Twenty-two patients with enduring pain after a rear impact car accident (Whiplash Associated Disorder grade II) and 14 healthy controls were investigated. Patients displayed significantly elevated tracer uptake in the neck, particularly in regions around the spineous process of the second cervical vertebra. This suggests that whiplash patients have signs of local persistent peripheral tissue inflammation, which may potentially serve as a diagnostic biomarker. The present investigation demonstrates that painful processes in the periphery can be objectively visualized and quantified with PET and that 11C-D-deprenyl is a promising tracer for these purposes.There are few diagnostic tools for chronic musculoskeletal pain as structural imaging methods seldom reveal pathological alterations. This is especially true for Whiplash Associated Disorder, for which physical signs of persistent injuries to the neck have yet to be established. Here, we sought to visualize inflammatory processes in the neck region by means Positron Emission Tomography using the tracer  Chronic musculoskeletal pain syndromes are common, cause extensive individual suffering and place a large burden on health care in society. Yet, pain remains notoriously difficult to visualize and diagnose objectively. Furthermore the pathophysiology of persistent pain is elusive and there is a great need for ways to visualize and quantify pain mechanisms. One common chronic pain syndrome is Whiplash Associated Disorder (WAD), where the onset is a soft tissue sprain of the neck caused by a rear end motor vehicle collision or a similar trauma. The injury is usually benign 11 (S-(+)-(d)-D-deprenyl, or DDE). High DDE retention has been demonstrated in the synovium in arthritic knee joints One candidate PET tracer for musculoskeletal injuries and inflammation is CTwenty-two patients  with chronic neck pain after car accident involving whiplash trauma were recruited through hospital patient registries. 12 of the patients experienced a rear end collision, 5 patients experienced combined lateral and frontal collisions. Detailed collision descriptions were not available from 5 patients. All patients were diagnosed with whiplash associated disorder (WAD) grade II Fourteen healthy control subjects were also investigated. Control group A consisted of eight healthy and pain free volunteers  recruited through advertisements. Control group B consisted of six patients with acute musculoskeletal pain (sprained ankle), but otherwise healthy and without current or prior neck pain . General exclusion criteria for patients and controls were psychiatric disorder, somatic disease, substance abuse and pregnancy. The study was approved by the local ethical review board  and the local radiation protection committee . All participants provided written informed consent before entering the study.Prior to the PET investigation, participants rated their pain intensity on a 0-100 visual analogue pain scale (VAS), and completed the state portion of the Spielberger State Trait Anxiety Inventory (STAI-s) Carbon-11 marked DDE was produced for each PET investigation at GE healthcare, Uppsala Imanet following published procedures During the course of the study, a new PET-CT  was installed and control subjects were investigated on this scanner, allowing for fusion of PET and CT images giving higher confidence in anatomical definitions. As different scanners were used for patients and controls, three of the 22 WAD patients were first investigated in the PET scanner and then also reinvestigated in the PET-CT scanner in order to ensure reproducibility across the two scanners. The time between these two investigations was about one month.Control group A  was investigated on the PET-CT scanner, which has 24 rings of bismuth germinate crystal detectors. This scanner enabled the acquisition of 47 contiguous planes of data with a distance of 3.27 mm, resulting in a total axial field of view of 15.7 cm Images were first analyzed visually and locations of elevated tracer retention were specified with guidance from anatomical atlases and CT images when available. To quantify DDE retention, a series of regions of interest (ROIs) were defined in each subject. ROIs of identical size and shape were delineated in (I) a sphere in the non glandular regions with the most elevated retention, , (II) a sphere left or right of the spineous process of axis , (III) a sphere in the spongeus bone of the cervical vertebra 2 through 6 (vertebra ROIs), and (IV) a larger prolate spheroid in muscle tissue region without visually evident elevated retention (muscle ROI), see Three patients were imaged with both the Siemens HR+ PET and the GE discovery PET-CT system. Reproducibility across scanner type and session was evaluated by extracting the SUV TAC data at the 12 time points for the above specified ROIs from each subject in both PET scans. In addition, two more peak ROIs were generated for the regions with the second and third highest tracer uptake in each patient. As a metric of test reproducibility, the Pearson product moment coefficient of determination was calculated for SUV values at all time point from the HR+ and the PET-CT system in each patient for each ROI. See Several soft tissue regions displayed visually evident elevated tracer retention in WAD patients, see WAD patients had significantly higher DDE retention than controls in tissue regions adjacent to spineous process of the second vertebra , in the normal muscle tissue (p = 0.027) and in the peak soft tissue ROI (p = 0.035), but similar levels in the in the spongeus bone of the cervical vertebra (p = 0.48) see Peak retentions were located around the spineous process of C2 in most patients, and also at the insertion of rectus capitis posterior major in the occipital bone, see Patients reported significantly elevated VAS neck pain ratings (patients (n = 22) mean ±SD = 47±21, control group A (n = 8)  = 4±8, p<0.001). Control group B (n = 6) reported pain due to their acutely sprained ankle pain at rest , but no neck pain. Patient's ratings of neck disabilities (NDI) were also significantly elevated (patients (n = 22) mean ±SD = 18±5, controls (n = 8)  = 2±1). No differences were found with respect to ratings of anxiety (STAI-s) at the time of investigation (patients (n = 22) mean ±SD  = 31±7, controls (n = 8)  = 30±5). Within the patient group, VAS pain ratings displayed a trend towards a positive correlation with DDE uptake in the C2 ROI .2) between the first and the second scan in SUV measures of the vertebra and muscle tissue was r2 ≥0.74 (p<0.001). In the presumably abnormal soft tissue peak ROIs, r2 was ≥0.63 (p<0.001). In the right and left C2 ROI's the lowest observed r2 was ≥0.93 (p<0.001) and r2 ≥0.86 (p<0.001) respectively. See also The test reproducibility between the two PET scanners used was high in all ROIs for the three subjects investigated on both scanners. The lowest observed coefficient of determination  from the dynamic PET-data show a rapid increase in the first frames followed by a plateau in the last frames as with specific binding or trapping of the tracer. In regions with high blood flow, such as the carotid artery , there w18F] fluorodeoxyglucose (FDG) PET is used to detect tumors, an uptake somewhat resembling the current finding has been observed in patients who are cold The main limitation of this study is that we do not yet know the exact mechanism of the increased DDE uptake. (S-(+)-(d)-D-deprenyl is the mirror isomer of L-deprenyl, a well established MAO B inhibitor also known as selegeline This study represents a phase 1 diagnostic study The present investigation demonstrates that painful processes in the periphery can be objectively visualized and quantified with PET and that DDE can be regarded as a promising tracer for these purposes. A large subset of patients with chronic pain after a whiplash injury displayed elevated DDE retention, suggestive of persistent peripheral tissue inflammation. The possibility to visualize and quantify sites of inflammation in chronic pain may be very useful in diagnosis and treatment monitoring. Inflammation imaging may also guide in examining and specifying mechanisms in debilitating chronic pain.Figure S1Patient A, B and C scanned in both the Siemens HR+ PET (top row) and the GE Discovery PET-CT (bottom row). Data is expressed as SUV values normalized for bodyweight and injected dose.(TIF)Click here for additional data file.


