Streams altered by pasture or sugar cane had wider and more overlapped trophic niches, which corresponded to more generalist feeding habits. In contrast, trophic groups in streams with native vegetation had narrower trophic niches with smaller overlaps, suggesting greater specialization. Pasture sites had greater ranges of resources exploited, indicating higher trophic diversity than sites with natural cover and sugar cane plantation. We conclude that agricultural land uses appears to alter the food base and shift macroinvertebrate assemblages towards more generalist feeding behaviors and greater overlap of the trophic niches.It is well recognized that assemblage structure of stream macroinvertebrates changes with alterations in catchment or local land use. Our objective was to understand how the trophic ecology of benthic macroinvertebrate assemblages responds to land use changes in tropical streams. We used the isotope methodology to assess how energy flow and trophic relations among macroinvertebrates were affected in environments affected by different land uses . Macroinvertebrates were sampled and categorized into functional feeding groups, and available trophic resources were sampled and evaluated for the isotopic composition of  Tropical streams are among the most threatened ecosystems in the world , especiaThose changes in turn have resulted in alarming losses of biodiversity in aquatic ecosystems, especially in tropical streams ,3. The CThe replacement, reduction or removal of vegetation cover, especially in riparian areas, leads to the degradation of physical habitat structure, increased sedimentation rates, hydrological changes, and water temperature oscillations ,10. ThesIn freshwater ecosystems, aquatic invertebrates are the main link between primary producers  and higher trophic levels . By breaking down organic matter, they contribute to litter decomposition and nutrient availability for other organisms ,14. AquaMacroinvertebrate assemblages are sensitive to environmental conditions and reflect the physical and chemical conditions of the ecosystem . Therefo13C and 12C  and of 15N and 14N  provide information that incorporates spatio-temporal scales and facilitates the analysis of food assimilation by consumers [The energy flow and trophic relationships among the organisms in an ecosystem may be assessed using stable isotope analysis (SIA) of carbon (C) and nitrogen (N) . The ratonsumers  and the onsumers ,22. Stabonsumers  to examionsumers , ecosystonsumers , to recoonsumers ,27 and tonsumers .δ13C and δ15N values in consumers reflect the C and N stable isotope ratios of the food sources [13C enrichment between food sources and consumers is usually low (0–1‰) [δ13C values typically differ among basal sources , δ13C is used as an indicator of C sources for certain consumers along food chains [δ15N usually varies from 2 to 4‰ at each trophic level [δ13C and δ15N in animal tissues reflect information on their use of physical habitats and trophic characteristics and are currently used to determine organic matter origin, trophic relationships and niche size and overlap [The  sources . The 13Cw (0–1‰) ,30. Becad chains ,20. In cic level ,30, faciic level ,31. Ther overlap ,28.The relative contributions of food resources to the diet of an animal may be calculated using isotope mixing models . HoweverThe isotopic C and N signatures of consumers in aquatic ecosystems may vary because of changes in riparian zones, which provide most of the organic matter used by aquatic communities . In addiIn this study, we evaluated how the energy flow and the trophic relationships among benthic macroinvertebrates were influenced by riparian land uses . Based on C and N stable isotope analyses, we compared isotopic niche breadth and the degree of niche overlap among macroinvertebrate trophic groups. We first tested whether anthropogenic activities in areas adjacent to streams can expand the trophic niche of macroinvertebrates and their degree of overlap. Then, we assessed whether anthropogenic land uses in riparian zones were associated with more generalist trophic groups and less specialized trophic groups compared with streams with native riparian vegetation.2 (25.4% of its area) [We studied sites in tributaries of the São Simão Hydroelectric Power Plant Reservoir, located in the sub-basin of the Paranaíba River, southeastern Brazil . The Parts area) .nd- and 3rd-order streams  located in the states of Minas Gerais and Goiás were selected from 110 previously investigated streams [Nine 2 streams . We used streams ) propose streams  to selec streams . For the streams . To illu streams .Each stream was subdivided into five equally sections. We collected five independent samples (one per section) of benthic macroinvertebrates from each site, along with five independent samples of food resources: CPOM, FPOM, filamentous algae, periphyton, leaves of the riparian vegetation  and suspended particulate organic matter (seston). Only one site had aquatic macrophytes; therefore, macrophytes were not considered as resources in the analyses. We collected algae, CPOM, periphyton, FPOM, leaves of native riparian vegetation and seston from sites in all three types of riparian environments, whereas pasture grasses were collected only in the pasture environment and sugar cane leaves only in the sugar cane environments .2 each) were taken per stream, one per section, totaling 0.45 m2 per site. All invertebrates sampled were stored on ice and after 2 days processed in laboratory. In the laboratory, the organisms collected were washed in distilled water, taxonomically identified [We collected benthic macroinvertebrates assemblages through use of D-frame kick-nets , following a systematic zig-zag pattern along the segments defined , coverinentified ,45,46 anentified –49. ShreAfter identifying and classifying in functional feeding groups, organisms were then oven-dried at 60° for 48 h, ground to a fine and homogeneous powder using a mortar and pestle and then stored in Eppendorf tubes for subsequent analysis of their isotopic compositions. Collector, filter-feeder, predator and scraper consumers were found at sites in all three types of riparian environments, whereas shrimp-shredders were not found in the sugar cane sites and insect-shredders were not found in natural cover sites. Each FFG sample was composed of only one specific family and different numbers of invertebrates were used for each sample to reach a minimal amount of material for isotope analysis .μm). Filamentous algae was collected manually in each segment, stored in plastic containers in ice coolers and then frozen. The FPOM samples were collected from sediment deposits revolving the sediment and passing a phytoplankton net (0.45 mm) in the material in suspension. After the material was stored in plastic containers and then frozen. Pasture leaves, sugar cane leaves, and leaves of the natural riparian vegetation were manually collected along the segments delimited in each sampled stream, with the most common species being prioritized at the site. Species priorization was made in compliance with the most common and abundant species in each segment. Five leaves were then collected from each of the five most common plants. We obtained samples of native riparian vegetation even at sugar cane and pasture sites. The CPOM was randomly collected from leaf litter deposits in the streams. All leaves were then stored in paper bags and kept in plant presses until processing in the laboratory. In the laboratory, all resource samples were dried in an oven at 60°C for 48 h and then ground with a mortar and pestle and stored in Eppendorf tubes. Approximately 2–5 mg of dried animal tissue and 5–10 mg of resources were used for the isotopic analysis.Sampling of resources was carried out in parallel to macroinvertebrates collecting along the segments defined in each site. Periphyton was collected by scraping rocks with a brush (three rocks per segment) and placing the material in a plastic container with distilled water . Seston δ13C and δ15N values. Analyses of isotopic ratios were processed through sample combustion under a continuous flow of ultrapure helium in an elemental analyzer  that was coupled to a Thermo Finnigan Delta Plus mass spectrometer for isotopic ratios. The results were expressed in delta notation (δ), in parts per thousand (‰), relative to standard international references (V-PDB–Vienna Pee Dee Belemnite for C and atmospheric air for N), and were calculated using the following equation:13C or 15N and R represents the isotopic ratios 13C/12C or 15N/14N [δ13C and δ15N were ±0.10 and ±0.11‰, respectively.All samples were sent to the Laboratory of Isotope Ecology of the Center for Nuclear Energy in Agriculture (Centro de Energia Nuclear na Agricultura—CENA), University of São Paulo (Universidade de São Paulo), Piracicaba, Brazil, for determination of the  15N/14N . The anaδ13C values of samples of the riparian vegetation and CPOM deposited in the streams were very similar among the environments; therefore, the values of the riparian vegetation samples were excluded from the analyses. The FPOM and the seston samples also had similar δ13C values and were grouped; hereafter, those groups are called fine particulate organic matter (FPOM).When using stable isotope data to reconstruct animal diets, the resources must have isotopically distinct signatures to ensure a sensitive interpretation of the results. If they are not significantly different and are somehow logically related , the resources may be combined and represented in the mixing model by a single set of isotopic values . The δ13We used the SIAR package for the analysis of stable isotopes ,52 in R δ15N and δ13C values. The fractionation values used were 0.5 ± 0.13‰ for C and 2.3 ± 0.18‰ for N [In the partition analysis, the food resources of each land use category were considered separately to determine the contribution of each resource for the consumers. The mean value of the food resources in all categories was used to visually represent the spatial distribution of the taxa according to their 8‰ for N . The tro8‰ for N . Those m8‰ for N . However8‰ for N  is that 8‰ for N .δ13C range (CRb) and δ15N range (NRb), which together indicate the variety of resources exploited by the assemblage. 2) The mean distance to centroid (CDb), which is the mean Euclidian distance of each assemblage component to the centroid, indicating the trophic diversity within the food chain. 3) The mean nearest neighbor distance (MNNDb), which is the mean Euclidean distance from each group to its nearest neighbor in the δ13C-δ15N bi-plot space (plotted based on their mean stable isotope signatures), an estimate of the total density and clustering within the assemblage. Low MNND values indicate an increase in trophic redundancy, i.e., the occurrence of many groups with similar trophic levels. 