required to have been diagnosed with disease-related pain for eligibility. Of the 13 RCTs, 4 investigated LBP , 40, andThe acupoints and needling techniques were judged as being adequate in all of the included trials. All of the trials included in this review were judged to be adequate in terms of the treatment duration, except for those by Maeda et al.  and YangOf the 13 trials included in this review, 11 , 41, 42 I2 = 85%). The results of the GRADE analysis indicated that the overall quality of evidence for this outcome was moderate as a consequence of uncertain risk of selection bias because of the nonavailability of detailed descriptions of sequence generation and allocation concealment (4 RCTs) and unclear risk of performance bias because of uncertain blinding (1 RCT).Real acupuncture showed a greater immediate pain relief effect compared to sham acupuncture . The results of the GRADE analysis indicated that the quality of evidence for this outcome was low as a consequence of a high risk of performance and detection bias because of inadequate data and the lack of blinding.The results of the comparative efficacy studies revealed that acupuncture was associated with statistically significantly greater immediate pain relief compared to analgesic injection with nonsteroidal anti-inflammatory drugs (NSAIDs) or local anesthetic . The results of the GRADE analysis indicated that the quality of evidence for this outcome was low as a consequence of a high risk of performance and detection bias because of inadequate data and the lack of blinding.P = 0.56, P = 0.92, I2 = 61%); however, the pooled result was not statistically significant when compared with that of penetrating sham acupuncture . For the duration of pain, the results showed that real acupuncture was statistically significantly more effective than sham acupuncture for chronic pain ; however, the pooled result was not statistically significant for acute pain .There was no statistically significant difference in the effect estimates between the two substrata for either of the clinical characteristics, that is, the type of sham (I2 = 68%) when the study by Zhang et al. [I2 = 75.8%, I2 = 0%, As shown in g et al.  was exclA total of 8 trials had included descriptions of adverse events associated with acupuncture , 40–42. This is the first systematic review and meta-analysis of RCTs on the immediate effects of acupuncture for the treatment of disease-related pain. We included a total of 13 studies in our review. The results showed statistically significant differences between the efficacy of real acupuncture and those of sham controls for all types of pain included in this review. The SMDs between real acupuncture and control sham acupuncture were lower than those between real acupuncture and a no-acupuncture control. In addition, acupuncture appeared to be more effective than analgesic injection  in reducing pain. The meta-analytic effect sizes were not similar across pain conditions. There was no evidence of any significant harm caused by acupuncture in any of the RCTs. However, it should be stressed that this lack of evidence is based on the results of a few small trials with a high risk of bias. Therefore, a careful interpretation is warranted before arriving at a positive conclusion.Compared with the assessment of the cumulative effects of acupuncture, the determination of the immediate effects could be relatively easy; that is, it is not necessary to consider the treatment endpoint or follow-up duration. Acupuncture also has a very low drop-out rate. For the systematic review and meta-analysis of the efficacy of acupuncture, various factors could affect the outcomes in the evaluation of the cumulative effects of acupuncture, including the total number of treatment sessions, treatment period, and variation in the end points, such as those of pain and function measurements at different times. Because of the exclusion or minimization of these variable factors, the evaluation of the immediate effect may closely reflect the actual analgesic effects of acupuncture stimulation.P = 0.46). However, our sensitivity analysis showed that real acupuncture was more effective than the sham acupuncture in reducing acute pain immediately, if excluding the study by Zhang et al. [Primary analgesic agents, such as morphine, can be used for the management of both acute and chronic pain. The peak effect of morphine is at around 20 min when administered intravenously and at 60 min when administered orally, while the duration of its effect is between 3 and 7 h , 53. Theg et al. . This miThe design of a control group is a continuing challenge for clinical trials of acupuncture. Many clinical trials were unable to detect statistically significant differences in the treatment efficacies between their acupuncture treatment and control groups in terms of any of the outcome measurements –58; the Our systematic review and meta-analysis study focuses on the immediate analgesic effect of acupuncture. This raises some interesting questions. The first question is whether the immediately analgesic effect following the first acupuncture treatment can be used as a predictor for the success of subsequent or long-term acupuncture treatment. Most clinical trials focused on analgesic effects after multiple acupuncture treatment. Few studies assessed analgesic effects after both immediate posttreatment and multiple acupuncture treatments. Therefore, further studies must be performed to clarify this issue. The second question is whether the immediate acupuncture analgesic effect and cumulative analgesic effects following repeated acupuncture treatments share common mechanisms. Thus far, there is no clear documentation in regard to the underlying mechanisms of these two analgesic effects. Based on the available data published, needle insertion of the local acupuncture points triggers the release of adenosine and changes of fibroblast organization at the loose connective tissue layer –61. The Our systematic review and meta-analysis study has several limitations. Only English and Chinese literatures were reviewed in present study and potential data from studies published in other languages might exist and were ignored, which decreased the credibility of the results in present study to some extent. We included RCTs evaluating various types of pain, including chronic neck pain, LBP, and knee pain. In fact, accumulated work has shown that acupuncture is beneficial in treatment of various pain syndromes. The effects of acupuncture on nonspecific pain may share the similar underlying mechanisms. In traditional Chinese medicine, disease-related pain results from stagnation of energy “Qi” flow within meridians. Pain is treated locally or distally via acupuncture points further along the meridian, drawing energy away from the pain. Recently, the neurophysiology of acupuncture has been investigated extensively. Local anesthesia at the needle-insertion sites completely blocks the immediate analgesic effect of acupuncture, indicating that these effects are dependent on intact neural conduction. The immediate analgesic effect on various types of pain may be involved in the nociceptive pathway, including descending noradrenergic and serotonergic pathways . In our In conclusion, this review facilitates a better understanding of acupuncture stimulation and its immediate analgesic effect for disease-related pain. The results of our systematic review and meta-analysis suggest that evidence of the immediate analgesic effect of acupuncture is encouraging, but not convincing. Nevertheless, our review has yielded interesting and innovative findings and provided impetus to further investigations. Further rigorous, high-quality, randomized controlled trials comparing acupuncture with nontreatment and sham acupuncture without skin penetration are required to evaluate the immediate analgesic effect of acupuncture.Supplementary material included the risk of bias in present study. Risk of bias summary of included RCTs ; The risk of bias assessment for each included study . As shown in Figures S1 and S2, all of the included trials were regarded as having a low risk of incomplete outcome data (attrition bias) and selective reporting (reporting bias).


Formica cinerea Mayr ants. In the first experiment, we determined the occurrence and characteristics of rescue behaviour directed towards nest mates with impaired mandibular gland communication. We did not observe any difference in rescue behaviour directed towards individuals who were untreated, treated with paint over the mandibles, or sham-treated with paint over the thorax. In the second experiment, we determined whether rescue behaviour would occur towards dummy ants coated with the contents of the mandibular glands. Compared with the control untreated nest mates, we found that rescue behaviour was not directed towards either the untreated dummy ants or the dummy ants covered with crushed mandibular glands. Our results indicated that the “call for help” signal does not originate from the mandibular glands. Therefore, we propose that gaster-tip glands represent a plausible alternative source of rescue-eliciting pheromone(s) for the F. cinerea ants examined in this study.Certain ants perform rescue behaviour for other ants that require help, and the expression of rescue behaviour is hypothesized to depend on signals  sent by the imperilled individuals. We studied whether the mandibular glands were involved in the elicitation of rescue behaviour in The online version of this article (doi:10.1007/s00040-017-0547-x) contains supplementary material, which is available to authorized users. Formica cinerea Mayr, showing rescue behaviour towards nest mates that require help  sent by the imperilled individuals  because of their involvement in the related functions, e.g., coordinating, alerting, and attracting  or the total duration of rescue . In the second experiment, we found that rescue behaviour occurred in 31 of 60 tests with the first untreated control group of ants, in none of the 60 tests with the third control group of dummy ants, and in 4 of 60 tests with the second gland-treated group of dummy ants. These results indicated that only the live ants in the first group elicited rescue behaviour (FET yielded a nonsignificant result for the comparison between the second and the third groups).In the first experiment, we found that rescue behaviour occurred in 16 of 30 tests with the first untreated control group of ants, in 12 of 30 tests with the second mandible-treated group of ants, and in 11 of 30 tests with the third thorax-treated group of ants. Based on these results, the frequency of rescue behaviour occurrence among the groups was not significantly different (FET yielded nonsignificant results for each comparison). In addition, differences were not observed among these three groups in either the latency to the first episode of rescue  on the body surface of trapped ants with blocked pheromone communication via the mandibular glands . However, the effects of pheromone residuals were unlikely, because in the second experiment, the contents of the mandibular glands did not elicit rescue behaviour towards the dummy ants. All four attempted rescues of the gland-treated group of dummy ants were weak and could have resulted from other substances transferred onto them during experimental procedures , including Pogonomyrmex, Wasmannia, and Myrmica. In addition, the mandibular glands in Formica workers contain low quantities of volatile materials .The current studies on the selected ecological and evolutionary aspects of rescue behaviour are strongly dependent on the hypothesis that individuals who require help emit “call for help” signals; however, this behaviour remains largely unknown


Diabetic nephropathy is the single most common cause of end-stage renal disease (ESRD) and accounts for significant morbidity and mortality. While the incidence of ESRD increased dramatically in the 1980s and 1990s, the U.S. Renal Data System (USRDS) 2005 Annual Data Report shows that 338 out of every million Americans had kidney failure in 2003, down slightly from 340 per million in 2002. This report shows that the numbers of people developing ESRD have stabilized despite the persistent increase in the number of people diagnosed with type 2 diabetes mellitus (T2DM). These data attest to both the efficacy of the currently available therapeutic regimens for the treatment of ESRD as well as better overall patient care. Unfortunately, these encouraging statistics do not apply to all patients. According to the USRDS report, the most marked ESRD decrease was seen in young Caucasian men (<40 years of age), while in other patient groups, particularly African Americans, ESRD has not changed much at all. These observations suggest that more in-depth studies, addressing specific issues, such as race, are needed to understand the disease process fully in order to create novel therapeutic strategies to eradicate the disease completely in all patient populations.The most commonly used therapeutic treatments for diabetic nephropathy are angiotensin-converting enzyme (ACE) inhibitors and angiotensin II (Ang II) receptor blockers (ARBs), implicating the importance of the renin-angiotensin-aldosterone system (RAAS) in the pathophysiology of diabetic nephropathy. The RAAS is not the only vasoactive hormonal system that is involved in the disease process. Over the past decade, studies have suggested that other vasoactive hormones, including endothelin, urotensin II, and the kallikrein-kinin system (KKS), are instrumental in mediating structural and functional alterations in the renal vasculature and parenchyma, leading to the development and progression of diabetic nephropathy. This review will summarize our current understanding of the contribution of vasoactive hormones in the pathophysiology of diabetic nephropathy with specific emphasis on the RAAS, especially the more recently identified components of this hormonal pathway.