An important challenge for conservation today is to understand the endangerment process and identify any generalized patterns in how threats occur and aggregate across taxa. Here we use a global database describing main current external threats in mammals to evaluate the prevalence of distinct threatening processes, primarily of anthropogenic origin, and to identify generalized drivers of extinction and their association with vulnerability status and intrinsic species' traits. We detect several primary threat combinations that are generally associated with distinct species. In particular, large and widely distributed mammals are affected by combinations of direct exploitation and threats associated with increasing landscape modification that go from logging to intense human land-use. Meanwhile, small, narrowly distributed species are affected by intensifying levels of landscape modification but are not directly exploited. In general more vulnerable species are affected by a greater number of threats, suggesting increased extinction risk is associated with the accumulation of external threats. Overall, our findings show that endangerment in mammals is strongly associated with increasing habitat loss and degradation caused by human land-use intensification. For large and widely distributed mammals there is the additional risk of being hunted. Today, as anthropogenic degradation of the world's ecosystems is causing the Earth's sixth mass extinction event The process of endangerment can start with a direct perturbation or threat to a species, which is then followed by a suite of secondary factors that may act additively or synergistically to cause final extinction In this study we search for evidence of generalized drivers of extinction in mammals, exploring the prevalence and aggregation patterns of multiple external threats and the relationship between threat aggregation and vulnerability status. The IUCN and the Conservation Measures Partnership have generated a threats classification scheme biological resource use includes both logging and hunting activities  number of studies per species used to populate the PanTHERIA database of mammalian trait data  with a multinomial distribution (cumulative logit link). We used taxonomic correction rather than phylogenetic correction because to our knowledge, there are not frequentist phylogenetic ordinal regression approaches currently available. The Red List uses five criteria to define status based on population reduction, distribution range size, population size and decline, and small or restricted populations. Species in naturally small fragmented ranges may be defined as threatened under criterion B because they are vulnerable to extinction even if they are affected by few or no extinction drivers. Therefore, we tested the relationship between Red List status and number of threats for all species and also for a subset that excludes those listed under criterion B. Data deficient species were excluded because of their unknown vulnerability status. Extinct and extinct in the wild species were not considered because, by definition, they have no listed ongoing threats.To address the issue that related species are not independent observations P-value of a likelihood ratio test comparing models with and without the fixed factor (but including taxonomic random effects). We also report the conditional deviance R2D(c) and marginal deviance R2D(m) calculated as 1-(full model deviance)/(null model deviance). The full model was the model including fixed and random factors. R2D(c) was calculated with a null model that includes random factors and thus, describes the improvement in model fit due to the fixed factor(s) alone. R2D(m) was calculated with a null model that only includes an intercept, and thus describes the improvement in model fit due to both fixed and random factors.For GLMM analyses we report the estimated regression coefficient (b), its standard error (SE), and the r10-transformed and standardized by subtracting the mean and dividing by the standard deviation. The first component (PC1) extracted from the PCA explains 59% of the variance in adult body mass and range area, with high values indicating large, widely distributed species . Differences in trait values among main threat combinations and group of species with different numbers of listed threats were tested using phylogenetic generalized least square (pgls) regression models. Pgls models control for the evolutionary relationships among species, thus addressing the issue that data from related species do not represent independent observations r estimating lambda with the maximum likelihood method F statistic for categorical dependent variables, in all cases we include the P-value and R2 estimated by ‘caper’.Using our threat reclassification we identified existing threat combinations among mammals and calculated their prevalence (how many species were affected by each threat and threat combination). We then selected the main combinations (combinations found in >10% of the species with the same number of listed threats) to explore if species affected by the same threats share common traits. In particular, we focused on two species' traits: distribution range area, which is generally associated with specialization 2. The size of the grid was selected in accordance to the resolution of the available distribution maps Finally, we characterized the terrestrial distribution of the most common threat combinations using the IUCN distribution range maps (including only regions where presence was described as “Extant” or “Probably Extant” and origin listed as “Native”) and assuming threats are homogeneously distributed throughout the range 2D(c) = 0.19, R2D(m) = 0.24; number of threats b = 0.88, SE = 0.030, P<0.001; data availability b = −1.31, SE = 0.082, P<0.001. 2D(c) = 0.17, R2D(m) = 0.28; b = 0.82, SE = 0.035, P<0.001. Vulnerability as defined by the IUCN Red List status tends to increase with the number of listed threats per species even controlling for the reduced data availability of more vulnerable species (GLMM R2D(c) = 0.07, R2D(m) = 0.08; b = 0.20, SE = 0.041, P<0.001. GLMM excluding species listed under criterion B, R2D(c) = 0.07, R2D(m) = 0.13; b = 0.17, SE = 0.053, P<0.001) and after excluding species without listed threats  = 0.08, R2D(m) = 0.11; b = 0.33, SE = 0.037, P<0.001. GLMM excluding species listed under criterion B, R2D(c) = 0.08, R2D(m) = 0.15; b = 0.42, SE = 0.045, P<0.001).Listing of threats may be less reliable for least concern species because these species are not considered to be at risk and thus, less emphasis is placed on reporting their threats. Similarly, species without listed threats may not have been adequately evaluated and thus, lack of threats may in some cases not reflect true conditions In all cases we find that critically endangered species tend to have fewer threats than those listed as endangered. As a species approaches its demise few threats may remain relevant or be perceived as such by evaluators. In addition, critically endangered species have small ranges that may overlap with fewer human activities or may be more actively managed leading to a true reduction of anthropogenic threats. Although there are no listed ongoing threats for extinct species, some extinct species have past threats listed. Among the 48 extinct species with listed past threats, the median number of described threats is low ( = 1), probably because only the final culprits are identified. As expected, species listed as data deficient also have few listed threats because little is known about these species and additionally because evaluators are not required to list existing threats for data deficient species.agriculture, exploitation, and logging .If all threats were equally likely to be listed, the expected proportion of species suffering from each threat when a single threat is listed would be on average 1/8, when two threats are listed the proportion would be 2/8, and so forth. Among species with a single threat we find three threats that affect more than 1/8 of the species:  logging . These tt status . In addi2) compared to those affected by combinations including only habitat loss and degradation . A phylogenetically-corrected model shows that these differences, as described by the first component of a principal component analysis, are statistically significant .To further describe existing threat combinations we focused on species with 1–4 listed threats. Few species have >4 listed threats and additionally this range matches the median number of threats of each of the Red List status going from least concern to endangered once species listed under criterion B are removed . We findN = 421, 39 species are not represented in the phylogeny; pgls b = −0.01, SE = 0.005, P = 0.005, R2 = 0.02) suggesting that range contraction could occur as the number of threats increases  while we only represent those affected by exploitation.Terrestrial mammals in the habitat loss and degradation group are found primarily in tropical areas which also have high mammalian richness comm disruption, may be relatively rare but could pose a great risk because they primarily affect already vulnerable species and have direct, and possibly devastating, effects on individual survival A limitation of our analysis is that IUCN threats are currently not rated in importance and thus, some listed threats may pose a relatively minor risk but the IUCN listing and, by necessity, our analyses treat all listed threats as equal. Our focus on main threats and a simplified scheme partly reduced the spurious effects of considering rare threats but we agree with Hayward logging or exploitation to which threats involving more intense habitat modifications are added. This possible sequence corresponds very well with the temporal changes in land-use intensity described by Foley et al. exploitation  and logging  of threats could be tested using temporal data on threats appearance, which to our knowledge are not currently available at a global scale. However, such temporal information could become available in the future if the IUCN reassesses the presence of ongoing threats for mammals using the same  threat classification system. The evaluations completed up to date have used different classification schemes limiting the use of these data to make temporal inferences. Nevertheless, our results offer some support to the interesting hypothesis that threats accumulate and intensify over time increasing extinction risk with different types of species experiencing different threat combinations and accumulation patterns. Whether these patterns truly reflect a temporal sequence and are also observable in other taxa remains to be explored.agriculture, logging and exploitation) are in effect For conservation practice our results at a global scale support the importance of first preserving natural habitats and then halting and reversing human land-use intensification. Intensifying habitat degradation is generally associated with higher vulnerability and its effects appear to be cumulative. In particular, there is a need to protect habitat in tropical areas where many small, narrowly distributed species occur but still have relatively low numbers of threats; thus these species may be effectively protected simply by preserving their remaining suitable habitat. Importantly, our results suggest that halting the land-use intensification process at any stage could be a good precautionary approach even in degraded areas because apparently greater intensification increases the risk by generating additional threats. Despite these general conclusions, particular actions should be defined after careful analysis of the threats affecting a given species locally, as well as evaluating the plausible measures that may be taken to stop or mitigate such threats. In certain regions, different threat patterns may be prevalent because globally rare threats are locally common. For example, invasive species are a serious problem in many islands Figure S1Frequency of each threat effect among species with different number of listed threats. Each panel represents species in each Red List status: LC (Least Concern), NT (Near Threatened), VU (Vulnerable), EN (Endangered), CR , DD (Data Deficient). Numbers in parenthesis indicate the number of species. Values that fall within the shaded area indicate fewer species than expected if all threats were equally likely .(TIF)Click here for additional data file.Figure S2Main threat combinations observed among mammals with distinct numbers of threats. Each combination is represented by a colored circle with size proportional to the number of species in that combination. For each combination we also plot the mean  and the standard error of the mean (error bars) of the adult body masses (left panel) and the distribution range areas (right panel) in the group. Red circles represent combinations we assigned to an exploitation-habitat loss group, and green circles combinations assigned to a habitat loss and degradation group. Threats are described in (TIF)Click here for additional data file.Table S1Description of the Red List status categories defined by the IUCN and used in the analyses.(DOCX)Click here for additional data file.Table S2Observed threat combinations for mammals with one listed threat.(DOCX)Click here for additional data file.Table S3Observed threat combinations for mammals with two listed threats.(DOCX)Click here for additional data file.Table S4Observed threat combinations for mammals with three listed threats.(DOCX)Click here for additional data file.Table S5Observed threat combinations for mammals with four listed threats.(DOCX)Click here for additional data file.


