To achieve robust sEMG measurements in an EMG prosthetic system, this study proposes a surface electromyogram (sEMG) sensor with a novel electrode structure composed of two-layered conductive silicone with different carbon concentrations. We hypothesized there is an optimal carbon concentration for achieving a large sEMG amplitude with robustness to external perturbation, and we empirically determined this optimal concentration. We produced fourteen sets of electrodes, with the weight ratio of carbon to silicone ranging from 1.7% to 4.0%. Using these electrodes, the user sEMG and electrical properties of the electrodes were measured. An external perturbation was applied on one side of the electrode to introduce a condition of unbalanced contact to the sEMG sensor. We defined an index of robustness for the sEMG sensor based on the signal-to-noise ratio in the balanced and unbalanced contact conditions. Based on the results of the robustness index, two optimal carbon concentrations, at weight ratios of 2.0%–2.1% and 2.6%–2.7%, were observed. Moreover, the double-peak property was correlated to the capacitance. Our results clearly demonstrate an optimal carbon concentration for robust sEMG measurements, and suggest that the robust measurement of sEMG is supported by the capacitance component of the sensor system. EMG is a biological electric potential generated when a muscle is activated. Conventionally, the magnitude4 or rate of change5 of EMG has been used to control a prosthetic hand. However, the conventional method can only control a few kinds of motion7; therefore, researchers have developed control methods based on pattern recognition9 and regression11. In any method, stable EMG measurements are important for stable control of an EMG prosthetic hand14. The EMG is generally measured by the following two methods: invasive and non-invasive. In the invasive method, the EMG is directly measured inside or near the muscle by inserting an electrode17. This method can precisely measure individual muscle activity without crosstalk, but the invasion is too substantial for users and is not suitable for long-term use. Therefore, the invasive method is not suitable for everyday-use devices. In the non-invasive method, the EMG is measured on the skin surface; hence, the surface EMG (sEMG) can be safely measured with a lighter burden for the user. Therefore, the non-invasive method is suitable for EMG prosthetic hand devices. In conventional physiological experiments in a laboratory or medical environment, a “wet electrode” (such as a silver/silver chloride (Ag/AgCl)-type electrode), is generally used to measure a high-precision signal19. The wet electrode reduces the electrical impedance  between the skin and the electrode to a smaller value than that of a “dry electrode.” For everyday-use devices, the wet electrode is not suitable because of the burden of attachment and running costs. Therefore, many commercially available sEMG prosthetic hand systems adopt a dry electrode. In many dry electrodes, a metallic material is exposed on the surface and makes contact with the skin20. An sEMG sensor with a metal electrode can be easily used in everyday life. However, long-term use of the metal electrode is difficult because of its rigidity, and some users are allergic to the metal. Therefore, a dry-type sEMG sensor without contact between the metal and the skin is required.Electromyography (EMG) is often used as an input signal to intuitively control a powered prosthetic hand24. We also developed sEMG sensors using electrodes composed of a conductive fabric25 and conductive silicone26; these sensors were applied to control numerous types of powered prosthetic hands and arms30. Electrodes composed of conductive fabric has superior flexibility but causes discomfort to the user, especially for children, because of a metallic element in the conductive fabric31. The electrode composed of conductive silicone was a hybrid type of electrode, and both conductive silicone and gold-coated metal wire contacted the skin. The hybrid electrode system can robustly measure the sEMG both under dry and wet skin conditions. However, the exposed gold-coated metal wire remains to present a drawback. Moreover, when the sEMG sensor is used in an EMG prosthetic hand system, the sEMG sensor is disturbed by vibrations in the socket and the body motion of the user, which reduces the controllability of EMG prosthetic hands depending on the pattern recognition technique34. The disturbance to the sEMG sensor also results in a change of contact force between the skin and electrode. This change of contact force results in an unbalanced contact for the electrodes and an unstable impedance balance38, leading to unstable sEMG measurements. To achieve stable sEMG measurement in the EMG prosthetic hand system, the problem of unbalanced contact in the electrodes must be resolved.Previously, researchers developed dry electrodes with soft materials39. To achieve requirement (2), the contact impedance should be larger than the change in contact impedance between the balanced and unbalanced electrode contact conditions. According to the above two requirements, an electrode suitable for sEMG measurement would have an optimal impedance under this trade-off relationship. Conductive silicone is composed of a mixture of silicone and carbon black powder26. Hence, changes in carbon concentration leads to changes in the electrical impedance of the conductive silicone. Metallic materials can be used to connect the conductive silicone and the amplifier circuit. However, we found that the contact impedance between the metal and the conductive silicone with low carbon