Ectopic xenografting of testis is a feasible option for preservation of male fertility and angiogenesis plays a pivotal role in xenograft survival and functionality. When compared to immature testis, the adult testis is unable to establish functional xenografts due to potentially lower efficiency to induce angiogenesis. The precise molecular mechanism, however, remains elusive. In the present study, we compared adult and immature testis xenografts for survival, maturation and germ cell differentiation. Further, we evaluated differential expression of angiogenesis signalling-specific proteins in adult and immature testis and their xenografts. Results showed that adult testis xenografts degenerated whereas immature testis xenografts survived and established spermatogenesis with the production of haploid germ cells. Protein expression analysis demonstrated that immature testis xenografts were able to establish angiogenesis either through eNOS activation via VEGF and PI3K/AKT or through EGFR-mediated STAT3 pathway. The role of ERK/MAPK pathway in xenograft angiogenesis was ruled out. The absence or reduced expression of angiogenesis-specific proteins in adult testis and its xenografts possibly resulted in poor angiogenesis and in their subsequent degeneration. This study provides insight into angiogenesis mechanism that can be utilized to augment testis xenografting efficiency. Testis tissue xenografting allows for modulation/modification of spermatogenesis by manipulation of recipient mice environment. Several factors such as the size of the tissue, temperature at the grafting site and period of hypoxia play a vital role in the success of xenografting. The most important event that ensures survival of grafted testis tissue is the induction of angiogenesis. Interestingly, testis from differently aged donors has different potential for development when grafted onto recipient mice2. Till date, only testis from sexually immature hosts have resulted in successful progression of xenografts to complete spermatogenesis and emerged as successful models for studying testicular development in vivo. Fresh immature testis tissues from several species such as monkeys4, bulls6, buffalo7, goats1, pigs1, dogs8, cats9, hamsters10, rabbits11 and mice11 have shown complete spermatogenesis following xenografting onto mice recipients.Ectopic testis tissue xenografting is a feasible technique for studying spermatogenesis and testicular maturation. This technique has been used for the production of mature gametes by grafting small pieces of testis tissue under the dorsal skin of immunodeficient mice recipients12 or in species with seasonal reproductive patterns outside of mating season13. Xenografting of adult testis can also be used for studying the mechanism that causes impairment of spermatogenesis14. However it has been observed that in the xenografted human adult testis, germ cells fail to differentiate16 and spermatogenesis was arrested at meiosis in grafted horse17, dog8 and in the photo-regressed hamster18. Similarly, testes from mature adult donor goat, pig and cattle have not supported germ cell differentiation in xenograft18. Complete spermatogenesis leading to formation of elongated spermatids in xenografted testis from sexually mature species has not been reported so far. The failure of sexually mature testis to establish functional xenografts is attributed to their greater sensitivity to ischemia when compared with immature tissue. Testis from sexually mature animals are potentially less effective in inducing angiogenesis19. The angiogenesis in grafted tissues is regulated by the expression of angiogenesis signalling-specific proteins in the donor testis. Expression of several angiogenesis signalling-specific transcripts have been reported in immature bovine testis however, their role in angiogenesis needs to be determined2. There is no report so far which specifically identifies differentially expressed key angiogenesis signalling-specific proteins in immature and adult testis xenografts.Despite poor outcome of ectopic xenografting of adult testis tissue, it could be a feasible option to maintain spermatogenesis in testis that is rendered azoospermic by disease or cancer treatment20. The survival and function of testis xenografts majorly depend on the rapid formation of a stable microvasculature between the host and the grafted tissue21. The grafted tissue must establish an efficient blood vessel connection to the host’s circulatory system rapidly to facilitate the supply of oxygen and nutrients22. A successful xenograft should have adequate angiogenesis for functional Sertoli cells, which in turn are necessary for functional germ cells2. It has been reported that host contributes towards the formation of major connecting blood vessels associated with the graft and host22. However, cell signalling in testis tissue xenografts that induces angiogenesis is not yet known.Angiogenesis or the formation of new blood vessels involves a complex and dynamic interaction between endothelial cells and the corresponding extracellular environmentThe objective of the present study is to evaluate the differentially expressed key-angiogenesis signalling-specific proteins in adult and immature testis xenografts to understand their role in angiogenesis.25. The average weight of seminal vesicles recovered from recipients grafted with adult testis tissues was significantly lower than from those grafted with immature testis and non-grafted intact control mice . The inability of xenografts from adult testis to produce sufficient testosterone could be due to lack of mature Leydig cells. These results contradict previous findings where restoration of seminal vesicle weight in mice27 and, serum testosterone level in rats28 following adult testis autografting has been reported. This inconsistency could be due to failure or delay in the establishment of angiogenesis in xenografted adult testis leading to loss of Leydig cells29. The recovery of seminal vesicle weight in recipients grafted with immature testis suggests that not only the spermatogenic but also the steroidogenic function of the immature rat testis was restored in the xenografts. Induction and maintenance of spermatogenesis require a continuous and controlled interaction of several hormones in the hypothalamic–pituitary–testis axis30. This indicates that a synchronized hormonal interaction was established between the recipient’s hypothalamus and pituitary and the xenografted immature rat testis, which induced spermatogenesis in the xenografted tissue. Thus, suggesting that Sertoli cells and Leydig cells present in donor tissue were able to respond to mouse gonadotrophin stimulation.At 8 wk post grafting, xenografts and seminal vesicles were recovered from recipients grafted with adult testis tissues or immature rat testes and weighed Table . The weiAverage graft weight and total graft recovery were significantly lower from recipients grafted with adult rat testis tissue than in recipients grafted with immature rat testis (P < 0.05). The lower graft recovery from recipients grafted with adult rat testis tissue can be attributed to complete degeneration of xenografts and lower xenograft weight to progressive disintegration of the testicular architecture and extensive degeneration of seminiferous tubules.18. Moreover, the adult testis tissue xenografts are also highly sensitive to ischaemia10 and have a high demand for oxygen because of ongoing spermatogenesis22. The survival of sperm cells in otherwise degenerated tubules is interesting. It is likely that spermatozoa being terminally differentiated cells are rather resistant to degeneration and are able to survive in degenerated tubules. We had observed similar findings in autografted adult mice testis27 and another report also confirmed similar finding16. Testis from immature donors could establish spermatogenesis following ectopic xenografting and meiosis was induced as indicated by the presence of pachytene-stage spermatocytes as the most advanced germ cells , its xenograft was unable to do so. This could be due to initial ischemia before the blood supply to the grafts is established leading to a delay in initiation of spermatogenesis. Whether a longer grafting period is required for spermatogenesis to get completed in xenografted immature rat testis needs to be evaluated.Histological evaluation of 10-wk-old donor rat testis showed intact seminiferous tubules with normal spermatogenesis whereas 6-day-old rat donor testis showed seminiferous tubules with spermatogonia as the most advanced germ cell in 100% tubules Fig. . Xenogralls Fig. . These r31. Sertoli cell proliferation, maturation and establishment of spermatogenesis further confirmed that a synchronized hormonal interaction was indeed established between the recipient’s hypothalamus and pituitary and the xenografted immature rat testis. However, a significantly lower number of PCNA-positive Sertoli cells in grafts than that in the adult testis implied incomplete maturation. This delayed maturation of grafts could be a possible reason for incomplete spermatogenesis in the grafted testis. In grafts from adult donor, no PCNA-stained cells were evident in the tubules  in the seminiferous tubules Fig. . In 6-dales Fig. . These r32 hence, xenografts were analysed 1 and 2 wk post grafting. Growth factors and their receptors play an important role in the initiation of angiogenesis and the generation of new blood vessels critical for graft survival. VEGF is a key regulator of both vasculogenesis and angiogenesis33. In the present study, the expression of VEGF protein was present both in testis of immature and adult donors but its expression level was significantly lower in adult testis  identified only alpha subunit (85KDa) in immature fresh testis but both forms of PI3K were absent from adult testis. Interestingly, gamma (55KDa) subunit of activated PI3K was present in xenografts of immature testis at both collection time points and in xenografts of adult testis at 2 wk only. It is reported previously that activated PI3K subunits, p55γ and p85α act in concert to mediate microtubule stabilization and rearrangement68. In this study, simultaneous expression of both the subunits was absent in the immature and adult testis as well as their xenografts. Expression of AKT, another subsequent downstream protein in PI3K pathway, was absent in both immature and adult testes. Nevertheless, the expression of AKT protein was present in xenografts of immature and adult testes at both the collection time points  pups at age 6 days postpartum (refer to as immature) and at age 10 wk (refer to as adult) were used as testis donors. Immunodeficient male nude mice  were kept under specific pathogen-free conditions, and food, water, and bedding were autoclaved before use. All the animals used were housed in 12 hours light: 12 hours darkness cycle at constant temperature and provided food and water ad libitum.All animal procedures were performed in accordance with the relevant guidelines and regulations approved by the Institutional Animal Care and Use Committee (IACUC) of the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, India (permit number 68/2015). Wistar rat . The testis pieces were placed on ice in Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12) HEPES  until grafting. Four rat testes (immature) or 4 pieces of testis (adult) were ectopically grafted