In PD group, PIGD severity was negatively correlated with MoCA score and Aβ1–42 level in CSF, and positively correlated with Hoehn-Yahr stage and P-tau181t level in CSF. In PIGD group, PIGD severity was negatively correlated with homovanillic acid (HVA) level in CSF, and HVA level was positively correlated with Aβ1–42 level in CSF. PIGD was significantly correlated with cognitive impairment, which underlying mechanism might be involved in Aβ1–42 aggregation in brain and relevant neurochemical disturbance featured by the depletion of HVA in CSF.Parkinson disease (PD) is identified as tremor-dominant (TD) and postural instability and gait difficulty (PIGD) phenotypes. The relationships between motor phenotypes and cognitive impairment and the underlying mechanisms relating pathological proteins and neurotransmitters in cerebrospinal fluid (CSF) are unknown. We evaluated the motor symptoms and cognitive function by scales, and detected the levels of pathological proteins and neurotransmitters in CSF. TD group and PIGD group had significantly higher levels of total tau, tau phosphorylated at the position of threonine 181(P-tau181t), threonine 231, serine 396, serine 199 and lower β amyloid (Aβ) Cognitive impairment of PD has been paid more attention to in recent years. However, motor symptoms and cognitive deficits in PD are heterogeneous in age of onset7The decreased levels of dihydroxyphenylacetate (DOPAC) and homovanillic acid (HVA) in CSF, the two metabolites of dopamine, have been observed in PD patients as compared to controls1819In total, 520 PD patients were recruited from the Department of Geriatrics and Neurology, Beijing Tiantan Hospital, Capital Medical University. Demographic information, including age, age of onset, sex, education, disease duration, predominantly affected side, disease severity, Montreal Cognitive Assessment (MoCA)-Beijing versionIn total, 28 age-matched controls from the Department of Geriatrics and Neurology, Beijing Tiantan Hospital were selected. The inclusion criteria were as follows: (1) no neurological symptoms and signs; (2) no intracranial diseases; (3) no essential tremor, PD, secondary parkinsonism, or Parkinson-plus syndrome; (4) no dysarthria or mental illness that affected expression; (5) no alcohol or drug abuse. All these controls had no family history of PD or tremor.The controls were also patients, but their diseases, such as peripheral neuropathy and headache caused by high intracranial pressure, etc, were not related to and had no effect on the results of this investigation.The severity of PD was assessed by Hoehn and Yahr (H-Y) stage.Each PD patient was evaluated using the items in the Unified Parkinson’s Disease Rating Scale (UPDRS) III, in which items 20 and 21 were for tremor, item 22 was for rigidity, items 23–26 and 31 were for bradykinesia, and items 27–30 were for PIGD. The score for each motor symptom was calculated by summing up the scores of corresponding items in UPDRS III.et al.Motor phenotypes were determined as either TD phenotype (n = 309) or PIGD (n = 211) phenotype following the classification algorithm proposed by Jankovic Cognitive function of PD patients was evaluated by the MoCA-Beijing versionBeijing Tiantan Hospital review board (KY2013-003-03) has approved this study. All participants completed the written informed consents. This study met the guidelines of Capital Medical University, which abode by the Helsinki Declaration on ethical principles for medical research involving human subjects.Before fasting, CSF (3 mL in a polypropylene tube via lumbar puncture) was collected from patients with PD whose conditions allowed us to withdraw the antiparkinsonian drugs for 12 to14 hours. Approximately 0.5 ml volume of CSF was aliquotted into separate Nunc cryotubes and kept frozen at −80 °C until used in assays. Each aliquot dedicated for each measure to avoid freeze-thawing and potential degradation of protein.1–42, T-tau, P-tau181t, P-tau396s and tau phosphorylated at the following positions: threonine 231 (P-tau231t) and serine 199 (P-tau199s), in CSF from PD patients and control participants were determined by using an enzyme-linked immunosorbent assay.The levels of pathological proteins, including Aβ1–42 and T-tau, respectively, were obtained from CUSABIO . KHB7031, KHB7041, KHB8051 and KHO0631 kits for measuring P-tau396s, P-tau199s, P-tau231t and P-tau181t, respectively, were obtained from Invitrogen .CSB-E10684h and CSBE12011h kits for measuring AβThe levels of DA and its two metabolites of HVA, DOPAC, Ach, serotonin (5-HT) and norepinephrine (NE) in CSF from PD patients were tested by high performance liquid chromatography (HPLC). Henomenex 150*2 mm, 150*3 mm chromatographic columns and LC-MS-MS 6410 chromatographic instrument were from Agilent Company (USA), and standard sample was from Sigma Company (USA).Statistical analyses were performed with SPSS Statistics 20.0 .1–42, T-tau, P-tau181t, P-tau231t, P-tau396s and P-tau199s in CSF were compared among control, TD and PIGD groups.Demographic information, motor symptoms and MoCA score were compared between TD and PIGD groups. The levels of AβContinuous variables, if they were normally distributed, were presented as means ± standard deviations and compared by ANOVA test. Bonferroni correction was performed in further comparisons between two groups. P value was significant when it was <0.017. Continuous variables, if they were not normally distributed, were presented as median (quartile) and compared by nonparametric test. P value was significant when it was <0.017 in further comparisons between two groups. Discrete variables were compared by Chi square test.1–42, T-tau, P-tau181t, P-tau231t, P-tau396s, and P-tau199s in CSF, between PIGD score and the levels of neurotransmitters in CSF, and between the levels of Aβ1–42 and P-tau181t and HVA level in CSF in PD group.Spearman correlation analyses were made between PIGD score and the levels of Aβ1–42 and P-tau181t were set as independent variables. P value was significant when it was <0.05.Multiple linear regression models were established, in which PIGD score in PD group was set as dependent variable, whereas age, age of onset, sex, education, predominantly affected side, MoCA score, disease duration, H-Y stage, AβAmong the 520 PD patients, 279 cases (53.63%) were male and 241 (46.37%) were female. The disease duration varied from 3 month to 30 years, with a median of 2.75 years [interquartile range (IQR): 4.0 years]. The average H-Y stage was 1.99 ± 0.80 stage. The demographic variables of PD patients were listed in PIGD group displayed significantly higher PIGD score (p < 0.001) and lower tremor score (p < 0.001) than the TD group, as well as higher scores of rigidity (p = 0.04) and bradykinesia (p < 0.001). PIGD group had significantly more patients at H-Y stage of 4–5 than TD group.The percentage of completely correct in the item of recall in MoCA in PIGD group was significantly lower than that in TD group (p = 0.04). No statistical differences were seen in age, age of onset, gender, education, predominantly affected side and LED. There were no significant differences in total scores of MoCA, MMSE and the score of each item in MoCA, as well as the percentage of completely correct in other items between PIGD group and TD group .1–42 level in CSF was prominently decreased and the levels of T-tau, P-tau181t, P-tau231t, P-tau396s and P-tau199s in CSF were all significantly elevated when compared with control group , and positively correlated with age, disease duration , H-Y stage  and P-tau181t level in CSF . Further multivariate linear regression models with adjustment for potential confounders revealed significant associations of PIGD severity with reduced MoCA score, decreased Aβ1–42 level and increased P-tau181t level in CSF , education  and Aβl in CSF .1–42 level  and higher P-tau181t level  in CSF. Further multivariate linear regression models with adjustment for potential confounders demonstrated that H-Y stage was the only influencing factor for tremor  , lower education level , lower MoCA score , longer disease duration , more advanced H-Y stage , lower Aβ= 0.015) .In total PD patients, there were no correlations between PIGD score and the levels of neurotransmitters in CSF. However, in PIGD group, PIGD score was prominently and negatively correlated with HVA level in CSF  .1–42, P-tau181t and HVA level in CSF. Correlation analyses showed that Aβ1–42 level was significantly and positively correlated with HVA level in CSF   .In this study, PIGD group displayed significantly higher scores of PIGD, rigidity and bradykinesia than those in TD group . Previou29In this study, the percentage of completely correct in the item of recall in MoCA in PIGD group was significantly lower than that in TD group , indicat33381–42 level in CSF indicated Aβ aggregation and deposition in brain1–42 level in CSF was related to cognitive impairment1–42 level in CSF prominently decreased at later stage in PD patients with dementia1–42 level in CSF, independent of age, education level, H-Y stage and cognition  and a mount of them (64.81%) were at the stage of no more than H-Y stage 2. We might, therefore, speculate that Aβ1–42 played a major role on cognitive impairment for PD patients at early stage, consistent with a recent study reporting that tau probably revealed the degree of neurodegeneration, whereas Aβ1–42 was a more specific marker for cognitive impairment.In this investigation, P-tau1–42 deposition might contribute to HVA dysfunction in brain related to both PIGD and cognitive impairment in PD patients.In summary, the findings of this study revealed a relationship between PIGD and cognitive impairment in PD patients. The results here provided a neuropathological mechanism underlying the correlation between PIGD and cognitive impairment. The decreased HVA level in CSF was closely associated with PIGD severity in PD patients. AβThere are following limitations in this study. Relatively few CSF samples were analyzed due to the difficulties of obtaining CSF from PD patients and controls, which might weaken the statistical power of the analyses, such as of the correlations between PIGD severity with the levels of 5-HT and Ach in CSF. Cognitive function evaluated by MoCA was relatively rough and a formal neuropsychological test battery containing detailed cognitive domains would be used in the future study in order to provide more information.How to cite this article: Zuo, L.