4) The standard deviation of the nearest neighbor distance (SDNND), which measures the uniformity of the groups in bi-plot space, where lower SDNND values suggest a more uniform trophic niche distribution [c, in ‰2) as a bivariate measure of the central mean of the isotopic niche [c enables calculating the degree of niche overlap of the assemblage  and may be used as a quantitative measure of diet similarity among the different groups [b”) to compare groups with different sample sizes. A small sample size correction (indicated by the subscript letter “c”) was applied to increase the accuracy of the comparisons, enabling the comparison of niches of groups with different sample sizes [A Bayesian approach recently developed for the aforementioned metrics enables the distribution of the sampling errors of the means estimated for the members of the assemblage. Using that approach, we generated a posterior distribution of the estimates of those metrics, providing a measure of uncertainty and allowing statistical comparisons among assemblages ,55. Thusribution . We calcribution  and refot groups . All meaδ13C values, whereas CPOM had the lowest values. The pasture and sugar cane plants exhibited the highest δ13C values, whereas CPOM and algae had the lowest δ13C values  based on the isotope ratio of trophic groups of macroinvertebrates differed in size, shape and position in the δ13C vs δ15N bi-plot space , followed by scrapers (3.29‰2), predators (3.01‰2), collectors (2.26‰2) and filter-feeders (1.42‰2).Sites with natural riparian vegetation cover exhibited very little overlap of isotopic niches  of the macroinvertebrate feeding groups . Small oc values were observed for the insect-shredders (19.2‰2), followed by the collectors (9.67‰2), predators (9.04‰2), shrimp-shredders (8.89‰2), filter-feeders (8.8‰2) and scrapers (6.9‰2).Considerable niche overlapping was observed among feeding groups in pasture sites , especiac (10.19‰2), followed by predators (8.23‰2), scrapers (4.96‰2) and filter-feeders (3.71‰2). Calculation of the SEAc for insect-shredders in sugar cane sites was not possible because we obtained only three samples of this group.The greatest niche overlaps among groups were observed in sugar cane sites . In thosb and CRb), trophic diversity (CDb), and trophic redundancy (MNNDb) and showed significantly lower group uniformity (SDNNDb) (b (The trophic niche metrics of the macroinvertebrate communities varied among land uses. Pasture sites had significantly greater ranges of resources exploited (NR(SDNNDb) . In contNNDb)  identification of the main resources consumed by benthic macroinvertebrates and (2) assessment of how the different land uses affected resource availability and trophic dynamics in tropical streams. Our hypotheses were corroborated because we observed macroinvertebrate assemblages with wider trophic niches and greater niche overlap in altered sites and greater resource specialization in sites with natural vegetation.δ15N values in sugar cane and pasture sites. Nutrients from agriculture and cattle strongly affect the waterbodies and may have been responsible for the high δ15N values found in those streams. On sugar cane plantations, vinasse, a byproduct of ethanol distillation, is the main fertilizer used, whereas chemical fertilizers and livestock manure are the main sources of residues and nutrients in pastures [15N enrichment [δ15N ratios [δ15N available in food resources and consumers [All trophic groups and virtually all resources evaluated had higher pastures . Fertilirichment . AgriculN ratios , and mosonsumers ,60.a priori, macroinvertebrates had more generalist feeding habits at pasture and sugar cane sites, whereas more specialization occurred in macroinvertebrate assemblages of sites with natural riparian vegetation. Benstead & Pringle [Although we categorized the groups  Pringle  reportedWe found some unexpected results in our study; for example, scrapers assimilated more CPOM than periphyton, a result found in pasture and natural vegetation sites. According to Marchese et al. , who obs13C in pasture and sugar cane sites, its contribution to the trophic chain was very low. The C4 plants  are considered to have low nutritional quality compared with C3 plants and are little used by aquatic consumers in many cases, either because of their physical or chemical characteristics that reduce consumption or because consumers are able to select other higher-quality resources [In contrast with observations made for some fish species in the same region  and in oesources . Althougesources  and Martesources  also fouδ13C and δ15N ranges. This wider range may result from reduced riparian canopy cover and, consequently, higher light input, promoting higher diversity and abundance of algae and periphyton species. Similar results have been reported by Turner & Edwards [δ13C and δ15N values. They argued that this variation might result from greater taxonomic diversity of the producers, which could lead to greater diversity in C and N metabolism.Pasture sites had autochthonous resources  with the widest  Edwards , where tConsumers from streams with natural riparian vegetation had the narrowest isotopic niches and the lowest niche overlaps. This pattern indicates that the macroinvertebrate functional groups in those sites had more selective feeding habits with a lower overlap of trophic niches and, consequently, less competition for resources. In their natural state, wooded riparian zones are effective in preserving the ecological integrity and trophic dynamics of aquatic ecosystems. Land use changes and the consequent shifts in the input of allochthonous nutrients and autochthonous production can reduce the balance between functional feeding groups  and, widδ13C and δ15N values of multiple individuals for each group in a trophic web, and such intraspecific variation was not considered in this analysis (i.e. species within the same FFG) [b and dCRb values observed in the pasture sites indicate that macroinvertebrates use a wider range of the available food sources in this environment, especially in contrast with the sugar cane sites. This result is consistent with the SEAc values, which were highest in pasture sites. The highest mean CDb value was also observed in pasture sites, indicating higher trophic diversity at those sites. The highest mean MNNDb values were observed for pasture sites, indicating low trophic redundancy. However, the highest SDNNDb values were also observed in the pasture sites, suggesting the existence of a less uniform trophic niche distribution, despite the low trophic redundancy. This is most likely a result of the greater availability of periphyton and algae in this environment, which are resources with wider δ13C ranges and which are used by many consumers. According to Layman et al. [b and SDNNDb increase because consumers have more distinct trophic positions (greater distance among consumers in isotopic space).The isotopic metrics calculated for the assemblages described in this study were based on the mean ame FFG) . The hign et al. , MNNDb aMacrobrachium amazonicus, considered a shrimp-shredder in this study, which is an alien species in the region found in pasture and in natural cover sites. The isotopic niche (SEAc) of shrimp-shredders was one of the highest compared with the other functional groups in those land uses. However, the niche of shrimp-shredders did not overlap with the others, suggesting that this group is exploiting resources that would otherwise not be fully exploited by the native fauna. In addition, the shrimp-shredders were the group least preyed upon, with virtually no contribution to predators. Recently in a global evaluation of the consequences of non-native species on the isotopic structure of freshwater fish communities, Sagouis et al. [Another noteworthy result is the occurrence of the crustacean, s et al.  found ths et al. ,69,70), We conclude that land use changes, such as sugar cane culture and livestock pasturing, may lead to benthic macroinvertebrate assemblages with more generalist feeding behaviors and higher trophic niche overlap. In addition, our results reinforce the idea that stable isotope analysis is a relevant tool for biomonitoring and evaluating the effects of land use changes on the dynamics and functioning of tropical streams.S1 TableThe numbers 1, 2 and 3 correspond to each of the three streams sampled in each land use category See . Order =(DOCX)Click here for additional data file.S2 Tablea and b indicate which signatures are different according to post hoc test. The letter “n” indicates the number of replicates used in each group analysis.The letters (DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


The present study uses electron microscopy to understand and quantify in vitro alterations in the ultrastructural composition of microtubules and neurofilaments within neuronal axons of rats following focal compression. Standard transmission electron microscopy processing methods are used to identify microtubules, while neurofilament identification is performed using antibody labeling through gold nanoparticles. The number, density, and spacing of microtubules and neurofilaments are quantified for specimens in sham Control and Crushed groups with fixation at <1min following load. Our results indicate that the axon caliber dependency known to exist for microtubule and neurofilament metrics extends to axons undergoing TAI, with the exception of neurofilament spacing, which appears to remain constant across all Crushed axon diameters. Confidence interval comparisons between Control and Crushed cytoskeletal measures suggests early changes in the neurofilament spatial distributions within axons undergoing TAI may precede microtubule changes in response to applied loads. This may serve as a trigger for further secondary damage to the axon, representing a key insight into the temporal aspects of cytoskeletal degeneration at the component level, and suggests the rapid removal of neurofilament sidearms as one possible mechanism.Although a number of cytoskeletal derangements have been described in the setting of traumatic axonal injury (TAI), little is known of early structural changes that may serve to initiate a cascade of further axonal degeneration. Recent work by the authors has examined conformational changes in cytoskeletal constituents of neuronal axons undergoing traumatic axonal injury (TAI) following focal compression through confocal imaging data taken  Traumatic brain injury (TBI) and spinal cord injury (SCI) affect over 1.7 million persons in the US annually and results in massive direct and indirect medical costs (over $77 billion in 2010) . TBI andThe cytoskeleton is an elaborate network of proteins, having a variety of configurations, whose purpose is to provide functional and structural stability to the cell. Major constituents of the cytoskeleton in CNS axons include neurofilaments (NFs) and microtubules (MTs). Neurofilaments provide mechanical strength, mechanical stability, and determine axon diameter, whereas microtubule functions include intracellular transport and provision of structural rigidity –28. NeurIn vitro approaches to understanding axonal injury include models based on transection, shear, compression, hydrostatic pressure, hydrodynamic changes, acceleration, and cell stretch [in vitro models, researchers are able to study how the mechanical insult applied can influence the acute mechanisms of axonal injury, where the aim is to mimic the structural consequences of the injury rather than replicating the mechanical loading conditions observed at larger length scales. Pettus and Povlishock qualitatively provide evidence of microtubule disruption and neurofilament compaction within 5min of fluid percussive injury using electron microscopy [ stretch . By desicroscopy . This incroscopy , 30, 