Ultrasonographic appearance of unorganized hyperechoic striations (UHS) has been observed in the canine gastric muscularis layer. The purpose of the study was to determine the prevalence, sonographic and postmortem histologic features, and to determine the clinical significance of canine gastric muscularis UHS. In the prospective study, 72 dogs were included. The presence of gastric muscularis UHS were reviewed to determine its distribution and location. In the retrospective study, 167 dogs that had both abdominal ultrasonography and necropsy were included.The prevalence of gastric muscularis UHS in dogs was 37.5% in the prospective and 5.4% in the retrospective studies respectively. The higher prevalence in prospective study was due to greater anticipation by the radiologists in search for gastric muscularis UHS. In the ventral gastric wall, the muscularis UHS were better defined when the gastric lumen was empty or non-distended, and were mostly parallel with the serosa when the gastric wall was distended (with gas or fluid). Visualization of the dorsal gastric wall was often obscured by gas shadowing from luminal gas. Histopathology was performed on eight dogs with gastric muscularis UHS, three of which had fibrous tissue observed with Masson’s trichrome stain.Presence of gastric muscularis UHS in dogs may have been attributable to presence of incomplete interfaces between the inner oblique, middle circular and outer longitudinal layers of the gastric tunica muscularis or due to presence of fibrous tissue within the gastric muscularis layer. The clinical significance of canine gastric muscularis UHS is uncertain. During routine ultrasonographic examination of the gastrointestinal tract (GIT), the GIT wall thickness and the 5-layered appearance, as well as the function (motility) are often assessed. The 5-layers of the stomach wall (from outermost to innermost) include the serosa, muscularis, submucosa, mucosa and mucosal surface. The alternating echogenicity appearance of these layers has been well-described in veterinary literature. The serosa, submucosa and mucosal surface are hyperechoic, while the muscularis and mucosa are hypoechoic . The hypThe aims of this study were to estimate the prevalence of canine gastric muscularis UHS, to characterize the ultrasonographic and histologic features, and to determine the clinical significance of canine gastric muscularis UHS wherever possible.This is a single institution, descriptive, cross-sectional study comprising of prospectively and retrospectively recruited sample populations of dogs.In the prospective part of the study, all dogs that underwent routine abdominal ultrasound examination during a 4-week period (March 2014) were included. There were no exclusion criteria. The standard care of the institute was provided to each animal. All ultrasonographic studies were performed by two board-certified radiologists (HGH & CKL) using the same ultrasound system  and machine settings. Both linear (5–12 MHz) and micro-convex (5–8 MHz) transducers were used. The choice of the transducer used depended on the body conformation and size of the dogs. The selection of the location of the focus point varied depending on the type of transducer used. The linear transducer was preferred whenever possible for superior image resolution. This ultrasonographic examination was part of the standard care for the diagnostic evaluation of the patients, thus owner consent was not deemed necessary. The radiologists were not blinded and therefore aware of the clinical presentation of each dog that was presented for abdominal ultrasound.The appearance of the gastric muscularis was evaluated to determine the presence of UHS. Still images and video clips were captured and reviewed by two board-certified radiologists (HGH & CKL) to determine the region of the stomach involved: (i) ventral wall, or (ii) dorsal wall, or (iii) both ventral and dorsal walls; and the distribution of UHS: (i) local if only one part of the fundus or body or pylorus in either of the ventral wall or dorsal wall is affected, or (ii) diffuse if more than one part of the fundus or body or pylorus of the ventral wall or dorsal wall is affected. Presence of gas or ingesta that may obscure visualization of the dorsal wall is also noted.In the retrospective part of the study, all dogs that had both abdominal ultrasound and necropsy from January 2011 to December 2013 were included, without any exclusion criteria. All ultrasonographic studies were performed using the same ultrasound machine and settings used in the prospective part of this study and reviewed by both radiologists to identify dogs with gastric muscularis UHS. Due to the retrospective nature of the study and limited images of the stomach  captured in routine abdominal ultrasound, the presence of gastric muscularis is remarked as present or not present. Once such cases were identified, the original histologic sections of formalin-fixed, paraffin-embedded stomach stained with hematoxylin and eosin (H&E) were reviewed by a board-certified veterinary pathologist (MAM). Additionally, Masson’s trichrome stain was used to identify fibrous collagen.The medical records of dogs with gastric muscularis UHS in both the prospective and retrospective parts of the study including the signalment , clinical signs at presentation, previous or current history of GIT problems, current and pertinent laboratory results (including histologic results whenever available) and outcome were reviewed by a board-certified internist (SS) to determine if a dog had any underlying GIT disease.A total of 72 dogs were evaluated during the prospective part of the study. Gastric muscularis UHS were observed in 27 dogs (37.5%). The entire stomach including the ventral wall and dorsal wall was visible in 10 dogs while only the ventral wall was visible for the remaining 17 dogs due to presence of intraluminal gas within the stomach. All 27 dogs with gastric muscularis UHS had diffuse distribution, involving all regions  of the ventral and dorsal stomach wall that were visible. Gastric muscularis UHS were best defined when the gastric lumen was empty or non-distended , all of which were within the ventral wall only. The dorsal gastric wall was not visible due to intraluminal gas. The nine dogs were comprised of four neutered females and five neutered males with a mean age of 9 years 9 months (ranges from 5 to 15 years old) and were of the breeds Yorkshire Terrier (n = 2) and English Bulldog, Welsh Corgi, Chow Chow, Beagle, Australian Shepherd, Labrador Retriever (one of each breed) and a mixed breed dog. The nine individual dogs with gastric muscularis UHS and their respective significant findings are shown in Table Eight of these nine dogs had postmortem histologic examination of the stomach. Histologically, one dog Dog 3) had mild lymphoplasmocytic gastritis and one dog (Dog 8) had mucosal mineralization. The muscularis layers of seven dogs were within normal limits in H&E-stained sections , except for one dog that had increased mature fibrous tissue throughout the muscularis layer. With Masson’s trichrome stain, however, fibrous tissue was observed in the gastric muscularis layer in three dogs  than in the retrospective study (5.4%) was expected because the prospective study was designed specifically to assess the presence of UHS in the gastric muscularis layer. In the prospective part of the study, multiple attempts were often made to capture optimal still images and video clips in order to demonstrate the presence of gastric muscularis UHS. In the retrospective part of the study, assessment for UHS in the gastric muscularis layer were only based on few (one or two) captured still images of the gastric wall, and some of these image quality may not have been optimized to illustrate the presence of gastric muscularis UHS. Gastric muscularis UHS were mostly observed in the ventral gastric wall because the evaluation of the dorsal gastric wall were often hampered by presence of artifacts originating from intraluminal gas. The gastric muscularis UHS and the diffuse distribution were best seen in a contracted (non-distended) gastric wall, possibly due to the increased thickness of the fibrous tissue Fig. .Based on the findings of this study, gastric muscularis UHS may be attributable to the presence of increased fibrous tissue within the muscularis layer. It is difficult to discern fibrous tissue in the gastric muscularis using conventional H&E-stain because smooth muscle and fibrous tissue have similar affinity for the eosin dye. Therefore, Masson’s trichrome stain was used in this study to differentiate blue-stained collagen fibers from red smooth muscle. With Masson’s trichrome stain, increased fibrous tissue was detected in three of the eight dogs where fibrous tissue was not detected with H&E stain. However, one may argue that fibrous tissue may not be the only possible explanation for presence of gastric muscularis UHS since five of the eight dogs did not have increased fibrous tissue within their tissue sections. The failure to detect an increase in fibrous tissue in the gastric muscularis in these five dogs could reflect sampling differences between the sonographic and the histologic examinations. Difficulties in a histologic search for sonographic lesions may also be due to small focal sample size (versus the entire stomach).Perhaps a more plausible explanation for the appearance of the gastric muscularis UHS would be the unique nature of three sublayers within the normal canine gastric tunica muscularis: (i) inner oblique layer (ii) middle circular layer (iii) outer longitudinal layer , 4. PresIn the canine stomach, altered echogenicity of the muscularis layer has not been correlated with any specific disease. However, altered echogenicity of the mucosa layer has been associated with disease. A hyperechoic line at the gastric mucosal-luminal interface is usually secondary to mineralization in dogs with uremic gastropathy . The preCanine gastric muscularis UHS were unlikely to be breed or gender specific as they were observed in large variety of canine breeds, ranging from small to large breed dogs, both in male and female. The mean age of the dogs with gastric muscularis UHS was about 10 years old. The lack of younger dog population in this study makes the correlation of age with canine gastric muscularis UHS impossible.The small number of patients in which histopathology was available was a limitation of this study, though approximately 1/3 of the histopathologic samples evaluated had fibrous tissue present in the muscularis layer.This study is the first to describe the appearance of gastric muscularis UHS in dogs. Presence of gastric muscularis UHS in dogs may have been attributable to presence of incomplete interfaces between the inner oblique, middle circular and outer longitudinal layers of the gastric tunica muscularis or due to presence of fibrous tissue within gastric muscularis layer. Based on this study, this finding appears to have no significant clinical correlation with primary GIT disease.


