During poultry production, some birds are killed humanely on farm, usuallybecause they are ill or injured. Recent European Union (EU) legislation has restricted the numberof birds that can be killed by manual neck dislocation to 70 birds per person per day. We examinedwhether this limit is meaningful by investigating the effects of repeated application of two methodsof killing . Twelve malestockworkers each killed 100 birds  at a fixed rate with eachmethod. Both methods were highly successful, and reflex and behaviour measures confirmed theycaused rapid loss of brain function. Importantly, there was no evidence of reduced performancewith time/bird number up to 100 birds with either method. The Cash Poultry Killer caused a morerapid death, but it was prone to technical difficulties with repeated use. Neck dislocation has theimportant advantage that it can be performed immediately with no equipment, which may make itpreferable in some situations. We present the first evidence that, at the killing rates tested, therewas no evidence to justify the current EU number limit for performance of neck dislocation to killpoultry on farm.Council Regulation (EC) no. 1099/2009 on the protection of animals at the time of killing restricts the use of manual cervical dislocation in poultry on farms in the European Union (EU) to birds weighing up to 3 kg and 70 birds per person per day. However, few studies have examined whether repeated application of manual cervical dislocation has welfare implications and whether these are dependent on individual operator skill or susceptibility to fatigue. We investigated the effects of repeated application (100 birds at a fixed killing rate of 1 bird per 2 min) and multiple operators on two methods of killing of broilers, laying hens, and turkeys in commercial settings. We compared the efficacy and welfare impact of repeated application of cervical dislocation and a percussive killer , using 12 male stockworkers on three farms (one farm per bird type). Both methods achieved over 96% kill success at the first attempt. The killing methods were equally effective for each bird type and there was no evidence of reduced performance with time and/or bird number. Both methods of killing caused a rapid loss of reflexes, indicating loss of brain function. There was more variation in reflex durations and post-mortem damage in birds killed by cervical dislocation than that found using CPK. High neck dislocation was associated with improved kill success and more rapid loss of reflexes. The CPK caused damage to multiple brain areas with little variation. Overall, the CPK was associated with faster abolition of reflexes, with fewer birds exhibiting them at all, suggestive of better welfare outcomes. However, technical difficulties with the CPK highlighted the advantages of cervical dislocation, which can be performed immediately with no equipment. At the killing rates tested, we did not find evidence to justify the current EU limit on the number of birds that one operator can kill on–farm by manual cervical dislocation. Poultry may need to be dispatched on farm for multiple reasons such as culling of individual injured and sick birds, and for stock management. The methods used to cull small numbers of birds on farm are different than those for slaughter ,2,3 or eThe uptake of killing methods for small numbers of poultry depends on their practicality, cost, and availability for rapid deployment, because the majority of emergency killing needs to be done immediately and on farm sites. Manual cervical dislocation is attractive because it requires no equipment and can be performed anywhere. Although mechanically-assisted cervical dislocation shares many of these advantages, it does require a tool or aid . AltThe welfare implications of both percussive and cervical dislocation techniques are reliant on skilful application by a human operator, with human error potentially having major welfare consequences. Variation in application of cervical dislocation  has been previously documented  and demoThe aim of this study was to assess the effects of repeated application (up to 100 birds) and multiple operators on the killing efficacy and welfare impact of two commercially relevant killing methods: the CPK ,23 and cMeleagris gallopavo), 800 layer hens , and 800 broilers . The study was conducted in collaboration with three commercial poultry producers, on their farms. All birds sourced for the experiment were either destined to go to slaughter (broilers and turkeys) or were being killed at the end of their productive life (end-of-lay hens). The birds were reared and housed commercially until killing occurred. The layer hens were housed in enriched colony cages  with 80 birds per colony. The broilers and turkeys were floor housed in a clear span shed with deep litter (wood-shavings). All birds had ad libitum access to food and water. The experiment was performed under the United Kingdom’s (UK’s) Animal Scientific Procedures Act, and as part of this, underwent review and approval by SRUC’s AWERB .A total of 2400 female birds were used; 800 turkey hens . Two types of cervical dislocation were used: manual cervical dislocation for broilers and layer hens and mechanically-assisted cervical dislocation for turkeys, which mimicked commercial practice and conformed to the EU Directive (EU 1099/2009). The manual cervical dislocation used was dependent on the stockworker’s previous training and standard operating procedures at each farm, and did not always follow Humane Slaughter Association’s (HSA’s) guidelines . VariatiThe second killing method was the Accles and Shelvoke Cash Poultry Killer .22 CPK 200, (using a 1 grain (65 mg) gunpowder cartridge), a non-penetrating captive bolt percussive device. To apply the CPK, the bird’s body was held in sternal recumbency by an assistant stockworker, with the bird’s head hanging freely. The beak tip was held by the stockworker applying the CPK, ensuring that free space was available for the head to swing away. The CPK was aimed at the top of the head, with the lower curved edge of the cowl midway between the eyes, with the device slightly angled towards the tail  volunteered for the trial and were pre-screened for experience in performing cervical dislocation and were deemed competent by their respective managers. All stockworkers completed a full training day in the CPK, provided and audited by the HSA in November 2012, and all were deemed competent by the HSA representative. Biometric data for each stockworker was recorded on site and included: age (years), height (m), weight (kg), arm length (cm), and hand span (cm). The stockworkers were also asked to provide a rough estimate of how many years of experience they had in performing cervical dislocation as an emergency killing method.On each farm, 100 birds were killed with each method  per stockworker. For commercial logistical reasons, the culling sessions for all four stockworkers per farm were done over a four day period. Each stockworker performed one killing method per day. The kill method order, day, time of day, and the stockman’s role (assist or kill) was allocated and balanced according to a Latin square design framework within each farm. On each farm, the four stockworkers were paired, where one acted as the assistant to the other (handling/catching birds), and the other conducted the killing (100 birds per session). After the completion of one session, the stockworkers swapped roles within their pairs, with a break between sessions. Within each kill session, the stockworkers were not permitted to have a break, however, a predetermined killing rate of one bird per 2 min was set in order to provide enough time for the recording of reflex and post-mortem measures for every 10th bird, to prevent kill rate becoming a confounding factor between individuals and to allow comparisons between stockworkers to be made accurately across farm and bird types. Fatigue could not be measured directly within stockworker, but was implied indirectly by kill sequence (1–100) per stockworker per session. All birds were weighed and identified with a numbered leg tag immediately prior to killing. Each killing session took approximately 3.5 h to complete without any breaks.Death was confirmed in every bird by an experienced poultry technician immediately post application of a method by assessment of two parameters: (1) cessation of rhythmic breathing and (2) absence of pupillary reflex ,26. The A gross post-mortem examination was performed on every bird immediately after the application of the killing method and confirmation of death by a trained poultry technician. General binary yes/no post-mortem measures were recorded for all birds: skin broken, external blood loss, and subcutaneous hematoma. Kill method specific post-mortem measures were obtained with regard to damage to specific anatomical areas related to target areas of the approaches. For cervical dislocation methods, four post-mortem parameters were recorded: cervical dislocation confirmation (y/n), the level of vertebral dislocation , spinal cord severed (y/n), and the number of carotid arteries severed . For the CPK method, seven gross post-mortem parameters were recorded: skull fracture (y/n), skull fracture location , damage For the first and every tenth bird within a session , reflex and behaviour latencies were recorded immediately post method application. Three cranial reflexes  spreadsheets and analysed using Genstat (16th Edition). Statistical significance was termed by a threshold of 5% probability based on F tests. Summary graphs and statistics were produced at the stockworker level. For all models, the random effects included the stockworker. All fixed effects were treated as factors and classed as categorical classifications.n = 12) and kill session (n = 24), and stockworker (n = 12). In the maximal models, fixed effects included killing method, bird type, bird order, session, and all their interactions. Co-variates included bird weight and number of years of stockworker experience.Generalized Linear Mixed Models (GLMMs) using logit link function and binomially distributed errors due to the nature of the binary data were used to statistically compare kill efficacy and post-mortem parameters across stockworkers. Dispersion was fixed dependent on the variable. Random effects included in models were day of kill (n = 264 birds), the presence/absence of each reflex and behaviour was summarized into interval counts , therefore, the data was summarized into means of the maximum interval counts at the bird level for each reflex, which were then converted back into the time dimension(s). GLMMs with logit-link function and Poisson distributed errors were fitted to the interval counts. Overall statistical comparisons across the killing treatments were conducted. Random effects included in models were day of kill, kill session, and stockworker. In the maximal models, fixed effects included killing method, bird type, bird order, and interactions between major factors. Co-variates included bird weight and stockworker experience. For both reflex data (264 birds) and post-mortem data , GLMMs were used to compare consistency across stockworkers. Model set-up was as described above for both types of measure, but stockworker was no longer included as a random effect and instead listed as a fixed effect.For the subset of birds subject to reflex measurements (Mean (±SE) bird weights across the three bird types were: broiler = 2.38 ± 0.9 kg, layer hen = 2.8 ± 0.9 kg, and turkey = 12.7 ± 4.0 kg. Variation between the stockworker biometric measures was minimal and not significant. The means (±SE) for key stockworker parameters were; age = 40 ± 1 years, experience = 20 ± 1 years, height = 1.8 ± 0.5 m, and weight = 92.2 ± 0.6 kg.A total of 2400 birds were killed within the trial. While every attempt was made to keep the kill rate within each session at two minutes per bird, due to unavoidable technical issues  the rate was not always consistent , and in p = 0.023), in that success improved as time went on within a session, with the CPK improving more than cervical dislocation methods (mean kill success rates for CPK: 98.4% (birds 1–20) to 99.6%  and 97.3% (birds 80–100)). There was no interaction between kill sequence and species. The main cause for kill success failures was multiple attempts ; cervical dislocation  = 97.3% (1200 birds), including manual = 96.9% (800 birds) and mechanically-assisted = 98.3% (400 birds)). There was no difference between the CPK and combined cervical dislocation methods in terms of kill success, or within cervical dislocation methods. There was no interaction between killing method and bird type. There was a significant interaction between kill success and kill sequence (location  