31.croscopy , 16, 21.The complex character of fiber families, fiber orientations, and fiber tract distributions in the brain ensure that macroscopic insults to the head result in the full gamut of deformation states at the axonal scale, including local tension, focal compression, kinking, and shear. Although the response to tension has been well studied, the consequences of focal axon compression are poorly understood and this state is considered in this paper.Our lab has recently demonstrated visualization and quantification of three-dimensional (3D) live subcellular populations under controlled focal compression using a confocal microscope at high magnification, where transmission electron microscopy (TEM) of fixed samples was utilized to corroborate the observed confocal changes with greater spatial resolution . Our resA previously designed axon injury micro-compression (AIM) platform is utilized for this study . The plaThe flow layer master template, where the neurons and media reside, is made from three aligned and stacked layers of silicon wafers  processed with 3005 and 3025 SU-8 . Once the master template is developed, the mold is spin-coated with a thick layer of PDMS pre-polymer and fully cured at 80°C for 20mins.The control layer master template, for controlling the injury pad, is patterned using a silicon wafer and 3050 SU-8  and is developed with a thick layer of PDMS. Once cured, individual devices are cut, and access ports for control fluidics are punched using sharpened gauge #23 needles .PDMS castings from both template layers are exposed to an oxygen plasma cleaner  and surface treated . Injury pad control layers are visually aligned with the flow layer features, brought into intimate contact, and baked overnight at 80°C to amalgamate PDMS layers. Composite devices are removed and access ports are created using a 3mm biopsy punch tool . Devices are sterilized by ethanol sonication, autoclaved, and sealed to a cleaned 50mm #1 glass-bottom petri dish  prior to use.2 incubator. The following day, devices are washed 3x with double deionized water (ddH2O) to remove unbound PDL and neurobasal media  is added for cells to be seeded.A 100μg/mL solution of Poly-D-Lysine , an extra cellular matrix that facilitates axonal growth diluted in molecular grade water , is introduced by way of access ports in the platform. The AIM devices are incubated overnight at 37°C in a humidified, 5% CO6 cells/mL in a mixture of neurobasal media , penicillin-streptomycin , B27 , hepes , and L-Glutamine . After a period of 6–8 days in culture, axons can be observed extending into the middle and distal chambers of the AIM device in sparse numbers to allow tracking of individual processes for subsequent experiments. Media composed of neurobasal media , penicillin-streptomycin , B27 , and hepes  is added every 3 to 4 days to maintain neuronal viability.Primary hippocampal neurons are isolated from E17 Sprague Dawley rat pups  following the specific regulations approved by the Johns Hopkins Institutional Animal Care and Use Committee (protocol RA12M213). Dissociated neurons are loaded into the somal compartment at a density of 25x102) pressure is manipulated with a Proportion Air electronic regulator  to apply the desired level of pressure between the glass substrate and injury pad.As reported in previous studies, dynamic compression is applied exclusively to axons of primary hippocampal neurons . Four inUsing a validated finite element model, geometric parameters for each platform provide the input fluidic pressure required to obtain the injury response observed in TAI . PressurDifferent approaches are used for preparing microtubule and neurofilament TEM specimens. The choice of approach is made by identifying what each group requires in terms of morphology and labeling. In our study microtubules are relatively easy to identify and quantify, so morphology is of paramount concern. Neurofilaments, by comparison, are harder to identify and require immuno electron microscopy with gold (Au) labeling using 2°NF-medium antibodies to confirm presence within processed TEM sections. Previous research regarding immunogold labeling using antibody specific binding has been shown to be effective in detecting the presence of a specified molecule of interest –35.One inherent limitation of using immunogold labeling is that the observed Au-nanoparticle is approximately 15–30nm from the primary binding site of the antibody . However2), and 3% sucrose buffer in three 10min rinses and stored in 1% osmium plus 0.8% potassium ferricyanide on ice and in the dark for 1hr, followed with three 0.1M maleate buffer rinses, 5min each. Cells are stained with 2% aqueous uranyl acetate  in 0.1M maleate buffer. Cells are dehydrated in a graded series of ethanol washes: 30%, 50%, 70%, 90% and 100% with 10min between washes.Microtubule TEM samples are fixed with a mixture of 2% paraformaldehyde, 2.5% glutaraldehyde, 0.1M sodium cacodylate (SC), and 1% sucrose for 1hr. Cells are washed with a 0.1M SC, 3mM calcium chloride  and 0.02% saponin in 0.1M SC buffer (1:100) is added, and cells are incubated overnight at 4°C. Cells are incubated back to room temperature and washed with 0.1M SC buffer six times in 10min rinses. A secondary antibody mixture of 6nm Au particles  and 0.01% saponin in 0.1M SC buffer (1:200) is added, and the cells are incubated at 4°C for 4hrs. Cells are incubated back to room temperature for 10min, rinsed with 0.1M SC buffer six times with 10min between rinses, and then 1% glutaraldehyde is added in 0.1M SC for 1hr. Cells are rinsed three times with ddH2O, with 10mins between rinses. 0.5% osmium tetraoxide in 0.1M SC buffer is added and cells are placed on ice and in the dark for 30min. Cells are rinsed with ddH2O 3 times, with 10mins between rinses, followed by dehydration in a graded series of ethanol washes: 30%, 50%, 70%, 90% and 100% with 10min between washes.Neurofilament TEM samples are fixed with a mixture of 6% paraformaldehyde, 0.5M SC, and 1.0M CaCl2, and embedded specimens are retrieved for post-embedding processing.Following the graded dehydration by ethanol, cells for both microtubule and neurofilament samples are embedded in a resin of Epon using DMP-30 as a catalyst and incubated overnight at room temperature. Cells go through a series of Epon + DMP-30 changes and are subjected to vacuum in an attempt to thoroughly embed cells in the resin. Cells are incubated overnight at 65°C. Cells are separated from the dishes through a process of thermal shocking, by placing samples into liquid N2O for one minute. Grids are then stained with aqueous 2% uranyl acetate  for 20min and rinsed with ddH2O. Finally grids are stained for 1min on 0.04% lead citrate (aqueous-filtered) and rinsed with ddH2O. Longitudinal and transverse TEM slices (60–80nm thickness) are obtained and serial sections are taken to address concerns of embedding and processing 3D embedded substructures [Samples are cut down to specified grids and go through a triple staining process with 1% tannic acid (aqueous) , filtered using 0.22μm filter, for 10min by flotation using formvar coated slot grids  before being rinsed with ddHructures .Our TEM measurements for substructure quantification utilize a series of longitudinal sections taken at the same magnification  and examine the entire segment under the compression pad . PreviouQuantification of TEM images requires metrics for assessing quantity, spacing, and density. Previous researchers indicate a relationship between the axon diameter and cytoskeletal measures within axons , 16. TheFor both microtubule and neurofilament groups, 15–20 axons were examined for each Control and Crushed axon subgroup. A minimum of 10 TEM images were required to construct a single axon spanning the area of the compression pad and 3 measurements of number, density, and spacing were taken from each image. The combined data resulted in 450–600 measurements for Control and Crushed axons of microtubules and neurofilaments groups.L) for a given TEM image is measured and then divided into quarters. Axon diameter (D) and the number of MTs (NMT) are measured at L/4, L/2, and 3L/4. The microtubule linear density across the diameter of the axon  at a given D is then related to the linear density and corrects for the non-zero thickness (t) of the microtubules:The average spacing for microtubules (NNF) and the area of the axon (A) using a code written in MATLAB :In contrast to the microtubule measures that can use linear metrics, neurofilament measurements depend on the quantification of the Au-nanoparticles over a specified area. The neurofilament measurements are made by dividing the axonal length into thirds for a given TEM image and measuring the diameter at the midpoint of each of these thirds. For each of the thirds the areal density (SNF) is computed from the average distance between a given Au-nanoparticle and its nearest Au-nanoparticle neighbor. The spacing is measured for all Au-nanoparticles in a given sample and assumes a proportional binding relationship where the observed Au-nanoparticles requires the presence of the NF-medium binding sites and infers the existence of NFs.The average spacing of NF (The collected data is divided into four groups: (1) microtubule Control cells, (2) neurofilament Control cells, (3) microtubule Crushed cells, (4) neurofilament Crushed cells. The data is further separated into bins covering the ranges of axon diameters used: <0.5μm, 0.5–1.0μm, 1.0–1.5μm, and 1.5–2.0μm to evaluate the relationship between axon caliber and cytoskeletal measures. Bonferroni’s multiple comparison tests are used to determine the significance (p < 0.05) between Control and Crushed groups, for a specific axon diameter bin.Microtubules are identifiable as long rod-like structures ~24nm in diameter within the axon . For theNMT), linear density (SMT) microtubules are the quantitative measures taken from axons fixed <1min after loading and compared for Control and Crushed groups . The number   . The ρLMer axons . The SMied load . A commoThe mean number of  are identified within treated axons . In thesNNF), areal density (SNF) neurofilaments (identified by Au-nanoparticles) are the quantitative measures taken and compared for Control and Crushed neurofilament groups . Our results, in terms of raw data, are shown in NNF) increases with axon diameter in the Control group and agrees with observations of the Control axon parameters in the literature s of TAI  [13, 15,SNF appears higher for Crushed groups, there is no statistically significant difference between axons of comparable axon diameters (SNF does not significantly change as axon diameter increases (unlike for Control) (The (p<0.05) . While Siameters . It is iControl) . As expeThe mean number of  with relevant applied loading rates and loads, we find appreciable changes in cytoskeletal spatial distributions within neuronal axons fixed less than 1min after compressive loading. These changes include decreases in the number and density of microtubules and neurofilaments, as well as increases in their spacing, following loading. Our findings suggest that early changes in the neurofilament structure, through sidearm removal or conformational shape change, may serve as a trigger for further secondary damage to the axon, representing a key insight into the temporal aspects of cytoskeletal degeneration at