The current interest in using nucleic acids as supramolecular scaffolds for the precise arrangement of chromophores is extremely high.N-(2-aminoethyl)glycine backbone introduced by Nielsen et al. was shown to bind strongly to DNA and preferentially to RNA with high affinity and sequence specificity.R,4′R) configurations in combination with cyclic β-amino acids including (R)-aminopyrrolidine-2-carboxylic acid (dapcPNA),S)-aminocyclopentane-(1S)-carboxylic acid (acpcPNA)S)-aminocyclobutane-(1S)-carboxylic acid (acbcPNA)15Peptide nucleic acid (PNA) is a class of DNA analogues that consists of nucleobases attached to a peptide-like backbone. The original PNA with a flexible DNA1 to DNA4 and the corresponding acpcPNA counterstrands, PNA1 to PNA4. These strands can form fully complementary DNA–PNA hybrids in the binding region  in which the two fluorophores are embedded in four different sequential contexts . Additionally, PNA–DNA hybrids or DNA duplexes with two mismatches can be formed . DNA9 to DNA12 represent reference oligonucleotides to measure the thermal stability of the unmodified acpcPNA–DNA hybrids .We applied, herein, dual-emitting oligonucleotides bearing ethynyl pyrene adjacent to ethynyl nile red to probe the differences in interactions in DNA duplexes compared to conformationally constrained acpcPNA–DNA hybrids. Using our published synthetic protocols,Tm), and steady-state fluorescence spectroscopy . Mismatch-containing DNA duplexes  show strongly reduced thermal stabilities (−9.3 °C to −27.9 °C), while mismatch-containing acpcPNA–DNA hybrids are not formed at all (e.g. DNA1–PNA2), as expected based on our previous results and as supported by the low absorption differences in the melting temperature experiments.10The optical properties of the acpcPNA–DNA hybrids in comparison with the corresponding DNA duplexes were characterized by UV/Vis absorption spectra, including melting temperatures  are higher compared with those of DNA2 and DNA4. In principle, ground state interactions interfere with energy transfer, because the latter process requires the excitation of an uncoupled chromophore as energy donor (ethynyl pyrene) and subsequent transfer of excitation energy to an uncoupled and unexcited chromophore as acceptor (ethynyl nile red). Hence, it is not surprising that those single stranded oligonucleotides (DNA2 and DNA4), in which the ethynyl pyrene is less coupled in the ground state (according to the ratios ΔA402/ΔA379), exhibit better energy-transfer efficiencies .The UV/Vis absorption of both the doubly modified acpcPNA–DNA hybrids and the DNA duplexes show the presence of both chromophores at ∼370–400 nm (ethynyl pyrene) and ∼620 nm (ethynyl nile red). If excited at 380 nm, which is highly selective for ethynyl pyrene, the various single strands, hybrids and duplexes show dual emission at ∼440 nm (ethynyl pyrene) and ∼665 nm (ethynyl nile red) as the result of an energy transfer between both chromophores. In the most interesting case from the supramolecular point of view, the contributions of the blue-green and the red emission are equal, and thereby an emission of white light is generated.DNA1 to DNA4 are annealed with the corresponding fully complementary counterstrands DNA5 to DNA8, the absorption side band at ∼402 nm gets intensified, and the ratios ΔA402/ΔA379 increase accordingly. This is not a new result and has been reported previously.without a corresponding increase of the ethynyl pyrene emission at ∼440 nm. The ratio of the fluorescence intensities I665/I440 are reduced to values between 1.7 (DNA4–DNA8) and 5.2 (DNA3–DNA7). It is important to note here that the excitonic interactions do not require the fully matched DNA architecture around them, as they are formed also in the mismatched DNA duplexes (DNA1–DNA6 to DNA4–DNA7). However, in the latter duplexes they do not interfere with energy transfer. Obviously, transition dipole moments of the nonstacked subensemble can—due to higher conformational flexibility—adapt an orientation that is more suitable for efficient energy transfer. Thus, fluorescence intensity ratios I665/I440 in the mismatched duplexes are similar to those of the corresponding single strands DNA1 to DNA4.If the single strands DNA1 to DNA4 are annealed with corresponding fully complementary acpcPNA strands PNA1 to PNA4, the optical properties change quite significantly compared with the DNA duplexes. Except for DNA1–PNA1, the I665/I440 ratios are generally lower than that of the corresponding DNA duplexes, ranging between 1.0 (DNA4–PNA4) to 3.6 (DNA1–PNA1). The fluorescence intensity ratio bears no relationship to the duplex stability as can be seen by the lower I665/I440 ratio, despite the lower thermal stability of DNA3–PNA3 compared with DNA3–DNA7. In addition, the absorption side bands of ethynyl pyrene at ∼402 nm get reduced and the absorption ratios ΔA402/ΔA379 get decreased accordingly. This observation indicates that the stacking interactions between the ethynyl pyrene chromophore and adjacent DNA bases are interrupted by the conformational constrain, which is introduced in acpcPNA–DNA hybrids. According to our discussion for the matched DNA duplexes above, this should yield stronger nile red fluorescence intensity at ∼665 nm compared with DNA double strands. However, the opposite effect is observed: the red emission is also reduced in the acpcPNA–DNA hybrids. This result can be explained by two different effects that are probably combined to various extents in the different acpcPNA–DNA hybrids. Firstly, except for DNA1–PNA1, the ethynyl pyrene fluorescence in acpcPNA–DNA hybrids increases slightly relative to DNA duplexes. The observed difference of the energy-transfer efficiencies is likely due to the change in the relative orientation of the chromophore dipole momentsDNA2–PNA2). This again supports ground state interactions between nile red and ethynyl pyrene, and thereby again interferes with ethynyl nile red emission induced by energy transfer.If the single strands Tm values) and the pronounced sequence selectivity including mismatch discrimination of acpcPNA provides added value to the basic concept of self-assembled nucleic acid-based nanostructures and multichromophore arrangements. Among the samples presented herein, especially hybrids DNA2–PNA2 and DNA4–PNA4 show nearly equal contributions of the blue-green and red emission and, thus, represent white-light emitters with better thermal stability than the previously published white light-emitting DNA (WED).The inspection of photophysical interactions in chromophore pairs is crucial for using the geometries of nucleic acid hybrids with artificial backbones for the development of multichromophore systems. From our results it becomes clear that the strong binding to DNA (high


Methylene blue (MB) is lately being proposed to be effective in treating Alzheimer’s disease (AD). Phase 2 clinical trials reported improvements in cognitive functions of AD patients after MB treatment. One of the main mechanisms of action that has been described for MB is inhibition of Tau aggregation [1]. Moreover, its antioxidant and mitochondrial protection have been previously described [2]. Only recently, a study using a triple transgenic AD mouse model has tested the mechanism of MB in vivo, showing improved cognition and reduced Aβ levels after MB treatment [3].Recently, the mitochondrial enzyme Amyloid binding alcohol dehydrogenase (ABAD) has been shown to bind Aβ inducing mitochondrial dysfunction, providing a direct relation between Aβ and mitochondrial dysfunction occurring in AD. Previous studies have shown that inhibiting ABAD protects mitochondrial functions and prevented Aβ-induced toxicity [4][5]. Taking into consideration the mitochondrial protective effect of MB and the recent data suggesting its ability to reduce Aβ levels, our aim was to investigate the effect of MB on ABAD and mitochondrial function in an LPS mouse model that has been previously described to induce memory impairment, with Aβ accumulation in hippocampus and cerebral cortex [6].in vivo model, mice were treated for 4 days with MB (I.P 4 mg/kg). After sacrification of the mice, mitochondrial associated ROS and cell viability of brain cells were measured using dihydrorhodamine probe and MTT assay respectively. Moreover, ABAD and Aβ levels were determined by western blotting in the brains of the treated mice compared to the control group.LPS mouse model was used (I.P 250 µg/kg LPS for 7 consecutive days) and accumulation of Aβ was assessed by immunohistochemistry. To Test the effect of MB on this The LPS mouse model used in this study showed brain accumulation of Aβ, decreased cell viability and increased mitochondrial associated ROS levels compared to the vehicle group. MB treatment significantly increased cell viability and reduced the LPS-induced increase in the ROS level. Moreover, brain Aβ level of the MB treated group was significantly decreased compared to the untreated group. Most interestingly, MB treatment was able to reduce the high level of ABAD that was found in the LPS mouse model.Taken together our results showed that MB decreased both Aβ and ABAD levels, while protecting the mitochondria from oxidative stress, consequently improving brain cell viability. Based on the previously reported role of the interaction between ABAD and AB in inducing mitochondrial dysfunction, the current study suggests a novel mechanism of action of MB, linking its mitochondrial protective effects to its lowering effect of both AB and ABAD which may decrease their binding and the resultant mitochondrial stress.