-J. et al. Phenotype of postural instability/gait difficulty in Parkinson disease: relevance to cognitive impairment and mechanism relating pathological proteins and neurotransmitters. Sci. Rep.7, 44872; doi: 10.1038/srep44872 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Augmenter of liver regeneration (ALR) exerts strong hepatoprotective properties in various animal models of liver injury, but its protective mechanisms have not yet been explored. Autophagy is a recently recognized rudimentary cellular response to inflammation and injury. The aim of this study was to test the hypothesis that ALR may protect against acute liver injury through the autophagic pathway.4). The effect of ALR on autophagy was analyzed in vitro and in vivo. After autophagy was inhibited by 3-methyladenine (3-MA), apoptosis and proliferation were detected in the mouse model with acute liver injury. The ALR and autophagic levels were measured in patients with liver cirrhosis (LC) and acute liver failure (ALF), respectively.The level and role of ALR in liver injury were studied in a mouse model of acute liver injury induced by carbon tetrachloride  ALR overexpression directly induced autophagy flux in vitro and in vivo; and (2) ALR treatment suppressed apoptosis and promoted proliferation in mice exposed to CClWe demonstrated that ALR ameliorated liver injury via an autophagic mechanism, which indicates a potential therapeutic application for liver injury. Acute liver injury is a liver function abnormality that results from multiple reasons, such as viral infection, abuse of drugs or alcohol, ingestion of toxic substance and so on , 2. SeriAugmenter of liver regeneration (ALR) is thought to be a hepatotrophic growth factor that is responsible for the extraordinary regenerative capacity of the mammalian liver . A recenAutophagy starts with the sequestration of a region of cytosol in double-membrane compartments followed by the formation of autophagosomes, the fusion of autophagosomes with lysosomes and degradation dependent on lysosome, which is regulated by autophagy-related genes (ATGs) , 11. Aut4)-induced acute liver injury model to explore the protective mechanisms of ALR and measured the expression of ALR and the autophagic protein in patients with liver injury.Given the above information, we proposed that ALR may play a protective role in acute liver injury by regulating autophagic pathway. To test this hypothesis, we used a carbon tetrachloride  and p62 have been widely used as markers of autophagy flux, in which the conversion of LC3I to LC3II indicates the closure of the autophagic vacuole and the p62 degradation indicates the fusion of autophagosome and lysosome . GFP-LC34 Figure . These rin vitro, we then investigated the role of ALR on autophagy in mice that had been exposed to CCl4. As shown in Figure 4-induced mice, which showed that ALR could increase autophagy in vivo. ALR treatment increased the expression of ATG5, ATG7 and Beclin-1 in mice exposed to CCl4. In addition, ALR treatment promoted LC3II conversion and p62 degradation in mice exposed to CCl4 , a class-III PI3 kinase (PI3K) inhibitor, was used to block autophagy because the complex of Beclin-1 and PI3K is required for autophagic vesicle nucleation . As repoy Figure . ALR was4 than those treated with CCl4 only, which suggested that ALR promotes DNA synthesis and mitosis. Moreover, the expression of proliferation cell nuclear antigen (PCNA) also revealed a similar trend as the cyclins. 3-MA addition significantly decreased the expression of PCNA and cyclins in mice treated with CCl4 and ALR, which indicated that the suppression of autophagy reverses the proliferative effect of ALR in acute liver injury  and acute liver failure (ALF). Relative to the normal controls, LC3II conversion and p62 accumulation were significantly increased in patients with LC and ALF. ALR level was decreased in patients with LC and ALF compared to normal controls Figure .Although ALR exerts strong hepatoprotective properties in various animal models of liver injury, its protective mechanism need to be further explored. The main findings of this study were that ALR promoted autophagic activity, which may have depressed hepatocyte apoptosis and increased hepatocyte proliferation, consequently resulting in liver protection.ALR was discovered and isolated from the cytosol of neonatal rat liver and regeneration liver after partial hepatectomy . ALR speet al found that HGF increased LC3II conversion in primary mouse nonparenchymal cells (NPCs), and this effect was inhibited by an anti-HGF neutralization antibody [et al found that HGF treatment increased LC3II levels in primary mouse hepatocytes [Some studies have shown that growth-factor-induced autophagy provides for cancer cells the important components of metabolism. Peng antibody . Toshimaatocytes . These fin vivo and in vitro. Collectively, our study revealed that ALR promoted autophagic flux in CCl4-induced acute liver injury. Interestingly, CCl4 exposure increased LC3II conversion and p62 accumulation, which suggests that autophagy may be induced during liver injury but that the fusion of autophagosomes with lysosomes may be blocked.The LC3II conversion and the p62 degradation are generally considered markers of autophagic flux . Beclin-et al also found that the inhibition of autophagy with CQ or siRNA significantly promoted hepatocyte apoptosis in an ethanol-induced model [et al found that autophagic activity was increased in mice after partial hepatectomy (PHx), and the DNA synthesis and cell proliferation were impaired in Atg5 KO mice after PHx [4-induced acute liver injury through an autophagic mechanism.Increasing reports shows that autophagy has a protective effect on liver injury –28. Ni efter PHx . In our et al also found that ALR mRNA levels in patients with acute liver failure were decreased [A previous study demonstrated that the levels of ALR in liver tissues were lower in patients with advanced alcoholic liver disease and nonalcoholic steatohepatitis than in controls . Yu et aecreased . Similarecreased , 30, ourTherefore, a clear understanding of how ALR influences autophagy can lead to the characterization of the underlying molecular mechanisms. Elucidation of the signaling cascades in ALR regulation and its mechanisms will be highly beneficial for the therapy and prevention of liver injury. In the future, a combined therapy regimen that includes ALR and autophagy may be a possible treatment strategy for patients with liver injury.Male BALB/c mice (aged 6-8 weeks) were provided by the Animal Center at the Academy of Military Medical Sciences . All of the animals were placed in a specific pathogen-free environment and received humane care according to the Capital Medical University Animal Care Committee guidelines.4, 50%) and oil (50%) at a dose of 2 mL/kg body weight. The normal control group received an intraperitoneal injection of the same volume of oil as the CCl4 group. The mice were scarified at 12 h, 24 h, 48 h and 72 h after the CCl4 injection. The ALR plasmid (10 mg/kg), which was a gift from professor An [4 exposure, and a pcDNA3.0  as a control vector was injected into the tail vein at the same time. The suppression of autophagy was achieved with a tail vein injection of 3-methyladenine  2 h before CCl4 exposure. Serum aminotransferase was analyzed by the Clinic Center of Capital Medical University. H&E staining was performed by the Clinic Pathologic Center of Beijing Youan Hospital.The mice received an intraperitoneal injection of a mixture of carbon tetrachloride , were cultured in RPMI-1640 medium with 10% fetal calf serum in a 37°C incubator with 5% CO2. The GFP-LC3 and ALR plasmids were cotransfected into AML12 cells with the X-tremeGENE HP DNA transfection reagent  according to the manufacturer's instructions. A pcDNA3.0 as a control vector was transfected into AML12 cells. Cells were then treated with carbon tetrachloride  for 48 h to induce injury.The patients were recruited from the Artificial Liver Treatment and Training Center of Beijing Youan Hospital from 2013 to 2014. Normal liver samples were collected from five donors undergoing hepatic resection for liver transplantation. Liver samples with liver cirrhosis (LC) or acute liver failure (ALF) were obtained from the eighteen patients undergoing liver transplantation. Ten patients with LC were diagnosed according to the guidelines for the prevention and treatment of chronic hepatitis B (2010 version) . Eight pAfter AML12 cells were transfected with GFP-LC3, GFP-LC3 puncta were observed in autophagic cells. To evaluate live tissues, the slides were incubated in an LC3 rabbit monoclonal antibody solution  overnight at 4°C and were then incubated in an Alexa Fluor 488 goat anti-rabbit IgG solution  for 1 h at room temperature. Apoptotic cells were stained with a TUNEL apoptosis detection kit . All images were obtained with an inverted fluorescence microscope .Live tissues were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). After dehydration, thin sections were cut and stained with uranyl acetate and lead citrate. Digital images were obtained with an electron microscope .via 12% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated overnight with rabbit antibodies against ALR , LC3B , p62, Atg5, Atg7, Beclin-1, CyclinA, CyclinD, CyclinE, and β-actin primary antibodies  at 4°C. Then, the membranes were treated with a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody  and developed with a chemiluminescent substrate . Densitometry analysis was performed using ImageJ software, and the relative levels of protein in each group were normalized to the loading control.After the designated treatments were performed, liver tissues and cell pellets were lysed with RIPA buffer supplemented with protease inhibitors. Total proteins (50 μg) were separated Total RNA was extracted using TRIzol reagent  according to the manufacturer's instructions. First-strand cDNA was synthesized from 5 μg of RNA , and the ALR and HPRT mRNA levels were estimated by QPCR using a SYBR Green PCR Kit  with a real-time PCR system . The relative quantity of the cycle threshold value was normalized to an internal primer.post hoc LSD test was performed to compare differences among multiple groups. P < 0.05 was considered statistically significant. All data were analyzed using SPSS 11.5 software.All data are expressed as the mean±SD. One-way analysis of variance (ANOVA) followed by the