The variability inherently present in biophysical data is partly contributed by disparate sampling resolutions across instrumentations. This poses a potential problem for statistical inference using pooled data in open access repositories. Such repositories combine data collected from multiple research sites using variable sampling resolutions. One example is the Autism Brain Imaging Data Exchange repository containing thousands of imaging and demographic records from participants in the spectrum of autism and age-matched neurotypical controls. Further, statistical analyses of groups from different diagnoses and demographics may be challenging, owing to the disparate number of participants across different clinical subgroups. In this paper, we examine the noise signatures of head motion data extracted from resting state fMRI data harnessed under different sampling resolutions. We characterize the quality of the noise in the variability of the raw linear and angular speeds for different clinical phenotypes in relation to age-matched controls. Further, we use bootstrapping methods to ensure compatible group sizes for statistical comparison and report the ranges of physical involuntary head excursions of these groups. We conclude that different sampling rates do affect the quality of noise in the variability of head motion data and, consequently, the type of random process appropriate to characterize the time series data. Further, given a qualitative range of noise, from pink to brown noise, it is possible to characterize different clinical subtypes and distinguish them in relation to ranges of neurotypical controls. These results may be of relevance to the pre-processing stages of the pipeline of analyses of resting state fMRI data, whereby head motion enters the criteria to clean imaging data from motion artifacts. The advent of open-access data repositories across various scientific fields has initiated new avenues with the potential for transformative discoveries. While poised for a rapid change in pace across many medical fields, particularly those related to the health and brain sciences, these new initiatives have also started to encourage novel exchange and reproducibility of results across labs worldwide. The field of autism research is among those beginning to greatly benefit from these new databanks. Scientists now have at their disposal the opportunity of uncovering new mechanisms and reporting new correlations in multi-modal data with high statistical power, owing this new possibility to the large number of available participants' data. Indeed, it is now possible to aggregate data from different sites and attain a very large number of subjects to build normative data sets from typical controls, as well as to examine pathologies of the nervous systems in relation to new standardized normative scales. Such new characterizations of mental illnesses respond to a recent paradigm shift in psychiatry neuroscience whereby neurodevelopmental disorders are now conceptualized as precursors of mental disorders  emerging later in life  and 19 sites in ABIDE II (http://fcon_1000.projects.nitrc.org/indi/abide/abide_II.html)  encompassing in 2017) imaging and demographics data, including 17 sites in ABIDE I  of the scanner, i.e. of the number of frames per unit time that the scanner captures, which are different in different sites of ABIDE. Since these differences in SR impact the variability of the speed-dependent data that is used to set thresholds for scrubbing, it is possible that different noise quality, denoting different types of underlying random processes  small rotations or displacements of the head with different frequencies per unit time , the mean and the variance characterizing the parameter of interest . This contrasts with assuming a given theoretical distribution  or a given feature of the random process . As a possible corollary of this proposition, we posit that within sites of similar SR, we may be able to use the noise range  to further characterize and differentiate neurodevelopmental disorders in the broad spectrum of autism relative to normative ranges. We report our results on combining and analyzing 2,154 participants from 28 sites of ABIDE I and II. Further, we provide evidence that different disorders can be well characterized by different noise qualities relative to age-matched typical controls.Given prior results from other fields concerning differences in signals representing different kinds of random processes  and ABIDE II (http://fcon_1000.projects.nitrc.org/indi/abide/abide_II.html). The work obeys Frontiers guideline on the use of human subject's data. To that end, citing from ABIDE “In accordance with HIPAA guidelines and 1000 Functional Connectomes Project / INDI protocols, all datasets have been anonymized, with no protected health information included.”All datasets included in this study are from the Autism Brain Imaging Data Exchange (ABIDE) databases: ABIDE I  lower than 1 Hz Autism Spectrum Disorder (ASD), Asperger's Syndrome (AS), Typical Development (TD), using estimation of noise signatures extracted from head excursion of individuals with a formal DSM-ASD, a DSM-IV-TR  Table Females vs. Males, using the above-mentioned metrics and selected across ABIDE based on the inclusion/exclusion criteria defined below.This study includes all sites publicly available through ABIDE I and ABIDE II (as of November 2017). Because datasets were independently collected at each side, varying types of phenotypic information was provided. For example, DSM-IV-TR diagnosis and medication intake were reported at some sites, but not at others. For this reason, the number of participants within each comparison group may be different from the reported amount  fMRI experiments. Motion extraction was performed using the Analysis of Functional NeuroImages (AFNI) software packages Cox, . Single-To ensure uniformity across all data sets, we resampled and truncated the time series data thus creating data sets of equal numbers of points and equal spacing between points Figure . AppendiTo obtain the head excursions we sum over the speed profiles thus yielding the path length of the linear displacements as well as the full excursion of angular displacements. This gives us a sense for the net amount of physical motion a person had. In both cases we used the same number of points for each participant.x, Δy, Δz) at each positional point of application  from frame to frame, for 370 frames (the same was done with the orientation trajectory). The scalar magnitude of the linear speed s was defined as for a common unit time:We computed the rate of change of linear displacement and angular rotation using the Euclidean norm to compute the magnitude of each 3-dimensional velocity vector displacement   and infer statistical features using empirical statistical estimation (see below).To ascertain the net physical head motions across all participants, we obtain the path length of the linear and angular displacements (as explained above). The empirically estimated mean was obtained using the continuous Gamma family of probability distributions for every group  of each subject resampled to the same rate for all participants and truncated to the same number of points for all are input to the DFA to obtain distributions of α-values per individual. These were pooled across each of the subgroups generated by the We first compare the SR0 and SR1 groups . Then we compare the different groups according to diagnosis, sex or medication  since the Gamma mean μΓ = a · b and the Gamma variance is We examine the frequency histograms of the α-values and use maximum likelihood estimation (MLE) to approximate the best fitting distribution encompassing all cases. To that end, we compare different families of probability distributions  and choose the best fit in an MLE sense. Owing to our prior works using the ABIDE sets  vs. processes that tend toward the Gaussian distribution (more predictable).In this sense, the Gamma parameter plane allows us to infer speed-dependent processes leading to higher noise levels Using the empirically estimated Gamma moments  which we then plot, for each pairwise-group comparison, as a four-dimensional parameter space using the x-axis as the mean, the y-axis as the variance, the z-axis as the skewness and the size of the marker as the kurtosis. We also plot the Gamma PDFs using the empirically estimated parameters.The comparison between data from the SR0 and SR1 sites yielded significantly different results when matching the ASD and the TD groups of each of the sites. The results can be appreciated in Figure Please refer to Figures DSM groups. Here the TD group is the larger of the two. As such, 500 points  had comparable mean to the TD centroid but on average lower variance and skewness than the centroid. The overlapping of the ASDDSM with a subset of the TD controls can be appreciated in Figure DSM and a subset of the TD controls. The estimated PDFs displayed in Figure DSM and the TD subgroups can be appreciated in the distribution of p-values displayed in Figure DSM can be seen at the rightmost tail of the distribution. These are the TD subgroups for which the two-sample Kolmogorov-Smirnov test using the empirical data yielded p ≥ 0.05 when comparing the alpha values to those of the age-matched ASDDSM set.The Figure p-values.The comparison between the AS and TD participants in the SR0 sites also yielded significant statistical differences. These are captured in Figure p-values in Figure Comparison between the ASD and AS groups whereby the ASD is the larger group confirmed the significance in statistical differences for the alpha range and the inherent variability of the underlying linear