the component level. Another unique result from our study comes from examining the dependency microtubule and neurofilament measures have on axon caliber. This dependence continues to exist immediately following load for most measures (the exception is neurofilament spacing) though the slope of these measures is modified in all cases. Finally the raw data in our study provides evidence that classical approaches of binning axon caliber linked cytoskeletal measures might mask mathematical methods of understanding cytoskeletal distributions.in vitro and in situ using the same testing platform [Data from this study provides measurable insights into cytoskeletal changes <1min after loading. Previous literature has focused on quantifying the spatial distributions of microtubules and neurofilaments as early as 15min after loading. Our data indicates metrics for neurofilament number and density are changing at higher rates than microtubules for the same time period. The literature, which focuses on later time points following injury, provides convincing evidence that microtubules are disrupted; however, little is known of early changes in structural components , 21. Ourplatform . In thatplatform . Thus thNMT and NNF on axon caliber. Additionally we observe a larger increase in NNF than NMT for increases in axon diameter of Controls, a trend supported by previous researchers [SMT has been linked with increasing axon caliber by previous researchers for Controls and is reflected in our data set [SNF and decreases in One goal of this study is to find measurable changes in cytoskeletal distribution and to link these quantifiable metrics to TAI. Previous researchers have indicated the number, spacing, and density of microtubules and neurofilaments in control axons to be measures with a strong dependency on axon caliber , 16, 21.data set , 16, 21.ol axons , 16, 21.Following loading, Crushed axons exhibited significant changes in the measures used, though the axon caliber dependency persisted for most. Our results for Crushed microtubules were supported by previous researchers, but were found to be in contrast for Crushed neurofilaments , 16, 21.One explanation for the observed changes in cytoskeletal components is the constituents are displaced into a plane above or below the TEM sections examined. To address this, serial sections were examined and transverse sections were taken. Previous researchers have noted the heterogeneous distribution of cytoskeletal components observed in transverse sections of TEM axon samples , 38. GivThe observed drop in the number of microtubules at larger axon calibers for Crushed axons could indicate the axon caliber is increased by the applied load and artificially increases the axon caliber for the associated cytoskeletal metrics. This limitation is supported by the lack of Controls for neurofilament at the axon caliber of 1.5–2.0μm, yet a Crushed population is observed for the same caliber.SNF and why the established relationship with axon caliber appears nullified in Crushed axons. This spacing is mediated by phosphorylation of carboxy-terminals of NF-medium and NF-heavy sidearms where, increases in the number of neurofilaments and carboxy-terminal phosphorylation increase axon caliber [An interesting question that arises from the spatial data is the mechanism governing  caliber . Previou caliber , 41. The caliber , 41, 42. caliber . However caliber .We posit that local modifications in conformation where NF-medium antibodies label are no longer accessible, due to sidearm removal, result in a decrease of observed Au-nanoparticles. Within the Crushed axon, local modifications to neurofilament sidearms result in a spacing mechanism where a baseline level of approximately 70nm exists regardless of axon caliber. Remarkably the 70nm value we observe for Crushed axon neurofilament spacing has been observed by researchers at later time periods following stretch loading , 21.Further research is needed to examine the early biochemical pathways initiated by neurofilament disruption, and how these disruptions affect microtubule functions over short time durations following loading (<15min). The mechanics of the applied load likely influence the whole cell response resulting TAI. Strain and strain rate, for example, have been implicated by several research groups for governing cytoskeletal responses to applied load , 45. An in situ and in vitro and may be critical in understanding mechanical failure and degeneration of the cytoskeletal system for neuronal axons undergoing TAI.The present study suggests changes in cytoskeletal spatial distribution can be captured within 1min of loading and the quantification of these distributions can provide insight regarding the temporal evolution of neuronal axons undergoing TAI. The raw data indicates a wealth of information regarding mathematical approaches to understanding cytoskeletal distributions is potentially being ignored by the classical approaches of binning data and insights might be developed by approaching the data in a more open format. The raw data results demonstrate that measurable changes in neurofilament distribution occur ahead of distinguishable changes in microtubule distribution. The results indicate microtubule and neurofilament spatial distribution dependencies on axon caliber continue to occur following applied loads inducing TAI, with the exception of neurofilament spacing. These metrics provide a pathway for connecting changes in cytoskeletal spatial distributions to previously observed changes in measured intensity using confocal microscopy with the same loading platform S1 Arrive Checklist(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Salvia officinalis (sage) has been used as menstrual cycle regulator. In the present study the effects of sage extract on breast tissue were examined. In traditional medicine Saliva officinalis hydroalcoholic extract (SHE)-treated rats that were gavaged with 30mg/kg/body weight of sage extract for 30 days. The estrus cycle changes were monitored by daily examination of vaginal smear. Whole mounts of right pelvic breast were spread on the slide and stained by carmine. The number of alveolar buds (ABs) type 1 and 2 and lobules of mammary gland were scored. Tissue sections of left pelvic mammary gland were prepared and its histomorphometrical changes were measured. Blood samples were taken from dorsal aorta and estradiol and progesterone concentrations were measured using radioimmunoassay. Fourteen female rats were divided into two groups: 1) Distilled water-treated rats (Con) that were gavaged with 1ml distilled water and 2) Estrous cycles decreased significantly in SHE-treated animals. The number of alveolar buds and lobules in mammary gland whole mount of SHE-treated group were higher than the Con group. The number and diameter of ducts in histological section of mammary gland in SHE-treated group increased as compared to the Con group. Sage promotes alveologenesis of mammary glands and it can be used as a lactiferous herb. A mammary gland is an apocrine gland that consists of alveoli lined with simple cuboidal epithelium surrounded by myoepithelial cells. These alveoli join to form groups known as lobules. Each lobule has a lactiferous duct that drains into openings in the nipple. Maintenance of the morphology of the lactiferous duct tree requires extracellular matrix (ECM) such as adipocytes, fibroblast, inflammatory cells, etc. that constitute mammary stroma  region, two pairs in intermediate  region and one pair in posterior  region of the body. Male rats do not have nipples.Salvia officinalis (sage), a plant belonging to the Lamiaceae family, chiefly contains monoterpenes such as α and β-thujone, α and β-pinene and camphor. In addition, Salvia officinalis ) S. officinalis leaves were obtained from Zardband Pharmaceuticals Company, Tehran, Iran. Sage leaves were powdered, and 100 g of powder was percolated with 800 ml of 70% ethanol for three days to obtain hydroalcoholic extracts. Subsequently, the mixtures were filtered and concentrated under reduced pressure using a rotary evaporator and vacuumed desiccator. The yield (w/w) of extraction method was 17% (g/g). Animal groupingFemale Wistar rats weighing between 150 to 200 g were obtained from Animal House of Razi Institute, Shiraz, Iran. The animals were adapted to the laboratory conditions for two weeks prior to the experiments. The rats were kept at a controlled temperature (22-24°C) and a 12:12 h light-dark cycle  with free access to food and tap water. Animal experiments were approved by the Institutional Animal Ethics and Health Committee of the Biology Department of Shiraz University, and were performed according to the principles of the care and use of laboratory animals established by the National Institute of Health. Vaginal smears were examined daily and normal estrous cycle female rats were selected. Estrous cycle lasted 4-5 days and was divided into threephases of proestrus (9-16 h), estrus (9-16 h) and diestrus (60-70 h) . For this purpose, serum samples were transferred to Department of Hormonal assay, Research Center of Namazi Hospital, Shiraz, Iran.Mammary gland whole mount preparationAt the end of 30-day experiments, rats from each group were dissected and the right mammary gland of pelvic region, free from skin, were removed from area close to the nipple towards the distal end of the gland. It was spread immediately onto a labeled glass slide, fixed with Carnoy's fixative  at room temperature (RT) for two days. Slides were washed with 70% ethanol for 1 h and distilled water for 30 min. Then, slides were stained with carmine alum and left for two days. Excess dye was washed using increasing concentrations of ethanol   and cleared with xylene and left at RT at least for two days. Mammary gland whole mounts were examined under light microscope  and the  Histomorphometrical studiesBancroft and Steven, 1991) from which 6 µm-thick sections were stained with haematoxylin-eosin. Histomorphometric studies examined 5 microscopic fields for each section . The number of ducts, lobules, branch of ducts and duct with deposits were recorded. Then, ductal and its epithelial and stromal diameters were measured using an ocular micrometer . Left pelvic mammary glands of rats were removed and fixed in 10% buffered formalin solution for one week. Then, paraffin blocks were prepared . Carnosic acid and rosmarinic acid are present at high concentrations in the extract of sage plants  of mammary glands of SHE-treated rats increased significantly as compared to the Con group. It may be due to either estrogenic activity of sage extract on stromal cells or anti-allergic response of stromal cells against antigenic activity of extract components. Mast cells increase in stromal microenvironment of mammary gland at puberty and breast morphogenesis when its alveolar buds are proliferated and its ducts are branched  on glandular secretion of breast in pre-and post-menopausal women have been reported (Petrakis et al., 1996S. officinalis hydroalcoholic extract, just similar to steroids hormones stimulated the growth of mammary gland alveolar buds and ducts and induced lobule formation in adult female rats. This medicinal herb showed no side effects on body weight, estrous cycle duration and sex hormones concentrations. Therefore, it can be considered as a lactiferous herb.