Contrast sensitivity (CS) is widely used as a measure of visual function in both basic research and clinical evaluation. There is conflicting evidence on the extent to which measuring the full contrast sensitivity function (CSF) offers more functionally relevant information than a single measurement from an optotype CS test, such as the Pelli–Robson chart. Here we examine the relationship between functional CSF parameters and other measures of visual function, and establish a framework for predicting individual CSFs with effectively a zero-parameter model that shifts a standard-shaped template CSF horizontally and vertically according to independent measurements of high contrast acuity and letter CS, respectively. This method was evaluated for three different CSF tests: a chart test (CSV-1000), a computerized sine-wave test (M&S Sine Test), and a recently developed adaptive test (quick CSF). Subjects were 43 individuals with healthy vision or impairment too mild to be considered low vision (acuity range of −0.3 to 0.34 logMAR). While each test demands a slightly different normative template, results show that individual subject CSFs can be predicted with roughly the same precision as test–retest repeatability, confirming that individuals predominantly differ in terms of peak CS and peak spatial frequency. In fact, these parameters were sufficiently related to empirical measurements of acuity and letter CS to permit accurate estimation of the entire CSF of any individual with a deterministic model (zero free parameters). These results demonstrate that in many cases, measuring the full CSF may provide little additional information beyond letter acuity and contrast sensitivity. The spatial contrast sensitivity function (CSF) represents a useful summary of functional vision by measuring the amount of contrast needed to detect or discriminate patterns across a range of spatial scales  and measures the smallest amount of luminance contrast needed to detect or discriminate the target using various tasks that include target detection, letter identification, and orientation discrimination. Optotype-based tests, prominent examples being the Pelli–Robson chart , a computerized sine-grating test , and quick CSF , which offers a novel adaptive approach to estimate functional parameters of the underlying CSF using a 10-alternative forced-choice task on narrowband filtered Sloan letters  were designated as having healthy vision with no history of ophthalmic disease, and 14 of the 43  had prior history of some type of ophthalmic disease . Despite the prior history and later age of some individuals, this group collectively had a relatively mild degree of functional impairment, evidenced by the fact that all subjects had better than 20/40 vision except a single individual with 20/50 acuity. All subjects gave written informed consent in accordance with the study procedures approved by the institutional review board at the Western University of Health Sciences, and all research was done in adherence to the tenets of Declaration of Helsinki. All examinations were performed binocularly and were repeated on the same or following day, with the exception of two participants who returned within 1 week of the first examination. The order in which the examinations were completed was determined pseudorandomly for each subject with a random number generator.Forty-three subjects  participated in the study and were recruited at the Eye Care Institute at the Western University of Health Sciences College of Optometry by use of advertisement with flyers and posters in the local campus community. Recruiting participants at the clinic offered the benefit of obtaining a relatively diverse sample of participants in terms of age (22 to 86 years), acuity (−0.3 to 0.34 logMAR), and history of ophthalmologic disease. While the study obtained a broad sample of subjects visiting the clinic for routine examination, it was not explicitly designed with the power to compare individuals with normal and low, due to the convenience sampling. Incidentally, 29 of the 43 subjects  for up to 5 s at a distance of 9 ft, and subjects verbally reported the perceived orientation. Using a staircase procedure, the test measured contrast thresholds for five levels of spatial frequency: 1.5, 3, 6, 12, and 18 c/°, presented in that order. Michelson contrast ranged from 100% to 0.5%. We used the standard settings associated with the built-in Sine CSF test without modification.http://www.vectorvision.com/csv1000-norms/) to translate scores, which range from 1 to 8, to logCS units for curve fitting and analysis. We used the standard recommended protocol associated with the commercial CSV-1000 test (without glare).The CSV-1000 is a printed chart-based test produced by VectorVision. The test presented sine-wave targets of varying spatial frequency  at a distance of 8 ft, with subjects making a forced choice between two targets presented on different rows . Contrast was adjusted using the method of limits, with eight discrete levels of contrast per SF, and the lowest contrast available on the chart was 0.5%. We used the table listed on the company's website (Quick CSF (qCSF) is a relatively new computerized test designed to measure a specific functional form of the CSF, the truncated log parabola, using Bayesian adaptive methods  and far (3 m) distances with printed ETDRS charts using standard procedures. LogMAR was scored letter by letter  is defined by the following equations:S′(f) defines the standard three-parameter log-parabola model in Data were analyzed using MATLAB . The outcome variable for each test was contrast sensitivity (1/contrast threshold) logarithmically transformed (base 10) to logCS units. For each of the three CSF tests, logCS measurements were fitted with a parametric form of the CSF , the trumax; units: CS); peak SF ; bandwidth , describing the width of the parabola; and truncation , describing the plateau of the function at the lowest SFs , we computed the Pearson correlation coefficient to indicate the strength and directionality of the linear relationships. Prior work has shown a negative linear relationship between high-contrast acuity and peak SF of the CSF  to retest . In terms of high-contrast visual acuity, logMAR values for distance acuity ranged from −0.3 (20/10) to 0.34 (20/45), and near acuity ranged from −0.2 (20/12.5) to 0.3 (20/40). The median for both near and far acuity was −0.06 logMAR—slightly better than 20/20 vision—indicating a sample population with generally little to no deficit in high-contrast visual acuity.Mean logCS values for the Pelli–Toronto letter test were similar from test  with very low bias (0.01). Generally, these levels of repeatability are comparatively similar to results of prior studies on the repeatability of other CS tests, which commonly range from about 0.15 to 0.5 logCS units , F = 39.4, p < 0.001, which was due primarily to much lower values for the qCSF in comparison to the other two tests. There was a significant effect for peak SF (sfmax), F = 127.8, p < 0.001, due to significantly higher values for the CSV-1000 test. The effect for bandwidth (β) was statistically significant, F = 29.2, p < 0.001, and the effect for low-SF truncation (δ) was also significant, F = 46.2, p < 0.001, with the Sine test showing significantly higher values and more variance than the other two tests. The AULCSF was also significantly different among the test types, F = 61.6, p < 0.001. In particular, the qCSF showed much lower AULCSF measurements in comparison to the other two tests. These analyses highlight the fact that the shape of the CSF differs systematically as a function of test type  = 5.38, p < 0.001, and peak SF, t(246) = 2.51, p = 0.012, had significantly worse RMSE values. Fixing bandwidth had a marginal impact, t(246) = 1.72, p = 0.09, and fixing truncation had a minimal impact on the RMSE of model fits, t(246) = 0.98, p = 0.33. The fact that model fitting is robust to cases in which select parameters are fixed to the group mean supports the idea that a template CSF may be adapted from group data to account for the global shape of the CSF and then subsequently adjusted according to just the two most relevant parameters  to potentially provide an accurate fit of individual data . We found a statistically significant correlation between letter CS and peak CS for two of the three tests, as shown in Finally, a central issue of the study is to assess the relationship between CSF model parameters and auxiliary measures of visual function. The bandwidth parameter was less consistent than peak CS and peak SF, and showed no relationship to auxiliary measures for the Sine and CSV-1000 tests—but interestingly, it did show a relatively high correlation to far acuity and letter CS for the qCSF test. The truncation parameter was also a mixed bag, showing some significant correlations among tests and measures, but was generally less consistent than peak CS and peak SF. The strongest correlations were found for summary measures—AULCSF and high-SF cutoff—likely because these measures represent the composite influence of all four parameters and are therefore less noisy than any single measure in capturing visual function. Each summary measure showed effects similar to peak CS and peak SF, namely, a negative relationship with acuity and a positive relationship with letter CS, particularly for the Sine and qCSF tests. By comparison, the CSV-1000 test was less consistent, perhaps because it produces highly quantized values of logCS, owing to the fact that it represented only eight distinct levels of contrast for each SF. We return to this point in the DThe results outlined so far can be distilled to three key insights. First, there are various approaches to estimating the CSF of an individual subject, and each type of test produces a distinct estimate of CSF parameters according to a test-specific template, as seen by group averages in n is the total number of subjects, subscript i represents the ith subject in the group, and subscript T indicates the parameter set defining the CSF template. Template parameters for peak CS and peak SF are not fixed to the group mean but instead vary according to independent measurements of letter CS and far visual acuity, respectively, following the linear models:xletterCS and xacuity represent empirical measurements of performance on the Pelli–Toronto letter CS and ETDRS high-contrast acuity tests, m is the slope of the best-fitting linear function, and c is the intercept