concentrations is much higher than that between the conductive silicones (see Supplementary Information). Therefore, conductive silicone with a high carbon concentration should be connected to the amplifier via a metallic material, and the conductive silicone in the electrode, which has a low carbon concentration, should come in contact with the skin and the conductive silicone with the high carbon concentration. We propose a two-layered electrode consisting of conductive silicone with different carbon concentrations. To develop the proposed electrode, we must find the optimal carbon concentration for the conductive silicone of the electrode under the trade-off relationship.For the electrode of the sEMG sensor to be suitable for an EMG prosthetic hand system, (1) the sEMG must be robustly measured when the contacts of both electrodes are stable, and (2) the measurement ability of the sEMG cannot undergo substantial changes, even for an unbalanced contact in the electrodes. To achieve requirement (1), the contact impedance between the skin and the electrode should be smallThe aim of this study is to design an sEMG sensor structure with two-layered electrodes and to find the optimal carbon concentration of the conductive silicone in contact with the skin. We produced electrodes with different carbon concentrations and measured the corresponding sEMG. The signal-to-noise (SN) ratio was defined as the ratio of sEMGs measured in relaxing and grasping states; the SN ratio is used to quantitatively evaluate the sEMG measurement ability of each electrode. To generate unbalanced and balanced electrode contacts artificially, we adopted conditions in which one electrode is or is not externally pressed. We evaluated the difference in SN ratio between the non-pressed and pressed conditions. An index of robustness for the sEMG measurement was defined using the value of the SN ratio and the change in SN ratio. We expected the index of robustness to show a single peak at a specific carbon concentration. Moreover, we measured the electrical properties of the electrode with skin contact and evaluated the relationship between the electrical properties and the robustness index.We developed an sEMG sensor with two-layered electrodes, as shown in Fig. Figure Ir. The results clearly show two peaks at 2.0%–2.1% and 2.6%–2.7%. The above results demonstrate that the contact electrodes with carbon concentrations of 2.0%–2.1% and 2.6%–2.7% can robustly measure sEMG.For the SN ratio, a peak was observed near concentrations of 2.1% and 2.6% Fig. . MoreoveFigure Figures r = 0.49), and the second highest value was observed for the change in capacitance between the balanced and unbalanced contact conditions . The third highest value was found for the capacitance under the unbalanced contact condition . The positive correlation between the sEMG measurement robustness and the capacitance for the balanced contact condition indicates that a greater capacitance corresponds to a more robust sEMG measurement. Remarkably, all three high correlation coefficients were observed for explanatory variables related to the capacitance. The results suggest that the capacitance is the cause of the double-peak nature of the sEMG measurement robustness.Finally, analysis results regarding the correlation between the change in robustness of the sEMG measurement Fig.  and chanIn this study, we assumed there is an optimal carbon concentration for conductive silicone in contact with skin based on the two requirements of sEMG sensors suitable for EMG prosthetic hands, and we experimentally determined the optimal concentration. We produced fourteen sets of electrodes with carbon concentrations ranging from 1.7% to 4.0% and measured the sEMG and electrical properties for each concentration. Moreover, we applied an external perturbation of 20 N to one side of the electrodes for an unbalanced contact with the electrodes of the sensor. The results demonstrate the existence of carbon concentrations that can robustly measure large sEMG amplitudes under the balanced contact condition of the electrodes Fig. , and thaInitially, we expected that a single carbon concentration peak would be observed based on the trade-off of two requirements for robust sEMG measurement. Contrary to our expectation, the results of the robustness index of sEMG measurement showed two peaks. This double-peak property Fig.  and the The proposed structure of the electrode is based on the assumption that the contact impedance is small between the conductive silicone components and relatively large between the conductive silicone and the metal. The achievement of a robust sEMG measurement at a specific carbon concentration implies the feasibility of our assumption. Remarkably, under the balanced contact condition, the electrode with a carbon concentration of 2.1% measured sEMGs at an amplitude equal to or larger than that at 4.0%, the highest carbon concentration Fig. . This re26.The electrode contacts the skin of the user to measure the sEMG. Hence, the electrode should possess non-allergic properties and flexibility for long-term use. In this study, the conductive silicone consisted of a mixture of silicone  and carbon black  was used for the electrodesupplementary discussion, we have proposed a two-layered electrode structure and a sEMG sensor system, as shown in Fig. Based on the We conducted experiments to determine the optimal carbon concentration of the contact electrode for sEMG measurement.One healthy male subject in his twenties participated in the experiments. The subject did not have any rashes on his skin. All experiments were approved by the ethics committee of the University of