speed parameter across these sets from the SR0 sites. These differences can be appreciated in Figure p-values can be appreciated in Figure Examination of the smaller group of females in relation to the lager group of males for the angular speed case yielded some degree of overlapping whereby the alpha values of the females fell within the range of the ASD alpha values in Figure p-values for the 50/100 groups show values over the 0.05 level of significance. Other comparisons from alpha values derived from the linear speed time series can be found in the Figure Bootstrapping method.The comparison of participants who reported medication intake  vs. those who did not report medication intake (NoMEDS-larger group) also yielded some overlap in the comparison of alpha values derived from the time series of the linear speed parameter. This can be appreciated in Figure p-values; Figures For more information about the comparisons between the different analyzed groups, refer Figures A summary of alpha ranges and noise types referring the reader to the classes of random processes cited in Appendix Figure This paper addressed the question of whether the disparity in sampling resolution across different sites in the ABIDE repository would affect the quality of the noise-to-signal ratios empirically derived from the fluctuations in the amplitude of head motion speed. The main motivation for this question is the common use of speed amplitude as criterion to threshold the scrubbing of the imaging slides early in the pipeline of fMRI analyses. The disparate SR resulting in statistically different alphas strongly suggests adhering to SR that are comparable when pooling data and thresholding motor artifacts before further statistical inferential analyses.To address this question, we used DFA to estimate the scaling  exponent values for the time series of linear and angular speed peaks defining the envelope (amplitude) of this signal. These were obtained from the head motions extractable from the rs-fMRI data using traditional methods from the imaging community. More specifically, such methods are commonly employed to detect excess in head motion and remove motion artifacts from the set of images acquired in the imaging session. This pre-processing stage eliminates “contaminated” frames and selects the remaining frames for statistical inference in subsequent image analyses . The methods presented in the paper also address the disparity in clinical group sizes within diagnoses of the ABIDE demographics, thus providing a way to enable appropriate statistical comparison of groups while using similar size and age composition according to the various DSM and phenotypical criteria listed in the demographics data of ABIDE.Given the wealth of information of the ABIDE repository, many more comparisons and other parameters could have been explored. Here we focused primarily on ASD (DSM-IV and non-DSM-IV), AS, TD, FEMALES, MALES and MEDS, No MEDS reports. These groups served to illustrate that:does affect the type of random process underlying the time series of imaging data and the parameters one can derive from it (in this example we used head motion in the form of displacements and rotations).The sampling resolution of the scanner Given comparable sampling resolution, the noise quality inherent in the time series of the head motion parameters and their overall stochastic signatures may serve to further characterize different diagnoses and sex- or medication-related data.The statistical estimation procedures using comparable group size and age composition uncovered a gradient of alpha values toward the ranges of fractional Brownian motion (fBm) for the sites with sampling resolution below 1 Hz (denoted SR0) Figure . Most vaIt is important to note that the biorhythms we extracted from head motion data obtainable from the rs-fMRI time series are merely one out of many possible read-outs from the nervous systems across the body . Other biorhythms are also reported in various data repositories. As such, the methods presented here may be of use when pooling data harnessed under different sampling resolutions. They include for example electroencephalography (EEG) and other morphological and structural parameters in cross-sectional and longitudinal data, expressible as time series of fluctuations. For a summary on various open access data repositories see  of beginning the steps to design research programs within the fields of Psychiatric and Psychology that follow empirical estimation procedures of the scientific method.CC: Wrote the methods, analyzed all data and produced the Supplementary Materials; SM: Extracted all head motion data from ABIDE I and ABIDE II, processed all the demographics for exclusion-inclusion criteria and implemented randomizer for bootstrapping; JV: Developed the Python code for DFA; ET: Designed study, created/implemented analyses and wrote paper; CC, SM, JV, and ET: Edited paper. All authors read and approved the last version of the MS.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.


In recent decades; scientists have provided encouraging evidence that probiotic microorganisms are valuable in the prevention and treatment of a variety of diseases. The use of probiotics is expanding dramatically because of our growing understanding of how these beneficial microorganisms exert their effects and which strains are effective for specific conditions.Microorganisms, Yamada and co-workers explored this field. Those authors previously showed that Lactobacillus gasseri PA-3 incorporates adenosine and its related purines and that oral treatment with the PA-3 strain diminished adenosine absorption in rats [L. gasseri PA-3 also incorporates inosine-monophosphate, inosine, and hypoxanthine, and whether it reduces their absorption in rats. Interestingly, the work reported that the oral administration of L. gasseri PA-3 reduced the absorption of the three studied compounds. The PA-3 strain may therefore protect individuals against elevated serum uric acid levels and could have a positive effect in the reduction of hyperuricemia and the prevention of gout [Several beneficial effects have been attributed to probiotic consumption in clinical trials as well as in vitro and in vivo experimental models. One interesting example is an application of these microorganisms as a non-pharmaceutical approach to help manage human metabolic disorders . In this in rats . Here, tLactobacillus delbrueckii subspecies bulgaricus and Streptococcus thermophilus have generally been used as starters for yogurt production. Yogurt is considered a dairy food carrying viable bacteria with health-promoting effects. In fact, some immunomodulatory properties have been attributed to yogurt. However, as the efficacy of probiotics was discovered to be strain-specific, researchers have questioned whether the beneficial effects of yogurt could be improved by the use of selected L. delbrueckii and S. thermophilus strains with specific properties [L. delbrueckii CRL423 and S. thermophilus CRL412, strains previously selected by their immunomodulatory capacities, was able to improve respiratory immunity and protect against Streptococcus pneumoniae infection [L. delbrueckii ME-552 and S. thermophilus ME-553. By performing in vitro and in vivo experiments, authors investigated the effect of both strains on T cell function. It was shown that the oral administration of the ME553 strain to C57BL/6 mice enhanced the amount of IFN-γ and IL-17 produced by CD4+ T cells from Peyer’s patches [The expanding knowledge of probiotic effects is also modifying the way in which we see classical functional foods such as yogurt or kefir. operties . In thisnfection . In thisnfection  aimed toProbiotics have been used not only to improve immunity but to modulate excessive or unproductive inflammation as well. Thereby, probiotics have found a potential place in the management of inflammatory diseases such as allergies. Intervention studies with probiotics have demonstrated beneficial effects on allergic sensitization and IgE levels, having an impact on eczema, wheezing illnesses and asthma . It is wIn addition to the application of probiotics in the prevention or treatment of different diseases, great advances have been made in the understanding of the cellular and molecular mechanisms of probiotic action. Whole-genome sequencing of commensal and probiotic microbes and the study of microbiota using advanced genomic techniques have allowed us to piece together the relationship between the host and bacteria within the complex ecosystems of the human body. In this regard, adhesion of microbes to the mucosal tissues has been considered one of the key initial events in the successful colonization of the gastrointestinal tract. Therefore, there is considerable interest in determining the molecular mechanisms that probiotic bacteria use to colonize their host, and a substantial amount of research has been performed in this filed. As discussed in the review of Nishiyama, Sugiyama and Mukai , probiotThe science of probiotics has proven that some of these beneficial bacteria exert their beneficial effects on the immune system through several molecules including cell wall, peptidoglycan and exopolysaccharides (EPS) that are able to interact with specific host cell receptors such as pattern recognition receptors. In the review of Laiño et al., , the curThe articles presented in this Special Issue investigate several aspects of probiotic interaction with the host and their beneficial impact on health. We expect that these articles will stimulate new discussions and investigations to cover the great diversity of probiotics, applications and mechanisms of action that still need to be addressed.