term. Least-squares regression is used to estimate the slope and intercept terms from group data, and hence used to predict template parameters of peak CS and peak SF for any subject not contained within the normative group data set. As such, the entire predictive model is determined by just six parameters—βT, δT, The procedure of the predictive model is to first establish a standard template CSF derived from normative group data that is distinct for a given test type (due to aforementioned mean differences among tests), and then to shift this template left, right, up, or down according to performance on other auxiliary measures of visual function. Here, the template CSF is defined by the truncated log-parabola model see  and 2, iTo evaluate the performance of the predictive model on each individual subject, we use a leave-one-out procedure that estimates six template parameters from the group cohort that includes all subjects except the one to be predicted. The model is then evaluated for the left-out subject according to that subject's individual measurements of letter CS and far acuity to predict parameters of the subject's CSF. By evaluating the parameterized log-parabola function at the SFs specified by the original test , we derive predictions to be compared directly to the raw logCS measurements.n is the number of measured frequency-specific data points , f is an integer index for stepping through SF levels, x(f) represents actual measured data, S(f) represents frequency-specific values of the fitted CSF function (see Stemplate(f) represents frequency-specific values estimated from the predictive model  in RMSEpredicted between subjects with a prior history of ophthalmologic disease  and subjects (n = 29) without a prior history . There are several examples of low- and high-error predictions within each subgroup, as shown by organization in rows within Not surprisingly, there was variability across the group in terms of how well the zero-free-parameter model could predict individual CSF curves. test-retest (the consistency of the test on repeated measurements), which would determine whether they are within the same range of reliability as the instrument or test itself, and RMSEfitted (with four free parameters to vary), which provides an effective upper bound on how well the truncated log-parabola functions can fit the data in the first place. The scatterplot in predicted fall above the unity line with RMSEfitted, highlighting the fact that the ability to predict individual performance with this model is constrained inherently by the quality of fit for the full four-parameter CSF model. In other words, individuals with high RMSEfitted will necessarily have greater RMSEpredicted values due to the fact that the zero-free-parameter model is couched in the same parametric form , and this model is shown to fit the particular subject only so well in the first place. The variability in the ability of the full CSF model to fit individual subjects is clearly a driving factor in explaining variability in the performance of the predictive model.As discussed previously, a key benchmark for interpreting the degree of error in model predictions across subjects is to compare them directly to both RMSEpredicted and RMSEtest-retest values are uncorrelated across individuals, and in fact have a similar underlying mean distribution. For example, for the Sine test the mean RMSEtest-retest across individuals was 0.25 (SD = 0.13) and the mean RMSEpredicted was 0.23 (SD = 0.08), and a t-test revealed no significant difference between these distributions, t(42) = 1.02, p = 0.32. Likewise, for the CSV-1000 the mean RMSEtest-retest across individuals was 0.15 (SD = 0.09) and the mean RMSEpredicted was 0.14 (SD = 0.07), and a t-test revealed no significant difference between these distributions, t(42) = 0.45, p = 0.65. For the qCSF test the mean RMSEtest-retest across individuals was 0.11 (SD = 0.07) and the mean RMSEpredicted was 0.14 (SD = 0.08), and a t-test revealed marginal statistical significance between the distributions, t(42) = −2.0, p = 0.051. Even accounting for the fact that error is introduced to model predictions directly via errors in parametric function fitting, as demonstrated in By comparison, as shown in not achieved via optimized least-squares fitting of the parametric CSF model to individual-subject data. In theory, the CSF for any number of future subjects can be predicted with this same model by simply collecting measurements for letter CS and high-contrast acuity under similar testing conditions and plugging them into Equations 3It is important to underscore the point here that this level of prediction accuracy is achieved with a template CSF that is shifted according to independent measures of visual function, and is This study was designed to evaluate several aspects of CS data within a heterogeneous cohort of subjects with minimal visual impairment, while focusing on three distinct tests designed to measure the CSF . Our analysis focused on several factors, including test–retest repeatability, parametric CSF model fitting and comparisons among test types, reliability of individual functional parameters for describing individual differences, and correlations among functional parameters with auxiliary measures of visual function, and culminated with the development of a predictive model built directly from collective findings in these analyses. This approach was motivated by very recent work by Chung and Legge , who proIn general, our choice of parametric curve to model the CSF  was suitable to account for the shape of the CSF derived from each test, and produced relatively small errors in curve fitting for most individuals, consistent with prior studies  As a practical matter, such a model could be used to predict CSF curves for any arbitrary CSF test in which normative data were available, potentially allowing better comparisons among data sets in the literature and in the clinic; and (b) as a theoretical matter, it would suggest that there is such redundancy between CSF measurements and other common clinical measures that it would obviate the need to spend time estimating the full CSF curve in many clinical situations.On this matter, our results affirm the notion that CSF tests are highly redundant and differ from each other primarily on the surface  but deliver essentially the same information as other tests that are easier to use and more readily available, such as high-contrast ETDRS acuity and letter CS estimated from single unfiltered optotypes. Yet CSF tests do differ from each other in practically relevant ways, such as test–retest repeatability and the precision of threshold estimates. That these results were found in such a diverse cohort of individuals suggests that these findings should extend to most people within the range of relatively normal visual function. The degree to which this modeling framework would apply well to particular patient populations with more severe visual impairment is an important topic for future study. However, the study by Chung and Legge  did use While the results in the present article do suggest that in many cases, estimation of the full CSF function may produce redundant information with the more quickly and easily measured assessments of acuity and letter CS, it would be inappropriate to assert that less will always be more. As can be seen in For instance, one potential limitation of the present approach is the inability to characterize subtle changes to the shape of the CSF, in particular midfrequency notches that may result from retinal or optic-nerve disease, multiple sclerosis, or even optical abnormalities such as astigmatism (Apkarian, Tijssen, Spekreijse, & Regan, Supplement 1Click here for additional data file.


This article provides LA-ICP-MS in-situ U-Pb zircon dates performed on single crystals from dacitic to rhyolitic ignimbrites of the Bükkalja Volcanic Field  temporally covering the main period of the Neogene silicic volcanic activity in the Pannonian Basin. The data include drift-corrected, alpha dose-corrected, Th-disequilibrium-corrected, and filtered data for geochronological use. The data presented in this article are interpreted and discussed in the research article entitled “Early to Mid-Miocene syn-extensional massive silicic volcanism in the Pannonian Basin : eruption chronology, correlation potential and geodynamic implications” by Lukács et al. (2018) [1]. The amount of xenocrystic zircons was minimized by separating zircon grains solely from pumice clasts of the pyroclastic rock (when available), while in case of lapilli tuff samples we attempted to remove all lithic fragments before zircon separation.3 was added. The beakers were placed in a high pressure Parr bomb and the zircons were etched at 180 °C for 12–15 h. The zircons were rinsed with H2O and acetone before being fluxed for 12 h in 6 N HCl at ~ 85 °C. The zircons were rinsed in H2O and washed with acetone.In order to minimize the effects of lead loss, chemical abrasion CA;  was emplThe separated zircon grains were mounted in 1 in. epoxy resin mount and polished to a 1 µm finish. Before dating, zircons were checked by optical microscopic and cathodoluminescence (CL) imaging. CL imaging was produced using an AMRAY 1830 SEM equipped with GATAN MiniCL and 3 nA, 10 kV setup at the Department of Petrology and Geochemistry, Eötvös University, Hungary and a JEOL JXA 8900 electron microprobe with 10 kV setup at the University of Göttingen.2.3Analyses were performed in two laboratories: Department of Earth Sciences, ETH Zürich and GÖochron Laboratories, University of Göttingen. Analytical setups of the laboratories are presented in 2.4We filtered out the data that was > 10% discordant determined by the following equation:σ) Validation reference materials were used to correct for alpha dose-dependent age offsets in non-CA treated zircons