onto the back of castrated nude mice . Briefly, mice were anesthetized with ketamine (0.1 mg/kg body weight (BW) and xylazine (0.5 mg/kg BW) in sterile physiological saline. For castration of recipient mice, a ventral medial incision was given in the abdomen and the testes were removed following which the peritoneum and skin sutured closed using absorbable suture . During the same surgery, each mouse received two incisions (~5 mm) on each side of the back . One piece of testis was inserted through each incision. The incisions were sutured closed and the mice were allowed to recover and returned to their cages.Testes were collected from immature (n = 44) and adult (n = 6) rats following euthanasia by CO2 inhalation. The fresh testis from immature and adult rats served as starting material . Xenografts were collected from mice at 1 and 2 wk post grafting to observe the expression of angiogenesis signalling-specific proteins by western blot analysis (n = 6 for each time point). At 8-wk-post grafting, the xenografts were recovered, weighed and taken for histological analysis. The weight of the seminal vesicle from recipient mice was measured as an indicator of bioactive testosterone production by the grafted tissue7. Average weight of seminal vesicle from non-grafted intact adult males  was taken as control. The percentage of recovered xenografts was calculated for mice in which xenografts were found. It was also recorded when no xenografts were recovered from a mouse. For histological analysis, xenografts and control testis tissues were fixed in Bouin’s solution followed by tissue dehydration with increased concentrations of alcohol in a series. After processing, the tissues were embedded in paraffin, sectioned (7 µm thick), stained with haematoxylin and eosin (H&E), dehydrated, mounted in Vectamount  and observed under a light microscope  for morphological evaluation. Establishment of spermatogenesis was assessed by morphological evaluation as previously described72. In xenografts where seminiferous tubules were observed, all tubules were analysed and classified either as degenerated or non-degenerated. A graft was classified as degenerated which did not contain distinct cell types and no tubules were found and, non-degenerated if it contained even a single seminiferous tubule with spermatogenesis. The percentage of seminiferous tubules and degenerated tubules within a graft was also calculated. Spermatogenesis was assessed by examining the most advanced germ cell type in the recovered xenografts.For the collection of xenografts and seminal vesicles, recipient mice were euthanized by CO2O2 for 10 min, washed three times with PBS, incubated with rabbit anti-mouse horseradish peroxidise conjugated secondary antibody  for 30 min at 37 °C, rinsed three times with PBS, incubated for 3 to 5 min using a DAB Substrate Kit (Vector Laboratories) according to the manufacturer’s instruction, rinsed thoroughly in distilled water, and mounted. Negative control sections were incubated in mouse isotype control antibodies  instead of primary antibodies at the same concentration, but all other procedures remained the same. PCNA-stained tubule cross sections were scored for the presence of PCNA-positive germ cells and Sertoli cells  in each section. An average of 50–100 tubules/cross-section and 15–30 random fields at 400x magnification per recipient mouse were examined.The tissue sections from xenografts collected at 8 wk post grafting were stained with proliferating cell nuclear antigen (PCNA) antibody to evaluate cell proliferation in seminiferous tubules. The fresh testis from immature and adult rats served as control (T0). Dilutions of primary and secondary antibodies were done in PBS with 1% BSA . Briefly, after deparaffinization and rehydration, sections were blocked with 10% FBS and 3% BSA in PBS for 30 minutes, incubated with mouse anti-PCNA antibody  overnight at 4 °C, washed several times with PBS, incubated with 3% HTotal protein from xenografts collected 1 and 2 wk post grafting was extracted upon homogenization by sonication in a dissolving buffer . Single-strength ProteCEASE-50, EDTA-free protease inhibitor  was added to the dissolving buffer before protein extraction. Protein from testis of immature and adult rats served as a representation of starting material (T0). The lysed samples (40 µg) were subjected to electrophoresis on 12% SDS–polyacrylamide gel. The gels were transferred onto PVDF membranes . The membranes were blocked with Starting Block TBS blocking buffer  for 1 h at room temperature. The blocked membranes were incubated with one of the following primary antibodies for overnight at 4 °C: hypoxia inducible factor1 alpha , epidermal growth factor receptor , angiopoietin receptor TIE2 , mitogen-activated protein kinase , phospho-p44/42 MAPK , serine-threonine protein kinase AKT , endothelial NOS , glyceraldehyde-3-phosphate dehydrogenase   vascular endothelial growth factor receptor 1 ; Angiopoietin 1  ; vascular endothelial growth factor , vascular endothelial growth factor receptor 2 , platelet-derived growth factor , platelet-derived growth factor receptor beta , AKT-pThr308 (1:100), AKT-pSer473 (1:500), PI3K-p85 alpha/p55 gamma(pY467/199) 1:500, mammalian target of rapamycin , mammalian target of rapamycin pSer2448 , phosphatase and tensin homolog , serine/threonine-protein kinase c-RAF , mitogen-activated protein kinase kinase 1/2 , signal transducer and activator of transcription 3 , STAT3-pY705 (1:100), janus kinase 1 . The membranes were then washed with TBS-T and incubated with goat anti-rabbit, goat anti-mouse or rabbit anti-goat HRP-conjugated secondary antibody  in TBS-T for 1 h at room temperature. After washing with TBS-T, immunoreactivity was detected by chemoluminescence using a C-DiGit Blot Scanner  against SuperSignal West Femto chemiluminescent substrate (Thermo Scientific), and the generated signal was analysed using a densitometer. To control protein loading on the gels, the membranes were further probed with GAPDH antibody.The results were presented as mean ± S.E.M. The statistical analyses were performed by ANOVA. Significant differences between the means were determined by analysing the data using the Tukey honest significance difference (Tukey HSD) test. The level of significance was set at P < 0.05.


Background. The presence of CSF oligoclonal bands (OBs) is an independent prognostic factor for multiple sclerosis (MS), but the difficulties in the standardization of the test and the interlaboratory variation in reporting have contributed to its limited use in the diagnosis of the disease. Standard nephelometric assays to measure free light chains (FLC) levels have been recently developed and the test may improve the detection of intrathecal B cells activity. Methods. The presence of OBs, kappa and lambda FLC levels, and standard indices of intrathecal inflammation were assessed in 100 consecutive patients, including patients with MS, clinically isolated syndromes (CIS), other inflammatory diseases of the CNS, and other noninflammatory diseases. Results. Both KFLC and LFLC correlated strongly with the presence of OCBs and with all common tests for intrathecal inflammation . KFLC and LFLC were significantly different in patients with MS and CIS compared to the other groups  and had a better diagnostic accuracy than all the other tests (area under the curve 82.3 % for KFLC index and 79.3 % for LFLC index). Conclusion. Nephelometric assays for KFLC in CSF reliably detect intrathecal immunoglobulin synthesis and discriminate MS patients. Various studies on the pathogenesis of multiple sclerosis (MS) have indicated that B cells, as the humoral component of the adaptive immune system, are active participants in the pathogenesis and lesion maintenance throughout the disease process . This hyThe earliest and perhaps still most consistent abnormal immunologic laboratory finding in MS is the increased concentration of Ig in the CSF and the presence of CSF-restricted oligoclonal bands (OCB), all of which constitute the pathophysiological evidences of ongoing inflammation within the CNS , 4. In pThe interpretation of results is rater-dependent, albeit quite high interrater agreement has been reported . SeveralIn this study, we aimed to validate formerly published results confirming that the KFLC and LFLC are valid biomarkers of intrathecal immunoglobulin synthesis and have a diagnostic value in CIS and MS patients.Between January 1, 2014, and December 31, 2014, CSF and serum samples were collected from 100 consecutive unselected patients who were admitted to our department for a suspected neurological condition and who underwent a lumbar puncture as part of their diagnostic work-up. Patient's demographics  and clinical information  were collected from hospital charts. The study was approved by Ethical Committee of San Raffaele Hospital, Milan, Italy. Written informed consent was obtained from all patients and controls.Paired CSF and serum samples from 100 patients were collected during diagnostic lumbar puncture and peripheral vein puncture, respectively, as standard practice. CSF and serum samples were centrifuged 10 min at 800 rpm and 10 min at 3000 rpm, respectively, and were stored at −20°C until the analyses were performed.CSF and serum concentrations for immunoglobulins and albumin were determined within the same analytical series by immunonephelometry using a BNII System  with calibrators and internal controls provided by Siemens, in accordance with the manufacturer's recommendations.(i)The CSF/serum albumin quotient, Qalb = albCSF/albserum × 1000, was used to assess the blood-CSF barrier function.(ii)Ig index (Link index) was calculated according to the Delpech and Lichtblau protein quotient QIg/Qalb  .(iii)loc) was calculated according to the Reiber-Felgenhauer Ig hyperbolic function Igloc = [QIg − Qlim(Ig)] ×  Igserum , with Qlim(Ig) representing the upper limit of the reference range [The absolute amount of intrathecally produced Ig (Igce range .