or misfip = 0.040), again suggesting that method success improved over time, but there was no effect of species or weight on method success.In all killing treatments, method success was numerically lower than kill success, however, a minimal difference was seen with the CPK, suggesting that when it was successfully applied, it produced optimal damage to the bird . There wp = 0.042) and method success (p < 0.0001), with both kill and method success being more likely with a higher dislocation (kill success (y) C0–C1 = 53.3%, C1–C2 = 29.6%, C2–C3 = 16.3%, >C3–C4 = 0.8%; method success (y) C0–C1 = 53.7%, C1–C2 = 29.5%, C2–C3 = 16.3%, >C3–C4 = 0.5%). There was also a significant interaction between dislocation level and species (p < 0.0001), with 83.5% of turkeys receiving a C0–C1 dislocation, while only 52–65% of broilers and layers received the same. However, it is interesting to note that no broilers received a dislocation lower than C2–C3, while the lowest recorded dislocation for hens and turkeys was C4–C5 (p = 0.036), with artery severance more likely in turkeys (mean (±SE) = 1.2 ± 0.1 carotid arteries) compared to layer hens (0.1 ± 0.1), however, bird type was confounded with cervical dislocationmethod. Broilers were removed from analysis as no carotids were ever severed, but stretch damage was noted. Stockworker had an effect on the mean level of dislocation achieved (p = 0.002), with stockworker B and D both on average achieving a C0–C1 in >96% of their birds, while the joint worst performing stockworkers (E and G) achieved a C0–C1 in less than 20% of their birds. However, the stockworker effect was confounded with bird type: both stockworkers B and D were from the turkey farm, while stockworkers E and G were from the broiler farm. Kill sequence had no effect on dislocation level, carotid artery severance, and skin damage.For birds that were successfully killed by cervical dislocation (97.3%), the majority received a C0–C1 dislocation (53.3%), did not have the neck skin torn (97%), and only 24.2% of birds had one or more of the carotid arteries severed. Dislocation level had a significant effect on kill success (as C4–C5 . Body weFor birds that were successfully killed by the CPK (99.1%), over 90% received damage to all regions of the brain assessed (forebrain (left (97.7%) and right (90.2%)), midbrain (left (99.2%) and right (99.2%)), and cerebellum (97%)). Statistical analysis could not be carried out on brain damage data because of its lack of variation and the low number of unsuccessful kills for comparison.p < 0.001); pupillary reflex could not be modelled as too few birds killed with the CPK showed this reflex). Jaw tone was abolished with all methods in a mean time of <6 s after method application, with cervical dislocation taking 1.7 s longer than CPK, and showing more between bird variation. There was no effect of kill sequence on any behaviour or reflex parameter.Of the birds whose reflexes were measured, no birds showed rhythmic breathing post successful method application for any method. In general, birds killed with CPK displayed fewer reflexes post successful method application than those killed by cervical dislocation , and thep < 0.001) and nictitating membrane reflex (p < 0.001) was reduced if one or more carotid arteries were severed. Dislocation level was also associated with time to loss of jaw tone (p = 0.009), pupillary (p = 0.003), and nictitating membrane (p < 0.001), with higher dislocations associated with more rapid loss of each reflex. Statistical analysis could not be carried out on reflexes in relation to brain damage data from CPK use because of its sparseness, lack of variation, or because reflexes were not seen.The mean durations of reflexes and behaviours post killing method application for cervical dislocation  and CPK Practical evaluations of on-farm killing methods in commercial environments are vital to accurately assess performance across multiple operators and in continuous use. In this study, both the CPK and cervical dislocation were highly successful with no evidence that one approach was significantly better than the other, in all three bird types. The main cause for kill success failures was multiple attempts , but also prevented a potential confound of some methods being faster to perform and potential fatigue effects. Thus, the experiment investigated the effects of repeated and sequential use of each method, which, while incorporating an element of fatigue, did not reflect maximal commercial killing rates with the methods. Nevertheless, there was no evidence of a reduction in kill success or method success with sequential use. In fact, the opposite was observed with some evidence of improved kill success with greater sequential use of the CPK, possibly reflecting within-session improvement with practice. This was not observed for cervical dislocation. However, the kill fail rate across all methods was very small (43/2400) and the The CPK was the most prone to technical issues, which resulted in the longest delays. Twice, the CPK jammed due to a build-up of carbon deposits; twice, delays were caused by mis-fires or, in one case, partial explosion of the cartridge. On three occasions, the CPK had to be swapped with the spare in order to continue the experiment . The CPKs were cleaned at the end of every day  by a trained individual, however, this did not prevent these issues from occurring repeatedly. It was noted that the recuperating sleeves which return the bolt to its initial firing position suffered higher than expected degradation after approximately 600 shots, and this may have contributed to failures. It is also essential to retrieve all fired cartridges, which can be difficult and time consuming, particularly in commercial settings on deep-litter floors. Another practical issue with the CPK is the requirement for the bird to be restrained, in this case, it was by another stockworker, however, in a commercial setting, this is unlikely and instead a killing cone or a rope would be used to hang and shackle the bird by the legs. This highlights that it is a potentially less convenient method than manual cervical dislocation, which requires no additional tools or support to apply.Although the mechanically-assisted cervical dislocation was a form of cervical dislocation, care should be taken (because of the techniques involved) when comparing the results from previous studies or grouping it with manual cervical dislocation. For this reason, we have reported the data with the two methods combined and separated. The primary issue identified with the dislocation methods is the variation in application between stockworkers, as seen in post-mortem measures of dislocation level which in turn was related to reflex responses. Variation in manual cervical dislocation performance across multiple operators has been observed previously . This emAs with trauma, abolition of reflexes and durations of behaviours were not affected by repeated application of the methods, suggesting that for both methods, killing up to 100 birds resulted in reliable and rapid kills (at least at the rates used). Overall, the CPK was associated with better welfare outcomes, with shorter durations of reflex and behaviour persistence, and fewer birds exhibiting them. Similar results have been reported previously, showing captive bolt methods to be humane when applied successfully ,14,24,25Both of the methods tested  are highly successful and acceptable on-farm culling methods for broilers, layers, and turkeys. Reflex and behaviour measures showed that both methods caused loss of brain function rapidly, however, there was more variation in birds killed by neck dislocation than CPK. High neck dislocation was associated with improved kill success and more rapid loss of reflexes compared to lower level dislocations for cervical dislocation. The CPK caused damage to multiple brain areas with little variation; post-mortem measures for neck dislocation showed greater variation as a result of the stockworker applying the technique. There was no evidence of a negative effect of sequential application of the methods on killing efficacy or welfare impact in this experiment, up to 100 birds at a killing rate of one bird per 2 min. However, it is important to note that technical difficulties with the CPK resulted in excessive delays, which could have compromised bird welfare in a realistic commercial setting.


VHL mutations were found to be universal throughout individual tumors when it occurred (ubiquitous), while the mutations in other tumor suppressor genes tended to be detected only in parts of the tumors . ITH has been studied mostly by DNA sequencing in limited numbers of samples, either by whole genome sequencing or by targeted sequencing. It is not known whether immunohistochemistry (IHC) can be used as a tool to study ITH. To address this question, we examined the protein expression of PBRM1, and PBRM1-related proteins such as ARID1A, SETD2, BRG1, and BRM. Altogether, 160 ccRCC (40 per stage) were used to generate a tissue microarray (TMA), with four foci from each tumor included. Loss of expression was defined as 0–5% of tumor cells with positive nuclear staining in an individual focus. We found that 49/160 (31%), 81/160 (51%), 23/160 (14%), 24/160 (15%), and 61/160 (38%) of ccRCC showed loss of expression of PBRM1, ARID1A, SETD2, BRG1, and BRM, respectively, and that IHC could successfully detect a high prevalence of ITH. Phylogenetic trees were constructed that reflected the ITH. Striking co-losses among proteins were also observed. For instance, ARID1A loss almost always accompanied PBRM1 loss, whereas BRM loss accompanied loss of BRG1, PBRM1 or ARID1A. SETD2 loss frequently occurred with loss of one or more of the other four proteins. Finally, in order to learn the impact of combined losses, we compared the tumor growth after cells acquired losses of ARID1A, PBRM1, or both in a xenograft model. The results suggest that ARID1A loss has a greater tumor-promoting effect than PBRM1 loss, indicating that xenograft analysis is a useful tool to investigate how these losses impact on tumor behavior, either alone or in combination.Recent studies have shown that intratumoral heterogeneity (ITH) is prevalent in clear cell renal cell carcinoma (ccRCC), based on DNA sequencing and chromosome aberration analysis of multiple regions from the same tumor.  Tumors are generally thought to originate from one or a few cancerous cells with founding mutation(s), with additional mutations occurring in later stages of tumor development to promote disease progression . During ITH poses a serious challenge to precision medicine. Precision medicine assumes that tumors in different patients have different genetic mutations, and the presence of specific mutations indicate sensitivities to certain treatments. Thus, treatment options must be individually tailored according to the mutation profile of each patient. Often a single biopsy is applied to assay the mutational profiles of the patients. If the tumors have regional heterogeneity, then the evaluation of a single site will likely miss many DNA mutations that are present in other regions of the same tumor and will also fail to pinpoint which mutation(s) is most prevalent and whether it should be targeted. Such incomplete information for a given tumor is likely to negatively impact the selection of therapeutic options. Effective therapeutic options might be overlooked, and wrong choice might be made.VHL, either through DNA mutations or promoter hypermethylation, is the founding tumorigenic event [PBRM1) tumor suppressor gene, which encodes a component of a SWI/SNF chromatin-remodeling complex [BAP1) or SET domain containing 2 gene (SETD2), encoding a histone deubiquitinase and a histone methyltransferase, respectively [PBRM1 mutations have been found to confer a slight increase of death risk, while SETD2 or BAP1 mutations were associated with serious death risks in ccRCC patients [In ccRCC, inactivation of the von-Hippel Lindau tumor suppressor gene, ic event , 11. VHLic event –14. Appr complex . Additioectively . Furtherpatients –17.SETD2, PTEN and KDM5C, suggestive of convergent phylogenetic evolution. Gene-expression signatures, allelic composition and ploidy profiling analysis all revealed extensive ITH. When all the changes were compared, chromosome 3p loss and VHL alterations were the only ubiquitous events in ccRCC, and they were defined as truncal losses [PBRM1 mutations, 50% of tumors with PBRM1 mutations were truncal [VHL, PBRM1, SETD2, BAP1 and KDM5C in multiple regions of the same ccRCC tumors [Gerlinger et al. firmly established that ITH in ccRCC was prevalent. Through exome sequencing of multiple intratumoral regions, they found that ITH preceded any treatment . They