Pain in masticatory muscles is among the most prominent symptoms of temperomandibular disorders (TMDs) that have diverse and complex etiology. A common complaint of TMD is that unilateral pain of craniofacial muscle can cause a widespread of bilateral pain sensation, although the underlying mechanism remains unknown. To investigate whether unilateral inflammation of masseter muscle can cause a bilateral allodynia, we generated masseter muscle inflammation induced by unilateral injection of complete Freund’s adjuvant (CFA) in rats, and measured the bilateral head withdrawal threshold at different time points using a von Frey anesthesiometer. After behavioral assessment, both right and left trigeminal ganglia (TRG) were dissected and examined for histopathology and transient receptor potential vanilloid 1 (TRPV1) mRNA expression using quantitative real-time PCR analysis. A significant increase in TRPV1 mRNA expression occurred in TRG ipsilateral to CFA injected masseter muscle, whereas no significant alteration in TRPV1 occurred in the contralateral TRG. Interestingly, central injection of TRPV1 antagonist 5-iodoresiniferatoxin into the hippocampus significantly attenuated the head withdrawal response of both CFA injected and non-CFA injected contralateral masseter muscle. Our findings show that unilateral inflammation of masseter muscle is capable of inducing bilateral allodynia in rats. Upregulation of TRPV1 at the TRG level is due to nociception caused by inflammation, whereas contralateral nocifensive behavior in masticatory muscle nociception is likely mediated by central TRPV1, pointing to the involvement of altered information processing in higher centers. Temporomandibular disorders (TMDs) are the most common orofacial pain conditions affecting musculoskeletal and joint tissue . TMD affTRPV1 plays a key role in peripheral inflammatory and neuropathic pain states . They arAlthough the hippocampus is not recognized as a major area in the brain involved in pain processing, there is possible hippocampal involvement in various pain conditions because painful stimuli can induce changes of neuronal activity, blood flow, and c-fos expressions in the hippocampus -30. HippDespite growing interest in understanding masticatory muscle pain conditions the pathophysiological mechanisms leading to the development of masticatory muscle pain are still poorly understood. It is not clear whether unilateral inflammation can induce bilateral nocifensive response, and if so, whether the changes in nociception are regulated by TRPV1 channel function. The aim of this study was to investigate the nocifensive behavioral response and changes in TRPV1 channel expression in TRG neurons both at the site of inflammation and at contralateral side following unilateral injection of CFA into the masseter muscle. Our findings show that the involvement of central TRPV1 in bilateral allodynia induced by unilateral masseter muscle inflammation.Adult male Sprague Dawley rats (250–300 g) with total of 46 were used in this study. Rats were housed in groups (two to five per cage) under a controlled temperature (22 ± 1°C) and humidity (50 ± 5%) environment with ad libitum access to food and water. Animals were maintained on a 12 h/12 h light/dark cycle. The animal experimental protocols were approved by the Animal Use and Care Committee of Peking University and were consistent with the Ethical Guidelines of the International association for the Study of Pain.Mycobacterium tuberculosis suspended in oil: saline at 1:1 emulsion) into the mid-region of right masseter muscle (n = 6), as described in previous studies [Each animal was anesthetized with sodium pentobarbital (50 mg/kg i.p.), and injected with 50 μL complete Freund’s adjuvant  with the tip size of 1.0 mm as previously reported in investigations of deep tissue inflammatory pain conditions ,38. InitFour days after CFA (n = 8) or saline injection (n = 8), head withdrawal threshold of the additional rats was measured. After behavioral assessment, rats were decapitated and both right and left trigeminal ganglia from CFA rats and control animals were dissected for RNA extraction, frozen in liquid nitrogen and stored at −80°C until processed. Bilateral masseter muscles were fixed in 4% paraformaldehide, embedded in paraffin, cut at 40 μm and stained with haematoxylin and eosin.-ΔΔCt method to compare expression of genes of interest with that of β-actin.Total RNA was extracted from trigeminal ganglion (TRG) with TRIzol and was digested with 6 μl Turbo DNase for 1 hour. cDNA was synthesized using oligo dT and M-MLV reverse transcriptase according to the manufacturer’s instructions (Invitrogen). Real-time PCR amplification mixture (20 μl) contained 1 μl template cDNA, 10 μl 2× SYBR® Premix Ex TaqTM (Takara) containing SYBR Green and 5 μM forward and reverse primers. The mRNA level for gene of interest was acquired from the value of threshold cycle (Ct) as a relative level of β-actin through the formula 2-ΔCt (ΔCt = β-actin Ct - gene of interest Ct). The sequences of these primers  were designed using Oligo software . The proTo test the effect of hippoampal TRPV1 on bilateral nocifensive behavioral response after unilateral masseter muscle inflammation, rats were anesthetized with sodium pentobarbital , and injected with 5-iodoresiniferatoxin  or vehicle  bilaterally into the CA1 region of hippocampus, according to our previous experimental procedures . All solThe effect of intrahippocampal injection of TRPV1 antagonist and vehicle on the baseline of head withdrawal threshold was initially examined. The head withdrawal threshold was measured for a period of 60 min after hippocampal injections. To examine the effect of intrahippocampal injection of TRPV1 antagonist on bilateral nocifensive response after unilateral masseter inflammation, all rats were injected with CFA into unilateral masseter muscle. Four days after CFA injection, bilateral head withdrawal threshold was measured to reconfirm development of bilateral mechanical allodynia. After behavioral assessment each rat was intrahippocampally injected with TRPV1 antagonist or vehicle, and head withdrawal thresholds were re-measured every 15 min for a period of 60 min. Rats were then decapitated and coronal sections (30 μm) of the brains were sliced on a cryostat, and the sections were examined under a microscope to confirm the placement of cannulas.t-test with Welch correction was used to analyze differences between the two groups. A probability level of less than 0.05 was considered for statistical significance.GraphPad Prism 4.0 software  was used for all statistical analysis. All data are presented as mean ± SEM. Two-way repeated measures ANOVA with treatment  as the independent factor and time as the repeated factor was used for statistical analyses of changes in mechanical head withdrawal thresholds for each side separately . Bonferroni was used as post-hoc test when the ANOVA indicated an overall significance. The Unilateral injection of CFA into masseter muscle induced edema on the injected side. This sign of edema appeared 4 hours after injection and persisted for at least 4 days. No edema was observed at contralateral masseter or masseter injected with saline. Histopathological examination showed qualitative differences between muscles injected with CFA and saline. Injection of CFA into the masseter muscle produced clear signs of inflammation characterized by extensive infiltration of granular leukocytes and numerous vacuoles at site of injection Figure A. In conp < 0.01), reached its peak at day 4 (p < 0.001), and returned to the baseline level at day 11 (p > 0.05) , also reached the maximum reduction at day 4 (p < 0.001), and returned to the baseline level at day 11 after inflammation  Figure B. The den Figure B. Mechanp < 0.0001; B, contralateral head p = 0.0016, as compared with control animals), reconfirming the development of bilateral allodynia after unilateral masseter muscle CFA injection , while there was no alteration in TRPV1 mRNA in the contralateral TRG . These results show that unilateral injection of CFA into masseter muscle up-regulates TRPV1 expression in ipsilateral TRG, and appears not to affect TRPV1 expression level in contralateral TRG.Real-time PCR was used to examine changes in TRPV1 mRNA expression level in ipsilateral and contralateral trigeminal ganglion (TRG) after unilateral injection of CFA. TRPV1 mRNA levels were measured at post-injection day 4 when the rats exhibited significant reduction in bilateral head withdrawal threshold   (Figure Intrahippocampal injection 0.1 or 0.5 nmol of 5-iodoresiferatoxin or vehicle did not affect the baseline of head withdrawal threshold before injection of CFA in masseter muscle (Figure The purpose of this study was to investigate whether unilateral inflammation of masseter muscle can cause a bilateral allodynia in rats and if so, whether TRPV1 channels may contribute to the bilateral allodynia. We took advantage of the CFA models of inflammation that have been used in studies investigating nociception in the orofacial region ,37,39. OIn this study we found bilateral nocifensive behavioral response 1 day and 4 days after unilateral injection of CFA. This result confirms induction of bilateral allodynia by unilateral deep tissue inflammation in orofacial region. Mechanical hypersensitivity developed almost immediately at site of CFA injection and the highest hypersensitivity reached at day 4. Contralateral behavioral response to mechanical stimuli follows the same pattern as the ipsilateral inflamed side, although not as immediately nor as robustly as the injection side. Head withdrawal threshold reached lowest and highly significant difference in both ipsilateral and contralateral masseter 4 days after CFA injection. Similar to this study in rats, clinical studies report bilateral pain in patients with myofascial temperomandibular disorder ,42. BesiPrevious studies from ours and others have reported the involvement of TRPV1 in orofacial pain, and its importance in muscle nociception and mechanical hyperalgesia in animal models ,18. In tIn conclusion, the results of this study support the possibility of bilateral changes in pain response after unilateral inflammation of masseter muscle in rats, and suggest the involvement of TRPV1 channels in masseter muscle pain conditions.The authors declare that they have no competing interests.SSK carried out behavioral assays, animal surgery, histological experiments and drafted the manuscript. XHZ performed behavioral experiments. WL, YWW and IV were responsible for performance of quantitative real-time PCR. KWW participated in the study design and wrote the manuscript. All authors have read and approved the final manuscript.