Drosophila metamorphosis, nascent testis myotubes migrate from the prospective seminal vesicle of the genital disc onto pupal testes and then further to cover the testes with multinucleated smooth-like muscles. Here we show that DWnt2 is likely required for determination of testis-relevant myoblasts on the genital disc. Knock down of fibroblast growth factor receptor (FGFR) heartless by RNAi and a dominant-negative version revealed multiple functions of Heartless, namely regulation of the amount of myoblasts on the genital disc, connection of seminal vesicles and testes, and migration of muscles along the testes. Live imaging indicated that the downstream effector Stumps is required for migration of testis myotubes on the testis towards the apical tip. After myoblast fusion, myosin II is needed for migration of nascent testis myotubes, in which Thisbe-dependent fibroblast growth factor (FGF) signaling is activated. Cadherin-N is essential for connecting these single myofibers and for creating a firm testis muscle sheath that shapes and stabilizes the testis tubule. Based on these results, we propose a model for the migration of testis myotubes in which nascent testis myotubes migrate as a collective onto and along the testis, dependent on FGF-regulated expression of myosin II.During  Summary:Drosophila testes and mammalian seminiferous tubules are surrounded by a muscle layer. Drosophila myotubes migrate towards testes in dependence of the FGF receptor Heartless, myosin II and Cadherin-N. Drosophila, various single as well as collective cell migration processes have been described, such as border cell migration in the ovary and embryonal mesoderm migration  in segments A8/A9/A10  signaling. The FGF receptor (FGFR) Breathless (Btl) and its ligand Branchless recruit larval mesodermal cells, which become epithelial and give rise to paragonia and seminal vesicles , becomes activated by its ligands Thisbe (Ths) and Pyramus (Pyr)   and is ius (Pyr) , where ius (Pyr) . Htl is us (Pyr) . Htl-depogenesis . During ogenesis , while nogenesis .Drosophila, cadherins such as Shotgun  and Cadherin-N (Cad-N) can mediate adhesion between neighboring cells. Catenins, such as beta-catenin, mediate the link between cadherins and the cytoskeleton  was expressed in the epithelium of the prospective seminal vesicle but was hardly detectable in myoblasts  with the driver line UAS-Dcr-2;;Mef2-Gal4 specifically in myoblasts. This resulted in a disturbed morphology of the testis. In the wild type, the adult testis is a long, thin tubule of 2.5 coils , Cad-N expression is largely restricted to FC-like myoblasts . mefGAL4-driven knock-down of Cad-N was efficient in FC-like myoblasts, whereas expression was hardly affected in myoblasts lying over the paragonia . At 44 h APF, cad-N knock-down testes displayed nascent myotubes, which were distributed all over the testes . In contrast to wild-type testes , the nascent myotubes in these cad-N knock-down testes are less elongated and their number seemed to be reduced . Nevertheless, adult males with this phenotype were able to produce offspring .Hence, we then knocked down .5 coils F. In theone coil G. The teone coil G′. Genitone coil . In diffDrosophila . Cad-N expression between adjacent myotubes was preserved upon arm knock-down, whereas no Cad-N expression was detected when testis myotubes did not adhere to each other , the beta-catenin homologue in osophila , specifiwo coils H. The tewo coils H′. Thesech other I.We conclude that Cad-N in cooperation with Arm is involved in testis myotube migration and is necessary both to stabilize the testis muscle sheath and to shape the testis.Many migratory processes depend on the actin-myosin network . Using mspaghetti squash (sqh), the regulatory light chain of non-muscle myosin II .When we myoblast-specifically knocked down yosin II . The tesyosin II A, and hayosin II C. The muyosin II C′ than iyosin II B. These zipper (zip), the non-muscle myosin II heavy chain . This was surprising because the sqh and zip knock-down phenotypes resembled each other and are components of myosin II. The observed differences in male fertility might be due to an unpredicted off-target in the sqh RNAi line.Testes morphology and musculature were also disturbed when vy chain , was dowvy chain D, compara sheath D′. In cosqh knock-down testes in more detail. Cad-N was expressed between the few remaining adjacent myotubes , which suggested that the muscle sheath defect was not due to faults in Cad-N-mediated attachment of single myotubes. Instead, it was likely caused by insufficient population of the testis with muscles. We further analyzed the status of the extracellular matrix by monitoring Terribly reduced optic lobes (Trol) , which indicated extracellular matrix integrity. At 44 h APF, pupal testes with myoblast-specific sqh knock-down were populated with fewer nascent myotubes than wild-type testes . However, the testis shape was not affected at this time of development.As an example, we first analyzed adult s (Trol) , which is (Trol) . ExpressTaken together, these data led us to conclude that non-muscle myosin II is required for testis myotube migration and that correct shaping of the testis depends on the presence of an intact tight muscle sheath.DWnt2 alleles in trans . By contrast, we observed very drastic defects when the FGFR Htl was knocked down. Specifically, all flies were pharate lethal, and most testes were not attached to the seminal vesicles and did not elongate . This might be due to myoblast determination defects, since htl knock-down genital discs 24 h APF contained fewer myoblasts than wild-type genital discs 24 h APF. This was especially the case in the posterior part of the genital discs, where myoblasts for the muscle sheaths of the ejaculatory duct and sperm pump are localized . At 30 h APF, prospective seminal vesicles of htl knock-down genital discs contained fewer nascent myotubes than the wild type . Myoblast-specific expression of a dominant-negative (DN) version of Htl lacking the kinase domain . The adult testes were elongated and coiled, but had bulky tips as observed with the DN-version of Htl . The testis muscles covered the organs except for the tips . To gain further insight into which FGF ligand activates Htl in nascent myotubes, we analyzed the testis shape and musculature of hypomorph e02026ths/Df(2R)ths238 mutants. Adult testes had approximately two coils and a very bulky tip . After stumps knock-down – in contrast to the htl knock-down  – we observed many myoblasts lying over the seminal vesicles . Stumps expression on htl knock-down genital discs 24 h APF remained unchanged in testis-relevant myoblasts  in our analysis, which resembles the Htl-DN  phenotype and the thisbe hypomorph mutant and thus had muscles on the testes. As RNAi-mediated knock-down of Stumps  was very efficient , we focused on stumps in our analysis in parallel to the htl-RNAi line BL35024.So far, we gained evidence for Heartless-dependent migration of nascent myotubes along the testes and showed that knock-down of Cad-N G-G′ and We observed Cad-N expression in nascent myotubes migrating along the testis and in the mature musculature D-E. We ahtl-RNAi knock-down situation, however, nascent myotubes were less densely packed and had started to stretch while encircling the testis tube. In this situation, Sqh expression was not obviously disturbed  resulted in adult testes that were not fully covered with muscles, which indicated that non-muscle myosin II is necessary for the migration of nascent testis myotubes. Myosin II is also required for proper detachment and migration of border cells during egg chamber development  After myoblast fusion, when Stumps and Cad-N are distributed along the plasma membrane of nascent testis myotubes that are in close contact to the adjacent epithelium of the prospective seminal vesicles, FGF signaling initiates collective migration of nascent testis myotubes from the prospective seminal vesicle onto the testis B. (ii) TWe propose that the ligand Ths and possibly also Pyr binds Htl in nascent myotubes and thereby activates FGF signaling via Stumps during both phases of migration; the source of these ligands needs to be determined. Downstream, components of MAPK signaling, such as Erk, could be activated, which might cause changes in transcription of different target genes. Indeed, we observed Htl-dependent expression of Sqh. We hypothesize that Thisbe/Heartless-induced MAPK signaling might link to the actin cytoskeleton via non-muscle myosin II, thereby facilitating cytoskeletal changes needed for cell migration B.Our data provide first insights into the Thisbe/Heartless-dependent regulation of myotube migration via myosin II expression from the seminal vesicle to and along the testes, which is dependent on successful connection of these tissues. Future studies aiming at identifying missing components and functions to fully elucidate testis myoblast specification and myotube migration will require the establishment of more specific tools, such as driver lines specifically active in testes myoblasts and myotubes.1118 (BL6326) was used as the wild-type reference. The following transgenic flies were used: Mef2-Gal4 , UAS-arm-RNAi , UAS-sqh-RNAi , UAS-zip-RNAi , UAS-btl-RNAi , UAS-htl-RNAi , and UAS-stumps-RNAi . We conducted all RNAi experiments with at least two different fly lines per gene; the differences were in knock-down efficiencies or effects of second site insertions, as described in Table S1. The ths mutant was generated by crossing e02026ths/CyO.actin-GFP (Df(2R)ths238/CyO.actin-GFP , and v flies were obtained from Vienna Drosophila RNAi Center .Flies were kept and RNAi crossings were carried out on standard medium at 25°C. wef2-Gal4 , Htl-Galctin-GFP  and Df over seven days. After two weeks, the efficiency of the matings was examined. All fertility tests were carried out at 25°C.One adult male of the F® 488 , anti-rabbit DyLight 488 , anti-rabbit DyLight 549 . For visualization of F-actin, we used Phalloidin-Atto 565 ; to visualize nuclei, Hoechst 33342 was used .Pupae selection, genital disc dissection, and immunofluorescence were carried out as described elsewhere  and were35mm,high  containing 2 ml culture medium Shields and Sang M3 Insect Medium  was made according to −1 Ecdysone . Culture medium worked successfully for up to 4 days when stored at 4°C. A Zeiss AxioObserver Z.1 inverse microscope was used for live imaging. A Z-stack was acquired every 15 min for up to 10 h.Male white prepupae were collected at 0 h APF and aged on a moistened filter. Genital discs and pupal testes were dissected in culture medium and transferred into a tissue culture treated µ-DishConventional fluorescent images and optical sections were captured with a Zeiss AxioObserver Z.1 inverse microscope with ApoTome function. Images were taken and processed with AxioVision LE64 , figures were assembled with Adobe Photoshop CS6 , and models were generated in Adobe Illustrator CS6 (Adobe Systems Incorporated). Charts were generated in Microsoft Excel 2016 .