(iv)Tourtellotte index was calculated according to the Ig synthesis rate formula [(IgCSF − Igserum/369) −  ×  × 0.43)] × 5  .Agarose isoelectric focusing (pH 3.0–10.0) followed by immunofixation with peroxidase labelled anti-IgG antiserum was performed to detect OCB [μg/L. Classification of the OCB pattern was performed according to the guidelines of an international consensus [Quantitative expression of intrathecal total IgG synthesis was based on the CSF/serum quotients QIgG, according to different formulas. CSF indexes were defined as follows:tect OCB . Serum sonsensus .KFLC and LFLC measurement were performed employing particle-enhanced immunonephelometry using the BNII System Siemens . FLC in CSF and serum samples were measured according to the manufacturer's protocol with calibrators and internal controls provided by Siemens. Serum samples were diluted 1 : 100 for KFLC and 1 : 20 for LFLC determinations; CSFs were analyzed undiluted as a starting point. According to the manufacturer, the lower detection limit for KFLC was 0.03 mg/L and for LFLC was 0.06 mg/L.KFLC and LFLC indices were defined as quotients of KFLC and LFLC concentrations in CSF and serum divided by the respective Qalb:KFLC index = (KFLC CSF/KFLC serum)/Qalb × 1000.LFLC index = (LFLC CSF/LFLC serum)/Qalb × 1000. t-tests. Nonnormally distributed variables were shown as medians with 25 and 75% percentiles, and nonparametric methods  were used to test for differences. Categorical variables were shown as proportions, and the differences were analyzed using χ2 tests. p values less than 0.05 were considered statistically significant. Receiver operator characteristic (ROC) curves were generated and the area under the curve (AUC) was calculated to compare the diagnostic accuracy of CSF indexes for predicting MS. Cut-off values with the highest accuracy were selected as the diagnostic cut-off points. Standard cut-off values for CSF indexes  were also tested. All statistical analyses were performed using the computing environment R .Normally distributed variables were shown as mean (SD), and differences between groups were analyzed using unpairedWe performed a meta-analysis that incorporated results from the current study into findings from previous studies of the diagnostic accuracy of FLC for MS. We searched Pubmed and EMBASE from inception until August 1, 2016, using the search terms free light chains combined with multiple sclerosis. Reference lists of pertinent articles were reviewed to identify further relevant studies. Studies were included if they used standard nephelometric methods for the quantification of FLC levels and if data reported allowed for the calculation of the following parameters: true positives (TP), true negatives (TN), false positives (FP), and false negatives (FN). The results of the literature search are presented in a flowchart following the PRISMA guidelines.The main outcome measure was the diagnostic test performance of the KFLC index for separating MS patients from controls , as KFLC index has been reported as the best index of intrathecal synthesis according to all studies. The following information was extracted from all studies: sensitivity (TP/(TP + FN)) and specificity (TN/(TN + FP)), names of the authors, year of publication, population characteristics . Data extraction was performed by two authors separately  to ensure accuracy and disagreements were discussed in a consensus conference. A bivariate approach with a linear mixed model has been used to estimate sensitivity and specificity across studies  accountiOne hundred consecutive patients who were admitted to our department for a suspected neurological condition and who underwent a lumbar puncture as part of their diagnostic work-up have been enrolled.According to their final diagnosis, we established several diagnostic subgroups: 34 patients fulfilled the criteria of dissemination in space and time for the diagnosis of relapsing-remitting multiple sclerosis according to latest criteria ; 22 patip < 000.1), and the test had a sensitivity of 71.4% (95% CIs: 57.8–82.7) and a specificity of 86.4% (95% CIs: 72.7–94.8).CSF-restricted OCB were present in 46 patients. Patients with MS and CIS had the highest prevalence of OCB positivity (79.4% and 59%), although these percentages are lower than those previously reported in the literature . PatientStandard CSF indices  were also significantly different in MS and CIS patients with respect to other inflammatory or noninflammatory CNS diseases . The besp < 0.001 and p < 0.001, resp.). The best cut-off values that maximized (sensitivity + specificity) in our sample were 2.43 for the KFLC index and 3.04 for the LFLC index.In patients with MS or at the onset of MS, we found high levels of both KFLC and LFLC in the CSF. There was no major difference between patients with either definite MS or a CIS. Median FLC values in CSF in MS and CIS patients were 9.1 mg/l (3.2–19.0) and 7.3 mg/l (1.4–15.2) for KFLC and 5.6 mg/l (2.2–9.3) and 4.5 mg/l (2.3–11.2) for LFLC. Patients with other inflammatory or noninflammatory diseases generally showed lower levels, both KFLC and LFLC levels in CSF . All patWe compared the performance of KFLC and LFLC indices with common tests for intrathecal inflammation in their ability to discriminate MS and CIS from other neurological disorders. According to the ROC curves  KFLC andSensitivities and specificities of all the tests are shown in p < 0.001) and 36 with elevated LFLC index (p < 0.001). There was high correlation also between LFLC and KFLC indices and common tests for intrathecal inflammation . KFLC index was helpful in the discrimination of MS and CIS patients from patients with other diseases, particularly in patients with negative OCB (Positive OCB correlated strongly with both KFLC and LFLC indices. Out of 46 patients with detectable OCB, 44 presented with elevated KFLC index (tive OCB .The initial literature search identified 95 studies of interest. After screening all studies and applying the inclusion criteria, five studies have been identified , 24–27. Increasing evidence suggests that B cells, as the humoral component of the adaptive immune system, are active participants in the pathogenesis of MS and lesion maintenance throughout the disease process. OCB, immunoglobulins generated by plasma blasts and plasma cells in the CSF or CNS compartment , have loWe applied here a standard immunonephelometric method to assess qualitative and quantitative CSF FLC levels, and our results show that FLC levels, particularly KFLC index, have a good diagnostic accuracy for MS. KFLC index well correlates with OCB status and seems to have a superior diagnostic accuracy compared with common indices of intrathecal synthesis, particularly in case of CSF-blood brain barrier damage. The significant correlation of KFLC index with OCB status supports observations from other studies that suggest CSF KFLC to be elevated in patients with intrathecal IgG synthesis, independently of the type of clonality. According to our results, KFLC allow the discrimination of MS-CIS patients from patients with other diseases, particularly in patients with negative OCB. Our results are consistent with recent reports on the use of such assays for the detection of FLC and validate their use for routine detection of intrathecal immunoglobulin synthesis –13. NotaOverall, presenting at least equal diagnostic accuracy KFLC determination has the potential to replace OCB during diagnostic work-up in suspected demyelinating CNS diseases. The nephelometric assay for the detection of FLC is methodologically easy to perform and to standardize and it is rapid, and as such it would be easily integrated into laboratory processes. Furthermore, interpretation of KFLC is unequivocal as it provides a quantitative measure and would allow easy following of changes in intrathecal immunoglobulin synthesis.Further, multicentric prospective studies enrolling a large number of CIS patients are necessary in order to evaluate the discrimination improvement offered by FLC to current MRI criteria and, ultimately, the utility of FLC in the diagnosis of MS.


Drug delivery to the central nervous system (CNS) is complicated by the blood-brain barrier. As a result, many agents that are found to be potentially effective at their site of action cannot be sufficiently or effectively delivered to the CNS and therefore have been discarded and not developed further for clinical use, leaving many CNS diseases untreated. One way to overcome this obstacle is intracerebroventricular (ICV) delivery of the therapeutics directly to cerebrospinal fluid (CSF). Recent experimental and clinical findings reveal that CSF flows from the ventricles throughout the parenchyma towards the subarachnoid space also named minor CSF pathway, while earlier, it was suggested that only in pathological conditions such as hydrocephalus this form of CSF flow occurs. This transependymal flow of CSF provides a route to distribute ICV-infused drugs throughout the brain. More insight on transependymal CSF flow will direct more rational to ICV drug delivery and broaden its clinical indications in managing CNS diseases. Central nervous system (CNS) disorders are currently estimated to affect hundreds of millions of people worldwide and represent a major public health problem . PharmacLocal drug delivery to the brain tissue or the ventricular system has been extensively used –7. The vDefined as a clear and bright fluid, CSF fills the ventricular system and subarachnoid space and has protective function since it serves as a hydraulic cushion for the brain and spinal cord . Being tIn humans, the normal CSF pressure is approximately 3 to 5 mmHg higher than the pressure in the venous sinuses, which creates a driving force for the outflow of CSF from the subarachnoid space across the basal membrane of the cells lining the arachnoid granulations and eventually across the apical membrane into the venous sinuses , 12. SevThe steady CSF flow, caused by the CSF production mainly in the choroid plexus of the ventricles, is superimposed by a pulsatile motion, which is synchronized with the cardiac cycle.During the systolic phase, the CSF flows in cranio-caudal direction from the lateral ventricles to the third ventricle via the foramina of Monro and through the aqueduct of Sylvius into the fourth ventricle where it leaves the ventricle via the foramina of Magendie and Luschka into the subarachnoid space. Conversely, during diastole, CSF flows in the same route but in reverse direction  Fig. 1)1). The uAccording to a time-resolved three-dimensional magnetic resonance velocity mapping study, the maximum peak value of the velocity vector for CSF flow in the lateral ventricles ranged between 0.3 and 1.3 cm/s (mean ± SD 0.7 ± 0.2 cm/s), while the mean value ranged between 0.1 and 0.3 cm/s (0.2 ± 0.1 cm/s) . This stIn the third ventricle, the CSF flows in a left-right direction when it comes from and goes into the foramina of Monro. This pattern was observed in the vortices of the posterior part of the third ventricle, but not in the central part. Inside the fourth ventricle, the CSF flow patterns tend to be more complex when compared with the lateral and third ventricles, mainly due to involvement of significant motion in all three dimensions and complexity of the fourth ventricle’s structure.Of note, the abovementioned CSF flow patterns are characteristic for young and healthy subjects. It has been shown that the magnitudes of the frequency components of CSF flow in the aqueduct differ significantly between young and elderly subjects, as do the frequency components of the cervical spinal CSF and the arterial flows , 18. In Besides the major pathway, a minor pathway has been described for CSF flow. The minor pathway is a route that the CSF makes through the ventricular ependymal layer, interstitial and perivascular space, and perineural lymphatic channels  Fig. 2)2). UnlikDespite being described as minor pathway, this channel of CSF circulation can play a significant role in ICV drug delivery, which is the main topic of this article. The existence of a semipermeable barrier between the CSF and extra-cellular spaces has been known for some years and allows mixing of interstitial fluid and CSF . This epThe use of CSF as delivery and carrier substrate for CNS drugs is a common practice in several pathologies. Eventually, the drugs infused intra-CSF are expected to reach their sites of action, most of the time, deep structures or tumors. However, how these infused compounds cross the ependymal layer and reach the parenchyma remains to be elucidated. Transependymal flow can simply be defined as a motion of fluids/molecules across the ependymal layer of cells that surrounds the ventricles and consequently penetration of this liquor into the brain parenchyma and adjacent