Mitochondrial (mt) genes are some of the most popular and widely-utilized genetic loci in phylogenetic studies of metazoan taxa. However, their linked nature has raised questions on whether using the entire mitogenome for phylogenetics is overkill (at best) or pseudoreplication (at worst). Moreover, no studies have addressed the comparative phylogenetic utility of mitochondrial genes across individual lineages within the entire Metazoa. To comment on the phylogenetic utility of individual mt genes as well as concatenated subsets of genes, we analyzed mitogenomic data from 1865 metazoan taxa in 372 separate lineages spanning genera to subphyla. Specifically, phylogenies inferred from these datasets were statistically compared to ones generated from all 13 mt protein-coding (PC) genes  to determine which single genes performed “best” at, and the minimum number of genes required to, recover the “supergene” topology. Surprisingly, the popular marker COX1 performed poorest, while ND5, ND4, and ND2 were most likely to reproduce the “supergene” topology. Averaged across all lineages, the longest ∼2 mt PC genes were sufficient to recreate the “supergene” topology, although this average increased to ∼5 genes for datasets with 40 or more taxa. Furthermore, concatenation of the three “best” performing mt PC genes outperformed that of the three longest mt PC genes . Taken together, while not all mt PC genes are equally interchangeable in phylogenetic studies of the metazoans, some subset can serve as a proxy for the 13 mt PC genes. However, the exact number and identity of these genes is specific to the lineage in question and cannot be applied indiscriminately across the Metazoa. PlasmodiumAll metazoans  possess a symbiotically derived genome within their mitochondria, the remnant of a bacterial endosymbiosis established early in eukaryotic evolution some two billion years ago http://www.barcodeoflife.org/). In this context and given that data from mt genes will continue to play a significant role in various biological disciplines, it is essential to: 1) identify whether each mt gene possesses the same phylogenetic signal and is thus interchangeable; 2) distinguish if particular genes perform better at representing the phylogenetic signal of the entire mitogenome; 3) determine just how many genes might be required to recreate the same topology as inferred from the complete mitogenome; and 4) establish whether any trends in #1–3 are lineage specific or can be generalized across metazoans. Answering these questions will shed light on mitogenome evolution, allow commentary on the robustness of previously published studies, and help shape future research directions.As all mt genes generally reside on the same  DNA molecule, it has often been implicitly or explicitly assumed that the mitogenome is a single “supergene”, with each gene possessing an identical, or highly similar, phylogenetic signal due to their linked nature Orcinus. The behavior of individual mt genes has also been assessed when recreating mitogenome phylogenies of well-known relationships  with mixed results Several studies have tested the assumption that a single PC gene can capture the phylogenetic signal of the entire mitogenome in clades where complete mitogenome sequences were available. In plethodontid salamanders, for example, ND4, ND2, and ND5 performed best at recreating the phylogeny inferred from complete mitogenomes While evaluating how individual mt PC genes (or some subset of them) perform in a phylogenetic context for specific groups and clades is interesting, no systematic survey has yet addressed this across a wide taxonomic range of metazoans, the largest eukaryotic group in which such genes are extensively utilized. Given the increased number of complete mitogenomes that have become available, such a study is now possible. Here, we computationally analyzed publically available mitogenome sequence data from across the major metazoan lineages  to: 1) iAmbystoma were downloaded and utilized in three separate datasets representing different taxonomic “lineages”: Ambystoma, Salamandroidea, and Amphibia. Construction of each dataset was based on two criteria. Firstly, datasets were restricted to taxonomic ranks lower than phylum since mitochondrial DNA is fast-evolving in many metazoan lineages (but see Cervus nippon subspecies) were included in some datasets. Generally, all OTUs with available and complete mitogenomes were used in phylogenetic analyses, with no OTUs spanning the different hierarchical datasets . However, several individual OTUs were excluded due to ambiguous or incomplete nucleotide sequences, errors in open reading frames following translation, or unconventional naming schemes that proved incompatible with our search strategies (see Complete metazoan mitogenomes were downloaded from NCBI's organelle genome resource database between 11 June and 24 June 2010 (see below) and parsed into different hierarchical datasets see . For exa but see  and may  but see . Secondlgies see .The 13 protein-coding (PC) mt genes for each dataset were retrieved from NCBI's organelle genome resource database as both amino acid (AA) and nucleotide (nt) sequences and parsed by gene using MitoBank.pl v2.0 Amino acid and nt alignments for individual mt genes were used to infer maximum likelihood (ML) phylogenies for a subset of the datasets (see Results) with the Pthreads version of RAxML v7.0.3 http://www.auburn.edu/~santosr/sequencedatasets.htm).Phylogenies were also inferred for the datasets using concatenation of mt PC genes. The mt PC genes for each dataset were concatenated using the script fastacat.py  and phylogenies generated under ML as above. All 13 protein-coding genes were concatenated to construct the “supergene” set. Additional concatenated alignments for all 372 datasets were also generated which included only the 12 longest PC genes, the 11 longest PC genes, and so on until the final alignment contained only the single longest PC gene. Genes lengths were ranked from longest to shortest  based on a “typical” metazoan mitogenome . Phylogenies were also inferred for a subset of the datasets using a concatenation of the three mt PC genes that individually performed “best” at recreating the “supergene” topology to determine whether they performed better than utilizing a concatenation of the three longest genes. Bayesian inference (BI) analyses were conducted with PhyloBayes v3.3 L) were also calculated between the “supergene” phylogeny and each of the phylogenies inferred from mt gene subsets. This was performed for phylogenies inferred using both ML and BI approaches as well as based on either AA or nt data. To complement the SH tests, the agreement metric For each dataset, phylogenies inferred from the 13 protein-coding genes  were statistically compared to those based on: 1) individual mt genes (for a subset of 63 datasets); 2) subsets comprised of the longest protein-coding genes , and; 3) the three “best” performing mt genes (for a subset of 67 datasets). In some lineages , nearly all taxa have lost the shortest protein-coding mt gene, ATP8. Given this, comparisons were made to the phylogeny inferred from the 12 protein-coding genes see  for theshttp://www.auburn.edu/~santosr/sequencedatasets.htm) as a resource for addressing additional questions . Furthermore, new mitogenomes are frequently deposited into NCBI  and the methodology presented here can serve as an outline for future work as novel datasets become available. As a “control” for our methodological approach, 46 datasets with published phylogenies based on complete mitogenomes  from across the Metazoa were compared to phylogenies inferred here for the same datasets utilizing all 13 PC genes  (see Based on the selection criteria (see Methods), the protein-coding (PC) genes from 1865 mitogenomes constituting 372 metazoan lineages ; set) see . In geneset) see .L) relative to the “supergene” phylogeny and statistically different topologies for 54 of the 63 (86%) datasets  topology, ML phylogenies were inferred from amino acid (AA) alignments of the individual mt PC genes and statistically compared to the “supergene” topology via Shimodaira-Hasegawa (SH) tests ts e.g., . While ls length . In contength . The “supergene” topology was recreated when using only the single longest gene (ND5) in 214/372 (58%) datasets, although such datasets had a significantly lower number of OTUs . The number of OTUs in a lineage was subsequently identified as a good predictor of the minimum number of genes required (R2 = 0.451). However, taxonomic rank encompassed in the dataset and average % sequence divergence among taxa within a lineage were not good predictors of the minimum number of mt PC genes needed to infer the “supergene” topology  were statistically compared to the “supergene” topology using SH tests. Averaged across all 372 metazoan lineages, 2.16±0.09 (S.E.M.) of the longest mt PC genes statistically reproduced the “supergene” topology based on ML analyses of AA alignments and changes in Ln L scores . SimilarNotably, there was considerable variation in the estimated minimum number of mt PC genes needed to recover the “supergene” topology, sometimes corresponding to specific metazoan lineages. For instance, turtle lineages  required the largest number of genes  to recreate the “supergene” phylogeny. Some lineages also required a larger number of genes despite having a relatively low number of OTUs. The turtle lineages are also an example of this since they possessed only 8–32 OTUs but required 10 genes . Additiodf = 1, 31, P = 0.40 between AA and nt data for the larger datasets) than the identified average across all datasets were needed for this subset of the data in order to infer the “supergene” topology based on ML and SH tests. Given that AA and nt data produced similar results, AA data were utilized in the below analyses. For the 32 lineages with > 40 OTUs, agreement metrics were consistent with results derived from SH tests and ΔLn L , additional analyses were performed only on datasets with at least 40 OTUs . These analyses included: 1) comparisons between ML phylogenies using the agreement metric nd ΔLn L . PhylogeL when compared to the “supergene” phylogenies  could recreate the “supergene” topology with higher success than when genes were chosen based solely on length, phylogenies were inferred from a concatenation of the three “best” performing mt PC genes . This was done for the 67 datasets that originally required more than three mt PC genes when those genes were selected by length (these included the 32 datasets with > 40 OTUs). In these cases, phylogenies inferred from the three “best” genes performed significantly better than ones from the concatenation of the three longest mt genes , as measured via decreases in Ln OrcinusAlthough single-mt gene phylogenies generally performed poorly in the analyses presented here, some mt protein-coding (PC) genes were significantly better at reproducing a topology consistent with that of the “supergene” set, with ND2, ND4, and ND5 being the most likely to do so . These rWhy did COX1 perform poorly here in contrast to previous studies? Two significant differences between these previous studies and the analyses conducted here are the taxa in question and the taxonomic “breadth” under examination. In our study, all major metazoan lineages were analyzed, whereas in previous studies, single lineages or exemplar relationships were explored. Thus, while COX1 may provide applicable data for particular lineages Although gene length was generally a good predictor of performance, among the longest genes those encoding proteins of the NADH dehydrogenase subunits (ND) tended to outperform those encoding the cytochrome carriers . One hypothesis, consistent with the results presented here, is that functionally different classes of mt genes possess their own distinctive phylogenetic signals due to varying evolutionary and/or selection pressures. Such a situation is apparent when comparing previous studies where comparable phylogenies were generated from different functional classes of genes. For example, within the Mollusca, phylogenies based on mt protein-coding genes recover a monophyletic Opisthobranchia The data presented here suggest a select subset of mt genes can serve as a proxy for all 13 PC mt genes when inferring complex metazoan phylogenies that include many OTUs. Moreover, these were not necessarily the longest mt genes. This demonstrates that sequencing more loci, one typical answer when addressing phylogenetic questions, may not produce a significantly better topology. In support of this, increasing the number of taxa has been found to be more effective than the inclusion of additional sequence data for taxa already present in data matrices When all 372 datasets are considered, only the ∼2 longest mt PC genes, on average, were needed to infer a topology statistically indistinguishable to the “supergene” topology based on all 13 mt PC genes. However, it should be noted that the high number  of datasets 1) requiring only ND5 to recreate the “supergene” topology and 2) with a low number of OTUs, biases this estimate. Support for this comes from the fact that for datasets with > 40 OTUs, the estimate increased to an average of ∼5 mt PC genes . Likewise, greater numbers of mt PC genes were required for some specific lineages  than were estimated based solely on number of OTUs. In any case, given that single mt PC genes are usually not representative of the mitogenome while a fraction of them provides an adequate proxy supports the argument that, in most instances, entire mitogenome sequences are unnecessary towards resolving phylogenetic relationships Taxonomic rank was not a good predictor of the estimated minimum number of genes required to reproduce a topology consistent with that of the “supergene” set. This supports the idea that the traditional Linnaean classification hierarchy is arbitrary and may not reflect actual evolutionary relationships Here, we examined the performance of individual mt PC genes as well as concatenated loci across all major metazoan lineages in recreating the topology inferred from the entire mt protein-coding gene set. From a phylogenetic perspective, not all mt PC genes were found to be equally representative of the complete set and a single gene cannot serve as an adequate substitute for many phylogenetic inferences. Moreover, COX1, probably the most commonly utilized mt PC gene in studies of metazoans Figure S1Comparisons between the “supergene” topology inferred here and 46 previously published mitogenome phylogenies (see Dataset S1). The similarity of the topologies was assesed qualitatively and then converted to a score of “1” (not similar) to “5” . To achieve a score of “5” (which was given in 16 out of 46 comparisons), all taxa shared between both studies must have had identical relationships . To achieve a score of “4” (which was given in 23 out of 46 comparisons), all shared taxa had to have identical relationships except for one to three clades at the tips of the trees involving no more than five taxa. A score of “3” (which was given in 6 out of 46 comparisons) were assigned to comparisons where shared taxa had the same relationships among major lineages, but greater than three clades at the tips of the tree showed differences, involving no more than eight taxa. A single comparison received a score of “2” because while all major lineages were recovered, the relationships between them were not. Scores of “1” were reserved for comparisons where a minority of the major lineages were recovered in our analyses. Similarity between the topologies was related to the proportion of taxa shared between the two phylogenies. For example, published phylogenies often had multiple outgroups that were not present in our analyses. Furthermore, our analyses often contained dozens of taxa for particular clades that may have been represented by a single or few exemplar taxa in the published studies. These account for all comparisons receiving scores less than a “4”. “Supergene” phylogenies utilized amino acid data and were inferred via maximum likelihood for the 46 lineages examined in this comparison.(TIF)Click here for additional data file.Figure S2Comparisons between using amino acid (AA) or nucleotide (nt) data from mt protein-coding (PC) genes. Average decreases (± S.E.M.) in Ln L between phylogenies based on concatenated alignments of the longest mt protein-coding (PC) genes relative to the “supergene”  topology, as in z = −1.34, df = 1, 371, P = 0.176 between amino acid and nucleotide datasets).(TIF)Click here for additional data file.Figure S3Residuals from . Outlier datasets requiring more mt protein-coding genes than the average predicted by the number of OTUs in the dataset have residuals < 0 while those requiring less than the predicted have residuals > 0. The turtle lineages are highlighted as an outlier (see text for details).(TIF)Click here for additional data file.Dataset S1Summary of data utilized in this study. First spreadsheet contains information on all 372 datasets used in the current study, including details of phylogenies built for each analyses. Second spreadsheet contains a summary of problematic OTUs (see Methods).(XLS)Click here for additional data file.Dataset S2Data files utilized and generated in this study. All AA and nt alignments, tree files, and scripts used in this study. Found at http://www.auburn.edu/~santosr/sequencedatasets.htm.(DOCX)Click here for additional data file.