Nose is one of the most important aesthetic unit of the face.Management of nasal trauma plays a significant role in the practices of the majority of facial and reconstructive surgeons. Replantation, although technically very challenging, is undoubtedly the procedure of choice following traumatic nasal amputation. Here we present an illustrative case report of the traumatic amputation of a nasal tip that was treated successfully with a paramedian forehead flap and further nasal reconstructive surgery. Use of the forehead flap was performed five hours after the occurrence of trauma and was followed by surgical repair about three weeks later. This case presents evidence that a forehead flap as a full-thickness composite graft can survive with an acceptable clinical outcome. In this particular case, the final result was satisfactory. Among the available methods, the median forehead flap has been used for centuries and remains a workhorse flap for reconstruction of major and complex nasal defects. The technique originated almost 3000 years ago in India, where nasal amputation was a common form of social punishment for various crimes. This brought about a significant rise in the group of individuals in need of total or subtotal nasal reconstruction.2Although nasal defects created by traumatic amputation are much less common than those created by the surgical excision of regional malignancies,3 Here, we present the case of recent traumatic nasal tip amputation during an altercation which was successfully repaired using the paramedian forehead flap technique and further nasal reconstructive surgery.In the late 18th century, Carpue found a description of this Indian technique, which gave rise to the modern era of nasal resurfacing with the use of a pedicelled forehead flap. His basic technique laid the foundation for modern nasal reconstruction for the next century. These techniques were later modified and popularized by other surgical pioneers.A 28-year-old male presented at Day General Hospital following traumatic complete nasal tip amputation after a personal altercation. The amputated tissue was detached about three hours previously at the scene of the conflict and was brought to the emergency department. It was preserved in frozen saline until the time of surgery, which occurred approximately five hours after the occurrence of the altercation. The amputated tissue measured about 2.5×2.5 cm and contained skin, alar cartilage and nasal mucosa . PhysicaDuring the first operation, the skin component of the amputated tissue was resected and the residual parts, including the nasal mucosa and alar cartilage, were re-implanted in their original sites using interrupted 5-0 vicryl suture. Afterwards, the reconstructive forehead flap was designed, incised, elevated and sutured onto the nose defect. The donor site (forehead dissection) was closed in three layers . Post-opThree weeks after initial repair surgery, the nasal part of the flap had become vascularized in the replanted area. At this time, extra granulation tissue of the forehead flap pedicle was excised and both the donor and recipient sites were repaired . 4 Forehead flaps and replantation can be utilized for reconstruction of extensive and deep nasal defects. Clearly, the forehead flap is a reliable, valuable and well-established reconstructive option for management of extensive nasal defects.5 This paper presents an evaluation of the esthetic and functional results of forehead flaps for reconstruction of extensive nasal defects.Although the most common etiology of nasal defects that require reconstruction is skin cancer, traumatic nasal accidents are the second most common cause. Reconstruction of extensive nasal defects including the nasal bone, septum and esthetically-defined units of the nose always present certain difficulties.6Carpue found a description of this Indian technique, giving rise to the modern era of nasal resurfacing with the use of a pedicelled forehead flap. Kazanjian advanced the development of the forehead flap by advocating primary closure of the forehead donor site. In the current case study, the ultimate deformity was smaller than the original defect and reconstruction with a midline forehead flap was successful.7,8 One of the greatest barriers to the success of this method was the establishment of adequate venous drainage and lack of venous outflow that resulted in graft failure due to tissue vascular congestion. The advantages of this method included the matching of the replanted tissue with the surrounding skin in terms of color and texture.9,10Documentation has advocated replantation for similar cases. Nasal replantation was first reported by Fioraventi in 1570, who described the case of a gentleman who had had his nose amputated by a Spanish soldier. In 1828, Hottacker, a German physician who attended dueling matches, similarly demonstrated the ability of cleansed, amputated nasal tissue to survive replantation. He reported the successful replantation of nasal tissue in 12 of 16 cases by simply securing the amputated tissue to the defect with tape.Several authors have described the benefits of forehead flap reconstruction over other reconstructive efforts, such as the use of cheek flaps, free flaps and replantation. Nasal amputations present difficult management problems that have prompted the development of various methods for restoring function and appearance. Here, we have described the successful case of midline forehead flap and reconstructive nasal surgery in a moderately-sized nasal tip defect containing several subunits. Several advantages exist in this method. Furthermore, there is an emotional and psychological component to replacing a missing body part that is difficult to quantify, but often significant. Poor results can cause permanent deterioration of personal and social interactions on the part of the patient. In the present case, based on existing documentation and our own previous experience, we recommended forehead flap reconstructive surgery in the appropriated amputated nasal tissue after proper irrigation and debridement with the understanding that further reconstructive efforts would most likely be required.The authors declare no conflict of interest.


Enamel formation requires consecutive stages of development to achieve its characteristic extreme mineral hardness. Mineralization depends on the initial presence then removal of degraded enamel proteins from the matrix via endocytosis. The ameloblast membrane resides at the interface between matrix and cell. Enamel formation is controlled by ameloblasts that produce enamel in stages to build the enamel layer (secretory stage) and to reach final mineralization (maturation stage). Each stage has specific functional requirements for the ameloblasts. Ameloblasts adopt different cell morphologies during each stage. Protein trafficking including the secretion and endocytosis of enamel proteins is a fundamental task in ameloblasts. The sites of internalization of enamel proteins on the ameloblast membrane are specific for every stage. In this review, an overview of endocytosis and trafficking of vesicles in ameloblasts is presented. The pathways for internalization and routing of vesicles are described. Endocytosis is proposed as a mechanism to remove debris of degraded enamel protein and to obtain feedback from the matrix on the status of the maturing enamel. Enamel formation is a unique process that coordinates the movement of proteins and ions between ameloblasts and the developing extracellular matrix  and cluster of differentiation 63 (CD63)  and observing the intracellular localization of HRP in cell organelles to which HRP is transported. For this technique, HRP is intravenously injected as a 5% solution and animals are sacrificed after 15–90 min Sasaki, . During Phagocytosis describes the uptake of a solid particle (>0.5 μm) by the cell to form an internal compartment known as a phagosome Gordon, . In unicAfter completion of the secretory stage, ameloblasts go through a brief shift to enter the maturation stage. This shift is accompanied by notable changes in ameloblasts morphology from tall polarized cells with a Tomes' process to shorter polarized cells without an apical process. In rodent incisors, this change occurs within 19 hours  have been identified as the molecular machinery that regulates membrane trafficking pathways Segev, . They taRab proteins constitute the largest branch of the Ras superfamily with over 70 members. They are small GTPases/GTP-binding proteins of 21–25 kDa and localize to the cytosolic periphery of the membrane. As the predominant regulators of trafficking of endocytic vesicles, they control the un-coating, tethering, and membrane fusion and are executed by different types of Rabs  is the site for sorting newly translated membrane and secretory proteins . The extreme extension of the ameloblasts facilitates the accommodation of a large number of pronounced cell organelles to synthesize and secrete enamel proteins and to support enamel mineralization. After transcription into mRNA in the nucleus and protein translation in the rough endoplasmatic reticulum, the synthesized enamel proteins progress through the centrally located and very large tubular-shaped Golgi apparatus for post-translational modification and packaging into secretory granules. The supranuclear cytoplasm between nucleus and the Tomes' process is filled with rough endoplasmatic reticulum, Golgi apparatus cisternae, and vesicles, forming an intricate network of membranes in constant exchange of membrane and cargo  containing amelogenin protein have been found with immune gold labeling techniques in secretory-stage, smooth-ended, and ruffle-ended ameloblasts (Nanci et al., Lysosomes originate from late endosomes. The transition between these two organelles is continuous, often forming an endo-lysosome before it transforms into a secondary or dense lysosome (Luzio et al., In enamel formation lysosomes are found in ameloblasts at presecretory, secretory and maturation stages (Katchburian and Burgess, Lysosomal enzymes present in ameloblasts include acid phosphatase, β-glucoronidase, and leucyl-naphthylamidase Beynon, . The disEnamel mineralizes and matures as a result of a balance between synthesis, deposition, degradation and internalization. The homeostasis between ameloblasts and matrix is critical for proper enamel formation. The intracellular transport of vesicles is important for polarization of epithelial cells, serving as a mechanism to regulate differentiation (Bryant and Mostov, Aside from releasing and internalizing vesicle content, the vesicle membrane may supply or reduce the plasma membrane surface when vesicles fuse with the membrane or are pinched off from the membrane. Thereby, the shape and size of the cell may be modified. During the life cycle of an ameloblast, the cell undergoes significant changes in cell shape. During the development of the