From the 1950s, the Po delta, one of the largest anthropogenic world deltas, has been subjected to a fast degradation and shoreline retreat due a marked reduction of sediment supply, mainly controlled by human impacts/factors, including subsidence. Through the interpretation of satellite images, coupled with the analysis of the flow discharge, and of the annual frequency of marine storms, we show that recently (>2010) the Po River has resumed delta progradation, especially in its northern portion. This happens after decades of erosion, followed by alternating regrowth and degradation phases, indicating conditions of substantial stability (1970–2000). Today the delta shows aggradation of new mouth-bars at the main distributary mouth, a clear evidence of active constructive processes. The ongoing trend marks a countertendency compared to many deltas worldwide. The Po delta is considered relatively young (~4–5 ky)1 and can be described as a delta dominated by both natural and anthropogenic process2. Historically, the delta developed through different branches, the position of which was quite different from the present one. The Po delta evolution has been driven by natural fluvial processes (floods/avulsions) and by a growing anthropogenic influence (e.g. engineering works and extensive land reclamation)2.The Po is the largest Italian river, draining an area of 70,091 kmth century, following massive hydraulic works such as the ‘Porto Viro” bypass. The works were made by the Venetian Republic in 1604 to divert the delta southwards in order to prevent the sedimentation generated by the northern branches into the Venice lagoon3. The recent human influence  has been shaping the delta and it is still a dominant controlling factor. Most likely, the Po delta can be considered among the best examples of man-made (anthropogenic) delta in the world4.The delta progradation near the current position started in the early 171, whereas during the 20th century, the delta progradation slowed down.The historical maximum sedimentation (and progradation) rates were associated with the Little Ice Age climatic transition2. This reduction in sediment yield, generally observed in all Italian rivers after World War II, was caused by different factors including bed material exploitation, dam construction, landslide stabilization, and a progressive reduction of agriculture on low mountain and hilly areas which was commonly followed by reforestation or by a natural regrowth of bush and low forest5. In the same period, a marked subsidence, due to extraction of methane and water from shallow aquifers, occurred. These actions resulted in a halt of progradation and a very rapid shrinkage of the deltaic body, as observed in other large delta systems around the world 6. The Po delta, therefore, turned into a fragile and vulnerable landscape, exposed to human modification and climate change impacts like sea level rise and coastal flooding7.The delta reached its maximum extension between 1930s–1940s; thereafter a significant retreat started at a rate of tens of meter per year, with a negative peak in the 1954–1978 interval3), the Po di Pila mouth erosion started in the 1950s and proceeded till today. The Pila mouth is estimated to deliver at least 70% of the whole Po sediment load9. The sediment input to the delta mouths was modulated also by the fast artificial subsidence experienced by the channel beds during the second half of the 20th century10 and caused by fluid withdrawal and by the changing flow proportions between the different distributary channels in consequence of river regulation and water pumping for irrigation.The post-1950 reduction in sediment supply generated an overall retreat of the coastline and of all the delta outlets, which was particularly severe in the Po di Pila mouth and the northern part of the delta , we investigated the coastal changes occurred in the Po Delta during the last phase of general reduction in sediment supply (1970–2000). Moreover, the recent enhanced satellite capabilities (e.g. Sentinel 2) permit to focus, in particular, on the most recent and on the current geomorphological evolution of the Po delta margins.While for the period 1950 onwards researchers had to rely on indirect sources like a not frequently updated cartography and sparse aerial photography, remotely sensed data offers a higher frequency dataset of direct observation.2 and, from summer 2015, through the Copernicus Program with the Sentinel-2a satellite, followed, in march 2017, by the Sentinel-2b, which allowed an almost continuous monitoring of the geomorphological evolution of the most active Po delta margins.With the technological progress of scientific optical satellite imaging and access policy, large public datasets are now available, starting from the 1970s through the Landsat missionsCompared with Landsat 8, Sentinel-2 has a better temporal resolution (e.g. it can archive on average at least one cloud free image every week), a better spatial resolution on VIS and NIR (10 m vs 30 m of Landsat) and a good radiometric capability (12 bit depth like the Landsat 8). For this study, images taken during extremely high or low tidal levels were not considered because they could give false information on the position of the shoreline. All the other images were considered acceptable, considering that in the Adriatic Sea the tide range is very limited (0.2–0.8 m). Moreover, some images, acquired with visible wave crests near the coast, underlined the presence of subaqueous landforms  3(560 nm) 2(490 nm). The false color compositions were digitally enhanced applying a Qk) was considered a good proxy of sediment yield. In a large river like the Po, sediment yield is controlled by a number of factors, the most important of which are sediment supply and flood frequency. The most downstream reach of the Po river, between the flow gauge of Pontelagoscuro, near Ferrara  is a useful datum that may well represent the fraction of the largest flows, i.e. the most effective for bedload transport. Though yearly variable, Q10d, is in fact close to the threshold conditions for bed material entrainment in sandbed rivers of the northern Adriatic coast11; moreover, that is the lowest duration flow data available in the official data records for the rivers of the region. On the base of yearly data, as the discharge exceeded 10 days a year (Q10d) is close to the maximum discharge (Qmax), and the higher is Qmax the larger amount of sediment is likely to be delivered at the mouth. These two characteristic discharges were combined to obtain a sediment transport capability discharge (Qk) in the following from:Qk was found to well explain the variability of the sediment yield of the Po R. on the base of the data published until 1984. A time series of Qk yearly values was constructed and plotted in the diagram of Fig. In order to get complementary information on the river delta dynamics, flow measurements carried out by the Po River Authority were obtained for the gauging station of Pontelagoscuro Fig. , approxiara Fig.  and the https://arpae.it/sim/?mare/boa), maintained by the Regional Environment Agency of Emilia-Romagna (ARPAe) and located about 100 km south of the Pila mouths. The data of this measuring station were used to analyze the annual frequency and mean duration of representative storms  aggraded above sea level at the secondary northern Pila mouth. From the summer/winter 2016 the islands and the mouth bars became evident with almost every tide level. This trend seems to continue until present as shown by the May 2017 Sentinel-2a digitally enhanced image , i.e. mainly following a longshore drift direction opposite to the dominant northern wind (Bora)13. Also the main channel outlet and the subaqueous mouth bar are oriented N-NW , based on the interpretation of at least one images every 7–10 years , the occurrence of a recent uninterrupted trend can be considered as a reliable evidence of delta progradation Fig. . This isDeltas are complex and dynamic landforms, with a vast submerged zone that is rarely visible in traditional air photography and/or surveyed during monitoring, especially its shallowest parts, where land surveys cannot extend and large survey vessels cannot access. Remotely sensed optical data allow detecting the changes of even the shallowest coastal morphologies (between tide levels ~0.8–1.5 m) but in the past had the limitation of a low frequency of data acquisition and an unsatisfactory on-the-ground resolution. The Sentinel platform revisiting capabilities permit the frequent comparison between images captured at the same tidal level and increase the possibility of finding acquisitions made during conditions of good water transparency, as in Fig. 10 found no evidence that in the last two to four decades the coastal sediment transport system and the wave activity have changed. As it can be seen in Table Two main factors generally control delta progradation: longshore sediment transport system and the amount of sediment supply by the river. Furthermore, storm-related erosive marine processes can play a fundamental role in contrasting the mouths progradation and aggradation. Previous works4, resulting in higher values of the potential effective discharge (Qk)  Fig. . In Fig.st century, has started again to prograde. In the most recent years (>2010) between the Pila mouths the process has been accelerating and now some progradation is taking place as showed by the growth of mouth bars in Fig. In conclusion, the results of this study point out that the Po delta, after a sediment budget crisis extended throughout the first years of the 216 and can be summarized as the result of sediment management policy paired by unexpected climate change positive feedbacks (e.g. accelerated spring ice/snow melting). In any case the area still remains highly vulnerable to sea-level rise as well as storm and river flooding due to the low elevation above mean seal level.The current trend marks a countertendency compared to many other world deltas


The pathophysiology of neurodegeneration is complex. Its diagnosis requires an early identification of sequential changes in several hallmarks in the brains of affected subjects. The presence of brain pathology can be visualized in the cerebrospinal fluid (CSF) by protein profiling. It is clear that the field of Alzheimer’s disease (AD) will benefit from an integration of algorithms including CSF concentrations of individual proteins, especially as an aid in clinical decision-making or to improve patient enrolment in clinical trials. The protein profiling approach requires standard operating procedures for collection and storage of CSF which must be easy to integrate into a routine clinical lab environment. Our study provides recommendations for analysis of neurogranin trunc P75, α-synuclein, and tau, in combination with the ratio of β-amyloid Aβ(1–42)/Aβ(1–40).n = 19). Variables included recipient type , tube volume, and addition of detergents at the time of collection. CSF biomarker analysis was performed with enzyme-linked immunosorbent assays (ELISAs). Data were analyzed with linear repeated measures and mixed effects models.Protocols for CSF collection were compared with CSF derived from subjects with normal pressure hydrocephalus , as compared to Aβ(1–42). For neurogranin trunc P75 and total tau, there is still an effect on analyte concentrations as a function of the tube volume. Protocol-related differences for Aβ(1–42) can be normalized at the (pre-)analytical level using the ratio Aβ(1–42)/Aβ(1–40), but not by using the ratio Aβ(1–42)/tau. The addition of detergent at the time of collection eliminates differences due to adsorption.Our study recommends the use of low protein binding tubes for quantification in CSF (without additives) of all relevant CSF biomarkers. Pre-analytical factors have less effect on α-synuclein, neurogranin trunc P75, and total tau, as compared to Aβ(1–42). The ratio of Aβ(1–42)/Aβ(1–40), but not Aβ(1–42)/tau, can be used to adjust for pre-analytical differences in analyte concentrations. Our study does not recommend the inclusion of detergents at the time of collection of CSF. The present results provide an experimental basis for new recommendations for parallel analysis of several proteins using one protocol for collection and storage of CSF.The online version of this article (doi:10.1186/s13195-017-0265-7) contains supplementary material, which is available to authorized users. The pathophysiology of the neurodegenerative process is complex. Neurodegeneration is characterized by sequential changes observed in the brain of affected subjects, starting several decades before the onset of clinical symptoms . It is cThe diagnosis of AD focused in the past especially on the use of proteins which have been identified in plaques and tangles, such as β-amyloid (Aβ) and tau . HoweverCSF biomarker analysis is already integrated in (research) criteria for the diagnosis of AD , 4, whilSome recommendations for collection and storage of CSF for protein analysis were published previously. The standardization efforts in the field of AD focused predominantly on CSF Aβ(1–42) , 9. TheyThe present paper extends for the first time observations made for Aβ species with respect to sample handling by the inclusion of data for total tau, α-synuclein, and neurogranin trunc P75. Data were analyzed with linear repeated measures and mixed effects models. Our study provides experimental evidence that it is possible to apply the optimized SOP for Aβ isoform analysis to SOPs for other CSF proteins. It will provide an experimental basis for new recommendations to improve the collection and storage of CSF for protein analysis in the field of AD.CSF was collected prospectively from patients undergoing lumbar puncture (LP) due to clinical suspicion of normal pressure hydrocephalus at the Memory Clinic, Skåne University Hospital, Sweden. In these patients, 40 mL of CSF