all losses . In the  truncal . The vas truncal . These IC tumors . CollectTo date, ITH in ccRCC has been mostly studied using Next Gen Sequencing, which revealed DNA mutations in exomes. ITH in ccRCC has also been studied based on the assessment of DNA copy number changes, gene expression or ploidy, but mutational analysis has provided the most detailed information to facilitate comparisons. While DNA sequencing provides high quality data and high resolution, the major drawback of this approach is that it is expensive and labor intensive, so the total number of the samples that are analyzed is generally low, thus lacking statistical power. Immunohistochemistry (IHC) was also used to probe the samples, but it was only used to characterize samples after ITH was identified by other techniques, not as a tool to reveal ITH in the first place.Since IHC is a mature technology that can be applied to human sample relatively inexpensively once a tissue microarray (TMA) is built, in this study we asked whether IHC on ccRCC TMAs could be used to detect ITH. Four spatially distinct regions from each ccRCC tumor were selected for TMA. Forty cases were chosen for each tumor stage, with a total of 160 cases. We decided to stain for PBRM1, ARID1A, BRG1, BRM, and SETD2 as they represent important players in ccRCC. We report that IHC can readily detect prevalent ITH in a relatively large series ccRCC, and striking co-losses among proteins were observed. Finally, we demonstrate in xenografts of ccRCC how losses of PBRM1 and ARID1A impact tumor growth.Written informed patient consent was obtained under a protocol (IRB#13–810) approved by the IACUC of Fox Chase Cancer Center (FCCC). All samples were collected in accordance with institutional guidelines and protocols.E.D.). Four different, well-separated areas from each tumor were selected for assessment of intratumoral heterogeneity. Eight TMAs were generated by the FCCC Biosample Repository.Altogether, 160 ccRCC patients with available archived formalin-fixed, paraffin-embedded (FFPE) tumor tissue were identified based on a search of the FCCC kidney cancer database; 40 cases from each of the 4 stages (Stage I-IV) were randomly selected. All cases were reviewed by the same pathologist  for a total application time of 64 min. Primary immunostaining was performed using antibodies against PBRM1 (1:50), ARID1A (1:250), BRM (1:50), BRG1 (1:200), and SEDT2 (1:100) using Ventana Antibody Dilution Buffer (Ventana cat. #ADB250) with a 44-min incubation at room temperature. Secondary immunostaining used a rabbit Horseradish Peroxidase (HRP) multimer cocktail (Ventana cat#760–500) and immune complexes were visualized using the ultraView Universal DAB (diaminobenzidine tetrahydrochloride) Detection Kit (Ventana cat#760–500). Slides were then washed with a Tris-based reaction buffer (Ventana cat. #950–300) and stained with Hematoxylin II (Ventana cat. #790–2208) for 8 min. The antibodies used were: PBRM1 , ARID1A , BRM , BRG1 , and SETD2 .W.J., T.P.) independently performed the scoring of the immunostained TMAs. The staining of tumor foci was scored as 2 if greater than 50% of tumor cells were positive, 1 if less than 50% but greater than 5% of tumor cells were positive, and 0 if less than 5% of tumor cells were positive. In the cases where the foci had different scores, two pathologists examined these foci together to reach a consensus. For each tumor, if one marker was scored as 0 in one focus, then that tumor is also considered to have a score of 0 for that marker.Two pathologists  supplemented with a protease inhibitor cocktail. The same total amount of each lysate was loaded and resolved by SDS–PAGE and analyzed by immunoblotting. The blots were developed with Super Signal Pico substrate  or Immobilon Western substrate . The antibodies were the same as the ones used for IHC.GCGCG CUUUGUAGGAUUCGTT; PBRM1-94: CCGGAGTCTTTGATCTACAAA; ARID1A-90 CCTCTCTTATACACAGCAGAT; BRM-329: GCTGAGAAACTGTCACCAAAT; BRG1-551: CCGAGGTCTGATAGTGAAGAA; SETD2-30: CCTGAAGAATGATGAGATAAT; SETD2-32: GCCCTATGACTCTCTTGGTTA.All the shRNA constructs except for SCR were obtained from Sigma . The sequences were: SCR: 7 cells were injected subcutaneously into the flanks of nude mice. Each mouse was injected with two cell lines, one on each flank. The mice were anesthetized with isoflurane before injection. The mice were monitored once weekly by an investigator from Yang lab during the experiment and daily by the staff of the animal facility. The mice were followed according to an early/humane endpoint protocol, with. euthanization of animals if the tumors become ulcerated, impede the mice’s ability to reach food and water, or cause weight loss of 20%. In our experiments, no mouse died before the experimental endpoint. The mice were maintained in groups of five per cage with 12 hours of light and 12 hour of darkness light cycle. Food and water were monitored daily by staff from the animal facility. Forty immunocompromised male nu/nu nude mice of four weeks old were purchased from Taconic or Jackson labs. All mice were sacrificed by CO2 inhalation 8 to 10 weeks after injection of cells, and tumors were excised and weighed.All animal experiments were conducted in accordance with a protocol (#01462-935A) approved by the IACUC of Thomas Jefferson University. The subcutaneous nude-mice xenograft assay was performed as described . For eacResults are reported as mean ± s.e. of the mean. Nine to ten pairs of tumors were compared. Results were statistically evaluated with Mann–Whitney U statistic analysis from SigmaPlot.The two-sided Fisher’s exact test was used to test the association between variables (PBRM1 vs. ARID1A etc.) in the 2x2 tables shown in PBRM1 (top left). The same antibody was used by others for IHC and the loss of IHC signal mostly coincided with DNA mutations in PBRM1 [ARID1A (top middle). This antibody has been previously used and validated by the Human Protein Atlas project [BRM (top left). This antibody was also used and validated by the Human Protein Atlas project [BRG1 (bottom left). This antibody was rigorously tested and confirmed by IHC staining SCCOHT tumors that contained either germline or somatic mutations in BRG1 [BRG1 mutations. The SETD2 antibody from Pro-Sci detected a band near 250 KDa, which was suppressed by two independent shRNA, constructs in the ccRCC cell line Ren-02 [bottom right). This antibody was shown by the supplier to detect an IHC signal in human placenta.In order to perform IHC on the TMAs of ccRCC tumor tissues, we sought to validate the specificity of each antibody we intended to use for scoring. The anti-PBRM1 antibody from Bethyl Laboratories revealed a major band of ~180 KDa which was diminished upon stable expression of a shRNA construct against in PBRM1 . The ant project , 23. The project , 23. It  project . The ant in BRG1 , 25. Thee Ren-02  indicates that 25% of tumors and 13% of all foci exhibited protein expression loss of the indicated marker. PBRM1 loss tended to increase with rising tumor stage, i.e., 25% (13%) for stage I, 30% (16%) for stage II, 18% (9%) for stage III, and 50% (29%) for stage IV . A similleft). In this case, losses of ARID1A and BRM were deemed to be truncal events, whereas losses of PBRM1 and BRG1 were considered branch events. Thus, a phylogenetic tree was built based on this interpretation (right). Using this method we created phylogenetic trees of tumors with protein losses in tumor stage I   . The oriIt is not known whether co-loss of these protein markers significantly impacts ccRCC tumor biology. To investigate this possibility, we next tested whether co-losses of PBRM1 and ARID1A affect tumor growth in a xenograft model when compared to single losses. We first verified that our shRNA constructs knocked down the expression of intended targets, as expected . The samITH presents an important challenge for the field of precision medicine. The goal of precision medicine is to treat patients with drugs that specifically attack the vulnerabilities conferred by the major driving mutations in a given cancer. For example, imatinib (Gleevec) is used to treat chronic myeloid leukemia  to blockITH is very prevalent in various human malignancies, and it potentially has a huge impact on how we mange and treat cancers. However, our understanding of ITH is still quite limited. DNA sequencing for mutations, either by whole exome scanning or by use of a targeted panel, has emerged as an excellent tool to study ITH. It provides a very accurate representation of ITH in tumors, and the data quality is very high. In the case of whole exome sequencing, profiles of different portions of a given tumor can provide a comprehensive view of the heterogeneity within it. However, the drawback of this approach is that DNA sequencing still tends to be expensive and labor intensive, so reports of these studies usually consist of a dozen or so tumors, even when only a panel of genes is sequenced. Such a low number of samples do not provide enough statistical power to establish an association between phylogenetic trees with clinical parameters such as patient survival and response to drugs. Regional ploidy analysis can reveal ITH but is lacking in other details, while karyotypic and chromosome microarray analysis are not convenient for large-scale analysis because of cost and/or labor. Thus, we sought to address this problem by investigating whether we could reveal ITH by performing IHC on TMAs, a very mature technique that can simultaneously analyze a large number of samples at a relatively low cost.However, a major concern for IHC is signal specificity, i.e., are the positive signals coming from the protein of interest or cross-reactivating proteins? In the case of loss of expression, did the IHC simply fail on the sample? Thus, the choice of antibodies to be used is crucial to the credibility of IHC study. We chose antibodies that have been validated in other studies as well as in our own hands. A key method to validate IHC signals is to compare them with DNA mutation profiles in the same tumor samples. It has been found that tumor samples harboring DNA mutations in a tumor suppressor gene also tend to lose expression of the encoded protein when examined by IHC, so we chose antibodies that were shown to pass this test where possible. We further examined the specificity of the chosen antibodies using shRNA and immunoblotting. Since these antibodies passed these tests, we are confident that the IHC signals are specific.Our IHC analyses revealed that ITH is prevalent in ccRCC. In most cases, lost of expression of any given protein did not happen in all four foci from the same tumor , similarThe close examination of the protein loss patterns revealed surprising co-losses of the markers , and ourPrevious studies have shown that when BRG1 is deficient, cancer cells become dependent on the presence of BRM for survival , 33. ConDNA sequencing of multiple tumor regions provides a wealth of high quality data, but the current costs and labor involved make it difficult to apply to studies of ITH on a large scale. IHC is a technique that is relatively inexpensive to survey large number of samples if TMAs are constructed. It is worth pointing out that alterations detected by sequencing and IHC are not always consistent with each other. DNA mutations do not necessarily lead to changes in protein levels, and loss of protein expression certainly may arise for reasons other than DNA mutations. Proteins are the executors of the biological actions while the DNA is the blueprint. Protein expression changes are more nuanced and fluid while DNA mutations are more definitive. Thus, these approaches are mutually supportive and should both be employed for ITH studies.ITH is a product of tumor evolution and can reflect adaptation to the microenvironment during the natural history of a neoplasm. ITH might also play a critical role in therapeutic efficacy and drug resistance. A multisite, dynamic survey of ITH within the same patients over time might elucidate how tumors evade treatments, and also provide clues about ways to modify treatments to manage a changing enemy effectively.IHC can successfully detect prevalent ITH at a large scale. We also observed striking co-losses of many markers. Using xenograft analysis, we provide evidence suggesting that ARID1A loss might be more potent in enhancing tumor growth than PBRM1 loss.S1 Fig(TIF)Click here for additional data file.S2 FigARID1A, PBRM1, or JARID1C were used for western blot analysis with indicated antibodies.Soluble lysates from 786-O kidney cancer cells stably expressing control shRNA or shRNA against (TIF)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table0 stands for expression loss and 1 for positive expression.(XLSX)Click here for additional data file.