Electro-Communications. The participant received explanations of the experimental procedure and gave written informed consent. In addition, all procedures were performed in accordance with the Declaration of Helsinki and the guidelines of the Japan Neuroscience Society and the Japan Society of Clinical Neurophysiology.We measured the sEMG signal and the electrical properties of the electrode using the apparatus shown in Fig. The measured variables included the impedance, resistance component, and capacitance component; the impedance was represented by an equivalent circuit of a serial connection of the resistance and capacitance components. In the electrical property measurements, the input voltage was 0.1 V, the input frequency was 300 Hz, and the sampling rate was 4 Hz. We produced fourteen sets of contact electrodes with different carbon concentrations; the weight ratios of carbon to silicone were 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 3.0%, 3.5%, and 4.0%. We produced four electrodes for each carbon concentration  and three electrode pairs for sEMG measurement . The weight ratio of carbon to silicone for the body electrode was 4.0%.The same subject participated in (1) the sEMG measurement experiment and (2) the electrical property measurement experiment on different days.(1) sEMG measurement: We measured the sEMG of the subject in a relaxed condition and while grasping the grip dynamometer. The task schedule is shown in Fig. In the unbalanced contact condition, the force tester was controlled at 20 N.(2) Electrical property measurement: We measured the electrical properties of the electrode as the subject performed a grasping motion while in a stationary condition for 3 s. The task schedule is shown in Fig. E, we calculated the root mean square (RMS), i.e., rectified sEMG data t denotes the time step. We defined Using the measured sEMG data In this study, we defined the SN ratio as the ratio of the sEMG signal for the grasping motion to that for the relaxing motion, calculated as follows:For the measured electrical properties, we calculated the average value for each trial. To assess which electrical properties affect the sEMG measurement robustness, we analyzed the correlation between changes in the robustness index and the measured electrical properties depending on the carbon concentration. We considered the electrical properties shown in Fig. Supplementary information


Since dysregulation in lipid metabolism accompanies abnormal amyloid formation, we quantified glycerophospholipids (GP) and sphingolipids (SP) in CSF fractions from participants with late-onset AD  or with Other Dementia  to determine if alterations in lipid metabolism account for pathological differences. Aβ42 and total Tau levels were determined using a sandwich ELISA. Liposomal-based fluorescent assays were used to measure phospholipase A2 (PLA2) and acid or neutral sphingomyelinase  activities. Supernatant fluid (SF) and nanoparticle (NP) lipids were quantified using LC-MS/MS. Although CSF Aβ42 and Tau levels are similar, phosphatidylserine (PS) in SF and ceramide (CM) levels in NP are significantly higher in OD compared with LOAD. The aSMase but not the nSMase activity is higher in OD. PLA2 activity in CSF from OD subjects positively correlates with several GP classes in SF and NP fractions but not in LOAD fractions. Our data indicate differences in CSF lipid metabolism between dementia variants. Higher levels of inflammatory and apoptotic lipids may induce faster neuronal death, resulting in the earlier cognitive decline in patients with OD phenotypes. Abnormal cerebrospinal fluid (CSF) levels of β-amyloid peptides (Aβ The pat2 levels ,7,8. HowIn addition to LOAD, Frontotemporal Dementia (FTD), Dementia with Lewy Body (DLB), and vascular dementia (VaD) account for a substantial amount of cases of other dementias. FTD constitutes 5–15% of all dementia cases and is the second most common cause of dementia for patients under the age of 65 . Unlike Lipids are involved in critical neurobiological functions, such as membrane formation, cellular binding and recognition, transport, energy, and signaling ,15. In ASeveral enzymes, as well as lipids, interact with Aβ metabolism ,30,31,32The huge role of lipid metabolism in dementia and differences in clinical manifestation of diseases led us to hypothesize that lipid metabolism may differentiate dementias. This project aims to determine if lipid metabolism can differentiate LOAD from other dementias (OD), such as FTD and DLB. This characterization will allow for further research on early biomarkers of dementia subtypes. It would provide insight into any possible metabolic pathways, early diagnosis, and effective treatment of these diseases.Human study participants were enlisted through newspaper advertising and visits to senior centers and assisted living residences. Criteria for inclusion required participants to be between the ages of 45 to 100 years old and diagnosed with LOAD , DLB, FTApoE genotyping was performed using a polymerase chain reaction mixture of primers specific for E2, E3, and E4, using the protocol described by Calero et al. . We crea42 and total Tau using a sandwich ELISA kit  as previously described [IT, Invitrogen/Molecular Probes, Eugene, OR, USA) using serum albumin (0–500 ng/mL) as a standard as previously described [A clinician obtained lumbar CSF between 8:00 am and 10:00 am after an overnight fast, and laboratory personnel removed cellular debris by centrifugation at 1000 g for 2 min. We measured CSF levels of Aβescribed . We deteescribed .2 (PLA2), acid sphingomyelinase (aSMase), or neutral sphingomyelinase (nSMase) activity in the CSF