Tomes' process in preameloblasts, the membrane surface is enlarged which could be supplied by secretory vesicles. During this event, secretion may dominate over endocytosis. Once the Tomes process has been established in secretory stage ameloblasts, secretion and endocytosis may take place in equilibrium. Given the intense protein synthesis activity of secretory ameloblasts, a mechanism is needed to attain homeostasis of membrane surface and the overall cell shape. The three-dimensional shape of the Tomes' process coordinates enamel ribbons in the characteristic rod and interrod enamel. The forming interrod enamel completely surrounds the protruded distal portion of the Tomes' process like prongs. The interrod enamel originates from the proximal portions of the Tomes' process. If ameloblasts were separated from the forming enamel, a cavity in which the Tomes' process resides would become visible from a baso-apical direction. The interrod enamel would be elevated and form a rim surrounding a cavity in the shape of the Tomes' process. The interrod is partially mineralized, and therefore may restrict the size and shape the Tomes' process is able to adopt. When a secretory vesicle undergoes fusion with the surface membrane of a cell, the vesicle experiences a gain in membrane and increases the overall surface of the cell. For the Tomes' process, a gain in membrane from secretory vesicles would alter and enlarge the shape of the Tomes' process. However, being encased by rigid rod and interrod enamel, an enlarged Tomes' process would collide with enamel ribbons. Endocytosis, on the other hand, removes plasma membrane from the cell surface when vesicles are pinched off and trim the contour of the Tomes' process. Invaginations of the plasma membrane of the Tomes' process present an elegant solution to offer surface membrane, but because it is shifted internally, variability in the shape due to membrane fusion from secretory or endocytic vesicles is bypassed. As a result, the surface membrane and the shape of the Tomes' process remain largely unaltered. When the ameloblast progresses from secretory stage to transition stage and maturation stages, the cell's dimensions are reduced and the Tomes' process is dismantled. A preference of endocytosis events would facilitate a reduction in cell surface membrane. Ruffle-ended maturation stage ameloblasts display high activity in endocytosis. Throughout amelogenesis, secretion and endocytosis are highly prominent operations in ameloblasts that may participate in the control of the cell membrane surface available to form and disassemble cell appendages and infoldings and invaginations.During their life cycle, ameloblasts adopt many different morphologically distinct appearances as preameloblast, secretory ameloblast, transition stage ameloblast, ruffle-ended ameloblast, smooth-ended ameloblast, and reduced enamel epithelium. The sites of endocytosis on the ameloblast membrane are different in each stage. Small processes are protruded into the basal lamina in preameloblasts. The Tomes' process of the secretory ameloblast is a very large projection from the apical membrane. The infoldings of the membrane distal portion form inwards in the Tomes' process. The ruffled border of maturation stage ameloblasts is formed much differently from the secretory stage infoldings associated with rod and interrod growth sites. They sink into the supranuclear cytoplasmic mass. The stage-specific mechanisms and pathways of endocytosis in ameloblasts are not well defined. While strong evidence is available to document vesicles generated during endocytosis, so far, only clathrin-mediated endocytosis and pinocytosis have been identified in ameloblasts as internalization routes. Lipid rafts or caveolae-mediated endocytosis as a clathrin independent form of endocytosis has not been described in ameloblasts. Future directions in understanding endocytosis in ameloblasts should address the feedback mechanisms, differences in endocytosis types depending on the vesicle origin from the infolding of the Tomes' process vs. ruffled border vs. smooth-ended border vs. lateral membrane and the trafficking of endosomes.CP and YC drafted the manuscript with help from CS. JH, JS, and CS planned experiments for Figure 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.


Embryos have evolved a remarkable capacity to find implantation site. The impressive navigation ability of natural blastocysts may rely on highly sensitive signals arising from embryos and specialized signal processing strategies in the endometrium. Navigation capabilities may be compromised in ICSI embryos because of altered biochemical signaling. The design and delivery of artificial blastocyst (AB) carrying strong chemical signals may allow ICSI embryos to more easily locate to and be retained in the implantation zone. ICSI embryos will attach easily to the implantation zone after it is found by the AB. Co-transfer of the AB together with the ICSI embryo may overcome potential difficulties in implantation due to impaired embryo-maternal communication in cases with implantation failure. Less is known about how human blastocysts sense the spatial characteristics of their environment. The spatial orientation and navigation of natural/naïve blastocyst (NB) inside ampulla rely on information of landmarks associated with the endometrium. NB produces specific signals as a navigation system that is not exhibited by ICSI blastocyst (IB). Navigational abilities require knowledge of the ampullary location in relation to the endometrium. This biochemical knowledge is formed from blastocyst-derived signals and endometrial signals. More than half of embryos transferred in the assisted reproductive technologies (ART) cycle are not implanted. This failure may be due to either molecular, genetic, anatomical factors or embryo navigation problems. Most of the embryos transferred during the ART cycle are found in the vagina several hours after transfer suggests that these embryos may have navigation problems –5.Artificial blastocyst (AB) equipped with powerful chemical signals are needed to solve the navigation problem of NB or IB. Replacement of receptivity molecules that mimic the effects of healthy blastocyst may enhance the implantation chance of poor quality embryos and may be a new treatment option. The delivery of AB and IB in the same catheter may allow genuine embryos to easily locate and be retained in the implantation zone. This paper builds on our clinical observations and isolated discussions of the navigation problems of naïve or ICSI embryos and the role of AB in triggering endometrium as well as improving navigation in women with implantation failure.Developing embryos emerge from ampulla and begin trans-tubal journey as they reach the morula stage. Little is known about how bidirectional communication between the embryo and endometrium function. Signals from embryo or endometrium function as trans-tubal or trans-endometrial navigational markers and elicit changes in the rolling direction of embryos. The ability of embryos and the endometrium to produce signals as navigational markers is an adaptive mechanism that helps embryos avoid straying from their intended path, which is not suitable for implantation. Sub-endometrial or tubal contractions or ciliar activity contributes to the rolling movement of embryos towards to the tubal ostiums, where they enter the fundal region . EmbryosOne critically important aspect of embryo migration is the use of signal information to identify and remain in the implantation zone. Another equally important migratory task is the determination of when and/or where the journey should end. A small deviation in time and place may lead the embryo to go a different region and result in death. NB may use different forms of chemical and physical signals as a compass to choose and attach to a desired course. NB detects and responds to signals from decidua to compensate for involuntary drift and correctly find their destination. NB may also possess a map sense to determine its anatomical location in the tuba uterina or endometrium suggesting NB is capable of “true navigation”. The bidirectional chemical signals are highly important for controlling embryo migration, but it is not unreasonable to imagine that the physical properties of the fallopian tubes and endometrium which are used to maintain the desired voyage direction . The curThe navigation behavior of IB is different from that of NB for the following reasons. (i) The entry speed of NB into the uterine cavity is different from that of IB. The negative uterine pressure and tubal contraction determine the NB’s entry speed. (ii) The medium type and quantity as well as the pressure applied to the transfer catheter determine the IB’s entry speed into the endometrium. (iii) Defects in the shape or volume of the endometrium or fallopian tubes may adversely affect the embryo’s entry speed into the endometrial cavity. (iv) Trans-cervical passage of the transfer catheter may adversely affect negative uterine pressure in connection with implantation. (v) The air inside the transfer catheter may degrade the communication between the embryo and receptive zone. The polyvinylpyrrolidone inside the transfer catheter may degrade the reliability of chemical cues for navigation. (vi) Multiple egg development and deactivation of the tubes in the ART cycle may adversely affect the signal communication between the embryo and endometrium .Ovarian and endometrial aging are the main causes of implantation failure . Few optWe can perform our artificial blastocyst hypothesis in three stages. (i) The first stage is the design phase of the containers. During this phase the design and development of synthetic containers having the same size of a human blastocyst are necessary. Containers should be biodegradable molecules of synthetic origin or immature eggs collected during oocyte pick up. Metaphase I, II or germinal vesicle stage oocytes or empty zona can be used as physiological containers. Synthetic containers may be in the form of nano-particles or melting beads. (ii) The second phase is the loading phase of the containers. Basic receptivity molecules such as bone morphogenetic protein 2, transforming growth factor beta 1, Activin-A, the gene encoding heparin binding EGF-like growth factor, insulin-like growth factor-1 and -2, cyclic adenosine monophosphate, cyclooxygenase 2, homeobox genes, leukemia inhibitory factor, IL-6, IL-11, integrins, and e-selectin are the first choice for loading of containers –12.  regulation of the expression of receptivity genes and molecules associated with implantation; (ii) mechanical or chemical induction of decidualization; and (iii) regulation of blastocyst adhesion and attachment. Co-transfer of an AB together with IBs may overcome potential difficulties in implantation due to impaired embryo-maternal communication in patients with implantation failure.