structures.Recently, the contribution of different structures in secreting and removing CSF was determined, and evidence of how fluid secreted into the brain may move through the brain parenchyma and thence exit to the periphery was collected . Two mai131I] serum albumin, which was slowly infused intraventricularly, and animals were subjected to autoradiography. After injecting the radiolabeled compound into the cisterna magna of normal animals, radioactivity was observed in the basilar cisterns that spread over to the cerebral cortex [131I] serum albumin across the ependymal layer of the ventricular system. Moreover, there was a reduced gradient of the isotope from the ependymal surface and the brain parenchyma of animals with chronic communicating hydrocephalus. In this condition, the ventricular ependymal layer suffers histological damages leading to changes in its permeability to large molecules. Based on these results, it was suggested that in experimental conditions, the ventricular walls might be an alternative route for CSF, although this idea had been brought up earlier as well [Transependymal CSF flow can be explained partially by the studies done on experimental hydrocephalus models during the 60s. Early evidence is provided by animal studies: using radioactive-labeled substances, it turned out that the infused compound crosses the periventricular ependymal layer towards the extracellular space , 23. Latl cortex . At necr as well . SeemingSince CSF drainage is impaired in normal pressure hydrocephalus (NPH), an alternative way is the leakage of CSF into the brain parenchyma, described as periventricular lucency . LucencySeveral speculations have been put forward to explain the mechanism behind lucency. The main theory suggests a disruption of the ependymal wall and extravasation of CSF . Recent Some other processes have been invoked to explain these findings, like the passive process of diffusion. Simple diffusion through a permeable ependymal layer cannot be the mechanism behind the transependymal crossing of the large albumin molecules. For instance, histological and ultrastructural investigation on the brains with periventricular oedema have shown that distinct dynamic processes are involved in CSF crossing through brain parenchyma . In a diThe process of CSF absorption from the ventricular system into the parenchyma was initially suggested by DeLand and colleagues  and lateMore recently, a study reported that low-dose acetazolamide, frequently used to treat cerebral oedema and increased intracranial pressure, was able to reduce periventricular lucency in idiopathic NPH . The redWith the advent of more advanced techniques, like magnetic resonance imaging (MRI), the dynamics of CSF could be studied in more detail and some new facts were brought to light. Clinical application of MRI demonstrated that under high intraventricular pressure, “water” penetrates into the periventricular tissue , 35. In To investigate regional distribution and kinetics in the brain, a physiologically based pharmacokinetic modelling approach was applied in rats, using acetaminophen as a test compound . This coIn this section, we will “revisit” previous studies and apply our current knowledge on transependymal CSF flow to elucidate their findings. As mentioned before, two main mechanisms are suggested to explain how fluid secreted into the brain may move through the brain parenchyma and thence exit to the periphery: diffusion and bulk flow. Although, diffusion can explain a part of the process of how substances can cross the ependymal layer, the movement of compounds across long distances still needs to be clarified. As an intermediate process, transependymal CSF flow has been hypothesized to be the main process that drives substances inside the brain, instead of just diffusion or bulk flow . This inThe ventricular ependymal layer contains fewer tight gap junctions, allowing the CSF and extra-cellular fluid to communicate and even albumin and other proteins to cross this layer . AlbuminMoreover, CSF is mainly produced in the lateral ventricles , which cSince the CSF pressure gradient is higher than the pressure in the venous sinuses , 12, CSFThis pressure gradient might empirically explain why infusion in the subarachnoid space is not as effective, since this compound is administrated against the stream. This eccentric pressure also supports and explains why ICV is a preferred approach over other ways of drug delivery. This higher CSF pressure, in comparison with the venous blood, might be a factor in supporting the CSF transport process proposed here, transependymal CSF flow.The high intraventricular pressure explains the accumulation or absorbance of substances around the horns of the lateral ventricles in hydrocephalus. However, based on MRI studies, high intracranial pressure cannot explain per se why CSF periventricular leakage is observed in patients with chronic obstructive hydrocephalus . This piThe physiological constitution and flow of CSF makes this fluid a suitable vehicle for drugs/compounds across the whole brain. Although transependymal flow has not been clearly defined, several clinical and research methods of ICV drug delivery such as antisense oligonucleotide (AON) or oligodeoxynucleotide (ODN) therapies have recently provided indirect evidences supporting the existence of a transependymal CSF flow.huntingtin throughout most brain regions and ameliorate Huntington’s disease symptoms in rodents and nonhuman primates by exploiting the natural flow of CSF to deliver AONs widely after ICV infusion. As a matter of fact, ICV infusion of the AON appears to be the most effective method and achieves a broad distribution in the nonhuman primate brain [For instance, RNA interference can silence mutant te brain .Our data shows that a single infusion of a fluorescent-labeled oligonucleotide into the lateral ventricle of naive mice is followed by its distribution in many brain regions after 24 h including the cerebellum, brain stem, striatum, cortex, and hippocampus  Fig. 3)3).Fig. 3In an experiment by Grzanna and co-workers, fluorescein-labeled ODN was delivered into the lateral ventricle in rats. Twenty-four hours after the infusion, the tissue adjacent to the lateral ventricle on the contralateral side was labeled, although less markedly than the ipsilateral side of the injection. In line with previous observations, the penetration of ODNs in the tissues around the third and fourth ventricles was less than around the lateral ventricles due to increased distance from the injection site . BesidesDiffusion might be the process behind the distribution of the labeled substances, and in general of the therapeutic agents, yet this would only be valid for short distances. What explains distribution of the substances in the outer layers of the cortex, cerebellum, etc.? After crossing the ependymal layer, the substances are carried throughout the parenchyma in a process similar to bulk flow, following the drainage route of the CSF in direction to the venous sinuses.All together, these studies point towards a common process, in which intraventricular delivery of the compound can eventually affect the target site, for instance striatum, hippocampus, etc. Seemingly, transependymal CSF flow is the most probable process to explain these results. The ependymal layer that surrounds the ventricles is permeable to AON and allows AON to cross and spread across the parenchyma. Moreover, it has less tight junctions than a normal ependymal layer would have . Such a Despite the positive results mentioned above, some limitations to the translational feature of this study were identified. These include a need to incorporate a device that allows repeated and chronic infusions of AONs into the CNS and also the necessity to distribute the compound properly in order to target regions in a larger brain . These pA compound delivered locally into CSF can be affected by the distance from the CSF to the site of action, even if there are only a few centimeters. If the drug is expected to diffuse/spread and penetrate the brain tissue, the infused dose needs to be sufficient to reach the site of action. Brain regions in contact with CSF and potentially other regions are best reached by repeating the infusions or continuous delivery of compounds into the lateral ventricle . BesidesDirect drug delivery into the CNS is achievable via three main approaches that are developed in order to circumvent the existence of the BBB: intrathecal, intraparenchymal, and intraventricular delivery. The last two approaches are frequently preferred over intrathecal injection, since this method does usually not allow deep penetration of the substance in the brain which is frequently ineffective and can cause serious side effects. Moreover, substances delivered intrathecally are distributed unevenly, slowly, and incompletely in the brain . As an aIn 1963, the Ommaya reservoir was described as a subcutaneous reservoir and pump for sterile access to ventricular cerebrospinal fluid. Even with empirical data, the use of this approach is recommended by stating that “distribution of substances in the CSF depends largely on the site of introduction: the substance mixes best when introduced into the lateral ventricles” [Another technique widely used in treating tumors that may provide some understanding on how drugs can be delivered locally is convection-enhanced delivery (CED). CED was developed by Edward Oldfield and colleagues in the early 1990s, as a method to distribute large and small molecular weight compounds across the BBB (reviewed by ). This mIt should be noted that similar to every surgical procedure, both techniques face some shortcomings in clinical application. Ommaya reservoir can lead to a slow rate of drug distribution within the CSF, enhanced intracranial pressure, or CSF leakage . When usCurrent methodological and scientific advances in ICV drug delivery together with abovementioned scientific data predicts an upsurge in implementation of this approach in clinical practice. The transependymal CSF flow seems to be the basis of this approach, even if not clearly recognized as a physiological process thus far. Directional flow of the CSF from the lateral ventricles—the main production site—might allow the infused therapeutics to be carried and distributed to distant brain regions from the site of injection.Taking into account, this process will pave the road to benefit from ICV drug delivery advantages, as such crossing the blood-brain and blood-CSF barriers with a minimal risk of systemic effects and uniform distribution of the compound in the parenchyma and subarachnoid space , 50.The development of local drug delivery into the CNS was born as response to circumvent the protective features of the BBB. What once was considered to be an obstacle turns out to be an advantage in ICV drug delivery. The BBB prevents the therapeutic from leaking out, reducing the possible risk of systemic effects by keeping the drug in its site of action.The aim of the present article was to provide a synopsis on the available evidence for a process of transependymal CSF flow. This form of CSF flow exists and plays a significant role in CSF physiology. It provides an attractive route for drug delivery. We believe that this approach will open new opportunities for investigating drug-based therapeutics for neurological and psychiatric disorders.