was collected as part of the clinical routine investigation. Anonymized CSF samples from 19 individuals were investigated. The detailed procedure for CSF collection and storage is shown in Fig. For each subject, 20 different SOPs were applied. Several vials with 5 mL of CSF were collected during the LP from the same subject in two types of tubes (low protein binding (LoB) or polypropylene (PP)) with or without additives  or Triton-X100 ). Each tube was graduated in order to obtain the exact collection volume. A possible interference by a gradient effect was overcome by randomization of the order in which the different samples were collected. In a subset of the collection tubes, 25 μl of 10% Tw20 or TrX100 was added . All vials were mixed by inverting them 25 times. The vials were kept at room temperature (RT) for 20 min, followed by centrifugation . From these original tubes, CSF was transferred into microtubes . After 1 week of storage at −80 °C, one 1.5-mL PP or LoB tube containing 1.3 mL CSF was thawed, aliquoted, and stored at −80 °C before testing. Among the tested procedures, there was no SOP using a collection LoB tube and a PP storage tube. All samples were analyzed after one freeze/thaw cycle. For the cases included in the study, CSF collection started on 23 September 2014 and ended on 2 December 2014. Sample analysis was performed in the first quarter of 2016. The measured concentrations for each analyte and subject included in the study are given in Additional file CSF proteins were quantified by a CE-marked enzyme-linked immunosorbent assay (ELISA) for total tau  and research ELISAs for α-synuclein or neurogranin trunc P75 . All assays were designed with a combination of two well-characterized monoclonal antibodies (mAbs). Some details of the test procedures are described in Additional file Before analysis, CSF samples were pipetted into pre-blocked 96-well polypropylene plates as described in detail previously . The impCSF and run-validation samples  were analyzed in duplicate in one lab (ADx) by one operator with one lot number of each ELISA. CSF samples from each individual subject were tested on the same 96-well plate in order to limit inter-plate variability. Reported values  values) were used to calculate concentrations using a four parameter logistic (4-PL) curve fit algorithm. Acceptance criteria for run validation included: (i) re-calculated values of each calibrator concentration not to exceed 15% of the nominal concentration; (ii) kit controls within pre-defined specifications; and (iii) the variability between replicates must be lower than 20%.The analytical specificity of the new neurogranin assay was verified in sandwich immuno-assay formats with synthetic peptides covering either the full length of the protein (Sequence: UniProtKB—Q92686) or peptides truncated at the amino- or carboxy-terminus. Phosphorylation at amino acid Serine 36 was included as an additional confounding factor. Several approaches were used to determine the assay selectivity, including but not limited to the replacement in the assay design of either the capture mAb or the detector mAb by a non-neurogranin-specific mAb or by the addition of proteins known to be present in CSF.Parallelism was verified according to CLSI guideline EP06-A. Two neat CSF samples were diluted up to 1/10 (in the working range of the assay) with sample diluent and tested in quadruplicates. Concentrations were calculated taking into account the dilution factor.Precision was evaluated using native CSF samples.The specificity of the assay for the detection of α-synuclein  was verified with recombinant proteins . Parallelism was verified according to EP06-A. Four native CSF samples were serially diluted with sample diluent up to 1/20 and tested in duplicate. Concentrations were calculated taking into account the dilution factor. Precision was evaluated using native CSF samples.The analytical performance characteristics for the total tau assay have been summarized previously .The relative difference in adsorption rate between analytes  were visualized by plotting the mean relative differences for all subjects when a comparison was made between the selected reference protocol (Protocol 7) and the protocol that always resulted in the highest relative difference (Protocol 6) or between the reference protocol and a protocol including CSF collection in PP tubes (Protocol 1).The SOP experiment provided repeated measures data. The different measurements recorded on CSF collected from the same subject form a multivariate response. Measurements made with samples of the same subject are correlated, while measurements between different subjects are considered independent (with CSF of different subjects tested in different assay runs). More details on the analysis are described in reference  and AddiAn overview of the performance characteristics of each immuno-assay is essential to qualify the value of the results obtained in this study on standardization of the process for collection and storage of CSF. Part of the assay performance characteristics for the neurogranin trunc P75 and α-synuclein assay are described in Additional file The addition of detergent Tw20, Trx100) in the assay during sample incubation affects the outcome of the assay (= concentration) differently as a function of the analyte, the detergent, or OD values for calibrators and samples , we performed the simultaneous incubation of the sample  with biotinylated antibodies dissolved in a buffer containing 0.05%  Tw20. We observed no (major) difference in function of the selected detergent.p values are described in Additional file p < 0.0001), 5.7%  for α-synuclein (p = 0.005), and 8.8%  for total tau (p < 0.001). In the presence of detergent at the time of collection  between SOP 11 and SOP 17. All other differences were not significant.Figure In addition, it was calculated that the observed differences of protocol 6 as compared to the reference method (= protocol 7) was not dependent on the protein concentration in the CSF.p = 0.030), 1.7% , and 0.5%  for neurogranin, α-synuclein, and total tau, respectively.In the second analysis , 2.4% , and 2.5%  for neurogranin, α-synuclein, and total tau, respectively.The transfer to a PP storage tube had a larger effect and decreased the concentration on average by 5.3%  whereas for a LoB storage tube the estimated decrease was only 1.2% . For total tau, a smaller volume of PP storage tube decreased the concentration significantly by 4.5%  whereas for a larger volume of the storage tube, the estimated decrease was only 1.6% ).Storage tube volume had no effect on the measured α-synuclein concentrations. For neurogranin and total tau, the effect of tube volume was dependent on the storage tube type, with larger and significant effects for PP tubes. For neurogranin, a large PP tube volume decreased the concentration by 8.1% . For α-synuclein and total tau, estimated differences in concentration after freezing were below 1%. For the larger storage tubes, the effect of an additional freeze-thaw cycle was a significant decrease in concentration of 7.7%  for neurogranin, 3.0%  for α-synuclein, and 5.8%  for total tau.For all three analytes, the effect of an additional freeze-thaw cycle was dependent on the storage tube volume. The effect was more pronounced for tubes with a larger volume. For low volume tubes, the concentration was significantly decreased by an additional freeze-thaw cycle only for neurogranin, amounting to a decrease by 3.4%  and the use of a PP storage tube increased the concentration by 1.9%  compared to a LoB tube.With the addition of detergent at the time of collection of CSF (SOPs 11–20), measured concentrations increased for all three analytes  are identical (the latter is quantified by replacement in the assay design of mAb 3D6 (specific for Aβ1) by mAb 1A1 ). Changes in concentration of neurogranin as a function of collection protocol are lower than for the Aβ isoforms. Effects are even lower for tau or α-synuclein, but are still significant. Adsorption problems linked to the use of Aβ(1–42) can be normalized in part by the ratio Aβ(1–42)/Aβ(1–40), but not by using the ratio Aβ(1–42)/tau.In the absence of detergent at the time of collection (SOPs 1–10), the biggest difference with collection in LoB tubes is obtained with Aβ(1–42), followed by Aβ(1–40) and Aβ(1–38) (data modified according to ). ResultIn the presence of detergent at collection (SOPs 11–20), all analytes or ratio of analytes show comparable results to those obtained with the reference protocol.This study describes the effects of pre-analytical variables on the quantification in CSF of several proteins which are directly involved in the neurodegenerative process. The recommended protocol as described in the study is applicable for each selected protein in the CSF biomarker panel. Our study results will speed-up the integration of biomarker panels into clinical trials.Recent late-stage failures of clinical trials in the field of AD will force the community to identify better tools to select drug candidates which might become successful as disease-modifying drugs. When using algorithms including concentrations of a number of different biomarkers  for decision-making, one needs to understand how each individual protein will be affected by changes in (pre-)analytical conditions. For example, Bjerke et al.  reportedThe SOP experiments were analyzed with linear repeated measures and mixed effects models. These analysis methods properly handle the correlations between the measurements and enable the combination of measurements from all samples to derive one average effect. An important added value of this versatile statistical approach is that it allows the breakdown of the observed differences among SOPs into effects caused by different factors and their interplay.Our data show that CSF concentrations of total tau, α-synuclein, and neurogranin trunc P75 are less affected than Aβ isoforms by changes in collection protocols. Although some statistical differences were detected in some cases, we consider them considerably lower (<10.0% difference in protein concentration versus the reference protocol) as compared to what was published previously for Aβ(1–42) (>50% difference in concentration) . The prePublications on (pre-)analytical standardization for analysis in CSF of neurogranin or α-synuclein are limited. Kruse et al.  reportedOur results provide experimental evidence that it is possible to extend the panel of CSF proteins, using the same protocol for collection and storage as for the classical CSF proteins, with synapse proteins. Synapse loss correlates with cognitive decline and is more associated within certain areas of the brain with the degree of dementia as compared to plaques and tangles . NeurogrThe visualization of adsorption rates as a function of procedure and analyte will allow researchers to focus on the most important factors during standardization of collection and storage of CSF. The optimal protocol for quantification of Aβ isoforms can be easily integrated into protocols for other CSF analytes. However, some factors, such as the storage tube or the number of freeze-thaw cycles, can still affect the concentration determination in CSF, especially when the volume of sample per tube is taken into account. The impact of these differences on individual patient management or clinical decision-making will have to be documented in future diagnostic studies since these markers are not as integrated yet into the field of neurodegeneration as the tau and Aβ proteins.The addition of detergents at the time of collection of CSF can reduce the observed differences in adsorption rate as a function of the collection and storage protocols. However, this will result in a higher order of complexity for the already required detailed SOPs for the collection of CSF. The latter is related to the selected detergent and its in-process quality control, the need to optimize the working concentration of the detergent as well as how the detergent is added to the sample (volume/volume ratio), the time of presence in the sample before freezing, and the need to standardize the collected volume of sample per tube. Studies will have to verify whether samples with detergents are still commutable with results obtained with the available reference method for Aβ(1–42)  or whethThere are some limitations to our study protocol. The study was performed with two different detergents. Since no significant differences were found between the selected detergents, data were analyzed assuming the detergents had an identical effect. The study was performed with an ad-hoc design that has limitations to study the interplay among several factors. Among the tested procedures, there was no protocol using a collection LoB tube and a PP storage tube. As such, the reported effect of the storage tube type should be interpreted as the effect after collection in a PP tube. The additional freeze/thaw cycle was only performed in protocols with the same collection and storage tube type. Optimization of the SOPs for collection and storage of CSF was performed using one specific assay format and test procedure.Standardization of pre-analytical and analytical variables, including the development of fully automated assays and the definition of global cut-offs, are essential to enable a path for success for CSF neurogranin and α-synuclein in the future.The study recommends the integration of the following topics into new guidelines for collection of CSF: use low binding recipients during sample handling; limit dead volume in tubes; use one collection protocol for analysis of Aβ(1–42)/Aβ(1–40) with other CSF analytes; do not include detergent at the time of collection of CSF. Taking into account their clinical value, the recommendations provided in the present paper will be able to speed-up the worldwide standardization and integration of CSF AD biomarker protein panels.