Amyotrophic lateral sclerosis (ALS) is characterized by a gradual muscular paralysis resulting from progressive motoneurons death. ALS etiology remains unknown although it has been demonstrated to be a multifactorial disease involving several cellular partners. There is currently no effective treatment. Even if the effect of exercise is under investigation for many years, whether physical exercise is beneficial or harmful is still under debate.G93A mice. At the early-symptomatic stage (P60), males were isolated and randomly assigned to 5 conditions: 2 sedentary groups (“sedentary” and “sedentary treadmill” placed on the inert treadmill), and 3 different training intensity groups . We first demonstrated that an appropriate “control” of the environment is of the utmost importance since comparison of the two sedentary groups evidenced an 11.6% increase in survival in the “sedentary treadmill” group. Moreover, we showed by immunohistochemistry that this increased lifespan is accompanied with motoneurons survival and increased glial reactivity in the spinal cord. In a second step, we showed that when compared with the proper control, all three running-based training did not modify lifespan of the animals, but result in motoneurons preservation and changes in glial cells activation.We investigated the effect of three different intensities of running exercises on the survival of SOD1G93A. Using the appropriate control, we then demonstrate that all running intensities have no effect on the survival of ALS mice but induce cellular modifications. Our results highlight the critical importance of the control of the environment in ALS studies and may explain discrepancy in the literature regarding the effect of exercise in ALS.We demonstrate that increase in survival induced by a slight daily modification of the environment is associated with motoneurons preservation and strong glial modifications in the lumbar spinal cord of SOD1 Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease characterized by a selective motoneurons death in the motor cortex, brainstem and spinal cord that leads to progressive muscular paralysis. Pathogenesis of motoneuron degeneration in ALS and mechanisms of selective vulnerability are still largely unknown although it has been demonstrated that ALS is a complex multifactorial disease  that involves, besides neurons, several cellular partners such as glial and muscle cells  exceeded that of the sedentary group (129 days) by 15 days (corresponding to an 11.6% increase), and Kaplan–Meier survival statistics revealed a significant group difference in survival time (p = 0.0015) . The exercise protocol started at the early-symptomatic stage (60 days of age) and ended when mice were unable to maintain the exercise regimens. Median survivals were of 132.5, 134.5 and 146.5 days for the 5, 10 and 21 cm/s respectively. When compared with the appropriate control, i.e. “sedentary treadmill” group, Kaplan-Meier survival statistics evidenced no significant difference for any of the trained groups  had an increased median survival than the lowest intensity group (5 cm/s) (146.5 vs 132.5 p = 0.0439).In a second step, to assess the effect of running-based exercise on the survival of SOD1. We thus monitored body weight changes in all groups (sedentary and exercise) from the beginning of the training protocol to the death of the mice. Animals subjected to the “sedentary treadmill” protocol maintained their body weight longer than “sedentary” mice (respectively until day 105 (p = 0.017) vs day 91 (p = 0.0102)). Mice subjected to high intensity exercise (21 cm/s) conserved their body weight longer than the “sedentary treadmill” group (respectively until day 119 (p = 0.026) vs day 112 (p = 0.0359)) .Over the course of the disease, all ALS mice lost weight as a result of muscular atrophy . We thugastrocnemius-soleus-plantaris complex in the two groups at P90. Similar analyses were then done for all trained groups.Differences in lifespan between the two sedentary groups suggest possible cellular modifications resulting from the “sedentary treadmill” protocol. Considering that body mass differences between “sedentary” and “sedentary treadmill” ALS mice started at 90 days of age, we characterized cellular elements of the lumbar spinal cord and the .We first quantified the number of lumbar motoneurons. We showed that “sedentary treadmill” mice exhibit a significant increase in motoneuron survival as compared to the “sedentary” animals (28.69±1.164 vs. 21.90±0.4089) gastrocnemius-soleus-plantaris complex at 90 days of age. We did not notice any statistically significant differences in NMJ’s number between all groups .Modifications of motoneuron survival in the lumbar spinal cord prompted the examination of neuromuscular junctions (NMJ) in the , characteristics of an inflammatory state. In parallel, GFAP staining was increased by ≈1.25 fold in the ventral grey matter of “sedentary treadmill” group as compared to the “sedentary” (2944±104.2 in the “sedentary” vs. 3666±136.2 in the “sedentary treadmill”) .There are growing evidences of the involvement of glial cells in ALS. To detect glial correlates of motoneuron survival in the “sedentary treadmill” group we investigated possible modifications of microglia and astrocytes at 90 days of age and at the end stage. To evaluate gliosis, we used an immunohistochemical approach to detect the microglial marker ionized calcium binding adaptor molecule 1 (Iba1) and the astrocytic marker glial fibrillary acidic protein (GFAP). At 90 days of age, we observed a marked increase in Iba1 in the ventral grey matter of “sedentary treadmill” animals as compared to the “sedentary” group. The mean intensity of labeling was indeed about 20% higher (2478±88.6 in the “sedentary” vs. 2999±82.32 in the “sedentary treadmill”) , Figure S3C). Moreover, microglia morphology of the 5 cm/s animals was similar to the one of the sedentary group .As for the sedentary groups we analyzed glial components at 90 days of age. The mean intensity of Iba1 microglia labeling was decreased in the 5 cm/s group as compared to “sedentary treadmill” mice (2621±54.49 vs 2999±82.32) . The labeling intensity of GFAP was similar in the “sedentary treadmill”, the 10 and 21 cm/s groups  .Astrocytes behaved similarly, indeed, the mean intensity of GFAP astrocyte labeling was decreased in the 5 cm/s group as compared to “sedentary treadmill” mice (2615±140.7 vs 3666±136.2)  has no effect on the survival of SOD1G93A mice as reported in a recent study that also used proper control mice The initial aim of this study was to carefully evaluate the impact of different running intensities on the survival of male SOD1gastrocnemius-soleus-plantaris complex.To tentatively correlate differences in survival induced by a slight modification of the environment with cellular alterations and to further analyze possible effects of exercise at the cellular level, we carried out a histological characterization of the lumbar spinal cord and the . Moreover, “sedentary treadmill” mice had a largest motoneuron soma  than exercised animals which may reflect a more advanced “dying-state” of motoneurons in trained groups. Thus, even motoneuron number was higher in trained animals, it most likely reflect only a slight delay in degeneration. This observation suggests that even if the 3 intensities of exercise fail to significantly modify SOD1G93A survival, moderate and high exercise intensities may delay motoneurons death.Analysis of the spinal cellular partners at 90 days of age demonstrated higher motoneuron number preservation in the “sedentary treadmill” group when compared to sedentary animals. Mice subjected to moderate (10 cm/s) or high (21 cm/s) exercise intensity presented a better motoneuron survival than animals subjected to 5 cm/s-exercise or the “sedentary treadmill” groups . MoreoveConversely, we did not find any difference in NMJ numbers between all groups but we cannot exclude that NMJ of “sedentary treadmill” and/or trained mice present a better functionality. These results are thus in favour of a central  initiation of neuroprotective mechanisms rather than the result of a centripetal muscular influence..At 90 days of age, analysis of the glial compartment allowed us to distinguish two groups according to their glial response. “Sedentary” and 5 cm/s animals showed weak microglia and astrocytes reactivity whereas “sedentary treadmill”, 10 and 21 cm/s mice harbor strong astrogliosis and a marked increase of microglial activation In particular, we evidenced an increase in GFAP staining in the lumbar ventral spinal grey matter of the “sedentary treadmill” group as compared to the “sedentary” one. This result is consistent with the finding that an enriched environment (EE) increases astrocytes number and GFAP expression in healthy and pathological conditions .EE and exercise stimulate the production of several growth factors. Amongst those, brain-derived neurotrophic factor (BDNF) level is increased in the dentate gyrus of animals housed in enriched conditions .Glutamate excitotoxicity is considered to play an important role in pathological processes of motor neurons degeneration in amyotrophic lateral sclerosis 3CL1-CX3CR1 pathway is known to provide a neuroprotective phenotype to microglia 3CL1 is increased in the hippocampus after running exercise .Besides increased astrogliosis, we also evidenced in the ventral horn of “sedentary treadmill” animals an increase in microglial reactivity. This finding is consistent with that of Williamson et al. that demonstrated Iba1 expression increase in the dentate gyrus of rats exposed to EE G93A mice. However, exercise induces modifications at the cellular level in the spinal cord. We also show that conditions of housing/handling can significantly affect the life span of SOD1G93A mice. This is of importance since it may not only explain previous outcome discrepancies reported in the literature on the effect of exercise in ALS but also, more generally, flawed results in the area of preclinical studies in CNS pathologies.To conclude, using a strictly controlled experimental protocols, we show that three intensities of running-based exercise failed to modify the lifespan of SOD1G93A. Further studies are required to identify mechanisms involved in motoneuron preservation and survival and to decipher the contributions of glial cells in ALS pathogenesis.We demonstrate that a protracted survival induced by a slight daily modification of the environment is associated with motoneuron preservation and glial modifications in the lumbar spinal cord of SOD1S2). All groups were tested between 9∶00 and 11∶00 am. To strictly follow the best