High-density surface electromyography (HD-sEMG) is to record muscles’ electrical activity from a restricted area of the skin by using two dimensional arrays of closely spaced electrodes. This technique allows the analysis and modelling of sEMG signals in both the temporal and spatial domains, leading to new possibilities for studying next-generation muscle-computer interfaces (MCIs). sEMG-based gesture recognition has usually been investigated in an intra-session scenario, and the absence of a standard benchmark database limits the use of HD-sEMG in real-world MCI. To address these problems, we present a benchmark database of HD-sEMG recordings of hand gestures performed by 23 participants, based on an 8 × 16 electrode array, and propose a deep-learning-based domain adaptation framework to enhance sEMG-based inter-session gesture recognition. Experiments on NinaPro, CSL-HDEMG and our CapgMyo dataset validate that our approach outperforms state-of-the-arts methods on intra-session and effectively improved inter-session gesture recognition. A muscle-computer interface (MCI)  is an inGesture recognition based on surface electromyography (sEMG) forms the technical core of non-intrusive MCIs. sEMG is a technique that measures a muscle’s electrical activity from the surface of the skin using one or more electrodes. Gesture recognition based on sEMG can be naturally framed as a pattern classification problem in which a classifier is usually trained through supervised learning. Existing gesture recognition approaches can be broadly divided into two categories: (1) methods based on sparse multi-channel sEMG and 2) methods based on high-density sEMG (HD-sEMG). Gesture recognition based on sparse multi-channel sEMG usually requires precise positioning of the electrodes over the muscle  methods , and is The problem becomes challenging in realistic situations in which the trained model is used to recognize gestures during a new recording session, because sEMG signals are highly subject specific and vary considerably even between recording sessions of the same user within the same experimental paradigm ,11. To aHD-sEMG signals characterize the spatiotemporal distribution of myoelectric activity acquired by the electrode pickup placed over muscles. These signals can also provide a global view of the varying states of electric fields on the surface of the sampled muscles via arrays of electrodes over a muscle region. In other words, the instantaneous values of HD-sEMG present a relatively global measure of the physiological processes underlying muscle activities at a specific time. Our previous study  revealedIn this work, we describe the CapgMyo database, which includes HD-sEMG data for 128 channels acquired from 23 intact subjects by using our newly developed acquisition device . The acqOur work consists of two major contributions.We provide a new benchmark database for HD-sEMG-based gesture recognition. The CapgMyo database consists of 3 sub-databases ; 8 isometric and isotonic hand gestures were obtained from 18 of the 23 subjects in DB-a and from 10 of the 23 subjects in DB-b, and 12 basic movements of the fingers were obtained from 10 of the 23 subjects in DB-c.We embedded a deep domain adaptation mechanism into the gesture classifier. When applied to new sessions/users, the adaptation starts working after the device is worn, and never stops until the user removes the device, going through the entire process of interaction (performing gesture recognition simultaneously).Experiments on three benchmark datasets  indicated that our approach outperforms other state-of-the-art methods with respect to both intra- and inter-session gesture recognition. To the best of our knowledge, our work is the first to address inter-session sEMG-based gesture recognition in an end-to-end framework based on instantaneous values of HD-sEMG signals.The muscle-computer interface has received much attention from scientific communities and has been investigated using sEMG electrodes ,2,20,21,The recent emergence of high-density surface electromyography (HD-sEMG) ,28,29, iGesture recognition in sEMG-based MCIs can generally be divided into two categories based on the density of the employed electrodes. The first category is based on sparse multi-channel sEMG, which is typically posed as a sequence classification problem in which contiguous sequences of sEMG signals are assigned gesture labels. The classifier can be based on feature vectors extracted from a window of sEMG data ,35,36, oThe second category is based on high density surface EMG signals. Rojas et al. ,7 used tThe aforementioned HD-sEMG-based gesture recognition methods required a handcrafted feature extractor to transform a window of sEMG signals into a feature vector or HD-sEMG map. Our previous study  has reveFrom an MCI application scenario viewpoint, there are two types of gesture classifications: (1) intra-session, in which the classification model is trained on part of the data recorded from the subject during one session and evaluated on the another part of the data from that same session, and 2) inter-session, in which the classification model is trained on the data from all but a few sessions and evaluated on the remaining test set. Inter-session is often referred to as inter-subject when the training data and test data are from different subjects. The sEMG signals recorded in different sessions differ because of electrode shifts, changes in arm posture and slow time-dependent changes such as fatigue and electrode-skin contact impedance ,11. To r inter-seFrom a machine learning viewpoint, one of the key issues in inter-session MCIs is domain adaptation, i.e., developing learning algorithms in which the training data (source domain) used to learn a model have a different distribution compared with the data (target domain) to which the model is applied . Domain We recruited 23 healthy, able-bodied subjects ranging in age from 23 to 26 years. Each subject was paid to perform a set of gestures with a non-invasive wearable acquisition device.The study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Zhejiang University, China. Written informed consent was obtained from all subjects.We developed a non-invasive wearable device  to colleOn the PC, our software displayed an animated 3D virtual hand  driven bBefore the acquisition, subjects watched a tutorial video to familiarize themselves with the experiment. During the acquisition, subjects sat comfortably in an office chair and rested their hands on a desktop. Their skin was cleansed with rubbing alcohol prior to electrode placement, as recommended in previous studies ,60,61. TAs shown in For each recording session, two additional max-force gestures were each performed once to estimate the maximal voluntary contraction (MVC) force level. In this study, we didn’t perform the registration with max-force data. We provided these two max-force gestures for the development of gesture recognition in inter-session and inter-subject scenarios in the future.The CapgMyo database was divided into three sub-databases  in terms of the acquisition procedure. DB-a contains 8 isometric and isotonic hand gestures obtained from 18 of the 23 subjects. The gestures in DB-a correspond to Nos. 13-20 in the NinaPro database. Each gesture in DB-a was held for 3 to 10 seconds. DB-b contains the same gesture set as in DB-a but was obtained from 10 of the 23 subjects. Every subject in DB-b contributed two recording sessions on different days, with an inter-recording interval greater than one week. As a result, the electrodes of the array were attached at slightly different positions each time. DB-c contains 12 basic movements of the fingers obtained from 10 of the 23 subjects. The gestures in DB-c correspond to Nos. 1–12 in the NinaPro database. Each gesture in DB-b and DB-c was held for approximately 3 s. To ensure lower skin impedance in DB-b and DB-c, the skin was abraded with soft sandpaper before being cleansed with alcohol.Whereas DB-a was intended to fine-tune hyper-parameters of the recognition model, DB-b and DB-c were intended to evaluate intra-session and inter-session/inter-subject recognition algorithms. Inter-session recognition of hand gestures on the basis of sEMG typically suffers as a result of the shifting of the electrodes ,10. DB-bPower-line interference was removed from the sEMG signals by using a band-stop filter  . The labhttp://zju-capg.org/myo/data. The format and content are described below.The data records are public available at sss_ggg.mat for the raw data and sss_ggg_ttt.mat for the preprocessed data, where sss is the subject ID, ggg is the gesture ID, and ttt is the trial ID. For example, 004_001.mat contains the data (including the rest posture) from subject 4 performing gesture 1, and 004_001_003.mat contains the preprocessed 3rd trial.The data records are in Matlab format. Each sub-database contains sss_ggg.mat and sss_ggg_ttt.mat are shown in The variables included in C channels in the training sessions and θ is the unknown parameter estimated from Let θ is first estimated from Let In this section, we briefly describe the structure of the ConvNet-based classifier ous work . The valG-way fully connected layer and a softmax function, where G is the number of gestures. We adopted (1) ReLU non-linearity  isThe NinaPro sub-database 1 (DB-1)  is for tM) was set to 10. The learning rate started at 0.1 and was divided by 10 after the 16th and 24th epochs. The weights of the ConvNet were initialized as described in [The deep-learning framework is based on MxNet , a multiribed in  when a pWe also developed a real-time gesture recognition system, shown in Our evaluation consists of two parts. The first part is the verification of the technical quality of our CapgMyo database. In this part, we compared the recognition accuracy with that of the NinaPro database, using the same classification scheme, to show that state-of-the-art classifiers can leverage HD-sEMG and obtain higher recognition accuracies, demonstrating the superiority of HD-sEMG. The second part is the inter-session evaluation on three databases. The second part is motivated by the huge difference of accuracy between intra-session and inter-session gesture recognition on CSL-HDEMG  and CapgTo verify that the data in CapgMyo allow the recognition of finger gestures, we applied our ConvNet-based classifier and four conventional classification methods to the instantaneous values of HD-sEMG signals in a intra-session setting. Moreover, we compared the recognition accuracy with that of the NinaPro database, using the same classification scheme, to show that state-of-the-art classifiers can leverage HD-sEMG and obtain higher recognition accuracies, demonstrating the superiority of HD-sEMG.The evaluation procedure followed that of our previous study , but theAs shown in Based on simple majority voting algorithm, we have observed very good recognition rate . The recWe have briefly reported the average intra-session recognition accuracy on the CSL-HDEMG database in our previous study . Here weWe used the same evaluation procedure as in the previous study . For eacThe recognition accuracy reached 