Preeclampsia, a hypertensive multisystem disease that complicates 5–8% of all pregnancy, is a major cause for maternal and fetal mortality and morbidity. The disease is associated with increased spontaneous and evoked preterm birth and remote cardio-renal disorders in the mother and offspring. Thus the ability to predict the disease should lead to earlier care and decreased morbidity. This has led to fervent attempts to identify early predictive biomarkers and research endeavors that have expanded as we learn more regarding possible causes of the disease. As preeclampsia is associated with specific renal pathology including podocyte injury, early urinary podocyte (podocyturia), or the podocyte specific proteinuria nephrin in the urine (nephrinuria), as well as the more easily measured urinary albumin , have all been suggested as predictive markers. We performed a prospective study recruiting 91 pregnant women (78 of whom were high risk) and studied the predictive ability of these three urinary biomarkers. The subjects were recruited between 15–38 weeks of gestation. Fourteen patients, all in the high-risk obstetric group, developed preeclampsia. The levels of podocyturia, nephrinuria, and albuminuria were variably higher in the high-risk pregnant patients who developed preeclampsia. The sensitivities and specificities for podocyturia were 70% and 43%, for albuminuria were 36% and 96%, and for nephrinuria were 57% and 58%, respectively. Also, abnormal nephrinuria (69%) and podocyturia (38%) were detected in low risk women who had uncomplicated gestations; none of these women exhibited albuminuria. In our study, none of the three urinary markers achieved the minimum predictive values required for clinical testing. The lack of excessive albuminuria, however, may indicate a preeclampsia-free gestation. Given a discrepant literature, further studies with larger sample size should be considered. Preeclampsia (PE) is a hypertensive complication of pregnancy and a major cause of maternal and fetal morbidity and mortality worldwide. The disease involves many organs and its life-threatening events include cerebral hemorrhage, hepatic failure, pulmonary edema, disseminated intravascular coagulation accounting to 10–15 percent of maternal deaths worldwide nd trimester to be 100% sensitive and specific in distinguishing subsequent development of PE from normotensive pregnancies, gestational hypertension, or from both groups combined Interest in using podocytes as a predictive and diagnostic marker for PE stems from the observations that podocyte-specific protein expression is reduced in renal biopsies of patients with PE We recruited both women with apparently uncomplicated gestation (“low risk”) and others at risk for pregnancy complications (“high-risk” group as described below) from the obstetric outpatient service at Jacobi Medical Center, Bronx, NY, USA. Random urine samples were obtained from the subjects in the second (weeks 15 to 26) or third trimesters (weeks 27 to 38) of gestation. Inclusion criteria for high-risk group were PE in a previous pregnancy, chronic hypertension (HTN), Type I and Type II diabetes mellitus (DM), gestational DM, chronic kidney disease (CKD), obesity (BMI>30), and systemic lupus erythematosis (SLE), all 18 years or older. Chronic HTN was defined as preexisting HTN or blood pressure of 140/90 mmHg before 20 weeks of gestation. Gestational DM was defined as any degree of glucose intolerance with onset of first recognition during pregnancy. All patients were managed until delivery and studied for the development of PE. Diagnosis of PE fulfilled the criteria of new onset of HTN with blood pressure of 140/90 mmHg after 20 weeks of gestation and proteinuria of >300 mg of protein in a 24-hour urine specimen or 1+ protein on a urinalysis sample without evidence of another cause. For superimposed PE in patients with chronic HTN or CKD, the diagnosis was made if there was a rise in blood pressure (BP) from baseline (30 mmHg systolic blood pressure or 15 mmHg diastolic BP) or if there was a 2–3 fold increase in proteinuria. This study was approved by the Internal Review Board of Albert Einstein College of Medicine.A single voided urine specimen (20 mL) was obtained immediately following recruitment. Its concentrations of albumin-to-creatinine ratio  were measured by clinical laboratories at Jacobi Medical Center, Bronx, NY, USA. And elevated level of alb/Cr was defined as ≥30 mg/g. An aliquot of the urine was frozen at −80°C for urine nephrin measurement. Urinary nephrin was determined by a competitive enzyme-linked immunosorbent assay using polyclonal antibodies against the extracellular domain (amino acids 23–322) of human nephrin . The assay was performed by Exocell Inc. according to manufacturer’s instructions. Briefly, urine samples were diluted in the range of 1∶10 to 1∶500 (depending on degree of proteinuria in the sample). A 50-µl diluted sample was added to each well already coated with rat nephrin followed by the addition of a 50-µl rabbit anti-nephrin antibody for an incubation of 60 minutes at room temperature. Plates were washed followed by the incubation with 100-µl of anti-rabbit HRP conjugate to each well for 60 minutes. After plates were washed and color developed, absorbance was read at 450 nm. An elevated level of urinary nephrin (nephrinuria) was defined as urine nephrin-to-creatinine ratio (nephrin/Cr) (mg/g) ≥0.1 mg/g. This value was based on 10 healthy controls who consistently exhibited nephrin/Cr <0.1 mg/g.g for 5 min. These pellets were carefully recovered by aspirating the supernatant, washed twice with PBS and resuspended in 1 mL of PBS. Aliquots of 100 µl of the resuspended sediment were centrifuged onto slides using the Shandon Cytospin 4 Cytocentrifuge , air-dried and fixed with 1∶1 acetone/methanol for 10 minutes. The slides were immersed with PBS/1% H202 for 15 minutes and washed with deionized water and blocked with 10% donkey serum in PBS and 2% BSA for 30 minutes. Slides were incubated overnight with monoclonal mouse anti-human synaptopodin antibody at 1∶1 dilution and rabbit polyclonal podocin antibody at 1∶500 dilution . This was followed by incubation with FITC-conjugated anti-rabbit and TRITC-conjugated anti-mouse secondary antibodies at 1∶500 dilution  for 30 minutes. The nuclei were counterstained with DAPI . Slides were then washed and mounted with Vectashield mounting medium . Normal kidney tissue biopsy specimen was stained in a similar manner to obtain positive control, while negative controls were carried out by incubation in the absence of the primary antibody. Staining was evaluated by using immunofluorescent microscopy (Zeiss Axioplane2 IE microscope) and the images were obtained using the Zeiss AxioCAm MRm camera (200X magnification). Images were analyzed by two investigators and podocytes were identified by co-localization of podocin and synaptopodin; these counts were expressed as number of podocytes per mg of creatinine (#pod/mg) (pod/Cr).Pellets containing cellular material were retrieved from an aliquot of the patient’s urine that had been centrifuged at 700 The protocol titled “Serum and Urine Biomarkers for Preeclampsia” which was approved by the Institutional Review Board of the Albert Einstein College of Medicine of Yeshiva University. This included a consent form signed by each volunteer.GraphPad Prism version 5.02  and SPSS (version 20) were used for statistical analysis and for the graphs. Mann-Whitney non-parametric tests were performed to assess the differences between two given groups. Spearman correlations were calculated to assess trends for continues variables. Data were expressed as median (interquartile range) or % as appropriate. A two-tailed P value of <0.05 was considered to be statistically significant.nd trimester, and 40 during the 3rd trimester, the distribution among women with low risk pregnancies were 7 recruited during the second and 6 in the third trimester respectively; each pregnancy was evaluated until delivery. The high risk women consisted of 15 women considered to be obese, 12 with Type I or II DM, 24 with gestational DM, 26 chronic hypertensives, 2 with SLE, and 1 patient with CKD. Urine alb/Cr and nephrin/Cr were measured in all volunteers, while only 72 of the 78 high risk patients had a urine pod/Cr determination. Fourteen high-risk women developed PE, while none of the low risk patients did. Among those who later had PE, the urine samples were collected an average of 13.8 weeks before onset of PE in the 2nd trimester, and 6.8 weeks before onset of PE in the 3rd trimester, with an average of 10.0 weeks of both trimesters combined.A total of 78 high risk obstetric patients as well as 13 women with low risk gestations were recruited. Clinical characteristics of patients presented according to the outcome of PE are seen in nd trimester and p = 0.71 for 3rd trimester) (Images of podocyturia are shown in imester) .rd but not 2nd trimester (p = 0.04 vs. p = 0.07 respectively)  . When dictively) .nd nor the 3rd trimester recruited patients showed significant differences between any of the groups (p = 0.15 and p = 0.14 respectively)  . Subgrouctively) .The test characteristics for all three biomarkers are listed in There has been substantial progress in research that focuses on the cause of PE (once dubbed “the disease of theories”). Though the exact causes have yet to be determined, identification of many possibly involved molecules