samples, we used a modified liposomal-based fluorescent assay with 7-hydroxycoumarinyl-arachidonate (0.3 mM), 7-hydroxycoumarinyl-linolenate (0.3 mM), hydroxycoumarinyl-6-hyptenoate (0.3 mM), 10 mM dioleoylphophatidylcholine (DOPC), and dioleoylphosphatidylglycerol (DOPG) (10 mM) as substrates [2 activity, we added CSF containing 10 µg total protein to 96 well plates containing 50 µL PLA2 substrates and 50 mM Tris HCl buffer (pH 8.9). PLA2 activity was measured using an excitation wavelength of 360 nm and emission at 460 nm, and specific activity was calculated as the relative fluorescent units (RFU)/µg protein/min) for CSF samples. To measure phospholipase Abstrates . To initaSMase and nSMase activities in CSF were determined using an Amplex red fluorometric assay following the recommended protocol obtained from the kit’s manufacturer . Activities are presented as relative fluorescent units per minute for a 10 µg protein applied in duplicates on 96 well plates . 4-PAF, 5 ng lysophosphatidylcholine (LPC) (11:0), and 5 ng D4-LPC) to 1 mL SF, lipids were extracted using a modified Bligh & Dyer protocol [2, we reconstituted lipid extracts in HPLC solvent before LC-MS/MS analyses.We prepared lipid fractions consisting of supernatant fluid (SF) and nanoparticles (NP), according to Harrington et al. . After tprotocol . BrieflyWe separated lipid extracts on a TSK-Gel Amide-80 Column (2.0 × 150 mm) using a binary solvent system as previously described ,53. AfteWe used the Qual Browser module of the Xcalibur software  for peak integration of GP and SP classes. All integrated peaks were normalized to the internal standard (IS), PC (11:0/11:0) for quantification. For quantification of lipids classes in CSF fractions, known amounts of lipids with a fixed 5 ng IS were run separately through the LC-MS/MS. Standard curves of the lipid amounts versus lipid/PC (11:0/11:0) intensities were acquired and used to quantify the lipids in the SF and NP fractions (ng/mL CSF) .t-tests were used to determine differences between Aβ, Tau, lipids, and proteins among the clinical groups. We used Fisher’s exact tests for categorical variables describing the presence or absence of risk factors in our study groups. We used Spearman ranked analyses to assess the correlation between GP and SP classes with Aβ42, Tau, PLA2, nSMase, or aSMase. All analyses were performed using GraphPad Prism software  and 0.05 significance level. All lipid data are presented as mean ± SEM (ng/mL). Student paired n = 29), while the rest were classified as other dementias (n = 10). The OD category was composed of DLB (n = 3), behavior variant FTD (n = 3), and vascular or mixed dementia (n = 4). Analyses of education and ApoE genotype numerical scale showed no group differences. Similarly, there was no significant difference in age, sex, body mass index (BMI), and neuropsychological classification between LOAD and OD  [Typical for dementias, both groups had reduced Aβctively) . DementiWe examined the frequencies of some AD risk factors in LOAD and OD to ascertain that these did not contribute to any differences in lipid metabolism. Our data show that the occurrence of the most common risk factors was similar in the LOAD and OD populations .p > 0.4) differ from that of OD . Similarly, the number of over the counter supplements were similar (p > 0.6), suggesting that differences in medication usage might not account for metabolic differences in LOAD compared with OD.We examined the number of prescription and over the counter medications in our study populations. The mean number of prescription medications in LOAD  did not significantly (p < 0.05) higher in OD compared with LOAD  levels were significantly . The ratio aSMase/nSMase was higher in OD compared to LOAD (p < 0.02). However, we observed significantly higher aSMase activity in CSF from OD participants compared with LOAD . In contrast, only LPC in SF positively correlated with Tau in OD . To determine any link between lipid metabolism and AD pathology, we performed Spearman correlation analyses between GP classes and Aβ42 or Tau with SF or NP sphingolipids from LOAD study participants. In contrast to LOAD, Tau negatively correlated with CM in the SF fraction  in OD. Also, Tau levels negatively correlated to SM levels in supernatant fluids .There were no statistically significant correlations noted between Aβ2 activity correlated with several SF and NP lipids in CSF from OD participants. For OD samples, LPC in SF positively correlated with PLA2 activity in CSF , but no such correlation was evident for LOAD. Similarly, xPE levels in SF correlated with PLA2 activity  in OD but not in LOAD. PLA2 activity in LOAD did not correlate with NP PC  with LPC/PC of OD (2 activity in LOAD did not correlate with platelet-activating factor-like lipids (PAF-LL)  (p < 0.05) with the activity of PLA2  e but neg(PAF-LL) f. The on < 0.04) g. NP NAP of PLA2 h. 42/Tau ratio was significantly related to nSMase activity . NP-SM was negatively related to the activity of aSMase , and NP dhCM was positively correlated with the activity of nSMase .We also determined the correlation of SP levels in CSF fractions from OD participants with aSMase or nSMase activities. AβOur objective is to determine if there are any differences in lipid metabolic pathways in CSF from subjects with LOAD compared with those with OD. In this study, the OD population is made up of participants with DLB, FTD, VaD, and mixed dementia that can often be clinically differentiated from LOAD by the earlier age of onset, step-wise progression, and shorter survival time . OD subjExamining GP classes, the only significant variation was PS that almost tripled