35.4% for a single frame of HD-sEMG signals, and it reached 58.9%, 69.3% and 75.4% with simple majority voting over 98, 307 and 539 frames, respectively, with a 2048 Hz sampling rate.We also evaluated a data augmentation scheme that randomly shifted the training images by one pixel in four directions and filled the borders with the marginal values. This augmentation raised the recognition accuracies using per-trial voting from 62.7% to 68.3% without adaptation, and raised it from 82.3% to 85.0% with adaptation. Unless explicitly mentioned, we did not augment the training set in the following experiments.In the above experiments, we used the entire unlabeled test set as the calibration data for adaptation. It is natural to ask how much data is required to obtain a stable recognition accuracy. Therefore, we randomly selected a subset  of the unlabeled test set to perform domain adaptation and then evaluate the performance. As shown in Our method also works if some categories weren’t performed yet during the process of interaction. The per-frame inter-session recognition accuracy of 27 gestures in CSL-HDEMG reached 31.3%  and 34.6%  with randomly selected 5 and 13 gestures for adaptation, respectively. The randomly selected 13 gestures are In this experiment, we evaluated inter-subject recognition of 8 gestures using the second recording session of DB-b and the recognition of 12 gestures using DB-c. We performed a leave-one-out cross-validation, in which each of the 10 subjects was used in turn as the test subject and a classifier was trained using the data of the remaining 9 subjects.Our adaptation scheme enhanced inter-subject recognition with an per-frame improvement of 3.7% for DB-b and 2.3% for DB-c shown in . For DB-We evaluated inter-session recognition for DB-b, in which the model was trained using data recorded from the first session and evaluated using data recorded from the second session. Our adaptation scheme enhanced inter-session recognition with an per-frame improvement of 6.2% shown in . The recLastly, we evaluated classical AdaBN and our multi-stream AdaBN on both CSL-HDEMG and CapgMyo. As shown in In this experiment, we followed the same inter-subject evaluation procedure as in a previous study . We perfIn this work, we provide a new benchmark database—CapgMyo—for HD-sEMG-based gesture recognition. The CapgMyo database contains HD-sEMG data for 128 channels acquired from 23 intact subjects by using our newly developed acquisition device. We verified the technical quality of CapgMyo by showing that state-of-the-art classifiers could achieve higher recognition accuracy using HD-sEMG signals of CapgMyo than that using conventional sparse multi-channel sEMG.We proposed a deep domain adaptation scheme for inter-session sEMG-based gesture recognition. Our multi-stream extension of AdaBN  improvesUnlike typical inter-session gesture recognition requiring an explicit calibration for retraining or model-transferring, our unsupervised (no labels of data) adaptation works on data domain (no fine-tuning on the learned model), incrementally making statistics of all run-time data/samples closely similar to the one for training. It goes through the entire interaction process, not at a specific stage. Thus our work provides a foundation for developing more fluid and more natural muscle-computer interfaces. In future work, we plan to extend our framework using temporal models such as recurrent neural networks and accommodate dynamic transitional motions, in addition to static gestures.https://github.com/Answeror/adamyo.The codes are available at


Age-related neuromuscular change of Tibialis Anterior (TA) is a leading cause of muscle strength decline among the elderly. This study has established the baseline for age-associated changes in sEMG of TA at different levels of voluntary contraction. We have investigated the use of Gaussianity and maximal power of the power spectral density (PSD) as suitable features to identify age-associated changes in the surface electromyogram (sEMG). Eighteen younger (20–30 years) and 18 older (60–85 years) cohorts completed two trials of isometric dorsiflexion at four different force levels between 10% and 50% of the maximal voluntary contraction. Gaussianity and maximal power of the PSD of sEMG were determined. Results show a significant increase in sEMG's maximal power of the PSD and Gaussianity with increase in force for both cohorts. It was also observed that older cohorts had higher maximal power of the PSD and lower Gaussianity. These age-related differences observed in the PSD and Gaussianity could be due to motor unit remodelling. This can be useful for noninvasive tracking of age-associated neuromuscular changes. Age-associated decline in muscle strength of the Tibialis Anterior (TA) is very significant and has been found to increase the risk of falls . A preceSurface electromyogram (sEMG) is an easy-to-record noninvasive signal of the muscle activity. It is generated by the superposition of the electrical potential induced by the motor unit action potentials (MUAP) in the muscle. Recent studies have demonstrated age-associated changes of the sEMG –6. One c m. biceps brachii, showing increasing Gaussianity of the sEMG with increasing force [Zhao and Li  used a sng force –11.Age-associated change in Gaussianity has been studied for the biceps brachii ; howeverThis study reports the age-associated change in the Gaussianity of sEMG of the TA muscles. It has developed the baseline of the age-associated changes in the sEMG of the TA muscle by experimentally investigating the difference in sEMG features between the younger and older cohorts. The experiments were conducted for different levels of contraction measured based on the individual's maximal voluntary contraction (MVC).Eighteen younger and 18 older volunteers , respectively. The angles were measured using generic goniometer. A force sensor, SM-100 type , was attached to measure the isometric force applied to the fixed footplate. The left leg was planted firmly on the ground. To ensure absence of foot or toe movement during dorsiflexion, the foot and ankle were secured with straps to the footplate  Figure .Myomonitor 4  was used to record the EMG activity which had a gain of 1000, CMRR of 92 dB, and bandwidth of 20–450 Hz, with 12 dB/octave roll-off. The sampling frequency was set to 1000 Hz with resolution of 16 bits/sample. The Delsys single-channel active differential silver bar (1 mm × 1 mm) surface electrodes with an embedded preamplifier and interelectrode distance of 10 mm were used. The skin at the electrode locations was shaved, abraded, and cleansed with an alcohol swipe. EMG and force recordings were performed concurrently during the experiment.The electrode placement and skin preparation were done in accordance with SENIAM recommendations . EMG actPrior to maximal voluntary contraction (MVC) measurement for isometric dorsiflexion (DF), participants were trained to elicit their true MVC with visual force feedback on the screen. After significant rest and self-administered massage, they repeated this twice and recordings were accepted if the difference was less than 5% from each other. Minimum two-minute rest period during which the subjects massaged the muscle was given between each trial. Subsequently, they were instructed to perform two isometric DF repetitions, each of 10, 20, 30, and 50% MVC, each for 5 seconds with a two-minute rest period between each trial, with the order being randomly generated.Gaussianity: bispectral analysis.Power spectral density.The following EMG features were computed to identify the age-related changes in the Tibialis Anterior muscle:(a) Gaussianity Measure. Reduction in the number of action potentials due to reduced number of active motor units will make the sEMG signal less Gaussian [Gaussian . The GauGaussian , probabiGaussian , and recGaussian , 21. In Gaussian  which teGaussian .P(ω) is the power spectrum.The algorithm developed by Hinich  tests whB, is nonzero, then the underlying process or signal is classed as a non-Gaussian process. The bicoherence, Bn, will be a nonzero constant if the process is linear and non-Gaussian. The non-Gaussianity test statistic (Sg) is chi-squared distributed with 2P degrees of freedom when the bicoherence is zero  Power Spectral Analysis. Neuromuscular changes have typically been studied using amplitude features such as root mean square (RMS) [re (RMS) , 23, 24,re (RMS) . The powre (RMS)  and can The power spectral density of the sEMG was calculated for epoch lengths of 512 points with a 25% overlap . The max young and old. This was repeated for each of the MVC levels: 10%, 20%, 30%, and 50%. Prior to ANOVA calculations, normality of the test data was performed using Shapiro-Wilk test at α = 0.05. If the test data was determined to be not normal, this was transformed using the Aligned Rank Tool (ARTool) developed by Wobbrock et al. [Statistical analysis was performed using MATLAB and Statistics Toolbox Release 2011a . One-way ANOVA was performed on the data with age as a factor with two levels:k et al.  to addreThe relationship of Gaussianity and maximal power of PSD for different levels of MVC and for the two age groups is shown in Figures p < 0.05). Therefore, the Sg values were transformed using the ARTool prior to ANOVA calculations [ulations .p < 0.05), 20% MVC (p < 0.001), and 50% MVC (p < 0.001) (< 0.001) .This paper has established the baseline for the age-associated changes to the sEMG of the TA muscle. This muscle was investigated because earlier studies have identified changes in this muscle to be very significant –30 and iMaximal power of the PSD is an indicator of increased size or firing rate of the motor unit. From the literature, it is evident that firing rate reduces with age , 33; thuIncreasing force demands are met through two schemes: recruitment and rate coding. Both of these will increase the MUAP content of the sEMG which will increase the PSD amplitude , 9 and GConversely, decrease in the number of active motor units would contribute a lesser number of MUAP to the EMG signal and lead to a narrower amplitude distribution centered about zero . Since GLikewise, the older cohorts' sEMG had higher maximal power of the PSD, in agreement with other studies on sEMG amplitude , 13, 35.This study has investigated the effect of age on the Gaussianity and the PSD amplitude of the sEMG for the Tibialis Anterior at different force levels. SEMG of the older cohorts had higher amplitude and more non-Gaussianity, which was especially evident at submaximal contractions. The combination of these features may be associated with motor unit remodelling and would be useful for noninvasively tracking neuromuscular changes with age and implementing rehabilitation strategies to prevent strength degradation. While this study has developed the baseline for age-associated changes of TA muscles, there is a need for identifying the difference between the active elderly and those at risk of falls, or the “easy fallers.” There is also a need for establishing the relative contribution of other lower limb muscles in the decline in strength during standing or walking.