has increased studies focusing both on prediction and verification of diagnosis The contrasting results between our findings and those reported by Craici et al might relate to methodologies and patient populations. We utilized the cytospin technique in which the urine samples are fixed immediately to avoid the errors that occur with cultivation of urinary cells. Growing urinary podocytes in cell culture is frequently limited by bacterial or fungal contamination. Furthermore, cells may proliferate, undergo apoptosis, or not attach to the culture dish, thereby falsely representing the true podocyte count As enticing as it may be to pursue a novel method to predict PE, the methodologic limitations of isolating podocytes in the urine persist. As mentioned above, there are practical concerns associated with isolating and identifying podocytes The strengths of this study are that it is prospective and includes a high risk patient population which may also present with podocyturia, such as DM and SLE Podocyturia, nephrinuria, or albuminuria does not appear to be reliable markers to predict PE, though a “normal” alb/Cr may be useful to help rule out disease. We realize our notation of a relatively low sensitivity and specificity is not conclusive, but given the discrepancy of these observations to studies published prior to them, we suggest further studies are warranted. Currently, some underscore complex pathophysiology of the disease, suggesting different pathomechanisms especially between early and late PE, and suggest it is unlikely that any single test or cell type will be able to predict PE, while others disagree


In a randomised cross-over design, 61 healthy subjects  31 ± 4 kg/m2) alternated either high oleic peanuts (15%–20% of energy) or a nut free diet for 12 weeks. Body composition and mass, waist circumference, C-reactive protein (CRP), lipids, glucose and insulin were assessed at baseline and after each phase. Repeated measures analysis of variance (ANOVA) compared the two diets. Consistent with other nut studies, there were no differences in lipids, CRP, glucose and insulin with peanut consumption. In contrast, some reports have demonstrated benefits, likely due to differences in the study cohort. Energy intake was 10% higher , following peanut consumption vs. control, attributed to a 30% increase in fat intake (p < 0.001), predominantly monounsaturated . Despite greater energy intake during the peanut phase, there were no differences in body composition, and less than predicted increase (0.5 kg) in body weight for this additional energy intake, possibly due to incomplete nutrient absorption and energy utilisation.Epidemiological evidence indicates an inverse association between nut consumption and obesity, inflammation, hyperlipidaemia and glucose intolerance. We investigated effects of high oleic peanut consumption  Nuts are rich sources of bioactive nutrients with potential to deliver metabolic and cardiovascular health benefits . DespiteEvidence suggests that both tree nut and peanut consumption is associated with lower body weight . Several2, and waist circumference of 1.25 cm [Similarly, evidence from intervention studies indicates that incorporating nuts into the diet has little impact on body mass and body fat . A syste 1.25 cm ; two of  1.25 cm ,16. HoweSeveral mechanisms outlined below have been proposed for the lack of weight gain observed with nut consumption, despite their high energy and fat content. These include reduced energy intake subsequent to increased satiety , energy Nut consumption has also been associated with a reduced risk of type 2 diabetes; evidence to support this comes from large epidemiological studies ,19. The C-reactive protein (CRP), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and interleukin-6 (IL-6). Weighted mean reductions in ICAM-1 (9%), VCAM-1 (6%) and CRP (12%) were found from analysis of 27 intervention studies with nuts in a literature review [Clinical and epidemiological evidence also indicate that nut consumption can enable improvements in inflammatory markers, with daily doses of 30 g able to confer benefits ,25. Medie review . StudiesClinical trials have clearly shown that intakes of several varieties of nuts can lower total and low density lipoprotein (LDL) cholesterol by 9%–16%, even in the context of healthy diets ,29,30,31The cholesterol lowering, anti-inflammatory and glucose regulating benefits of nuts have been attributed to their high content of unsaturated fat, possible weight reduction effect and bioactive compounds, including plant sterols, dietary fibre and antioxidants . Whilst This study aimed to investigate the effect of adding high oleic peanuts to habitual diets of healthy overweight adults on cardio-metabolic measures , body composition and anthropometric measures and to determine if there are any relationships between these outcomes.2. Smokers, regular nut consumers and people with cardiovascular disease, hypertension (>160/100 mmHg), a thyroid condition or nut allergy or those consuming ≥40 g of alcohol/day were excluded. Restrained eaters  participant data base, newspaper advertisements, flyers and word of mouth. The inclusion criteria were healthy overweight males or post-menopausal females aged between 50 and 75 years with self-reported stable weight and BMI ≥ 25 kg/mionnaire ) were exionnaire   or 12 weeks consumption of high oleic peanuts for 6 out of 7 days each week. Subjects were provided with 4 day physical activity diaries  and weigAt week 6, subjects attended the research centre for measurements of body mass waist circumference and were provided with a further set of diaries to complete prior to the next visit. At week 12 measurements were taken of body composition using dual-energy X-ray absorptiometry (DEXA), body mass, waist circumference and blood samples were taken. Subjects were provided with a further set of diaries to complete 4 days prior to the next visit and then crossed over to the to the alternate treatment arm with repeat visits at weeks 18 and 24.Roasted, unsalted high oleic  peanuts with skins on were provided in 28 g sealed foil packets by the Peanut Company of Australia ; their profile is shown in Weight was measured to the nearest 200 g using an electronic scale  with subjects wearing light clothing and without shoes. Two measurements were taken with the average used. If measures differed by more than 1% a 3rd measure was taken. Height was measured to the nearest 0.1 cm using a wall-mounted telescopic stadiometer  with subjects in stockinged or bare feet and two measurements taken and the average used. Waist circumference was measured with a metal anthropometric tape measure  at the natural waist or narrowest part of the torso to the nearest 0.1 cm with two measurements taken and the average used.® Model GE, Madison, WI, USA). Subjects were required to remove all items of jewelry, over clothes and under garments with metal fixings and any surgical devices or prosthesis were noted. The machine was calibrated prior to each scan with details of subjects’ body weight, height and date of birth entered. Subjects were scanned supine, positioned centrally on the mat and restrained at the ankle to minimise movement. DEXA is limited to subjects under 135 kg hence those over this weight were excluded. Previous reports from our research centre have found the standard error of the measurement for assessment of body composition by DEXA were 0.87% for percentage body fat, 0.53 kg (1.6%) for fat mass, 1.05 kg (2.3%) for lean mass, measured on consecutive days [Body composition measuring total body fat mass, percentage body fat and lean tissue mass was performed by DEXA (Luna Corp Prodigy Advanceive days .Fasting blood samples were obtained by venepuncture after subjects were seated for more than ten minutes. Plasma from EDTA (Ethylenediaminetetraacetic acid) was stored at −80 °C until analysis. Fasting plasma triglycerides, total cholesterol, HDL and glucose concentrations were determined using a commercial assay kit with a Konelab 20XT clinical chemistry analyser . These tests have good precision and accuracy with coefficient of variation within and between assays (<5.0%). Low density lipoprotein cholesterol (LDL-C) was calculated using the Friedewald equation . Fasting® Nutritional software .A set of food scales was provided with instructions on how to accurately weigh (to the nearest gram), measure and record intake in a food diary for 3 weekdays and one weekend day prior to the next visit. Weighed or measured food intake was recorded for 4 days prior to each assessment visit at baseline and weeks 6, 12, 18 and 24. Subjects were instructed to maintain similar patterns of physical activity throughout the study and 4 day physical activity diaries  were comAll data were checked for errors, hs-CRP levels >10 ml/L were removed from analysis (indication of a possible acute infection). General linear repeated measures analysis of variance was used to compare the mid-point data  and at the end of each with order included in the analysis and baseline used as a covariate, using SPSS version 21 . Body composition, anthropometric measures, energy and macronutrient intakes and cardio-metabolic measures were correlated where a relationship was predicted.2 and mean age of 65 ± 7 years at baseline. Sixty one subjects  completed the study, with a mean BMI of 31 ± 4 kg/mBaseline energy and nutrient intakes are presented in r = −0.264, p = 0.042).A