practices and recommendations for preclinical studies using SOD1 mice, the age of death was recorded when mice were unable to right themselves 30s after having been pushed on their side Experimental procedures followed the European legislative, administrative and statutory measures for animal experimentation (86/609/EEC) and the Declaration of Helsinki. The study was approved by the “Direction des Services Vétérinaires de l'Hérault”, France (authorization number 34118) and ratified by the “Préfecture de l'Hérault”, France. Every effort was made to minimize the number of animals and their suffering. Transgenic mice carrying the G93A human SOD1 mutation B6SJL-Tg (SOD1-G93A) 1Gur/J (ALS mice) were purchased from The Jackson Laboratory  and bred on a B6SJL background. Transgenic mice were identified by PCR and housed in controlled conditions . Only males were used and litter-matching between groups and experiments were done as much as possible. A treadmill  was used to exercise the mice 15 min/day, 5 days a week. At day 60, males were isolated in individual cages (without any objects in their home cage) and randomly assigned to 5 different conditions: 2 sedentary groups [“sedentary” mice (n = 14) and “sedentary treadmill” mice placed on the inert treadmill (similar to trained mice but without any movement of the treadmill) (n = 13)], and 3 different training intensity groups 5 cm/s (n = 10), 10 cm/s (n = 10) and 21 cm/s (n = 10). Electrical shock system that encouraged the animals to run was disconnected to avoid bias due to stress. To accurately evaluate specific effects of the inert treadmill on the one hand and training intensities on the other hand, “sedentary” mice were handled similarly to other groups i.e. they were picked up by the tail and returned to their cage at the same time and frequency than other groups , and perfused intracardially with cold PBS followed by cold 4% paraformaldehyde . Tissues were removed and post fixed for 24 h in 4% paraformaldehyde. Samples were cryoprotected in sucrose 30%, included in Tissue Teck , frozen and kept at −80° until processing.2O2. Sections were then transferred for 1 hour in blocking solution  and Triton X-100 (0.1%) ) and then incubated 48 hours at 4°C with either Iba1  or GFAP  primary antibodies diluted in the same solution.Free floating spinal cord transverse sections (20 µm) were placed for 30 min in PBS containing lysine  and then for 15 min in 1% H202 0.1% as a substrate. Quantifications of the mean intensity labelling were done separately and blindly by two experimenters using the Adobe® Photoshop® software .Secondary rabbit peroxydase-conjugated antibody was used . Sections were then washed in TRIS buffer and enzymatic revelation was done with diaminobenzidine and HLumbar spinal cord sections were stained with Mayer hematoxylin solution for 15 minutes. Cells identified as motoneurons by their size, shape and location, were counted blindly for all groups. We quantified motoneurons soma areas using ImageJ v1.45 software . Only motoneurons with an identifiable nucleus were included in the surface analysis. One section out of twenty (i.e. each 400 µm) was used all along the lumbar spinal cord segment.gastrocnemius-soleus-plantaris muscular complex on transverse sections (16 µm). NMJ quantification was done blindly every 3rd section.Neuromuscular junctions (NMJ) labelling was done following the protocol of Karnovsky and Roots Survival was analysed using Kaplan–Meier curves and log-rank test. For the quantification of glial cells and NMJ, comparisons between “sedentary” and “sedentary treadmill” animals were done using t-test . Experiments were designed to reach a 95% power to detect a 10% or greater difference between each exercise group and the sedentary treadmill group.Figure S1“Sedentary treadmill” condition. Mice are placed on an inert treadmill 15 minutes per day, 5 days a week.(MP4)Click here for additional data file.Figure S2“Sedentary condition”. Sedentary mice were handled similarly to other groups i.e. they were picked up by the tail and returned to their cage at the same time and frequency than other groups (5 days a week).(MP4)Click here for additional data file.Figure S3Statistics. (Table A) - Table of p-values for the survival analysis. (Table B) - Summary of p-values for motoneurons quantification. (Table C) - Table of p-values for Iba1 quantification. (Table D) - Table of p-values for GFAP quantification. * = significantly different as compared to the sedentary treadmill group ; # = significantly different as compared to the 5 cm/s group ; NS = non significant.(DOC)Click here for additional data file.


Entangled quantum states of channeled proton trajectories are mapped in transverse and angular phase space of 29Si 29Si. Quantum discretization of density of states (DOS) was performed by the Monte Carlo simulation method using numerical solutions of proton equations of motion. Distribution of gaussian coherent states is obtained by continuous modulation of individual spin phase and amplitude. Obtained results allow precise engineering and faithful mapping of spin states. This would provide the effective quantum key distribution (QKD) and transmission of quantum information over remote distances between quantum memory centers for scalable quantum communication network. Furthermore, obtained results give insights in application of channeled protons subatomic microscopy as a complete versatile scanning-probe system capable of both quantum engineering of charged particle states and characterization of quantum states below diffraction limit linear and in-depth resolution.Selective coherence control and electrically mediated exchange coupling of single electron spin between triplet and singlet states using numerically derived optimal control of proton pulses is demonstrated. We obtained spatial confinement below size of the Bohr radius for proton spin chain FWHM. Precise manipulation of individual spins and polarization of electron spin states are analyzed via proton induced emission and controlled population of energy shells in pure PACS numbers: 03.65.Ud, 03.67.Bg, 61.85.+p, 67.30.hj Major progress of experimental techniques as well as theoretical models during the last few decades, has made possible the comprehensive analysis of the ion beams collision dynamics For transverse energy Accordingly to equations (1.31), when the ion beam incident angle is close to zero 29Si nanocrystal. We analyze precise control of entangled proton trajectories and discrete quantum states of phase space in connection to selective spin manipulation. The harmonic motion of highly correlated channeled protons is tuned by external RF field, by varying the CP energy and tilt angle relative to main In this paper we present theoretical study of localization and coherent control by superfocused channeled protons, CP beam induced polarization of individual electron spins in pure According to Liouville's theorem The subsequent parts of paper are organized as follows:Section 2 following the recent experimental attempts to realize electron spin processor in silicon capable of quantum information processing introduces quantum model for excitation and coherent control of electron spin states via entangled proton trajectories. Exchange coupling is analyzed under quasiharmonic approximation of interaction potential taking into account the constraint of singular proton flux density. The theoretical model is further explained by Moliere's approximation of the Tomas Fermi interaction potential. This formalism comprises Liouville's theorem to give simple explanation for mapping procedures for proton beam transformation matrix in configuration and angular phase space. Section 3 compares and discusses profiles of proton density distributions for transverse position plane (configuration space) and scattering exit-angle plane or angular space ., gives Coherent manipulation and precise control of single electron spin rotation represents first step toward quantum information processing (QIP) We have investigated the proton flux profile in scattering angle plane and transverse position plane. The boundary conditions of nonuniform density distributions are analyzed in case of small impact parameters along main g and ng are electron and nuclear effective g factors, A and B are secular and pseudosecular hyperfine couplings terms, respectively, oB is static magnetic field along z-axis. Under static magnetic field the singlet The corresponding Hamiltonian of rotating frame system , where   couples Namely, up to 1 MeV the external field induces coupling of nonparalell nuclear spin quantization axis to electron spin states and it allows the anisotropic pseudosecular term for universal control, otherwise the pseudosecular term is suppresed. Hence, adding a stronger external field to the quartic potential , alters Instead of applying the oscillating RF field to spatially resolve and manipulate spin resonance frequencies (or in order to measure response of the quantum dot by current flowing through the dot or by near quantum point), the induced transition can be generated upon CP excitation of the spin system. Thus, excited spin system displaces the center of the electron wavefunction along the oscillating superfocused CP field direction and change its potential depth. As a result the electron wavefunction frequency can be spatially distorted in order to coincides/shifts with applied CP field.A single spin excitation is then polarized along z axis coinciding with proton beam alignment. In addition, the resultant mixed state conserves the total angular momentum of the exchange Hamiltonian along z axis:In present case, numerical solutions of entangled proton trajectories, for different reduced crystal thicknesses and tilt angles, correspond to short range correlated proton - lattice interaction potential in vicinity of The interaction between the proton and the crystal's atoms includes elastic collisions, assuming classical, small-angles model of channeling ith atomic string, We have modeled the system considering the Lindhard continuum approximation for axial channeling The specific electronic energy loss is determined by equationThe angular frequency of the electron oscillation induced by the channeled proton isFurther calculations take into account the proton beam divergence before its interaction with the crystal The Monte Carlo simulation method has been used for parameterization of entangled proton trajectories. Obtained numerical solutions of channeled protons equations of motion correspond to their angular and spatial distributions. The phase space density, according to the Liuoville's theorem, cannot be changed in conservative