Certain disadvantages of the standard hematopoietic stem and progenitor cell (HSPC) mobilizing agent G-CSF fuel the quest for alternatives. We herein report results of a Phase I dose escalation trial comparing mobilization with a peptidic CXCR4 antagonist POL6326  vs. G-CSF.Healthy male volunteer donors with a documented average mobilization response to G-CSF received, following ≥6 weeks wash-out, a 1–2 h infusion of 500–2500 µg/kg of balixafortide. Safety, tolerability, pharmacokinetics and pharmacodynamics were assessed.Balixafortide was well tolerated and rated favorably over G-CSF by subjects. At all doses tested balixafortide mobilized HSPC. In the dose range between 1500 and 2500 µg/kg mobilization was similar, reaching 38.2 ± 2.8 CD34 + cells/µL (mean ± SEM). Balixafortide caused mixed leukocytosis in the mid-20 K/µL range. B-lymphocytosis was more pronounced, whereas neutrophilia and monocytosis were markedly less accentuated with balixafortide compared to G-CSF. At the 24 h time point, leukocytes had largely normalized.Balixafortide is safe, well tolerated, and induces efficient mobilization of HSPCs in healthy male volunteers. Based on experience with current apheresis technology, the observed mobilization at doses ≥1500 µg/kg of balixafortide is predicted to yield in a single apheresis a standard dose of 4× 10E6 CD34+ cells/kg from most individuals donating for an approximately weight-matched recipient. Exploration of alternative dosing regimens may provide even higher mobilization responses.Trial Registration European Medicines Agency (EudraCT-Nr. 2011-003316-23) and clinicaltrials.gov (NCT01841476) Most autologous and 80% of allogeneic hematopoietic stem cell transplantations (HSCT) are currently performed with mobilized peripheral blood stem cells . These cThe ability of CXCR4 antagonists to rapidly dislodge stem cells from the marrow has been recognized for many years . PreviouThe synthetic protein epitope mimetic (PEM) peptidic CXCR4 antagonist POL5551, a close analogue of the clinical stage compound balixafortide (POL6326), shows a very wide pharmacodynamic range in preclinical models and at optimal doses even mobilizes more efficiently than G-CSF . In thisVolunteers were healthy male HSC donors from the German Stem Cell Donor Registry (DSSD) who had received a 5-day course of filgrastim  for matched-unrelated stem cell donation and shown a grossly average mobilization response (121.6 ± 8.6 CD34+ cells/μL). Additional eligibility (inclusion) criteria for treatment with balixafortide were the same as for G-CSF mobilized stem cell donation . Betweenclinicaltrials.gov as NCT01841476.Written informed consent was provided prior to performing any study related activities. The study and all related documents were approved by the local Institutional Review Board (IRB) (#324/11) and the federal medicines agency BfArM . The trial was registered with the European Medicines Agency as EudraCT-Nr. 2011-003316-23 and on Study drug was administered on an in-patient basis in the phase I clinical trial unit of Goethe University Medical Center, the ‘Klinisches Studienzentrum Rhein-Main’. Volunteers were discharged 24 h after treatment, to return for a follow-up appointment 8–14 days thereafter.This was a prospective Phase I open label dose escalation trial; The study design is summarized in Table Vital signs were monitored immediately prior to and in the first 24 h after the start of the infusion of balixafortide; serial blood samples were drawn for biochemical safety profiling and pharmacokinetic/pharmacodynamic analyses. Given the cationic nature of the compound  the riskPrimary outcome parameters were safety and tolerability of balixafortide when compared to G-CSF, pharmacodynamics of mature and immature blood cell mobilization, specifically the intra-individual comparison of balixafortide- and G-CSF-induced mobilization of HSPCs. Secondary objectives included pharmacokinetic analyses and identification of a suitable window for HSPC apheresis.Plasma samples were collected at the indicated times and kept frozen until immediately before analysis.Blood samples were collected at the indicated times Figs. , 5 and 6Circulating colony-forming units-culture (CFU-C) were quantified by plating aliquots of lysed peripheral blood in commercial cytokine-replete methylcellulose media .Unless otherwise mentioned, data are expressed as mean ± SEM. Descriptive statistics and Student’s t-test for paired or unpaired analysis (as appropriate) were calculated using Excel. A p < 0.05, Bonferroni-corrected for multiple testing if appropriate, was considered statistically significant.A summary of adverse events that were documented throughout the trial is shown in Table Three adverse events (AEs) were considered possibly related to study drug: mild bone pain (1 subject), an unexplained elevation in serum creatinine kinase (2 subjects), and a systolic blood pressure reading of >150 mmHg (2 subjects).Constant-slope infusion, tested at the 2000 µg/kg dose level in three volunteers (group 6), as well as increased infusion rates , tested in paired analyses in three volunteers at the 1000 µg/kg dose level, did not influence the tolerability of the agent  , was detStem cell mobilization was accompanied with mixed leukocytosis affecting all cell lineages. It followed the same kinetics as stem cell mobilization and was dose-dependent as well as short-lived. At balixafortide doses of 1500–2500 µg/kg white blood counts (WBCs) of 25.3 ± 1.4 × 10*3 WBC/µL were reached, i.e. balixafortide mobilized approximately half as many mature cells as G-CSF Fig. a. The liWe performed a Phase I clinical trial to directly compare the novel CXCR4 antagonist balixafortide with the standard mobilizing agent G-CSF, with regard to the following parameters: safety and tolerability, pharmacokinetic profile, and pharmacodynamic effects; the latter were defined as mobilization volumes of immature and mature blood cells. At the doses tested, balixafortide was associated with few adverse effects, and none of these were dose limiting. Skin symptoms compatible with possible local histamine release syndrome were observed with some regularity but could easily be managed with routine co-administration of anti-histamines.Mechanistically, mobilization with CXCR4 antagonists like balixafortide involves a rather short-lived interference with stem cell retention in the bone marrow . TherefoThe higher balixafortide doses tested in this study mobilized 38.2 ± 2.8 CD34+ cells/µL. In view of the currently available technologies , 26 thisThe comparator agent in our study was G-CSF given in split doses as is routine practice in our center ; the ratHigh frequencies of a population presumed to represent precursors of plasmacytoid dendritic cells (pro-pDCs) were previously detected in plerixafor-mobilized blood  and wereAs all other mobilizing regimes, mobilization by balixafortide was associated with marked leukocytosis; the-fold difference for mature and immature cell mobilization between G-CSF and balixafortide was quite similar. However, the distribution of leukocyte subtypes was markedly different; specifically the virtually diagnostic left-shifted neutrophilia in G-CSF treated volunteers , 5 was nOne of the desired features of a mobilizing agent is predictability of efficacy. Mouse data clearly indicate that mobilization efficiency is dominated by genetics , 38 but We have demonstrated that HSPC mobilization with balixafortide can be both efficient and predictable; more potent mobilization may be achievable with higher doses of this agent, as future studies may be able to show. Balixafortide treatment was safe and well tolerated. Because of its mechanism of action along with its rapid elimination, this stem cell mobilizing agent can be considered an option for many of the patients and donors with contra-indications to G-CSF. Its brisk mobilization after one single dose is also highly convenient for donors and apheresis centers. In aggregate, balixafortide could be developed as an alternative single-agent mobilizing agent for patients and donors alike. By extension, our work also demonstrates the potential of PEM technology for rational drug design.


We hypothesized that soil Po would initially increase with paddy management and then would persist under steady-state conditions. We analyzed soils from a 2000-year chronosequence of a rice-wheat rotation and an adjacent non-paddy 700-year chronosequence in Bay of Hangzhou (China) for their Po composition using solution 31P-NMR after NaOH-EDTA extraction. Land reclamation promoted Po accumulation in both paddy and non-paddy topsoils (depths ≤ 18 cm) until steady-state equilibria were reached within 200 years of land use. Greater Po concentrations were found, however, in the non-paddy subsoils than in those under paddy management. Apparently, the formation of a dense paddy plough pan hindered long-term Po accumulation in the paddy subsoil. The surface soils showed higher proportions of orthophosphate diesters under paddy than under non-paddy management, likely reflecting suppressed decomposition of crop residues despite elevated microbial P compounds stocks under anaerobic paddy-rice management. Intriguingly, the composition of Po was remarkably stable after 194-years of paddy management and 144-years of non-paddy management, suggesting novel steady-state equilibria of P dynamics had been reached in these man-made ecosystems after less than two centuries.The contents and properties of soil organic phosphorus (P In undisturbed terrestrial ecosystems, organic P (Po) accumulates under nitrogen (N) limitation in the early stage of ecosystem development, while at advanced stages of ecosystem development Po concentrations decline under conditions of P limitation7. Much less is known, however, about human impacts on temporal soil P dynamics. These impacts might be particularly large in anthropogenically-modified soils such as paddy soils. More than 50% of the world’s population relies on rice as a dietary staple and about 163 million ha of land is used as flooded lowland rice fields8. These paddy systems are known to accumulate organic matter and microbial N residues14, and thus may potentially accumulate Po3.Phosphorus is an essential macronutrient for all life on earth. In soil, there is increasing evidence that the P pool changes its composition, abundance, and bioavailability during soil development in natural and agricultural ecosystems15. These processes result in accelerated soil weathering15 so that after only 700- to 1000- years, the soils are decalcified and have significant iron (Fe) oxide transformations, which can alter the sorption of specific Po forms16.Paddy soils environments likely behave differently from natural wetlands, because of the addition of chemical and organic  N and P fertilizers, removal of nutrients during harvest, intensive puddling, and alternating flooding and draining regimeso is comprised of a variety of compounds that differ in their stability and biological availability in environment17. The variation of P composition over pedogenic time scales has ecological significance5, as it may alter species composition of the seedling community, change the competitive ability of adult plant species18, and modulate soil microbial P cycling20. To reveal changes in Po composition, solution31P-nuclear magnetic resonance (31P-NMR) spectroscopy after NaOH-Na2EDTA extraction has been the method of choice23. This allows the identification of specific Po species, such as deoxyribonucleic acid (DNA), α- and β-glycerophosphate, mononucleotides, phosphonates, and even inorganic long-chain polyphosphate and pyrophosphate structures, including compounds originating from living microbial cells and fungal tissue26. However, little information