Phenotyping was performed by determining growth rates when seeds were germinated at 25° and 15°C in order to separate the germination vigorousness from cold tolerance effects. As expected, the ability to produce viable seedlings varied widely among rice cultivars and also depended greatly on temperature. Furthermore, we observed a differential response during seed germination and in coleoptile elongation. Faster development at 15°C was observed in seeds from varieties traditionally used as cold tolerant parents by breeders, along with other potentially useful cultivars, mainly of Italian origin. When phenotypic data were combined with the panel of SNPs for japonica rice cultivars, significant associations were detected for 31 markers: 7 were related to growth rate at 25°C and 24 to growth rates at 15°. Among the latter, some chromosome regions were associated to LTG while others were related to coleoptile elongation. Individual effects of the associated markers were low, but by combining favourable alleles in a linear regression model we estimated that 27 loci significantly explained the observed phenotypic variation. From these, a core panel of 13 markers was selected and, furthermore, two wide regions of chromosomes 3 and 6 were consistently associated to rice LTG. Varieties with higher numbers of favourable alleles for the panels of associated markers significantly correlated with increased phenotypic values at both temperatures, thus corroborating the utility of the tagged markers for marker assisted selection (MAS) when breeding japonica rice for LTG.A pool of 200 traditional, landraces and modern elite and old cultivars of rice, mainly  Quantitative trait loci (QTL) mapping and genome wide association studies (GWAS) have been used with genomic data, mostly single nucleotide polymorphisms (SNP), to dissect the genetic architecture of complex traits. While QTL mapping is generally based on biparental populations, GWAS identifies QTL based on the historic recombination in a panel of diverse germplasm, via the presence of linkage disequilibrium between SNPs and QTLs. The impact of these strategies in practical breeding programs, however, has been smaller than initially envisaged, mainly because the detected QTLs generally explain only a little portion of the phenotypic variance. Most traits are affected by large numbers of small-effect genes, and then negative QTL x QTL, QTL x genetic background or QTL x environment interactions may reduce the efficiency of marker assisted selection (MAS). Epistasis is also a factor accounting for this fail in the genetic gain, but conversely MAS can also be the only way of selecting favourable epistatic combinations . More reOne interesting trait for rice breeding in temperate regions is cold tolerance, since rice cultivation in these regions deals with low temperature stress as a main yield and flowering limitation . Temperajaponica usually more cold tolerant than those from the indica group, probably as a result of the process of adaptation of japonica rice to temperate areas from its tropical region of origin . In ourifferent  referredifferent , did notjaponica types represented 90% of genotypes. Differences in model efficiency to detect false associations have been reported in association analyses performed in rice [japonica rice seeds germinated at suboptimal temperatures, and 21 of them were consistently detected when the non-japonica cultivars were also included in the analysis. Two different groups of markers were significantly associated to phenotypes determined in germination experiments performed at 25° and at 15°C. Some chromosome regions were associated to the ability to germinate at 15°C (V1514d) and others to the elongation of the coleoptile (V153w), all contributing to faster development of seeds at low temperature conditions. Similar results were referred by Jiang et al. [2 population derived from the cross between USSR5 and N22, but the number of significant QTL and their magnitudes varied at the different germination times between 7 and 15 days.Using phenotypic data for the four variables and a panel of 1672 SNP markers, GLM and MLM-based association analyses identified markers significantly associated to these phenotypic traits, although allele effects were generally low. We detected markers associated to genomic regions with positive and negative effects on the analysed phenotypes. Significance of some marker associations varied when analyses were corrected with genetic structure as a covariate, especially for the complete data set of 200 cultivars, in which  in rice . To avoihttp://archive.gramene.org/qtl/) we also found five QTLs associated with low temperature germination (qLTG) that were mapped in chromosomes 2, 4, 5 and 11 in a F2 family derived from the cross Nipponbare x Kasalath [http://qtaro.abr.affrc.go.jp/qtab/table) describes two QTLs: qLTG-5 in the cultivar Milyang23  and qLTG-7 in cultivar Kinmaze . The regions tagged in our analyses are located at different positions to the referred for these QTLs, most of them detected in mapping populations derived from japonica x indica crosses and using other traits to characterise low temperature tolerance, as germination rate or seedling survival rate. However, some QTL regions identified by Pan et al. [http://rice.plantbiology.msu.edu/index.shtml), we found enzymes involved in mechanisms of stress tolerance, such as thioredoxins (i.e. Os01g07376), which have been related to oxidative stress response, and leucine rich repeat proteins which mediate in disease resistance (i.e. Os12g37280). Furthermore, two gene products located at chromosomes 3 and 6, Os03g12820 and Os06g06400, were referred to be differentially expressed in rice seedlings subjected to different abiotic stresses [As mentioned, several QTLs related to LTG  have been previously mapped in the rice genome –34. FurtKasalath . The Japn et al.  in chromstresses . Other pstresses . TEs coustresses . In thisjaponica rice at LTG. To validate these panels, we have obtained several families of plants derived from crosses between deficient Spanish cultivars and donor varieties identified in this work, that will be used in future studies. We aim also to compare the effect of the introduction of these QTLs from different donor genotypes into different recipient genomes.We finally investigated whether the combination of a higher number of favourable alleles in the panel of associated markers, considering both positive and negative effects as suggested by Pan et al. , correlaS1 Fig(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


The purpose of this prospective, randomised study was to interocularly compare the visual performance after implantation of two different toric IOLs with different haptic design.  59 subjects with corneal astigmatism greater than 1.25 diopter (D) were implanted with an AT TORBI 709M IOL (Carl Zeiss Meditec AG) in one eye and with a Tecnis toric aspheric IOL  in the other eye. Observation procedure was performed 12 months postoperatively. Main outcome measures included uncorrected distance visual acuity (UDVA), manifest refraction, IOL rotation, and IOL position. p = 0.3). The postoperative spherical equivalent values were significantly lower in the AT TORBI group. Mean toric IOL axis rotation was 3.0 ± 2.26 degrees for AT TORBI eyes and 3.27 ± 2.37 for Tecnis eyes (p = 0.5). The mean vertical IOL tilt and vertical decentration values measured with the Visante OCT were significantly larger in the AT TORBI group (p < 0.05).  Mean UCDVA was 0.04 ± 0.14 logMAR for AT TORBI eyes and 0.06 ± 0.15 logMAR for Tecnis eyes (NCT03371576. Both the Tecnis and the AT TORBI toric IOLs successfully reduced ocular astigmatism. Emmetropia could be better achieved with the AT TORBI IOL, whereas the Tecnis showed better positional stability. This trial is registered with ICMJE  Toric intraocular lenses (IOLs) were developed to correct the preexisting astigmatism induced by corneal toricity after cataract surgery. The goal is to provide complete visual rehabilitation for distance vision . New desSeveral reports have proven the interocular symmetry of biometric parameters like axial length, anterior chamber depth, and lens thickness between right and left eye, which all play an important role in the postoperative refractive outcome , 7. In ot-test with a two-sided significance level of 0.05. The sample was powered to detect the difference in the rotational stability of the two IOLs.In this prospective, randomized study, 118 eyes of 59 patients with age-related cataract and corneal astigmatism over 1.25 D on both eyes were included. According to a previous sample size calculations, the study had 80% of power using a paired μm), whereas the AT TORBI is a SA neutral IOL (SA: 0.0 μm).Each patient received, according to a randomization list, based on random permutation numbers one of the two lenses in one, and the second lens in the other eye. One of the lenses was the Technis toric aspheric IOL , which is a one-piece, loop-haptic, aspheric hydrophobic acrylic intraocular lens with three-point fixation and an anterior offset haptics. The other lens is the AT TORBI 709MP , which is a plate-haptic, bitoric, aspheric hydrophilic acrylic IOL. The Tecnis toric IOL aims a full correction of mean spherical aberration  of the WHO, as well as with applicable country and local requirements regarding ethics committee/institutional review boards, informed consent, and other statutes or regulations regarding protection of the rights and welfare of human subjects participating in biomedical research.Five experienced surgeons performed all cataract extractions under local anaesthesia. Preoperative, marking of the horizontal axis in seated position was performed. The self-sealing 2.4 mm superior-temporal incision (at 120 degree), injection of viscoelastic substance, capsulorhexis (with 360° overlapping edges), phacoemulsification, irrigation/aspiration of cortical material, and injection of viscoelastic substance into the capsular bag were performed as standard procedures. The IOL was implanted via injector into the capsular bag followed by thorough aspiration of the viscoelastic substance from the eye. The IOL axis was positioned on the planned meridian using a Mendez Marker Ring.Preoperatively, all patients had a complete ophthalmic examination. Biometry using the IOL Master (Carl Zeiss Meditec AG) was performed. The ULIB optimized IOL constant was used to determine IOL power for the Tecnis lens (A). The IOL power corresponding to the smallest minus refractive error, based on the IOL Master measurements, was chosen. The IOL cylinder power of the Tecnis lens and both the spherical and cylindrical power of the TORBI lens were calculated using the online Toric Calculators provided for each lens by its manufacturer, based on anterior corneal astigmatism values. Follow-up assessments were performed 1 week and 1, 3, 6, and 12 months postoperatively.Axial alignment of the toric IOL was measured using the tilted narrow light of the slit lamp , which was adjusted to the marking on the IOL, and the degree of axial alignment was read by the examiner.IOL tilt and decentration were measured with the Visante omni anterior segment OCT (Carl Zeiss Meditec AG) as described by Kumar et al. . PostopeTotal, corneal, and internal optical aberrations were measured using the iTrace VFA Visual Function Analyser . This device measures keratometry, autorefraction, pupil diameter, topography, and wavefront aberrations simultaneously on the same axis , 10. ImaPre- and postoperative cylindrical refractive errors were expressed as power vectors: as Jackson crossed cylinder J0 with axes at 90 degrees and 180 degrees and as J45 with axes at 45 degrees and 135 degrees as described by Thibos and Horner .p values for group comparisons [Statistical analyses were performed with SPSS 19.0 . Univariable and multivariable regression analyses via generalized estimating equation (GEE) models were used to compare visual, refractive, aberrometric, and IOL alignment parameters between the study groups. In GEE models, data from the two eyes of the same subject were statistically analysed as repeated measures. Thus, this analysis takes into account the correlated nature of data from the two eyes of the same patient and provides valid parisons .The study enrolled a total of 118 eyes of 59 patients with age-related cataract and corneal astigmatisms over 1.25 D. Mean age was 66.83 ± 11.54 years in the study group, and the female/male ratio was 33/26. Twelve months postoperatively there was no significant difference in manifest refractive cylinder between the two groups .  