in the SF of OD compared with LOAD. Typically found in the inner leaflet of plasma membranes and mitochondrial-associated membranes, PS only constitutes 2–10% of total lipids ,58. Alth42 and Tau metabolism. In regards to the correlation in LOAD between Aβ42 and LPC/PC in SF, alteration in lipid metabolism may influence the proper clearance of Aβ42, resulting in the pathological features associated with Aβ42 aggregation [42 in CSF have a lower disturbance of lipid metabolism. In contrast, highly perturbed lipid membranes do not only result in abnormal clearance of Aβ42 but increase the susceptibility of amyloid precursor protein (APP) to digestion by secretases [2-catalyzed lipolysis, induces myelination, and disrupts the blood-brain barrier [2 isoforms contribute to AD pathology [Our data also indicate that there is a disruption in GP metabolism in LOAD and OD that may impact Aβregation . The indcretases ,63. We a barrier , these dathology ,65,66,67SPs are another group of lipids essential in brain function and implicated in AD pathology ,68. Of tFurther evidence for a role of SPs in OD is shown when Tau levels negatively correlate with CM and SM in SF. How Tau interacts with lipids has not been widely studied, but one can conclude that SP metabolism associates with Tau and this interaction is different in LOAD compared with OD. 2 is notable for its role in Alzheimer’s disease and other neurological disorders, due to the release of the inflammatory precursor arachidonic acid [2- and COX-derived products are crucial in regulating an inflammatory response in LOAD [2 and also stimulate the de novo synthesis of ceramide and PAF [2 activity in CSF from OD participants correlated with levels of several GPs; LPC and xPE in SF, and PC, PAF, NAPE, and LPC/PC in the NP fraction. PAF is an inflammatory GP produced and metabolized through two variations of PLA2—cytosolic and Lp-PLA2 [2 with these lipids suggests that hydrolysis may be linked with destructive neuro-inflammation in subjects with OD. Several enzymes modify GPs and SPs to form lipid mediators of inflammation or lipids that modify neuronal function. PLAnic acid ,71. Once in LOAD . Inflamm and PAF ,74,75,76 Lp-PLA2 . LPC int Lp-PLA2 . LPC app Lp-PLA2 ,79. A hi2 observed in LOAD and OD reflects a disturbance in neuronal membrane lipids. NAPE is a significant component of intracellular membranes and has been shown to accumulate under pathological conditions that involve the degradation of membranes ,81. NAPEThe activities of aSMAse and aSMase/nSMase ratio were higher in CSF from subjects with OD compared to LOAD. aSMase and nSMase are both involved in the degradation of SM to form CM, a known pro-apoptotic biolipid ,82,83. FThis pilot cross-sectional study shows that lipid metabolism in CSF fractions differs between LOAD and OD. The small OD sample size limits our ability to examine previously described lipidomic variations between mixed dementia phenotypes . These s


Reduced levels of Aβ1–42 are indicative of AD, but significant variation in the absolute concentrations of this analyte has been described for both healthy and diseased populations. Preanalytical factors such as storage tube type are reported to impact Aβ recovery and quantification accuracy. Using complementary immunological and mass spectrometry-based approaches, we identified and characterized preanalytical factors that influence measured concentrations of CSF Aβ peptides in stored samples.Amyloid-β 1–42  at different stages of the CSF collection and aliquoting process and then measured with the mass spectrometry assay.CSF from healthy control subjects and patients with AD was aliquoted into polypropylene tubes at volumes of 0.1 ml and 0.5 ml. CSF Aβ1–42 concentrations measured from 0.5-ml aliquots were higher than those from 0.1-ml aliquots. Tween-20 surfactant supplementation increased Aβ1–42 recovery but did not effectively resolve measured concentration differences associated with aliquot size. A CSF Aβ peptide mass spectrometry assay confirmed that Aβ peptide recovery was linked to sample volume. Unlike the immunoassay experiments, measured differences were consistently eliminated when aliquots were denatured in the original sample tube. Recovery from a panel of low-retention polypropylene tubes was assessed, and 1.5-ml Eppendorf LoBind® tubes were determined to be the least absorptive for Aβ1–42. A comparison of CSF collection and processing methods suggested that Aβ peptide recovery was improved by denaturing CSF earlier in the collection/aliquoting process and that the Aβ1–42/Aβ1–40 ratio was a useful method to reduce variability.Two distinct immunoassays demonstrated that CSF Aβ1–42 measurements. Sample denaturation during aliquoting increases recovery of Aβ peptides and improves measurement accuracy. The Aβ1–42/Aβ1–40 ratio can overcome some of the quantitative variability precipitated by preanalytical factors affecting recovery.Analyte loss due to nonspecific sample tube adsorption is a significant preanalytical factor that can compromise the accuracy of CSF AβThe online version of this article (10.1186/s13195-018-0445-0) contains supplementary material, which is available to authorized users. According to the manufacturer’s documentation, the intra- and interassay CVs for this assay are < 15% with an accuracy of 98%. CSF (10 μl) was removed from the original sample tubes with protein low-binding tips  and was diluted 1:8 with 70 μl of assay diluent in 0.6 ml MAXYMum Recovery™ low-retention tubes . Plates were blocked with assay diluent for 1 h and washed with PBS containing 0.2% Tween-20, then CSF and calibrators were added to each well where Aβ1–42 peptides were captured