system, but one can manipulate with the form and position of the phase space elements. We can use the phase space transformations to improve the channeling efficiency.Discrete map of quantum states of channeled proton trajectories, their point transformation in the spatial (transverse) and angular phase space are presented in the following vector basisAlthough the phase space is six dimensional we consider four subspaces of the transverse and angular phase space. Correspondingly, the mapping of beam parameters and quantum discretization of entangled states is conformally rescaled through phase space transfer matrix In that sense we consider local symplectic condition for the realizable transfer matrix of the Hamiltonian systemAccording to the Liuoville's theorem the conservation of the phase space volume results from the statement: Complete characterization of the phase space volume is achieved over the second order moments of transfer beam matrices:X basis of electron spin system to diagonal SZ basis We reduce system dimensionality by decoupling the 4-d phase space on 2-d: configurational Under CP interaction In order to determine the matrix elements of CP  lattice confinement potential for singlet and triplet functions we use two single electron eigenstates denoted by spatial electronwaive functions φ, Λ) and tilt angleφ. Upon the excitation energy is applied to the quantum dot inside the Bohr radius, it is shown that spin system energy cost for adding an extra electron starts from state S , as indicated by dotted black line, where  J(ε) and EST denote the charge state with n and m electrons, exchange and splitting energy, respectively. The energy cost for reaching  is (nearly) independent of the spin configuration.Applying the inhomogeneous CP field along main crystal axis, i.e., involving x and y phase space components of tilted CP beam, if the energy difference of triplet and singlet electron states is close, they became strongly mixed. The triplet However, the energy cost for forming a singlet state S  is much lower than that for forming a triplet state (not shown in the diagram). This difference can be further exploited for spin initialization and detection.a , b , c ) gives 3-d representation of channeled protons contour plots for 92 nm φ = 0.05cψ, φ = 0.15cψ and φ = 0.20cψ, n-discretized 2-d potential: φ≤0.05cψ, φ = 0.05cψ and φ = 0.15cψ shows strongly picked circular cross section. Only a slight variation in pattern sharpness can be seen in density profile edges. DOS analysis for 0.20cψ reveals further effects of strong perturbation, as presented in Relative change of 5% for crystal tilts leads to strong yield redistribution in angular distribution profiles and it mostly affects phase profile central parts. The analysis in configuration space for tilts: φ = 0.05cψ, 0.10cψ and 0.15cψ. In L = 1.69 µm and 0.00–0.300, for L = 99.2 nm, respectively. These dependencies determine the focusing region, i.e. specify the proton beam full with at half maximum (FWHM).φ, Λ and amplitude maxima in configuration plane is presented in A comparative analysis for the same values of cψ in transverse and angular phase plane. The normalization and boundary condition are restricted to effective Bohr radius: L = 99.2 nm gives the non-monotonic dependence to exchange coupling energy  overlap , 17.φ = 0.00, 0.05cψ, 0.10cψ, 0.15cψ, 0.20cψ, 0.25cψ, 0.35cψ, 0.50cψ. Inside the Bohr radius at distance The quantum proton trajectory evolution with various tilt angles is calculated for Λ = 0.25 in configuration space, i.e. Λ = 0.50 in (mapped) angular space. We analyze eight characteristic tilt shifts: cψ are attributed to stronger interaction influences of higher anharmonic terms in Observed amplitude dependences of proton yield for tilts ≥0.10Amplitude decreasing and changes of peaks FWHM  are indicators for the state of strong system perturbation, i.e. due to the effect of quartic anharmonic terms in the exchange interaction, cψ is disregarded, i.e. the main contribution to the superfocusing effect comes from the crystal tilts below 20% of the critical angle for channeling.The observed modulation of DOS states for tilts ≥0.20φ = 0.00cψ, The analysis of the asymptotic behavior of the channeled protons axial yield for proton distributions: This behavior is confirmed for energy range from several eV up to several MeV which can be employed for PIXE analysis. To facilitate and control close encounter collision processes in order to induce nuclear reactions one can also use this method as intermediate process for nuclear collision cascade. However, for the proton beam energies above 100 MeV the FWHM of the channeled proton peaks in density profiles is highly narrower To provide a transition to triplet state Dependency of the field caused by lifted degeneracy of triplet state further decreases the separation between energy levels, while the exchange coupling increases the Gaussian of orbitals overlap .  shows the CP simulation patterns in transverse position plane for fixed value of Λ = 0.175. The proton trajectory, shifts with tilt angle along y = 0 axis and spreads from intersection area of x-y plane into cusped elongated deltoidal pattern, in, in+n] between two nearest neighboring fractal points Λ(n) = Lαn, n→∞, where fractal dimension for α<0 draws logarithmic singularity for proton density distribution.The shifts are governed by atomic strings repulsive potential. Even a small change of tilt angle causes a strong system perturbation evident in 29Si nanocrystal. It includes atomic strings on three nearest square coordination lines of the Simulation model considers cubic unit cell representation of the isotopically pure The proton trajectories are generated from the sequences of binary collisions via the Monte Carlo simulation method using the screened Moliere's interaction potential. The crystal is tilted in angular space along the axis x and y projection of scattering small angle with respect to the 1 and Z2 denote the atomic numbers of the proton and the atom, respectively, The motion of ions in the continuum model is determined by the Hamiltoniani, j sites isn is Born exponent. Coefficients B and n are experimental fitting parameters determined from ion compressibility measurements Correspondingly, the potential between two The measure of the orbitals overlap corresponds to2 (2 s)2 (2p)6 (3 s)2 (3p)2 are calculated in Hartree unit 1 Hartree = 2 Rydberg = 27.210 eV φ.In the rotating frame, the reduced form of protons equation of motion, considering small angle approximation in transverse position plane The phase space transformations are determined via Jacobian:13 Hz. It is determined from the second order terms of the Taylor expansion of the crystal continuum potential in vicinity of the channel axis The one-dimensional thermal vibration amplitude of the crystal's atoms is 0.0074 nm The components of the proton scattering angle, The components of the proton impact parameter are obtained randomly from the uniform distributions inside the channel. The transverse components of the final proton velocity, 7 traces.In order to quantify the read out fidelity the informations from entangled quantum trajectories are sampled from To summarize, our calculations and simulation results demonstrate a hybrid proton-electron quantum interface for multipartite entanglement under constraint metric of uncertainty principle. We established the correlation between electronic spin states and off-diagonal hyperpolarized nuclear spin states under CP induced field. We used axial configuration of Si By calculating the electron spin and CP field eigenstates via full density matrix we established the proof of non-orthogonal mixed quantum state. Upon hyperpolarization sequence, the increased sensitivity of nuclear spin subspaces dependence to electron spin states reduces the linear spin entropy and leads to maximized entanglement of mixed states in density matrix. We have shown that stability dependence of nuclear field results from anisotropic term of the hyperfine coupling, here regarded as a tunable parameter for unitary spin control. It can be chosen to enhance the feasibility of producing entangled mixed states. A resultant mixed quantum state that we demonstrated in S-T systems represents important step toward realization of scalable architecture for quantum information processing. Complementary, a scalable network of entangled electron-nuclear states would form a basis for a cluster state of quantum processors integrated in silicon. In addition, generation of entanglement process comprising the network of such correlated spin states would enhance the quantum error correction beyond any separable state and extend the precision in quantum metrology. That would allow implementation of quantum error correcting techniques (QEC codes) directly to perfectly entangled mixed states and direct protection of quantum states from interaction with environment without prior entanglement purification protocols (EPP). In that context, the off-diagonal electron-nuclear eigenstates as mixed quantum states are not longer invariant under unitary spin operations and represent observables in density matrix.Finally, the controllable addressing of single spins in quantum networks, the individual control of unitary spin precessions (electron-nuclear spin phase rotations) in combination with local g-factor engineering would provide a scheme for deposition of multipartite entangled states and manipulation of quantum memory and quantum key distribution (QKD) based on transmission of gaussian-modulated individual coherent states.Another possibility for further exploration points toward active control of the channeled proton beam properties in the superfocusing effect, revealing the important role of mutual contribution of the harmonic and anharmonic terms. This emphasizes the importance in careful selection regarding the appropriate combination of the crystal tilt angle value with crystal thickness in order to gain high spatial resolution and localization accuracy. As a result the implementation of such nano-scale precision scanning method could produce a detailed map of discrete inter-atom positions, and create a highly resolved image, built-up through a process of the proton beam focusing.This behavior is confirmed for energy range from several eV up to several MeV which can be employed for PIXE analysis. To facilitate and control close encounter collision processes in order to induce nuclear reactions one can also use this method as intermediate process for nuclear collision cascade. However for the proton beam energies above 100 MeV the FWHM of the channeled proton peaks in density profiles is highly narrower