Figurewhole eye aberrations and image quality characteristics of the two groups are presented in p > 0.05). The ocular vertical tilt (Z1−1) was significantly lower in the Tecnis group whereas the ocular horizontal tilt (Z1 [3−1) was significantly lower in the Tecnis group as well, but not the ocular horizontal coma (Z3 [p = 0.07). Due to the different asphericity of the two IOLs, the spherical aberration (Z40) of the AT TORBI group was significantly larger. The average height of the ocular MTF curves for higher-order aberrations was significantly higher in the Tecnis group than in the AT TORBI group, whereas the MTF for all aberrations did not differ.The postoperative al tilt Z−1 was siThe IOL rotation measured at the 12th month follow-up visit was not statistically different between the two study groups . The meaThe mean vertical IOL tilt and vertical decentration values measured with the Visante OCT were significantly larger in the AT TORBI group. Horizontal tilt and decentration showed no difference between the two groups. p < 0.001) deeper in the Tecnis group  as in the AT TORBI group . Postoperative anterior chamber depth values measured with the Visante OCT at the first, third, sixth, and twelfth month visit were all significantly  and also necessitates the preoperative anterior chamber depth value and estimates the postoperative ACD. Another difference between the two methods is that the Zeiss algorithm uses meridional analysis for calculating the spherocylindrical IOL power. This means that IOL power is calculated separately for both the steep and flat corneal meridians instead of using the mean keratometry value. According to Fam and Lim , this meSeveral studies have addressed the importance of estimated effective lens position (ELP) on IOL power prediction accuracy, which is also influenced by the haptic design –21. The Other than the Tecnis IOL with its anterior offset haptics, the plate haptic TORBI lens with its broad haptic-optic junction does not bend the posterior capsule. This explains our finding that the ACD in the Tecnis group was significantly deeper as in the AT TORBI group, mean value of this was 4.58 ± 0.31 mm. In a similar setting, Hirnschall et al.  measured1−1) and coma (Z3−1). The Strehl ratio and the height of the modulation function (MTF) curve for higher-order aberrations were both larger in the Tecnis group, indicating a better optical quality. Due to the different asphericity of the two IOLs, the spherical aberration (Z40) of the AT TORBI group was significantly greater. Aberrations is correcting aspheric IOLs as the Tecnis aim a full correction of the mean corneal spherical aberration (SA), which leads to an improved contrast sensitivity and optical quality. As corneal asphericity was similar in the groups, the better Strehl ratio and MTF values are probably due to the IOL.Further analysing the distribution of postoperative ACD values of the two IOLs , the deeOur study has confirmed the previous findings that both lenses have a high degree of rotational and centration stability while the refractive outcome was better in the AT TORBI group and the SA correction was better in the Tecnis group.There are some limitations of this study. The IOL constants of the formulas calculating the spherical power of the Tecnis lens were not personally optimized, which would avoid systematic errors in the preoperative measurements, instead they were taken from the ULIB database . Further


The effect of RHI on ptau pathology was partially mediated through increased CD68 positive cell density. A binary logistic regression demonstrated that a diagnosis of dementia was significantly predicted by CD68 cell density  independent of age , but this effect disappeared when ptau pathology was included in the model. In conclusion, RHI is associated with chronic activation of microglia, which may partially mediate the effect of RHI on the development of ptau pathology and dementia in CTE. Inflammatory molecules may be important diagnostic or predictive biomarkers as well as promising therapeutic targets in CTE.The chronic effects of repetitive head impacts (RHI) on the development of neuroinflammation and its relationship to chronic traumatic encephalopathy (CTE) are unknown. Here we set out to determine the relationship between RHI exposure, neuroinflammation, and the development of hyperphosphorylated tau (ptau) pathology and dementia risk in CTE. We studied a cohort of 66 deceased American football athletes from the Boston University-Veteran’s Affairs-Concussion Legacy Foundation Brain Bank as well as 16 non-athlete controls. Subjects with a neurodegenerative disease other than CTE were excluded. Counts of total and activated microglia, astrocytes, and ptau pathology were performed in the dorsolateral frontal cortex (DLF). Binary logistic and simultaneous equation regression models were used to test associations between RHI exposure, microglia, ptau pathology, and dementia. Duration of RHI exposure and the development and severity of CTE were associated with reactive microglial morphology and increased numbers of CD68 immunoreactive microglia in the DLF. A simultaneous equation regression model demonstrated that RHI exposure had a significant direct effect on CD68 cell density ( Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disease associated with a prolonged history of repetitive head impacts (RHI) including concussive and subconcussive hits , 2. AthlGrowing evidence suggests that cumulative RHI exposure is associated with the development and increased severity of CTE , 5–8. WeAlthough mild traumatic brain injury has been associated with multifocal axonal loss and microglial activation , the facHere, we test the hypothesis that increased neuroinflammation, defined by an increased astrocyte and microglial number and an upregulation of the inflammatory/phagocytic marker CD68, is associated with a history of longer exposure to RHI, increased CTE ptau pathology, and increased risk of developing dementia in a cohort of American football players and non-exposed control subjects.A total of 66 brains from former American football players with a history of RHI from the VA-BU-CLF Brain Bank were neuropathologically evaluated for the changes of CTE, as well as other neurodegenerative conditions using previously published selection criteria and protocols . SubjectInstitutional Review Board approval for post-mortem clinical record review, interviews with family members, and neuropathological evaluation was obtained through Boston University School of Medicine. RHI history, concussive events, athletic or military service history, history of cognitive, mood, and behavior changes, and clinical status leading up to death were assessed with post-mortem interviews with informants and through online surveys and medical record review as described previously . RHI expNeuropathological processing followed the procedures previously established for the BU ADC and CTE brain bank, which included a comprehensive analysis designed to screen for neurodegenerative conditions , 17. TheAll brain tissue was processed identically by fixation in periodate-lysine-paraformaldehyde and stored at 4 °C. A tissue block from the dorsolateral frontal cortex was taken perpendicular to the superior frontal sulcus, embedded in paraffin and cut at 10 μm (AT8) or 20 μm . Antigen retrieval was performed by boiling sections in citrate buffer (pH 6.0) for 10 mins. Sections were incubated at 4 °C overnight with antibodies to anti-Iba1 , anti-GFAP , anti-PHF-tau (AT8) , and anti-CD68 . Sections were treated with biotinylated secondary antibodies then labeled with a 3-amino-9-ethylcarbazol HRP substrate kit (Vector Laboratories). Sections were counter stained with Gill’s Hematoxlin (Vector Laborities H-3401) and coverslipped with Permount mounting medium. For fluorescent stains, antibody visualization was performed using secondary antibodies bound to Alexa fluorophores (Invitrogen) at a dilution of 1:500. Sudan black at a dilution of 0.1 % was used to quench autofluorescence.The sulcal depths of the dorsolateral frontal (DLF) cortex are often the first regions involved by CTE pathology , suggestStatistical analysis was performed with SPSS version 20.0 (IBM inc.), Prism v6 (Graphpad Software), and SAS version 9.4 (SAS Institute). CD68 density, AT8 immunoreactive NFT density, and AT8 density were log transformed to normalize for regression analyses. A one-way ANOVA was used to compare Iba1 and GFAP cell density among control, RHI, and CTE groups. CD68 density was compared using a Kruskal-Wallis test due to the non-normal distribution of the data. Age at death was included in all regression analyses to control for age-associated changes. Since the outcome variables (CD68 cell density and AT8 density) are correlated, the use of independent regression models for each outcome would give biased estimates. To avoid this endogeneity problem, simultaneous equation modeling was used to determine direct and indirect effects of predictor variables (age at death and years of RHI exposure) on outcome variables (CD68 cell density and AT8 density). Since we have two exogenous variables (age at death and years of RHI exposure) for two outcomes (CD68 cell density and AT8 density), the model is just-identified. Years of RHI was used as a metric of exposure as opposed to number of concussion due to the previous finding CTE tauopathy stage was significantly predicted by years of contact sports play, but not number of concussions . To ascep < 0.001) but not mild CTE (p = 0.120). Furthermore, the RHI without CTE group had significantly less years of exposure to RHI than both mild (p = 0.002) and severe (p > 0.001) CTE. No significant difference was observed for number of reported concussions between the RHI exposed groups , but not number of concussions . This is consistent with previously published data [p < 0.0001) and CD68 cell density . Comparing the standardized effects demonstrated that the effect of RHI exposure on AT8 tau density was 86 % direct and 14 % via increased CD68 immunopositive cell density (Table p < 0.0001) but not CD68 cell density . In addition, CD68 cell density had a significant direct positive effect on AT8 tau pathology . AT8 density also had a direct positive effect on CD68 cell density , suggesting a positive feedback loop. The magnitude of the standardized effect of CD68 cell density on AT8 density was 3.5 times greater than the effect of AT8 tau pathology on CD68 cell density. Overall, duration of RHI had an effect on severity of AT8 tau pathology that was both direct and via increased CD68 cell density. Furthermore, the observed increase in CD68 cell density was independent of age and directly associated with duration of exposure to RHI and severity of AT8 immunopositivity  equal to 0.99. Years of RHI was used as the exposure metric instead of the number of concussions due to linear regression modeling demonstrating CD68 having a significant relationship with RHI . A probability curve was generated based on the logistic regression and is plotted in Fig. p = 0.0213) (green line) and separately, neurofibrillary tangle (NFT) density  (red line)  independent of age . Interestingly, when ptau pathology was included into the model, NFT density  significantly predicted dementia independent of age , but CD68 density  was no longer a significant predictor. This suggests that the effect CD68 has on dementia is mediated through the development of tau pathology.To test if neuroinflammation was a predictor of dementia, a binary logistic regression demonstrated dementia was significantly predicted by CD68 . Last, we found that increased neuroinflammation was related to the risk of a subject being diagnosed with dementia and that the relationship was mediated through ptau, independent of age. Altogether, this suggests that increased neuroinflammation as a consequence of prolonged RHI exposure may play an important role in the development of ptau pathology in CTE. To our knowledge this is the first data directly linking neuroinflammation to the development of ptau pathology in CTE.Although moderate/severe TBI has been observed to result in long lived neuroinflammation , even miNumerous studies have demonstrated that neuroinflammation occurs acutely following TBI , 28–30. The simultaneous equations regression analysis is a form of structural equation modeling that incorporates feedback loops  and is tThere are several limitations to our study. There is selection bias in an autopsy-based study of individuals whose brains are donated by the family, and the subjects may not represent the population as a whole. Additionally, clinical and RHI exposure histories are obtained retrospectively and are subject to bias. Future longitudinal, prospective studies will be needed to verify these results. For the simultaneous equations regression analysis, CD68 and AT8 densities were treated as both outcomes and predictors to avoid the “endogeneity” problem  a linear regression model that has independent variables as predictors would give invalid inference due to the significant correlation between the predictor and the error term, and (2) their coefficients would be biased) . To addrHere, we showed for the first time that neuroinflammation was associated with greater ptau pathology in CTE. Furthermore, we demonstrated that the duration of RHI exposure predicted increased activated microglial cell density that, in turn, was directly associated with ptau pathology Fig. . Lastly,


MIR < 0.58 (in the maturity range of 0 to 1.0) and CIIR < 42% .Cotton fibers are natural plant products, and their end-use qualities depend on their stages of development. In general, the quantity of cellulose in cotton fibers increases rapidly, thus it leads to compositional, structural and physical attribute variations among the fibers with shorter and longer growth periods. This article discusses recent progress in applying the Fourier transform infrared (FTIR) spectroscopic technique to characterize these differences, to discriminate immature fibers from mature fibers, to assess fiber maturity and crystallinity and also to unravel the band assignments in crystalline and amorphous celluloses. The results were achieved through the use of various strategies, including wet chemical analysis, principal component analysis (PCA), simple algorithm development, two-dimensional correlation analysis and other independent fiber tests. Of particular interest is that, in general, immature fibers might have the characteristics of less than 21–28 dpa,  Cellulose I (β 1→4 linked glucose residues) is a major compositional component in mature cotton fibers. Its quantity impacts the end-use qualities for yarn and fabric products. In general, cotton fiber cellulose development is considered to include at least four overlapping, but distinctive, phases: initiation, primary wall formation (elongation), secondary cell wall thickening (cellulose synthesis) and maturation [Regarding the degree of fiber maturity, cotton fibers are simply classified into two categories of immature and mature fibers . A typicCompositional and structural differences between immature and mature fibers, as well as their physical properties and end-use qualities, have been investigated considerably over the years by diversified and comprehensive techniques, including wet chemistry, microscopy, X-ray diffractometry, molecular spectroscopy and well-defined fiber testing methods in the cotton industry ,12,13,14It is well-known that glucose units form cellulose macromolecules through polymerization reaction, during the period of cotton fiber development. This biogenesis process results in a co-existence of fiber cellulose and numerous non-cellulosic components in cotton fibers ,15.et al. [Gossypium hirsutum L. cv. TX19 and TX55). As anticipated, percentages of four sugars decrease significantly for two cultivars during the fiber development, while the amount of cellulose increases as a result of secondary cell wall development. Noticeably, TX19 fibers were observed to have a rapid cellulose synthesis from 10.7% to 80.4% between 14 and 24 dpa, with a clear increase at 18 dpa (56.9%). Relatively, cellulose content in developmental TX55 fibers is 9.1% between 10 and 20 dpa, raises to 34.1% at 21 dpa and to 68.3% at 24 dpa, before staying nearly unchanged between 27 and 56 dpa. Earlier start of cellulose synthesis in TX19 fibers (18 dpa) than in the TX55 cultivar (24 dpa) could have a significant impact on fiber maturity at the end of the growing season. They attributed the six day difference to more elevated enzymatic activities in TX19 fibers than in TX55 fibers. Recently, Abidi et al.  determinet al. [−1, they monitored the dpa-dependent IR intensity variations between two cultivars (TX19 vs. TX55). One of the notable views was to examine the relationships between the 1627 cm−1 band (adsorbed water) or the 710 cm−1 band (CH2 rocking vibration in cellulose Iβ) against fiber growth. For fibers from the TX19 cultivar, the amount of adsorbed water decreases between 10 and 18 dpa, and no significant changes are observed after 19 dpa; while intensity of the 710 cm−1 band increases linearly between 10 and 30 dpa, and no major change is noticed between 30 and 56 dpa. Among TX55 fibers, the amount of adsorbed water decreases linearly until the fibers reach 24 dpa, and a major intensity change of the 710 cm−1 band happens only at 21 dpa. Probably, the reduction of the adsorbed water amount could originate from the decrease of the surface area and the minimized accessibility of water molecules to the internal hydroxyl groups, mostly due to the formation of cellulose macromolecules that induce the cellulose re-organization and also increase the ordered  cellulose portion from shorter to longer dpa fibers.As a complementary and independent approach, Abidi et al.  applied etc.) that represent the most common variations to all data. It has been used as a tool to interpret the samples on the basis of both their spectral characteristics and their logical assignment. Abidi et al. performed PCA processing of respective ATR-FTIR spectra from TX19 and TX55 cultivars [Principal component analysis (PCA) is a very effective variable reduction technique for spectroscopic data. It decomposes the spectra into mathematical spectra  for their discrimination [R1 values increase with R2 values among a data set consisting of 402 seed bolls  immature fibers and 190 of 201 (94.5%) mature samples were correctly classified. By setting an R2 threshold value at 2.24, only six immature fibers and 10 mature samples were misidentified, yielding an overall 96% accuracy in their correct differentiation. Mature and immature fibers are determined easily from conventional microscopy, but the degree of maturity is difficult to assess. This is because the microscopic procedure is subjective and depends on one’s judgment to assign the fibers into the appropriate class of either immature or mature fibers. On the ATR-FTIR spectral intensity differences between immature and mature fibers, Liu mination . They obed bolls . With anMIR) by representing the R1 values. In this concept, the MIR values of 0.0 and 1.0 were assigned to the most immature and mature fibers, respectively. Thus, immature fibers whose R1 < 0.40 correspond to a MIR < 0.58 in the maturity range of 0 to 1.0, and vice versa. In order to validate the efficiency of accessing the MIR from direct ATR-FTIR measurement, cotton fibers with various maturity readings as determined from traditional image analysis (IA) and advanced fiber information system (AFIS) were taken. Within small and selected samples, strong correlations between MIR against referenced IA and AFIS maturity readings were reported. Further, they proposed a formula to estimate the degree of cotton cellulose maturity (CI) has been used to describe the relative amount of crystalline portion in a simple two-phase model . In practice, CI of cotton fiber cellulose is determined predominantly by a curve-fitting process that extracts individual crystalline peaks from the X-ray diffraction (XRD) intensity profile. In general, XRD determination of cellulose CI provides a qualitative or semi-quantitative evaluation of the amounts of either crystalline or amorphous components in a sample [Numerous hydroxyl groups in fiber cellulose are involved in complicated intra- and inter-molecular hydrogen bonds, which yield high-order  and low-order (amorphous) regions. Crystallinity index  that related the XRD intensity with the crystalline information [CIIR from ATR-FTIR spectra and R3 from XRD pattern showed a high correlation of R2 > 0.90  at 34 dpa for Acala fibers. Given the limited number of cotton bolls in this study, it is inappropriate to draw any solid comparisons about the three varieties examined, because fiber growth depends on a number of factors, such as genotype, grown locations and crop managements.arieties . Hsieh eMIR < 0.58  and CIIR < 42%  [Careful examination of results from different strategies by independent investigators suggests that, in general, immature fibers could have the characteristics of less than 21–28 dpa, to 100%) ,14,16,18Strength of cotton fibers is one of several important end-use attributes. To measure cotton strength at the level of either single fiber or flat bundle fibers, a number of testing devices have been in practice. A traditional laboratory testing method known as Stelometer is one of them, which is a different and also complementary tool to the automatic high volume instrument (HVI™) system . With thet al. reported the optimal PLS model performance from the 1800 to 800 cm−1 region [−1 revealed a remarkable improvement in model performance. The result suggested the potential of the ATR-FTIR technique in the quantitative determination of fiber strength. Undoubtedly, more studies are necessary to unravel the strength quality trait and to elucidate the strength mechanism.By comparing the statistics of optimal results in calibration and validation sets from individual spectral region, Liu 1 region . Howeveret al. probed the relationship between single fiber strengths and crystalline structures of greenhouse-grown Maxxa and Acala cotton fibers at varying growth stages [Hsieh h stages . They foTwo-dimensional correlation (2D-Corr) spectroscopy, a universal and modern technique of vibrational spectral analysis, was originally developed as 2D IR correlation spectroscopy by Noda . Years lMajor advantages of 2D-Corr analysis include the enhancement of spectral resolution by spreading peaks over the second dimension, the band assignments through the correlation analysis and probing the complex sequence of events arising from the changes in a system. To obtain 2D-Corr spectra and also to interpret them reasonably, a limited number of spectra were arranged in an increasing or decreasing variable. On the other hand, one of specific challenges might be how to implement the 2D-Corr analysis in large and diverse spectral sets that are common in model development and include multivariate variations in chemical and physical properties. Being a method to explore the variations within a diverse data set, PCA was attempted to classify the spectra of samples . −1 fingerprint region, their spectral features have not been well understood, mainly due to the complexity of IR bands from a mixture of constituents in cotton fibers, slight spectral difference between fiber cellulose and noncellulosic polysaccharides , as well as between the amorphous and crystalline celluloses in the 1500–1200 cm−1 region [et al. explored 2D-Corr spectroscopy to analyze the ATR bands in the 1800–650 cm−1 IR region for spectral differences and characteristic band assignments between immature and mature fibers [Despite the effort of extracting specific information from relatively sharp IR bands of cotton fibers in the 1800–600 cm1 region . In ordee fibers . Example−1 region between immature and mature fibers. With respect to cotton growth, cellulose concentrating induces fiber density augmentation and cellulose chain rearrangement through the formation of effective inter- and intra-molecular hydrogen bonding and also inter-chain van der Waals interactions. Clearly, the cellulose chains occur in amorphous and crystalline regions. Hence, a spectral intensity decrease in the 1050–1000 cm−1 region might be associated with the functional groups in amorphous regions, and spectral intensity increase in the 1000–950 cm−1 region could arise from crystalline regions.