with an antibody specific for Aβ1–42  that was immobilized to the plate. After 1 h, the unbound analyte was washed away, and a detection antibody  that recognizes the N-terminus of Aβ peptides was added to each well. After 1 h, wells were washed again, an ECL reagent was added, and then the ECL intensity was quantified. Aβ1–42 concentrations were determined by interpolation from a seven-point, four-parameter logistic regression curve. All determinations were made in duplicate. The lower and upper limits of quantification for the assay were 3 pg/ml and 2000 pg/ml, respectively.CSF Aβ+, 3.0 mM K+, 1.4 mM Ca+ 2, 0.8 mM Mg+ 2, 1.0 mM PO43−, 155 mM Cl−; DURECT Corporation, Cupertino, CA, USA), 4 mg/ml human serum albumin, and 50 μg/ml immunoglobulin G. The samples were then mixed with 150 μl of 6 M GuHCl, incubated at 37 °C for 75 min, and then diluted with 200 μl of 4% H3PO4. Analytes were isolated from the pretreated CSF sample using an Oasis® MCX strong cation exchange mixed-mode μElution 96-well plate . After sequential washes with 4% H3PO4 and 10% acetonitrile (ACN), the analyte peptides were eluted in a small volume of ACN/water/NH4OH (75:15:10 vol/vol/vol). The extract was then diluted to 37.5% ACN with water. The relatively high organic and ammonium hydroxide composition of the extract was optimized to maintain solubility of the peptide analytes and minimize carryover effects.A 20-μl aliquot of 10 ng/ml internal standard working solution containing stable isotope-labeled internal standard Aβ peptides was added to 100-μl samples of human CSF or analyte-fortified artificial CSF (aCSF) containing buffers and electrolytes specified in the ALZET® formulation , the technique of at-column dilution was used to ensure that the peptide analyte bands were efficiently focused on the precolumn. The solvent stream (comprised of 40% mobile phase B1 (ACN/methanol/trifluoroethanol 75:25:5 vol/vol/vol) carrying the injected sample plug from the autosampler was diluted fourfold with 0.3% aqueous ammonium hydroxide (to a weaker ~ 10% organic level) just prior to entering the trapping column . The focused analyte band was then back-transferred from the head of the precolumn onto the analytical column , and the separation was carried out at 50 °C using a 3-min gradient from 20% to 45% mobile phase B2 (ACN/water/trifluoroethanol 90:5:5 vol/vol/vol). The eluted analyte peaks were detected by multiple-reaction monitoring (MRM) in positive electrospray ionization mode using a Xevo TQ-S mass spectrometer (Waters Corporation). The source temperature was 150 °C, the desolvation temperature was 600 °C, and the capillary voltage was 3.5 kV. MRM transitions, cone voltages, and collision energies for each peptide and internal standard are summarized in Additional file 1–34, Aβ1–37, Aβ1–38, Aβ1–40, Aβ1–42, and the internal standard peaks. A calibration curve was constructed with the peak area ratios of the calibration standards by applying a linear, 1/concentration squared weighted, least-squares regression algorithm, and then all concentrations were calculated against the calibration line. The lower and upper limits of quantitation were 50 pg/ml and 5000 pg/ml for Aβ1–34, Aβ1–37, Aβ1–38, and Aβ1–42. The lower and upper limits of quantification for Aβ1–40 were 100 pg/ml and 10,000 pg/ml.The analytes were separated under high pH, NHt tests were used to compare Aβ1–42 concentrations recovered from matched 0.1-ml and 0.5-ml aliquots of CSF. Figures were generated with Spotfire® version 4.0.2  and Prism 6.0e  software.Paired two-tailed Aβ concentrations recovered in condition 2, 3, or 4 were expressed as a percentage of Aβ concentrations recovered in condition 1 because the amount of Aβ recovered in condition 1 was the highest of the four conditions Fig. a. The daIntrasubject percentage CV was calculated according to the following formula  and patients with AD   . There were no correlations between the Aβ1–42 measurements and age, gender, weight, apolipoprotein E genotype, or study site (data not shown). In contrast, Tau and pTau181 concentrations were not impacted by aliquot size  were assayed with the Meso Scale Discovery® human Aβ1–42 assay (MSD), a plate-based ECL immunoassay specific for Aβ1–42. In this subset, the mean AlzBio3-measured Aβ1–42 concentration in 0.5-ml aliquots was 61.5% greater than the mean concentration measured in 0.1-ml aliquots (p < 0.0001) (data not shown). In the MSD assay, the mean Aβ1–42 concentration measured from 0.5-ml aliquots in this subset was 108% greater than the mean concentration measured in matched 0.1-ml aliquots (p < 0.0001) . Aliquots of 0.5 ml from each donor were analyzed in pairs; one aliquot was pretreated with Tween-20 and then sampled for analysis, and the second aliquot was left untreated and then sampled for analysis. After the initial sampling, the remaining CSF was removed, the empty tubes were washed with 0.5 ml of artificial CSF containing Tween-20, and then the washes were sampled for analysis. The CSF and washes were measured concurrently with the AlzBio3 immunoassay, and concentrations reported for samples containing Tween-20 were adjusted to account for the dilution caused by the addition of the reagent (+ 2%).To determine if the observed sample volume-associated differences in measured Aβ1–42 concentration between patient aliquots increased 53.1%, from 367 (±167) pg/ml to 561 (±249) pg/ml, when the CSF was pretreated with Tween-20. The peptide was undetectable in the washes obtained from the Tween-20-pretreated samples, but it was abundant in washes obtained from originally untreated aliquots (mean concentration = 216 [±126] pg/ml) (data not shown). Although these changes