change in body mass was observed with consumption of high oleic peanuts, with a small significant increase at 6 weeks (0.4 kg) and at 12 weeks (0.5 kg) with peanut consumption compared with the control phase . No chanC-reactive protein data was analysed in two cohorts: Subjects with baseline CRP levels ≤ 3.5 mmol/L and those with baseline CRP > 3.5 mmol/L (reflecting inflammation). There were no differences in either of these sub-groups between the two treatments. Similarly there were no differences observed in lipids or glucose regulation between treatments .eatments . An incrThis is the first study to investigate the effect of Australian high oleic peanut consumption on cardio-metabolic measures, body composition, body mass or waist circumference. In addition, no previous peanut studies have measured inflammatory markers and no intervention studies have measured their effect on glucose regulation. The food diaries revealed ~50–70 g daily intake of peanuts, consumed by both men and women (6 days per week). The expected increase in energy intake from this amount of peanuts was 1400 kJ per day (assuming no other changes were made to the diet). The actual increase in energy intake was ~850 kJ  indicating that some of the peanuts were substituted for other foods in the diet. Additional monounsaturated fat consumed during the peanut phase accounted for ~90% of the additional energy consumed. The predicted weight gain for this increase in energy reported was 0.9 kg over 6 weeks and 1.9 kg over 12 weeks . The actual average weight gain over the whole period was only 0.5 kg (26% of expected), and no changes in waist circumference or body composition measures were observed. However, it is acknowledged that this predicted weight gain calculation does not take into account the dynamic nature of weight loss hence, may be overestimated.i.e., as energy changed body weight would expect to change proportionally. This was not observed; for every kJ consumed during the peanut there was less body weight change compared with the control phase. Similarly, eight weeks consumption of 89 g/day of peanuts compared with a nut free diet for 8 weeks by healthy subjects with no dietary compensation demonstrated a weight gain of only 1 kg which was 28% of the predicted 3.6 kg [Energy expenditure from physical activity remained the same for both phases so this did not contribute to the findings. Calculations revealed ~−1.1 g difference in body mass per unit (kJ) of total daily energy intake with peanut consumption compared with the control phase. The body mass per kJ would be expected to be the same for each phase, d 3.6 kg . A possid 3.6 kg  which isd 3.6 kg .In addition the body fat storage may have been limited as a result of the unsaturated fatty acids being oxidized. Unsaturated fats and have a greater thermogenic effect  and are Interestingly, in this study MUFA intake was inversely associated with body fat mass. It has been suggested that fat quality may have a stronger correlation with weight gain than fat quantity . Studies2 and had on average healthy baseline blood lipid levels. Nut consumption has also been found to lower triglyceride levels predominantly in individuals with hypertriglyceridemia [et al. [Surprisingly, lipid levels were not altered with high oleic peanut consumption, contrary to many other nut studies . Howevereridemia . Similar [et al.  also rep [et al. ; in the This study demonstrated no differences in glucose or insulin levels with consumption of high oleic peanuts. Similarly several other studies have not shown benefits with consumption of pistachios, almonds and walnuts on fasting glucose or insulin ,59,60,61The effects of nuts on insulin sensitivity are influenced strongly by changes in body weight which may have accounted for the changes observed in one of these studies. As there were no changes in body composition in this current study, improvements in glucose regulation were less likely. Improvements in glucose regulation have also been demonstrated when nuts are included as part of an intervention diet such as the Nordiet  or a MedC-reactive protein (CRP) when compared with the nut free diet. Similarly, 12 weeks of hazelnut consumption (60 g) resulted in minimal effect on inflammatory markers and cell adhesion molecules in this group of healthy, normocholesterolemic overweight and obese individuals [Consumption of high oleic peanuts in the chronic study did not affect the inflammatory marker ividuals . In addiividuals , we idenividuals . In addiividuals . Both maividuals ,73. It iA minimum nut dose of 30 g may be required to elicit benefits for inflammatory markers . The curA strength of this study was the randomised, controlled cross-over design which assessed the effects of high oleic peanuts in the same individuals, hence reducing between subject variability. Another strength was high compliance with the consumption of peanuts and with the nut free diet, as assessed by food diary entries and returned peanut packages. The study also controlled for physical activity levels, with subjects requested to maintain similar physical activity levels throughout the study.One of the limitations of the study was the inability to double-blind the intervention; however, data collection and data entry was blinded to minimise investigator bias. Another limitation was the high dose  of peanuts used as proof of concept. A dose of 42 g/day recommended by The American Heart Association for reduction of cardiovascular disease (CVD) may still provide some cardio-metabolic benefits; however, this needs to be confirmed with further investigation. This study did not include a washout period; however, the control phase was a habitual (nut free) diet for 12 weeks, allowing sufficient time for reversal of any effects of peanut consumption . AnotherDue to budget constraints, it was not possible to measure fat excretion and energy expenditure directly, which may have provided information on a possible mechanism for the observed weight maintenance and body fat despite an increase in energy intake. Future studies should consider these outcomes.After 12 weeks of incorporating high oleic peanuts into the diet, only a small increase in body weight was observed, despite a large additional amount of energy consumed from the peanuts, with the weight increase being far less than predicted. The dose of ~60 g/day current study was substantial, much greater than the mean nut intake of 5 g/day consumed in Australia . Cardio-


As we known, Traditional Chinese Medicine (TCM) helps to prevent the relapse of drug addiction. However, the scientific basis of TCM remains unclear because of limitations of current reductionist approaches. We aimed to explore the possible mechanism of how ANKK1 TaqIA (A1/A2) [rs1800497(T/C)] affects the relapse of opioid addiction on the perspective of Chinese traditional medicine..The ANKK1 TaqIA (A1/A2) [rs1800497(T/C)] of the dopamine D2 receptor (DRD2) polymorphisms were genotyped in a case–control sample consisting of 347 opioid addicts and 155 healthy controls with RT-PCR and the TCM pathological factors were collected by means of Syndrome Elements Differentiation in the case–control sampleP < 0.05).DRD2/ANKK1 TaqIA Polymorphisms has no relation with opioid addiction relapse; but for those who were diagnosed with phlegm syndrome, DRD2/ANKK1 TaqIA Polymorphisms affect the replapse of apioid addiction (DRD2/ANKK1 TaqIA is associated with opioid addict and it is obvious in opioid addicts who suffer from the phlegm syndrome. Opioid addiction, a kind of chronic brain disease relapses, which is caused by abuse of heroin, morphine, interacts with the reward system of brain . An inteTaqIA, TaqIB, and TaqID SNPs based on restriction digestion of this region with the TaqI enzyme. Early studies indicated the A1 allele of TaqIA as a risk factor of substance abuse, alcoholism [TaqIA restriction fragment length polymorphism (RFLP) is located approximately 10 kilobases downstream from the DRD2 gene, in exon 8 of the ANKK1 (ankyrin repeat and kinase domain containing 1) gene [TaqIA polymorphism, causing an amino acid change in ANKK1 (Glu713Lys), seems to have a significant effect on the specificity of substrate binding. The protein product of the ANKK1 gene was considered as a negative regulator of the NF-κB (Nuclear Factor-KappaB) transcription factor [TaqIA variants in an in vitro luciferase system [The dopamine D2 receptor (DRD2) was characterized previously with coholism  and herocoholism , but some system . Since De system , 13, it e system .But not all the results of study with DRD2/ANKK1 TaqIA and drug addiction are the same. Apart from the reason of gender, race, the individual reflection environment, natural condition may be one of the main reasons, and leads to different results. TCM theory is a holistic view. The doctor of traditional Chinese medicine will consider what you eat, what you do, and how you feel before giving treatment. Carrying out a study by using traditional Chinese medicine methodology, we may discover some different results from previous studies.Traditional Chinese Medicine has been practiced in China for more than 2000 years, and for the past 200 years, it has been used in treatment of opioid addiction. It is the fact that TCM is effective in controlling opiate withdrawal symptoms and preventing opiate relapse with fewer side effects. In Traditional Chinese Medicine (TCM) theory, the common TCM pathological factors