The conference focused on innovation and risk management in early clinical drug development. Among other innovations, immunotherapy in oncology and inflammatory diseases were discussed as well as the importance of adaptive trial designs in early clinical drug development. Consideration was given to assessing and mitigating risk in early clinical drug development, and included a preconference workshop. Different measures to minimize risks in healthy volunteers and patients in first-in-human trials were discussed in addition to the importance of non-clinical data, the need for reliable biomarkers, improved communication on adverse events (AEs) and well-trained study sites with ready access to intensive care units and clinical specialists. The need for a European-wide system for prevention of over-volunteering was also discussed. The conference provided opportunity to discuss these developments and concerns and the changing regulatory environment with stakeholders from academia, industry, and regulatory agencies including the European Medicines Agency (EMA). Presentations given by invited speakers are published on  Its first formal conference held in London focused on innovation and risk management. This report summarizes the key learnings from an audience perspective derived from the conference.The EUropean Federation for Exploratory MEdicines Development  welcomed attendees and explained how the focus of the workshop topic was a consequence of the Bial incident in France  emphasized the key role of the toxicologist during drug development, how timely communication between all disciplines is mandatory and how non-clinical and clinical development remain closely intertwined. She concluded that ongoing risk assessments should integrate all data as they become available, including those from the public domain.in silico prediction of off-target activities and how data derived from integrated in silico methods are gaining regulatory acceptance. As it is impossible to screen for all off-target effects and since all methods have their limitations, combining methods can provide a greater spread of information.Friedemann Schmidt  described computational systems toxicology and Marc Pallardy  spoke about determination of the first dose for multispecific monoclonal antibodies. Side effects from the broad range of biologics in development are very difficult to predict. Low doses have been implemented in some clinical trials, but these doses are far from clinically active levels.Bruno Boutouyrie-Dumont  discussed dose selection based on the minimal anticipated biological effect level (MABEL) CHMP, . It was Philippe Grosjean and Eric Legangneux  challenged the usefulness of the maximum tolerated dose (MTD) and its value in developing medicines for humans. It was questioned why doses must be escalated so high; why take the risk? Is this approach ethical?Workshop attendees were privileged to hear from representatives of regulatory agencies from UK, Belgium, and Germany. Thomas Sudhop  provided a regulatory update on the new EU guideline on First-in-Human clinical trials CHMP, . The 200Jan de Hoon focused on how incidents have shaped drug regulation. Most recently, the death of a healthy volunteer in the Bial trial  spoke about the revision of the EMA's guideline on strategies to identify and mitigate risks for early clinical trials CHMP, . When apAnnick Peremans  asked how a European-wide system could be established to prevent over-volunteering. Some healthy volunteers travel from one country to another, risking drug interactions, uncontrolled radiation exposure, and unknown consequences for their future health. There are currently two national databases in Europe: UK (TOPS) and France (VRB). Belgium/Germany/Netherlands use a privately owned database (VIPCHECK), but this database is not used by all institutions. Only a European subject database would be able to raise the ethical standards and safety of clinical research in Europe.Philippe Danjou  discussed minimizing risk in early development of central-nervous-system (CNS) medicines in the context of the death of a healthy volunteer in the Bial trial, where the subject was over 50 years of age and had an underlying pathology that was only detectable on autopsy. The first option would be to monitor the primary effect or target occupancy through molecular imaging. However, this is impossible when relying on animal data alone. Additional barriers include the availability of human ligands and cost. If molecular imaging is not possible, a downstream biomarker is another option, although the current state of knowledge and validation of assays may prove to be difficult. Conducting trials solely in a patient population is a third but unrealistic option. Healthy subjects frequently tolerate and overcome AEs and toxicities better than older, co-medicated patients with multiple pathologies.in vitro absorption, distribution, metabolism, and excretion data along with a plausible biology. It can also confirm mechanisms governing (non-linear) pharmacokinetics during dose escalation in the first-in-human ascending dose studies. It provides an ability to anticipate the impact of genetic polymorphisms on the pharmacokinetic profile and to anticipate drug interactions.An Van den Bergh  noted that the utility of physiology-based pharmacokinetics in risk mitigation in early studies lies in predicting the time profile of drug concentrations based upon Elaine Holmes  spoke about the interaction of an individual's genetic blueprint, the genetics of the individual's microflora, and the environment, which could take the form of food, food additives, drugs, and contaminants. It is becoming clear that the microbiome plays an important role in human health. Metabonomic profiling of biofluids  will help elucidate the functionality of the microbiome and identify associations with disease and disease risk.http://www.frontiersin.org/books/EUFEMED_2017/1195.Researchers were given the opportunity to submit abstracts to be displayed as posters. A selection was presented as oral presentations. All abstracts are published in Jorg Taubel  described how integrated adaptive Phase-I clinical trials are safer, take less time and cost less than traditional alternatives. He advised that adaptive designs can establish a “playing field” with set boundaries on various factors including starting dose, maximum exposure limits, numbers of subjects, procedures, samples, and ‘inconveniences.’ They also provide flexibility within the ‘playing field's’ boundaries to respond to challenges arising during the trial, all overseen by a safety review committee. Considering the worst case scenario, adaptive designs are possible with careful planning and expertise on the part of all stakeholders.David Jones  explained that it was considered necessary to revise the 2007 Risk Mitigation guidelines  presented innovative Alan Boyd  provided some examples of innovation and risk management. These included a gene therapy based product , molecule repurposing (ACE inhibitors in cancer cachexia), and an immunotherapy . Interestingly, none of these examples gained marketing authorization. Alan Boyd reported how the number of new drug applications has been declining steadily over the last 15 years and concluded that it is crucial to advance new strategies to reduce the development time frame, decrease costs, and improve success rates.14C microdosing were discussed, covering misplaced concerns over exposure to radioactivity. It was concluded that although there are challenges yet to be addressed with microdosing, it has the potential to provide critical information that will empower a more informed approach to medicine development in children.Saskia de Wildt  gave some historical examples of how poorly we understand the absorption, metabolism, and clearance of medicines in children. The benefits of An Oxford debate addressed the question of whether the development of pediatric medicines should be limited to pharmacokinetic bridging trials alone. The motion was defended by Clair Aubrey  and Christoph Male  countered the motion. The audience felt that pharmacokinetic bridging can be useful to predict the dose, but this does not necessarily extrapolate to the effect a drug may have. Overall, using all the information available was felt the optimal way to determine the best dose to be used in children.An integrative approach is needed when considering the various data from non-clinical testing. Consideration was given to the values and limitations of commonly determined parameters such as the MTD, NOAEL, and safety ratio as well as pharmacokinetic parameters. Emphasis was also placed on contextualizing findings. The example was that of patients with neurologic diseases who often tolerate considerably higher doses than healthy subjects. Finally, an exercise on choosing the starting dose based on non-clinical data was given. Safety margins should take into account possible differences in pharmacokinetics (such as drug absorption and protein binding) and pharmacodynamics (including susceptibility to develop AEs), between the animal species and humans.The changing face of oncology studies was reviewed in the context of adaptive trial designs, the use of biomarkers and novel immunotherapies. It was considered how designing trials intelligently can facilitate the smooth transition through dose selection, expansion cohorts and indications—all within a single adaptive-design Phase-I trial. Development is enhanced if biomarkers are identified to monitor the activity of a drug. It might be possible to further refine the study population with genetic testing, where markers of specific susceptibility can be determined. Identifying the right dose for cancer patients remains challenging. Should it be dependent on the size of the tumor? Labeling and imaging techniques can better identify drug distribution and delivery to the target site.The increasing number of clinical trials and targets being developed are making recruitment of patients to test new agents increasingly challenging. The possibility of including healthy volunteers remains limited. In all cases the value of the trial's outcome is limited by how dose-limiting toxicities and anti-tumor activity are assessed.Focus was first placed on ICH E6(R2), Christian Blank  took the audience on a historical journey from the 1890s and the ‘fathers’ of the tumor immunology concept to the early 2000s, covering the theory of immune-surveillance, the start of clinical trials with anti-‘cytotoxic T lymphocyte-associated antigen 4’ (CTLA-4) molecules and improved survival of patients with metastatic melanoma with ipilimumab. The action of the “programmed death-ligand 1” (PD-L1) and its upregulation induced by interferon-producing tumor-infiltrating CD8 T cells was used to introduce the concept of adaptive immune resistance, the cancer immunogram, and personalized immunotherapy. The presentation concluded that combinations of checkpoint inhibitors with short-term targeted therapies will be the approach most likely to prove successful in the future.Ioanis Karydis  looked at mitigating immunotoxicity in early phase trials in oncology. Animal models used to predict toxicity are problematic as they do not necessarily reflect the host system and there is a pressing need for predictive biomarkers. Patients are usually highly motivated and well-informed as well as prepared to accept significant risk of morbidity and mortality. Consequently, there is a potential to under-report adverse events. In addition, patients usually have been heavily pre-treated and express long-term toxicities, making monitoring challenging. Appropriate care should be taken not only in designing trials, preferably involving adaptive designs, but also in selecting study sites. These sites should have competence and experience in dealing with immune response AEs, and sufficient resources to deal with the increased burden associated with adaptive/multi-arm clinical trials, and with a high level of cross-speciality support.Ann Gils  reviewed immunotoxicity of IMPs for inflammatory disease. Monitoring of IMP concentrations using enzyme-linked immunosorbent assays (ELISA), factors influencing their pharmacokinetics and the incidence of anti-drug antibodies (ADA) were reviewed. Consideration was given to the differences between non-neutralizing and neutralizing ADAs, the development of drug tolerant ADA assays and symptomatic AE profiles associated with ADA detection using different assays. Early treatment optimization based on detection of ADAs was discussed in light of the concept of transient and persistent antibodies. However, the process is still not fully understood and detection of ADAs is assay dependent and not always associated with loss of response to treatment. Formation of ADAs can be mitigated by using de-immunizing molecules, adding comedication and increasing drug exposure.Geoff Hale  advised how the guidelines issued by the EMA and the Food and Drug Administration (FDA) are not fitted to the needs of a rapidly changing field of study. The example of polyethylene glycol (PEG) was presented with reference to the unexpectedly high frequency and titre of immunoglobulin G (IgG) anti-PEG antibodies observed in healthy populations as a consequence of the ubiquitous nature of PEG in our environment. This may have important implications in terms of the use of PEG in many drug conjugates and the utility of recombinant control agents. It was concluded that traditional cut-point approaches to the assessment of pre-existing antibodies are of little value and alternative approaches are necessary. Preventing unwanted antibodies via tolerance induction was discussed and a preview of the positive results of a proof of concept study inducing tolerance to alemtuzumab was given.The 2017 EUFEMED conference was well-attended and facilitated valuable cross-discipline interaction. It has achieved its objective of focusing on early clinical drug development in a changing regulatory environment. In closing the meeting, the president elect, Hildegard Sourgens, summarized how the topics discussed served to foster a shared appreciation of the innovative nature of the early clinical development space, and welcomed the commitment of all parties to addressing concerns over risk and improving our understanding of the challenges ahead.This work is written from an audience perspective. All authors were part of the scientific and/or organizing committee and contributed to the content of the conference. They all were able to interpret the data, critically revised the manuscript, gave final approval and agreed 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. LV, TH, and SS drafted 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.