is available on the degree and rates at which different Po species reflect impacts of prolonged paddy or non-paddy management on the overall Po status of the soils.Soil Po compounds accumulated in soil. For this purpose, we extracted Po with NaOH-Na2EDTA and characterized compounds by solution 31P-NMR spectroscopy. Samples originated from a unique 2000-year-old chronosequence of paddy soil management; adjacent sites under non-paddy management for 700 years served as a reference. All soils had developed from tidal wetland sediments in the Yangtze River delta, China. We hypothesized that paddy management accelerated the accumulation of Po compounds in soil, which may then persist for several hundred years under steady-state conditions.The objective of this study was to evaluate how prolonged paddy management influenced the degree and rates at which various P27. With increasing time of paddy soil development, as these Ardp horizons mature, topsoil surfaces may become increasingly decoupled from subsoil biogeochemistry14. In order to understand the role of paddy management in the biological cycling of P, particularly when compared to native ecosystems, we thus have to understand both the transformation of P with increasing soil depth as well as with time of management.During paddy soil management, the puddling of surface soil accompanies the formation of a dense plough (Ardp) horizon, which potentially restricts root growth into the deeper subsoil−1 in the 100-year-old paddy topsoils and from ~1100 mg P kg−1 in the respective non- paddy topsoils down to 40–50 cm soil depth (600–690 mg P kg−1) and then remained relatively constant in the subsoil  and inorganic P (EP-Pi) in these soils showed a similar trend (Table o (Ep-Po) to total P in the topsoil of the paddy soil were almost double those under non-paddy management, whereas those in paddy subsoil were reduced compared with subsoil under non-paddy management (Table p-Po declined with depth while that of Ep-Pi increased (Table o species in the paddy subsoil (from 30 to 100 cm) showed very low 31P-NMR signal intensities, and the spectra were dominated by orthophosphate P  in our study of P transformations along the chronosequence.Above 30 cm depth, orthophosphate diesters and −1 in surface soil (Table −1) in the surface horizon. With prolonged arable management, total P concentrations increased, reaching on average significantly greater concentrations in the non-paddy topsoils (average of 1128 ± 198 mg P kg−1) than in those under paddy management .The tidal wetland (TW) sediment, i.e. the parent material of soil formation for paddy and non-paddy soils, had a total P concentration of 760 mg P kgil Table . After l2EDTA extracts of the youngest soil were extremely low  than in non-paddy topsoils, but differences in rate constants (k) were not significant when including the uncertainty of k into the assessment of Xe  by those for EP and factoring in changes in bulk density gave results in kg P ha−1 (Table p < 0.001) with increasing duration of paddy rice cultivation. The concentrations of all other P compounds followed a similar trend through early stage of pedogenesis, i.e. being low or undetectable at the onset of soil development in TW and SM, but increasing thereafter in the paddy soils rapidly to a maximum that was obtained after no more than 300 years  or glycerophosphates + mononucleotides even tended to decline, whereas those of other compounds could be sustained even after prolonged land use , clays, and P-sorbing Fe oxides3. These processes likely led to the increase in P in non-paddy soils compared with soils under paddy management  extractable Fe in paddy and non-paddy chronosequences did not show large differences. However, greater concentrations of oxalate-extractable Fe  in the paddy topsoils than in the non-paddy soils supported previous findings of enhanced redox cycling and accelerated weathering , magnesium (Mg), Fe, manganese (Mn), and aluminum (Al) decreased with cultivation time in the paddy topsoils, so that the greatest concentrations of these elements were found mainly in the younger surface soilsng Table . The gre2EDTA extracted 12–31% of P-total in the paddy and non-paddy soils. We tested extraction methods to try to improve P recovery for these soils ; however, we found that a sequential repeated extraction did not increase the proportion of extracted Po and caused hydrolysis for some orthophosphate diesters (data not shown). The low P extraction yield in these soils may be related to the marine sediment parent materials and inputs of riverine sediments; most labile P was probably removed by sea water, leaving the majority of P occluded within Ca minerals. Low P recoveries  have been reported for NaOH-EDTA extracts of Ca-rich sea sediments31, and similar low recoveries of P from Ca-rich soils have been reported for NaOH-Na2EDTA extracts33 and for NaOH-NaF extracts34. The total P recoveries in NaOH-Na2EDTA extracts of the youngest soils without pronounced pedogenesis were extremely low  into the forest/shrub soil chronosequences in New Zealand than into the studied paddy soils2.Unlike the forest/shrub soil chronosequences in New Zealand with abundant inositol hexakisphosphate51 of P transformation predicts that with increasing time of pedogenesis there will be a loss in total P, a depletion of primary mineral P pools, but an accumulation of Po stocks in early stage of soil development followed by a slow, subsequent decline with time. Our sites are different to those studies by Walker and Syers51 in that our soils are fertilized to supplement P losses; besides, our land-use sequence covers a much shorter time scale to the chronosequence of Walker and Syers51. Our detailed assessment of Po species using31P NMR, including individual Po compounds and functional groups along 2000 years of rice paddy and non-paddy soil development demonstrated that: (i) the chemical nature of Po changes during pedogenesis, and (ii) soil paddy management strongly influences Po composition. All Po species accumulated rapidly in the early stages of pedogenesis. Thereafter, a plateau that is typical for steady-state conditions was reached. Studies in natural ecosystems7 noted that Po stocks that accumulated during early soil development stages would decline gradually in later stages of the ecosystem, as a response to nutrient limitation. For example, soil Po accumulated under N limitation but declined under P limitation in natural ecosystems2. In our arable study sites, concentrations of available N were low in the young paddy systems13, so the initial gain in Po may be explained by similar processes as described by Turner et al.2; in addition, the mere accrual of organic matter14 co-accumulated Po. In older systems, however, nutrient limitations have been counterbalanced by fertilization, so Po losses in response to P limitation do not occur because these ‘old’ systems do not become P limited.The Walker & Syers model52. A sample of the marine Yangtze River sediment in a tidal wetland  was taken in the Bay of Hangzhou as substrate reference. After 30 years of land embankment behind the youngest dike, built in 1977, an initial surface soil was considered as the starting point (0 years of cropping) for our land use with the growth of salt-tolerant shrubs on an initial salt marsh . The soil chronosequence contained replicated sites under paddy cultivation for 50, 100, 300, 700, 1000, and 2000 years (PR50–2000), as well as adjacent replicated sites under non-paddy cultivation for 50, 100, 300, and 700 years (NPR50–700). Additionally, the soils with 100 years paddy and non-paddy managements (i.e. PR100 and NPR100) were sampled by soil horizon to around 100 cm for elucidating changes in Po composition with soil depth; for the other soils we only analyzed the top A horizons (~0–15 cm depth). More details about these soil samples are shown in Table et al.13 and Lehndorff et al.28. It should be noted that the traditional paddy management practice in this region in China is a crop rotation of rice in the wet season followed by wheat or other upland crops in the dry season53. Total soil P concentrations were determined by melting a mixture of soils and lithium borate at 1000 °C for 30 min, followed by inductively coupled plasma optical emission spectrometry (ICP-OES)54.The studied soils are located in the Bay of Hangzhou near the city of Cixi , Zhejiang Province. Dike building over last 2000 years for land reclamation resulted in a chronosequence containing paddy and non-paddy soils. More details about the sites are available in other publications31P-NMR spectroscopy of alkaline extracts is commonly considered to be a reliable method for the quantification of Po40. Soil samples from each of the replicated plots at each location were extracted by shaking 4 g of air-dried soil with 40 mL of a solution containing 0.25 M NaOH and 0.05 M Na2EDTA for 4 h, followed by centrifugation at 10,000 × g for 30 min55. A 2-mL aliquot of each supernatant was used to determine total extracted P by ICP-mass spectrometer (MS); the remaining solutions were frozen at −80 °C, lyophilized, and ground. Each freeze-dried extract (~100 mg) was re-dissolved in 0.1 mL of deuterium oxide and 0.9 mL of a solution containing 1.0 M NaOH and 0.1 M Na2EDTA, and then immediately transferred to a 5-mm NMR tube.Solution 31P-NMR spectra were obtained using a Bruker 600-MHz spectrometer equipped with a prodigy-probe (a broadband CryoProbe that uses N-cooled RF coils and preamplifiers to deliver a sensitivity enhancement over room temperature probes by a factor of 2 to 3 for nuclei from 15N to 31P). Extracts were measured with a D2O-field lock at room temperature, and chemical shifts were referenced to 85% orthophosphoric acid (0 ppm). The NMR parameters generally used were: 32 K data points, 2.6 s repetition delay, 0.7 s acquisition time, 30° pulse width and 10,000 scans. The delay time used here allows sufficient spin-lattice relaxation between scans for P compounds in NaOH-Na2EDTA 1. The orthophosphate peak in each spectrum was standardized to 6.0 ppm during processing58. Identified compounds and compound classes include orthophosphate (6 ppm), pyrophosphate (~−5 ppm), polyphosphate , orthophosphate monoesters , orthophosphate diesters (3 to −4 ppm), and phosphonates (7 to 50 ppm). Because α- and β-glycerophosphates and mononucleotides, which result from degradation of orthophosphate diesters during 31P-NMR analysis59, were detected in orthophosphate monoester region, we assigned these compounds to orthophosphate diesters rather than to monoesters60. The concentrations of individual P species were calculated by multiplying 31P-NMR proportions by the total NaOH-Na2EDTA extractable P concentration. The percentage of each P compound or compound class was calculated manually by integration across the entire spectrum and also by integrating smaller regions within each spectrum. The high-molecular weight (HMW) Po recently identified by McLaren et al.61 is included with the unknown peaks in the P-mono-other category  by t test, one-way ANOVA was used to test significant differences of P compound concentrations among soil chronosequences, and regression functions of concentrations and distributions of various Pi and Po during pedogenesis were calculated. All statistical analyses were conducted using Sigmaplot 12.5 for Windows. To estimate accumulation rates, a mono exponential regression model was used:Xt is the parameter of issue at cultivation time t (years), Xe is the parameter stock at absolute equilibrium, X0 is the initial parameter concentration in the tidal wetland (t = 0), and k is a rate constant13.The total P concentrations between paddy and non-paddy soils were tested for significant differences .supplementary information