Immune system activation is involved in Huntington’s disease (HD) pathogenesis and biomarkers for this process could be relevant to study the disease and characterise the therapeutic response to specific interventions. We aimed to study inflammatory cytokines and microglial markers in the CSF of HD patients.CSF TNF-α, IL-1β, IL-6, IL-8, YKL-40, chitotriosidase, total tau and neurofilament light chain (NFL) from 23 mutation carriers and 14 healthy controls were assayed.−4) after adjustment for age.CSF TNF-α and IL-1β were below the limit of detection. Mutation carriers had higher YKL-40 (p = 0.003), chitotriosidase (p = 0.015) and IL-6 (p = 0.041) than controls. YKL-40 significantly correlated with disease stage (p = 0.007), UHDRS total functional capacity score , and UHDRS total motor score (r = 0.59, p = 4.5*10YKL-40 levels in CSF may, after further study, come to have a role as biomarkers for some aspects of HD. Further investigation is needed to support our exploratory findings. HTT gene and  and HTT ctivation are incrctivation, 10. Onectivation, 12Biomarkers reflecting these peripheral and/or central derangements of neuroinflammation could be useful to better characterise disease progression and the therapeutic response to specific interventions. Cerebrospinal fluid (CSF) is a relatively accessible body fluid, enriched in brain-derived proteins, that haIn this work, we explored immune-associated substances previously reported to be altered in CSF or plasma in HD, namely proinflammatory cytokines–TNF-α, IL-1β, IL-6 and IL-8 –and microglial markers–YKL-40 and chitotriosidase–in the CSF of well-characterised patients, to determine what markers are capable of predicting clinical severity in HD. By measuring total tau and neurofilament light chain (NFL)–established markers of neuronal cell death, 19–in tEthical approval was given by the joint University College London/ University College London Hospitals ethics committee. All patients gave informed written consent before enrolment. Patient consent, inclusion and exclusion criteria, clinical assessment, CSF collection and storage were as previously published. In briefCSF TNF-α, IL-1β, IL-6 and IL-8 were measured using a Meso Scale Discovery antibody-based tetra-plex array with electrochemiluminiscence detection according to the manufacturer’s instructions . CSF TNF-α and IL-1β concentrations were below the lower limits of detection (LODs) of the assay for all samples. CSF IL-6 and IL-8 concentrations were above the LODs  in all samples, except for one in which IL-6 was <0.32 pg/mL. CSF YKL-40 concentration was measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions . This assay has an LOD of 6.25 ng/mL and all samples were well above this limit.CSF chitotriosidase was measured using an in-house enzyme activity assay, as previously described. Two sampStatistical analysis was performed with Stata 14 software . We used one-way ANOVA to assess baseline intergroup differences. Potentially confounding demographic variables  were examined in preliminary analyses and those found significant were included as covariates for subsequent analyses. Samples below the LOD were assigned the LOD concentration. The distributions of all tested molecules were tested for normality. Two-group comparisons were tested using unpaired two-tailed t-test or Wilcoxon log-rank test. Stepwise forward logistic regression analysis was used to find combinations of molecules with better diagnostic performance, and these combinations’ receiver operating characteristics were compared with the best molecule alone. To test associations with disease progression we calculated Pearson’s and partial correlations coefficients. Where appropriate, bootstrapping with 1,000 repetitions was applied to non-normal variables. Significance level was defined as p<0.05. Where there was concern about a single participant group or outliers unduly influencing the analysis, a sensitivity analysis was conducted, repeating the tests with those datapoints excluded.Thirty-seven cross-sectional CSF samples were obtained from 14 healthy controls, 3 pre-manifest gene expansion carriers (HDGECs), and 20 manifest HDGECs. Details are given in −12 for YKL-40 and r = 0.50, p = 0.002 for IL-8) so age was used as a covariate for these analyses. No other associations with age were found.Medians and interquartile range (IQR) of CSF concentrations of quantified substances are shown in The concentration of CSF haemoglobin was not found to be associated with the concentrations of any analytes, and therefore we assume any minimal blood contamination did not interfere with quantification of the analytes.CSF YKL-40, chitotriosidase and IL-6 were significantly elevated in HDGECs compared with controls , and also after controlling for disease burden score , indicating that CSF YKL-40 can independently predict severity of motor manifestations beyond the known predictive value of age and CAG repeat length. Chitotriosidase, IL-6 and IL-8 were not statistically significantly associated with TMS .CSF YKL-40 was significantly positively associated with TMS  and NFL , which remained significant after controlling for age , and disease burden score , again suggesting that YKL-40 level can predict these markers of neuronal death, independent of their mutual association with age and CAG repeat length. IL-6 did not show any such association with tau or NFL .Because they had been found to be associated with measures of clinical severity, YKL-40 and IL-6 were further examined for association with CSF tau and NFL levels, each of which has been shown to be associated with neuronal death in neurodegenerative and other conditions.–26 ThereIn this exploratory cross-sectional study using CSF samples collected and processed under rigorously controlled conditions, we show that CSF concentrations of the microglial markers YKL-40 and chitotriosidase, and the pro-inflammatory cytokine IL-6, were elevated in HDGECs compared with healthy controls.YKL-40 was most robustly associated with clinical severity: age-adjusted YKL-40 CSF concentrations in gene expansion carriers were associated with disease stage and with clinical measurements of disease progression—UHDRS TFC and UHDRS TMS. These latter associations remained statistically significant after adjustment for disease burden score, indicating that CSF YKL-40 has independent clinical predictive power, beyond its association with age and CAG repeat length. To our knowledge, the only other CSF substance for which such independent predictive power has been reported is the pathological agent itself, mutant huntingtin protein (mHTT).Furthermore, CSF YKL-40 was associated with the CSF markers of neuronal death tau and NFL; and retained this predictive power after adjustment for the mutually-associated predictors age and CAG repeat length.YKL-40, also known as chitinase 3-like protein 1 (CHI3L1), is a member of the 18 glycosyl hydrolase family, without enzymatic activity and with poorly understood function. During nIL-6 in CSF–previously shown to be elevated in HD blood plasma–was found to be associated with disease stage but not with more granular clinical measures of severity or neuronal death. IL-6 is known to cross the blood-brain barrier so it seTo our knowledge, this is the first study describing the potential of CSF YKL-40 as an independent predictor of clinical severity and neuronal death in HD. Our findings differ from those of Vinther-Jensen et al, who did not find a statistically significant association between CSF YKL-40 and disease stage or severity. DifferenNotwithstanding the striking findings in respect of YKL-40, this exploratory study requires validation in larger studies such as the HDClarity initiative. The longWe conclude that IL-6, chitotriosidase and YKL-40 show disease-related elevations in CSF in HD, affirming the role of microglial activation and the innate immune system in the disease. CSF YKL-40 in particular can independently predict clinical severity and neuronal death, and may be a useful targeted biomarker for the contribution of microglial dysfunction to disease activity.S1 Appendix(XLSX)Click here for additional data file.