−1) at the diagonal position indicates that spectral intensities of these two bands vary greatly with the growth of cotton fibers. The positive cross-peaks (solid lines) at the off-diagonal position are observed between the 1048 cm−1 band and the two bands at 1152 and 1110 cm−1, while the negative cross-peaks (dotted lines) are found between the 1048 cm−1 band and the 968 cm−1 band. Hence, the spectral intensity at 968 cm−1 increases, while those at 1152, 1110 and 1048 cm−1 decrease, as the cotton fibers grow.−1 could be related to the C–O stretching mode of primary alcohols (–C6H2–O6H) in crystalline regions of fiber celluloses. On the other hand, the reduction of spectral intensity at 1048 cm−1 might arise from at least one of two sources, either the C–O stretching mode of primary alcohols (–C6H2–O6H) in amorphous regions of fiber celluloses or the C–O vibrational mode of such noncellulosic components as sugars.With the biosynthesis of cellulose from small and non-cellulosic components , fiber cellulose is deposited, and subsequent concentrating might produce a relative increase of crystalline regions and simultaneous decrease of amorphous regions through more effective hydrogen bonding. Apparently, IR absorptions of cellulose chains in amorphous regions differ from those in crystalline regions. To this point, ATR intensity variations observed in −1 and also several cross-peaks associated with them. Both autopeaks shift to lower frequencies compared to those at 1048 and 968 cm−1 in −1 band intensity is positively correlated with the intensity decreases at 1152 and 1110 cm−1 and also negatively correlated with the increase in the band intensity at 956 cm−1. This suggests that the 956 cm−1 band is associated with the C–O stretching mode of primary alcohols (–C6H2–O6H) in crystalline celluloses, and the 1042 cm−1 band might come from the C–O stretching mode of primary alcohols (–C6H2–O6H) in amorphous celluloses. An apparent distinction between −1 in −1 band is mainly due to the antisymmetric C–O–C stretching mode of the glycosidic linkage, while the 1110 cm−1 band can be attributed to the C–O stretching mode of secondary alcohols (–C2–O2H). Two cross-peaks at 1152 and 1110 cm−1 band in −1 band in −1 from the 950 to 850 cm−1 region, suggest the intensity increase of the 895 cm−1 band with fiber development. This is anticipated, since intensity increase of the 895 cm−1 band could be indicative of the increase in glycosidic linkage in fiber cellulose. In addition, negative cross-peaks between the 1048 cmet al. [Cotton growth in greenhouse, boll picking, fiber preparation, sugar and cellulose content determination, as well as ATR-FTIR spectral acquisition and interpretation have been described in detail by Abidi et al. ,12.Two types of cotton samples were used in this study. The first group consisted of a total of 402 mature and immature seed and dried cotton fibers, representing different varieties, locations and crop years. The condition of mature and immature bolls was identified subjectively by their appearance, size and shape. The second group included two sets of lint cottons from diversified sources, and their maturity indices were measured by routine AFIS and IA procedure. All ATR-FTIR spectra of both seed and lint fibers were collected with an FTS 3000MX FT-IR spectrometer, and the data was loaded into Microsoft Excel 2000 to execute simple algorithm analysis . Eighteen seed and dried cotton bolls from 3 cultivars  were collected at 6 developmental stages of 21 through 60 dpa. The ATR-FTIR spectra of all 18 bolls were acquired with an FTS 3000MX FT-IR spectrometer, and XRD data of only 10 bolls were obtained using a Rigaku Ultima IV Diffractometer. Two data sets were exported into Microsoft Excel 2000 for simple algorithm analysis [Stelometer bundle strengths (or tenacities) of lint cottons in great diversities were obtained by following the standard protocol. The ATR-FTIR spectra from broken specimens were collected and then spectral response was correlated with Stelometer strengths through PLS regression in the PLSplus/IQ package . −1 and then were loaded into the PLSplus/IQ package for preliminary PCA characterization. Next, normalized spectral sets representing immature and mature fiber clusters were created with the ordered sequence of increasing PC1 scores, and subsequent 2D-Corr analysis was conducted [The ATR-FTIR spectra of immature and mature seed cottons were simply offset to zero at the wavenumber of 1800 cmonducted .Cotton fiber cellulose is accumulated during the secondary cell wall biogenesis. Both cellulose content measurement and the ATR-FTIR spectroscopic technique are suitable to monitor the different transition phase between the primary wall and the secondary cell wall for two cultivars, with TX19 fibers occurring between 17 and 18 dpa and TX55 fibers between 21 and 24 dpa. MIR and CIIR, as well as CIXRD determination, indicates that, in general, immature fibers could be distinguished by the characteristics of less than 21–28 dpa, MIR < 0.58  and CIIR < 42% . Comparison of comprehensive observations on developmental fibers from cellulose content measurement, PCA pattern, −1 region. Intensity increase of the 956 cm−1 band in the 900 and 1000 cm cm−1 region, together with the intensity decreases of several bands in the 1000 and 1100 cm−1 region, is a mainstream of cotton fiber development and maturation. These unique bands could be useful in interpreting two key bands at 1032 and 956 cm cm−1 that were utilized to develop a simple algorithm for the classification of immature fibers from mature ones.The 2D-Corr characterization clearly indicates the IR intensity increase or decrease of the bands ascribed to different C-O confirmations of primary alcohols from immature to mature fibers in the 950–1050 cmApparently, the algorithm approach in ATR-FTIR spectral sensing is the most attractive and interesting, since, in its simplest form, there is no calibration model that is commonly built from a large sample set. In addition, the use of intensities at unique wavenumbers could reduce the influences from other components in diverse samples; hence, it could be universally applied for fast, accurate, routine and direct determination of cotton fiber maturity and crystallinity, simultaneously. This procedure avoids the need to perform any pretreatments of cotton fibers, analyzes a small amount of fibers (as little as 0.5 mg) and requires only a short time (less than 2 min) for sample loading, spectral acquisition and subsequent data reporting.


In this study, we isolated the neointimal cells and uninjured residential vascular smooth muscle cells by laser micro dissection and carried out single-cell whole-genome methylation sequencing. We also sequenced the bisulfite converted genome of circulating bone-marrow-derived cells such as peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC). We found totally 2,360 differential methylation sites (DMS) annotated to 1,127 gene regions. The majority of differentially methylated regions (DMRs) were located in intergenic regions, outside those CpG islands and island shores. Interestingly, exons have less DMRs than promotors and introns, and CpG islands contain more DMRs than islands shores. Pearson correlation analysis showed a clear clustering of neointimal cells with PBMC/BMMC. Gene set enrichment analysis of differentially methylated CpG sites revealed that many genes were important for regulation of VSMC differentiation and stem cell maintenance. In conclusion, our results showed that neointimal cells are more similar to the progenitor cells in methylation profile than the residential VSMCs at the 30 Vascular smooth muscle cell (VSMC) accumulation in the neointima is a common feature in vascular diseases such as atherosclerosis, transplant arteriosclerosis, and restenosis. In all of the phenomena, one of the characteristic changes is the accumulation of VSMCs within the neointima. The question to be addressed in this work is where the atherosclerotic cells come from, which is very important for the prevention and treatment of atherosclerosis.Generally, there are two hypothetical sources of the cell origins for atherosclerotic cells, the residential vascular smooth cells –9 and th2, blood was collected from the heart via intra-cardiac puncture; and the injured and normal arteries were exposed, arteries were collected and fixed in 4% formalin. Arteries were embedded in the paraffin block, cut into 10-µm sections, and then mounted onto glass slides and PET FrameSlide . The sections were briefly stained by hematoxylin/ eosin and air-dried at room temperature.Wild-type (WT) inbred Sprague Dawley (SD) rats were purchased from Charles River Laboratories. Animal procedures were approved by the Institutional Committee for Use and Care of Laboratory Animals at Morehouse School of Medicine and conform to the Guide for the Care and Use of Laboratory Animals published by US National Institutes of Health. Male Sprague-Dawley rats of 6–10 weeks old were anesthetized with isoflurane. The left carotid artery was exposed via a vertical midline incision and an arteriotomy was performed. The left common carotid artery was injured with an embolectomy balloon catheter  by inflating to 1.5 atmospheres and retracting to the arteriotomy site three times to assure a good vascular injury . The woug) for 5 minutes, and stored at –20°C. In the injured arteries, we used the laser to specifically isolate VSMC-like layer in the neointima leaving the endothelial layer intact; we also isolated the resident VSMC population in the medial layer as the control.A Leica LMD 7000 Laser Microdissection System (Leica Microsystems) was used to isolate cells from the tissue sections. The tissues were visualized under a microscope, target cells were selected and encircled on the computer monitor using a mouse, and then the computer program guided a UV laser (337-nm wavelength) to cut the slide foil with the target cells . The anaThe bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs) were isolated from whole blood and bone marrow aspirate by centrifugation through density gradient centrifugation using Histopaque 1083 (Sigma-Aldrich) as described previously. To isolate PBMCs, about 0.4 ml of blood was collected from each rat using a syringe via aortic puncture. A 21-gauge needle was directed into the thoracic cavity under the xiphoid cartilage while