that may induce addiction relapse are yang deficiency, blood heat, and phlegm, among which the most prominent one is phlegm , while iParticipants were recruited from November 2008 to December 2012 in Fuzhou City, Fujian Province, China. We recruited 347 opioid addicts from two drug rehabilitation centres (167 subjects came from the drug rehabilitation centers under the jurisdiction of the Department of Public Security of Fujian Province and 180 subjects came from the drug rehabilitation centers under the jurisdiction of the Department of Justice of Fujian Province). The experimental procedures and protocol were approved by the Ethical Committee of Fujian University of Traditional Chinese Medicine. Informed consent forms were signed by all subjects before the study. Figs. All these opioid addiction subjects met the Diagnostic and Statistical Manual of Mental Disorders-IV, followed completion of a 3- month detoxification-treatment, and had no history of mental illness, agreed to participate in this survey. All of them could understand and correctly answer the various assessment tools, and cooperated to complete the questionnaires. Those with liver, kidney, cardiac insufficiency, and those psychopaths who could not cope with the survey were excluded.P < 0.05)   Fig. .Fig. 4ThAll subjects participated in a 12-h overnight rapid blood draw. Trained research assistants then completed a survey of physiological characteristics for all participants, and the participants completed questionnaires immediately after breakfast.TCM Syndrome differentiation  is a process of analyzing data collected through four combined diagnostic methods: inspection, auscultation and olfaction, inquiry, and the palpation is a distinguishing feature of TCM. According to TCM theory, Prof. Wenfeng Zhu established the Syndrome Elements (ZHENG Elements) Differentiation method , and it What are TCM syndrome elements? For example, if a patient is diagnosed as syndrome “liver Yin deficiency”, according to this method, we could get two TCM syndrome elements: one is Liver ,the other is Yin deficiency. Prof. Zhu Wenfeng calculated the percentage of each symptom appearing in certain TCM syndrome elements through epidemiology studies of large sample, by investigating TCM disease cases books in history and consulting prestigious TCM doctors by Delphi method and then concluded how important a symptom can contribute to certain TCM syndrome element by weighting each symptom with certain integral. Hence, in this method, you can find a certain integral of each symptom which is related to a certain TCM syndrome element. Once the sum of integrals of all symptoms relating to certain syndrome element is equal to or above 100, certain syndrome element is accepted as part of diagnosis for a patient. For example, the patient’s syndrome is yellowing of the skin, , so it is 40 of symptom “yellowing of the skin”, which contributes to syndrome element “Liver”, according to Wenfeng III Auxiliary Diagnosis and Treat System of TCM –19. OnceDrug Abuse and Opioid Addiction Severity Inventory (OASI) [The Syndrome Differentiation Significance of 600 Kinds of Common Symptom [Syndrome Elements Differentiation method was used for reference. Through preliminary experiments, “the TCM syndrome scale for Drug abusers in Fujian province” was designed by trained and professional personnel to investigate objects, item by item.y (OASI)  and The  Symptom  were useDNA was extracted using standard Methodology. The assay reagents for SNP genotyping consisted of a mix of PCR primers and TaqMan MGB probes (FAM and VIC labeled) that were obtained from Applied Biosystems . TaqMan SNP Genotyping Assays (ABI) were used for the polymorphisms ANKK1 rs1800497. Probes and primer for the DRD ANKK1 rs1800497 polymorphism were designed in-house (Probe1: FAMCAC AGCCATCCTCAAAGTGCTGGTCGAGGCAGGCGCCCAGCTGGACGTCCA (5′-3′);Probe2:VICCACAGCCATCCTCAAAGTGCTGGTCAAGGCAGGCGCCCAGCTGGACGTCCA (5′-3′). Each assay enables scoring of both alleles in a single well within a 384-well plate. All assays were optimized to work with genomic DNA and TaqMan Universal Master Mix from Applied Biosystems. Forty cycles of PCR were performed. Plates were analyzed using an ABI Prism 7900HT Sequence Detection System from Applied Biosystems.The questionnaire data entry was processed by Access 2000 software, and a database was set up, then the calculation of the allele and genotype frequencies and Hardy-Weinberg equilibrium test. Chi-square test was analyzed by SPSS 19.0 software, and statistical analysis of differences in each group allele and genotype frequency distribution was carried out, then came correlation between genotype and syndrome factor using a stepwise logistic regression mode.The main Syndrome Elements we collected is shown in Fig. P = 0.000, 95 % CI:,0.708 to 0.962). This shows that people with phlegm syndrome element  or qi deficiency syndrome element have stronger addiction intensity and are more likely to relapse. Damp evil, blood stasis, lung and heart emotion are the protective factors of addiction relapse. According to the result, phlegm, qi deficiency, damp evil, blood stasis, lung and heart emotion are the TCM pathological factors which affect the relapse of opioid addiction.As shown in Table P > 0.05), and the distribution did not differ between the females and males, the opioid addicts and the health group, respectively. To further determine differential distribution in opioid addicts, we carried out Pearson Chi-square Test. We find there is no difference in opioid addicts who have different Addiction intensities  relative to TaqIA genotype(A1A1). The influence of the heterozygote(A1A2) was he same with genotype(A1A1), so we could find A2 is an risk factor of addiction relapse, when phlegm exists.As we known, the distribution of DRD2/ANKK1 TaqIA has no difference in opioid addicts who have different addiction intensities, and we find there is no relationship between the DRD2/ANKK1 TaqIA and Syndrome Elements by using one-way analysis of variance Table . FurtherRelapse is a complex pathological behavior, caused by the combination of the pathological psychological, pathophysiological and social environmental factors . BesidesTCM is a holistic and systematic medicine, and Syndrome Elements Differentiation method based on TCM Syndrome Differentiation was used to extract TCM pathological factors. Our statistics results suggest that in both group1 and group2, phlegm is a risk factor for addiction relapse. And qi stagnate, damp are protection factors, which is consistent with our previous findings .P > 0.05), which is contrary to the results of previous studies. The reasons we assume are as follows: first, there are varying gene frequency distributions among different geographic regions; second, different drugs using and short-term drug dependence might deviate the experiment result.Dopamine receptors belong to the G protein coupled receptor family. Studies have shown that DRD2 gene knockout mice had failed to react to opioids reward , showingLong-term opioid abuse caused Qi-blood disturbance, and Qi and blood were consumed, then leading to dysfunction of the zang-fu organs, and producing all sorts of Traditional Chinese Medicine pathological factors. The performance of opioid addicts show that their oral secretions increased, be easy to fatigue, in the bad emotional state, and so on, which is related with phlegm, qi deficiency, damp evil, and anther Traditional Chinese Medicine pathological factors, especially phlegm. Yawning, runny nose, tears, and frequently increasing oral secretions arose during Drug addiction episodes. That is the same with the performance of phlegm syndrome. And the previous data mining research on the relationship between the loci and addiction relapse shows once phlegm symptom was present, the addiction of patients who carry the A2A2 allele have more possibilities to relapse than those with genotype A1A1. According to our former research, phlegm syndrome is a major cause of addiction relapse, but the mechanism about how phlegm is involved is still a question. From the results of the current data, we found A2A2 had no effect on addiction relapse when TCM factors are not involved. However when DRD2/ANKK1 TaqIA stay with phlegm, it becomes an important factor for the addiction relapse. And A2A2 is a risk factor for addiction relapse. According to the above results we explain as follows: first, phlegm causes addiction relapse, but has no relation with the genotype. The other explanation is phlegm syndrome may affect the expression by modifying the allele through epigenetic mechanisms, thus leading to relapse. In modern research, investigators found opioid receptor gene (OPPM1) was highly methylated in the blood and semen of patients with opioid addiction . This inIn this research, we also found other TCM pathological factors associate with addiction relapse, and phlegm is not an independent risk factor. But in this paper; we discussed phlegm syndrome only, the mechanism of the other TCM pathological factors is still unclear.DRD2/ANKK1 TaqIA polymorphism is associated with opioid dependence risk, when individual is diagnosed with phlegm syndrome. We suspect that plegm syndrome could lead to form a particular environment in the body that is conducive to epigenetics of DRD2/ANKK1 TaqIA, further caused the addiction relapse. Further studies are needed to discover the specific mechanism. Epigenetic experiment is also necessary.