Transcranial direct current stimulation (tDCS), a non-invasive brain stimulation technique, modulates neuronal excitability by the application of a small electrical current. The low cost and ease of the technique has driven interest in potential clinical applications. However, outcomes are highly sensitive to stimulation parameters, leading to difficulty maximizing the technique's effectiveness. Although reversing the polarity of stimulation often causes opposite effects, this is not always the case. Effective clinical application will require an understanding of how tDCS works; how it modulates a neuron; how it affects the local network; and how it alters inter-network signaling. We have summarized what is known regarding the mechanisms of tDCS from sub-cellular processing to circuit level communication with a particular focus on what can be learned from the polarity specificity of the effects. Transcranial direct current stimulation (tDCS), a safe  effects change in the activity of individual neurons: changes in neuronal firing rate and pattern or modulations in synaptic release probability, uptake and sensitivity  through scalp electrodes  whereas cathodal tDCS (ctDCS) reduced the spontaneous firing  parallel to the current field were activated by atDCS and inhibited by ctDCS  of neurons affects the magnitude of the effect. Polarity specific modulation was higher in pyramidal neurons than non-pyramidal neurons in feline encephale isole preparation   blockers, Ca2+ dependent expression of early gene (c-fos) on the atDCS side was absent, except around the polarized point itself . Recently, Christie et al.  facilitation by atDCS  concentration  enhanced GABA release  dependent cortical plasticity. This notion was validated by an Other neuromodulators appear to have more complex effects. For instance, a dopamine (DA) agonist turned the atDCS facilitation of motor cortex into inhibition and prolonged the usual ctDCS inhibition  gamma oscillations in rodent hippocampus  and/-or with ctDCS of the competitive loop (the circuit whose activity hinders a behavior)  effects are crucial for effective intervention. Thus, the effectiveness of tDCS is questioned not only in terms of its specificity but also in terms of the extent of offline effects.2+ concentration  brain states and regional associations. Such an experiment has yet to be performed, but the recent achievements in this direction reviewed here, give cause for hope that the next couple of years will see significant progress in this endeavor.SD, PH, MF, and OD drafted the manuscript. All authors thoroughly went through and approved the current version of this paper.SD, MF, and OD were supported by Marie Curie ITN initiative C7 Research Grant Award. PH is funded by the Marie Curie ITN initiative C7 and Kreitman Post-doctoral fellowship.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.


Plant metabolism is compartmentalized at the level of organs, cells, subcellular compartments and molecular assemblies. The rapid progress of in-depth metabolite profiling and kinetic flux analysis has elucidated the environmentally dependent dynamics of metabolism. However, for a full understanding of metabolism and compartmentation, including modelling and simulation (e.g. of photosynthesis), metabolome analysis must consider subcellular localization. Here, the advantages and drawbacks of methods developed to analyze this are assessed. It is argued that the selection of method depends on the aim of a particular study, and results (using different methods) should ideally be compared: no single method is best.The methods that have been developed to scrutinize subcellular compartmentation for metabolome analysis include aqueous isolation of organelles, non-aqueous isolation of chloroplasts and non-aqueous fractionation of tissues.Aqueous fractionation necessarily processes tissue in an unquenched metabolic state. Two major potential drawbacks for metabolome analysis following this process need to be considered: the continuation of metabolism and the loss of metabolites to the suspension medium. Metabolites may be lost during irreversible or reversible permeabilization of the organelles and by physiological transport mechanisms present in the boundary membranes. Thus the experimentalist takes advantage of (i) low temperature, usually close to 0 °C, to slow down metabolism and transport; and (ii) fast processing to minimize the time elapsed between mechanical homogenization or maceration of the tissue and isolation of the final fraction for analysis.Aqueous isolation of intact chloroplasts with subsequent sedimentation and freezing can be achieved in less than a minute . Howeverin vivo. Homogenization in organic solvents such as petrolether or hexane combined with tetrachloromethane (or nowadays the less-toxic tetrachlorethylene), followed by fractionation in density gradients made from these organic compounds at proper mixing ratios, allows the isolation of a chloroplast fraction which can further be freed from lighter and heavier cellular material by sedimentation centrifugation. The non-aqueous chloroplast fraction obtained is highly enriched and a very good proxy for the metabolite contents of the chloroplast in vivo. The accuracy can be further increased by introducing corrections for contamination based on marker enzymes. The stability of the selected marker enzymes during lyophilization and fractionation should be tested, as any differential loss could affect the final results. Thus up to 80% of NADP-GAPDH activity, a commonly used chloroplastic marker, can be lost during lyophilization and a further 60% during fractionation. The loss of hydrophilic metabolites is extremely low and even lipophilic metabolites leak out slowly, probably due to their incorporation into precipitated, denatured protein particles. Non-aqueous chloroplasts have also been used to dissect dynamic processes of metabolites of low abundance and high turnover  of interest. This approach was first developed by ex vivo and characterizing the chloroplast metabolome as it varies with different physiological conditions . These cell types have distinct metabolic programs, accumulate unique metabolite profiles and participate in whole-tissue metabolite exchange at different rates. Thus barley leaves accumulate 5–15% of the leaf glucose and fructose pool in the epidermis during the day, while sucrose remains absent from the epidermis (3 photosynthesis). (B) Subcellular compartments in a barley mesophyll cell. (C) Relative cellular volumes of different cell types in barley and spinach leaves (above) and compartments/organelles in their leaf mesophyll cells (below). The schematic is based on the electron micrograph shown in (A) Cross section showing the distribution of different cells in a typical Arabidopsis leaf . However, these represent significant volumes in the cells and also accumulate specific metabolite profiles. The omission is necessary with the methodology currently available, but weakens the accuracy of iterative approximation of metabolite distribution. It would be extremely valuable to develop methods which include peroxisomes in such analyses, such as in the context of assessing metabolite exchange during photorespiration .An iterative search for the best solution of the metabolite-to-marker distribution in subcellular compartments is a reasonable approach when using the fractionation method. However, it should be kept in mind that this will only provide an approximation of the real distribution in the light of the many variables and the limited number of analytical fractions. An exact mathematical solution is impossible.From these considerations it is concluded that there is no single and exclusively valid method for subcellular metabolomics. It is necessary to compare the results obtained with different and competing methods to allow dispassionate scrutiny.in vivo metabolite state of additional cellular compartments. However, the reliability of data must be considered in the light of the critical points discussed.The choice of method must depend on the aims of any particular study. Naturally, this choice also depends on the feasibility and availability of resources in a particular laboratory. The isolation of highly purified non-aqueous chloroplasts can be established in most biochemical labs within short periods of time, and allows a good approximation of metabolite changes in the chloroplast. By contrast the analysis and evaluation of continuous non-aqueous gradient fractions requires extensive optimization and considerable resource investment. The advantage is that this method approximates the