were not statistically significant when the patients were analyzed as a group, they were significant at the individual level. A representative result from one of the patients is shown in Fig. 1–42 recovered from the Tween-20-treated aliquot (402 pg/ml) was 41.7% higher than that from the untreated aliquot (284 pg/ml) (p < 0.0001)  was almost equivalent to the amount of Aβ1–42 initially recovered from the aliquot pretreated with Tween-20 (402 pg/ml)  were assayed on the AlzBio3 and MSD Aβ1–42 immunoassay platforms to determine if Tween-20 pretreatment would improve Aβ1–42 recovery and eliminate sample volume-associated quantitation differences. As measured by the AlzBio3 immunoassay, Tween-20 pretreatment increased the mean Aβ1–42 recovery in 0.1-ml and 0.5-ml aliquots by 96.2% (p < 0.0001) and 39.8% (p < 0.0001), respectively  in 0.1-ml aliquots and 88.9% (p < 0.0001) in 0.5-ml aliquots. Unlike the AlzBio3 immunoassay, however, there was no substantial reduction in volume-associated differences in Aβ1–42 recovery  were taken from two patients with AD unique to this substudy and were aliquoted into 1-ml-capacity Matrix® tubes , 1.5-ml-capacity LoBind® tubes, and MAXYMum® Recovery tubes at volumes of 0.1 ml and 0.5 ml. The aliquots were sampled in pairs, either untreated or after Tween-20 pretreatment. Measured Aβ1–42 concentrations from Matrix® tubes and MAXYMum® Recovery tubes increased significantly after Tween-20 pretreatment, demonstrating that Aβ1–42 adsorbed to these tubes  was identified that denatures Aβ in CSF with a strong chaotrope (GuHCl) and that was recently qualified for simultaneous measurement of five Aβ isoforms  [1–42 (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA), is the most hydrophobic isoform analyzed, and we hypothesized that the shorter isoforms would be less susceptible to preanalytical adsorption.We sought an alternative assay method because Tween-20 pretreatment failed to consistently resolve discrepancies in Aβ Aβ1–34) . The muln = 7) unique to this substudy were selected. Following the qualified preanalytical assay workflow, a known volume of CSF was removed from the original aliquot and then transferred to a fresh tube containing an equal volume of denaturant (GuHCl). When 0.1-ml and 0.5-ml aliquots of AD CSF were processed by this method  . Results for Aβ1–34 were inconclusive because concentrations were below the limit of detection for two and four subjects assayed under each condition  with the original sample preparation method  with the modified sample preparation protocol  Fig. . Aβ1–40,1–42 recovery from aliquot tubes would impact the capacity of this biomarker to discriminate between YNC and patients with AD, sets of 0.1-ml CSF aliquots from YNC (n = 17) and patients with AD (n = 20) were prepared for analysis with each of the sample preparation workflows  in YNC and 107% (p < 0.0001) in patients with AD relative to matched aliquots processed with the original sample preparation workflow . ROC curves were generated to determine whether the capacity of Aβ1–42 to discriminate between YNC and patients with AD was impacted by increased recovery. Although values increased in both YNC and AD when GuHCl was added directly to CSF aliquots, there was no significant difference in the diagnostic performance of this biomarker. The AUC was 0.8588  when GuHCl was added directly to CSF aliquots, and the AUC was 0.8453  when GuHCl was added after the CSF was transferred out of the original storage tube  were not significantly impacted by tube type or GuHCl denaturation  and patients diagnosed with dementia of unknown origin (n = 10) was collected into 15-ml polypropylene tubes. The CSF was immediately divided into three workflows: GuHCl denaturation in the 15-ml polypropylene tube immediately after collection  relative to condition 1   less than condition 1, but 9% (p = 0.0025) higher than condition 3   reduced differences in recovery between collection conditions  Fig. a, recove1–42 levels as diagnostic for AD [1–42 has been reported [1–42 [Rigorous biomarker development efforts have validated reduced CSF Aβc for AD , and recc for AD . In spitreported . Althouged [1–42 , the ide1–42 recovered from matched 0.1-ml and 0.5-ml aliquots of CSF measured with a Luminex® immunoassay . In contrast, CSF Tau and pTau181 measurements were stable, regardless of sample volume, suggesting that the effect was specifically associated with the hydrophobicity of Aβ1–42 and its tendency to nonspecifically bind (adsorb) to polypropylene sample tubes [1–42 concentrations recovered from 0.5-ml aliquots are consistently higher than those from 0.1-ml aliquots, independent of immunoassay platform. We replicated this finding a third time by measuring a subset of 0.1-ml and 0.5-ml aliquots with an absolute quantitative 2D-UPLC-MS/MS assay. The MRM method used for these measurements was developed in association with the Global Biomarkers Standardization Consortium of the Alzheimer’s Association and quantifies five Aβ peptides simultaneously  [1–42 alone, and it enabled us to observe whether different forms of Aβ may be less impacted by preanalytical factors. This effect declined progressively as the length and hydrophobicity of the measured peptide decreased, supporting the concept that tube adsorption is a function by hydrophobicity, and this finding is consistent with recently reported results using multiplex immunoassays [In the first part of study, we observed significant differences in the amount of Aβle tubes , 26, 34. Aβ1–34) , 35, 36,1–42 concentrations increased following pretreatment, similar to previous reports [1–42 population was tube-bound and therefore unmeasured by the assay, not