raising the cartilage slightly with the index finger, at approximately a 30–35° angle slightly to the left of the midline. The plunger pulled back on the syringe to create a vacuum. Blood was collected into 8-ml tubes (Becton Dickinson Vacutainer Systems) containing EDTA. Whole blood was centrifuged at 3,500 rpm for 5 min. The entire plasma layer except 5 mm (0.5 mL) was removed. The collection tube was then tilted to a 45° angle and PBMCs was removed from 2 mm above the interface between buffy coat layer and red blood cells (RBC) layer. It was then added to 3.5 mL of PBS in 15 ml centrifuge tube and mixed gently. About 5 mL of dilute buffy coat was then layered on top of 5 mL of HISTOPAQUE 1083 surface in a separate 15 mL centrifuge tube.g and 4°C. The supernatant was carefully discarded and the pellet resuspended in 6 mL of MACS buffer at room temperature. About 6 ml of the homogenized bone marrow cell suspension was then layered onto the HISTOPAQUE 1083 surface in a separate 15 ml conical tube.To isolate BMMCs, the tibia and femur bones were exposed and cut at the articulatio coxae and talotarsalis, leaving both condyli intact, and keeping the femur and tibia connected. The isolated bone was transferred into a sterile 6-cm Petri dish, filled with 6 mL of cold PBS. The femurs and tibias were then separated by cutting the bone at the articulatio genus, leaving the condyli of both the tibia and femur intact. The ends of the tibia and femur were then cut away. Bone marrow cells were harvested from the male inbred SD rats by flushing tibias and femurs with cold PBS using a sterile 21-gage needle, the samples were passed through a 20.5-gage needle to disperse clumps of cells, and then through a 70-µm filter to further remove bone fragments and cell clumps. Bone marrow cell suspension was transferred to a 15- mL conical tube in cold Magnetic Affinity Cell Sorting (MACS) buffer and spun for 5 min at 675 g for 7 min, and the cell pellet was resuspended in 10ml of MACS buffer.Histopaque layered buffy coat suspension and bone marrow aspirate suspensions were centrifuged at 400 g for 30 min. Then the upper layer was aspirated, then the tube was tilted at 45° and the mononuclear cell layer was taken and then added to 10 mL of MACS buffer + 2% FBS in 15 mL centrifuge tube and gently mixed. Tubes were spun at 300 7 BMMCs were centrifuged at 300 g for 10 min at 4 °C. The supernatant was aspirated and cell pellet was resuspended into 100 µl of cold MACS buffer. Then 2 µl of biotin conjugated rabbit anti-CD14 antibody  was added, and incubated at 4°C for 10 min. Cells were washed with 2 ml of cold MACS buffer and centrifuged. The supernatant was aspirated completely and the cell pellet was resuspended in 80 µl of cold MACS buffer. Magnetic labeling of CD14 labeled BMMCs with Anti-Biotin MicroBeads  and positive selection of CD14+ cells was performed following the manufacturer’s instructions. The isolated cell fraction was passed over a new, freshly prepared column according to Miltenyi protocol to increase the purity.BMMCs were further isolated by CD14 positive using Magnetic Affinity Cell Sorting (MACS). All reagents and supplies for MACS separation were purchased from Miltenyi Biotec, Bergisch-Gladbach, Germany. MACS buffer and instruments were pre-cooled to 4°C prior to use. About 1×10® ViaCount® assay was used to measure the number of BMMCs and PBMCs. During density gradient centrifugation, BMMCs were re-suspended in 10 mL of MACS buffer, and 20 µl of cell suspension was added to 180 µL of ViaCount solution (Guava Technologies 4000-0040) in a 96-well tray removed for ViaCount. The samples were vortexed and incubated, shielded from light, for 8–10 minutes at room temperature.The GuavaGenomic DNA was extracted from PBMCs and BMMCs using the Qiagen DNeasy Blood and Tissue Kit  according to the manufacturer’s instructions. DNA concentration was measured by the Nanodrop spectrophotometer (Thermo Scientific). The collected neointimal cells and residential VSMCs were directly processed to bisulfite conversion without DNA extraction. The EZ DNA Methylation-Direct™ Kit  was used to perform bisulfite conversion. For the microdissected samples, 13 µl M-Digestion Buffer and 1 µl Proteinase K were added to 12 µl of sample for digestion. The sample was incubated for 4 h at 50°C. For the BMMCs, we employed 350 ng of genomic DNA for optimized bisulfite conversion. The manufacturer’s protocol for bisulfite conversion was then followed for all samples. After bisulfite conversion, the DNA was bound to a Zymo spin column and desulfonated on the column using M-desulfonation reagent per manufacturer’s instructions. The bisulfite-converted DNA was eluted from the column in 10 µl of water. Bisulfite converted DNA was amplified with The Single Cell Bisulfite Whole Genome Amplification & Library Preparation Kit (Omigenomics) following the manufacturer’s procedure. The products are sequencing-ready library for Illumina sequencer GAII and HiSeq2000. Quantitative PCR was used to measure the concentration of viable sequencing template molecules in the library prior to sequencing. Libraries were quantitated and spiked in 40% Phix prior to loading on an Illumina paired end flow cell (v4), and sequenced on the Illumina Genome Analyzer IIx (Illumina) at a concentration of 11 pM with 101 bp paired read length (paired-end sequencing PE100) as per manufacturer’s instructions. Image analysis and base calling was performed by Illumina Real Time Analysis (RTA) v1.9.35 and output of RTA was demultiplexed and converted to Fastq format with Illumina CASAVA 1.8.2.http://genome.ucsc.edu). Sequence reads were mapped to the rat converted reference genome using the software package BSMAP [We downloaded the rat genome sequence (RGSC 5.0/rn5 assembly) from the University of California Santa Cruz Genome Bioinformatics Site , peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs). We generated totally 13,968,170 reads, 13,197,332 reads, 9,067,668 reads, and 11,116,242 reads for BMMCs and PBMCs, neointimal cells, and VSMCs, respectively. We counted the conversion rates at non-CpG cytosines of those uniquely mapped reads. The results are 98.6% , 98.4% (VSMCs), 99.3% (BMMCs), and 99.2% (PBMCs). Whole genome methylation sequencing was complemented by publicly available whole-genome bisulfite sequencing data, including mammary tissue, brain and liver.In general, the neointimal cells showed an overall global methylation percentage similar to the BMMC/PBMC samples . AlthougWe determined which cell types resembles the neointimal cells the most on the methylation profile. We calculated pairwise Pearson correlation coefficients between the neointimal cells and residential VSMC, BMMC and PBMC. The Pearson Correlation coefficients were then used to perform hierarchical clustering of samples based on their molecular signatures. Samples were clustered hierarchically using “correlation” as the distance metric and “Ward’s method” as the agglomeration method in the clustering algorithm. The results showed that the epigenetic signature of neointimal cells were more similar to the BMMC and BMC than to residential VSMC . It is nWe searched for differential methylation sites (DMS) by comparing the β-values  in the neointimal cells with residential cells, PBMC and BMMC samples. For calling a CpG site as differentially methylated , we required a minimum absolute Δβ -value of 0.5 and a false discovery rate (FDR)-adjusted Wilcoxon rank-sum P -value of <0.01 for the difference. The relatively specific SMPs for different cell types are shown in . In totaTo explore putative functional roles for the DMS, we performed gene set enrichment analysis using Ingenuity Pathway Analysis tool. Among those genes containing DMS between the neointimal cells and residential VSMCs, 61 DMS were found to be associated with 38 genes, mainly in functional classes, such as cardiovascular system development, cell growth and proliferation, cell death and survival, embryonic development, cellular movement, and skeletal and muscular system development . The neoBecause the PBMCs and BMMCs had very similar DNA methylation profiles, they were combined and analyzed as a single set. We interrogated the non-repetitive part of the genome for regions containing at least five consecutive and consistently differentially methylated CpGs and in which DNA methylation levels of the flanking CpG sites differed significantly between the two samples . Using these criteria, we identified 662 DMRs between neointimal cells and residential VSMCs, 69 DMRs between neointimal cells and BMMC/PBMC . DMRs weP value)  . Other hP value) .30. These cells retain remarkable plasticity and can undergo profound phenotypic transition in response to local environmental changes14. When blood vessel is injured, VSMCs dramatically increase their proliferation, migration, and synthetic capacity; however, the high degree of plasticity can also lead to an adverse phenotypic switch and vascular diseases, such as atherosclerosis, restenosis, cancer, and hypertension [Klf4 plays a critical role in the phenotypic transitions of VSMCs that have favorable effects in inhibiting plaque pathogenesis [in vitro and in vivo [Myh11-CreERT2 ROSA floxed STOP eYFP Apoe−/− mice for SMC lineage tracing showed that >80% of SMCs within advanced atherosclerotic lesions are phenotypically modulated and that traditional methods for detecting SMCs based on immunostaining for SMC markers fail to detect >80% of SMC-derived cells within advanced atherosclerotic lesions, indicating that the contribution of SMCs to atherosclerotic plaques has been greatly underestimated by conventional techniques [In this study, we compared the DNA methylation profiles between neointimal cells, residential VSMCs and bone marrow derived circulating progenitor cells. Our results clearly showed that the atherosclerotic cells keep their epigenetic memory of the bone-marrow derived progenitor cells rather than the residential VSMCs. We believe that these discoveries may guide the development of intervention methods based on the migration of BM derived cells to the site of vascular injury and the proliferation of SMCs after coronary artery bypass surgery or angioplasty. It is known that adult vascular smooth muscle cells (VSMCs) proliferate at a very low rate, exhibit very low synthetic activity, and express a unique set of markersrtension  VSMC pheogenesis ; and a m in vivo . There a in vivo –38. Furtchniques .th day). The third caveat is that the small sample size, although we isolated 50–100 cells, but they were from one rat, so we cannot eliminate the personal bias from rat to rat. Next caveat is the low coverage, which is similar to an array study, only a small number of CpG sites were studied instead of all CpG sites in the whole-genome, it may cause biases because some CpGs may be more important than others regarding the differentiation or transition during vascular remodeling. In conclusion, we have specifically isolated neointimal cells and compared their methylation signature with the other two hypothesized cell origins; the residential VSMCs and the circulating progenitor cells. To our knowledge this is the first time that laser micro dissection and methylation signatures have been used to address this issue. Although the question about the origination of neointimal cells can only be completely addressed after migration and trans-differentiation are directly observed, our study showed that neointimal cells have a more similar methylation profile to the circulating bone marrow derived cells than the residential VSMCs. The origin of atherosclerotic cells may guide us where our therapeutic strategies should target.On the other hand, several studies have suggested that circulating BM derived cells contribute to neointima formation ,39,40. ISuppl Table 1Suppl Table 2Suppl Table 3Suppl Table 4Suppl Table 5Suppl Table 6Suppl Table 7