masked by matrix interference as reported by others [1–42 from the walls of emptied tubes by washing with aCSF and Tween-20. Because Tween-20 had such a profound effect on recovery, we hypothesized that sample pretreatment might be an easy way to limit peptide adsorption and eliminate volume-associated quantitative discrepancies. In our hands, however, this led to inconsistent results, and we hypothesized that Tween-20 might be interfering with immunoassay components as reported by Vanderstichele et al. [To determine the extent to which tube adsorption was a factor in these observations, CSF aliquots were pretreated with Tween-20, a polysorbate surfactant capable of disrupting hydrophobic interactions. Aβ reports , 27, 28,y others , 19. Had1–42 adsorbed to the tubes, the original 2D-UPLC-MS/MS sample preparation workflow were modified to denature CSF in the original sample tube and maximize recovery by solubilizing tube-bound peptides. When matched pairs of 0.1-ml and 0.5-ml aliquots of AD CSF from the same donors were prepared in this manner, measured concentrations of Aβ1–42 increased, and sample volume-associated differences were eliminated. Similar results were also observed for Aβ1–40, Aβ1–38, and Aβ1–37. These results demonstrate that it is possible to eliminate inconsistencies in CSF Aβ1–42 quantification due to preanalytical tube adsorption with a simple modification to an existing sample preparation procedure, rather than introducing substances that could interfere with assay components.Knowing from our Tween-20 recovery experiments that Aβ1–42 biomarker assays across all technologies [1–42 recovery measured by AlzBio3 from untreated CSF in 1.5-ml Eppendorf LoBind® tubes exceeded untreated recovery and approached or exceeded Tween-20-facilitated recovery from the other tube types. In the 2D-UPLC-MS/MS assay, regardless of whether CSF was denatured directly in the tube or transferred to a fresh tube containing denaturant, Aβ1–42 recovery from 1.5-ml Eppendorf LoBind® tubes exceeded recovery from polypropylene tubes. Both our immunoassay and 2D-UPLC-MS/MS results suggest that 1.5-ml-capacity Eppendorf LoBind® tubes be considered for the storage of CSF intended for Aβ1–42 analysis and are consistent with recommendations by Vanderstichele et al. [There is a limitation to the approach of denaturing CSF in the original sample tube: Concentrations calculated using the 2D-UPLC-MS/MS assay are dependent on the accuracy of the sample volume processed for the method. In a routine clinical setting, it may be difficult to ensure that a precise and accurate volume of CSF is collected into the tube, which may limit the broad adoption of this approach. Because the LC-MS platform is routinely used for absolute quantitation, this approach could be effectively applied as a reference or comparator method. The multiplex LC-MS assay used in our work was developed in collaboration with the Global Biomarker Standardization Consortium of the Alzheimer’s Association and the International Federation of Clinical Chemistry and Laboratory Medicine Working Group on CSF Proteins. It was used during global interlaboratory studies conducted to develop and qualify candidate LC-MS-based reference methods and establish values for human CSF-certified reference materials produced by the European Commission Joint Research Centre to aid in harmonizing Aβnologies , 35, 36.e et al. .1–42. For example, if CSF Aβ1–42 peptides from patients with AD are more adsorptive than peptides obtained from healthy control subjects, differential tube retention might explain why measured concentrations differ between control subjects and patients with AD by as much as twofold [1–42 recovery in both patients with AD and control subjects, and ROC curve analysis did not indicate that there was an impact on the diagnostic power of the analyte, suggesting that the discriminatory power of Aβ1–42 is not dependent on tube adsorption.Improved recovery could negatively impact the diagnostic power of CSF Aβ twofold . However1–42 recovery when denaturation was initiated sooner, with the highest recovered concentrations detected in samples denatured immediately after collection or immediately before subaliquoting. These results demonstrate that analyte loss also occurs during the collection and aliquoting process. From a clinical assay perspective, the option to delay GuHCl denaturation until subaliquoting provides valuable flexibility; rather than commit an entire CSF draw to a specific assay such as 2D-UPLC-MS/MS, an investigator can postpone GuHCl denaturation until the subaliquoting step without risking Aβ loss, creating samples both with and without denaturant, thereby allowing additional biomarker measurements on platforms that may not be GuHCl-compatible.With experimental evidence that Aβ peptide adsorption occurs in sample aliquots and additional published data demonstrating that it occurs within 30 s of exposure to an adsorbent , it is l1–42/Aβ1–40 ratio strongly correlates with brain amyloid load and is considerably more effective at diagnosing clinical AD than CSF Aβ1–42 alone [1–42. These reports have generated substantial interest in the field of AD biomarkers, and the potential impact of this ratio continues to be explored. Beyond diagnostic applications, Aβ peptide ratios have also been applied to preanalytical questions and are reported to be useful in ameliorating the effects of inconsistent recovery due to adsorption, although there has been some inconsistency in the results [1–42 and Aβ1–40 concentrations from the same aliquot simultaneously, thereby eliminating intra-aliquot, interassay, and interplatform inconsistencies that could potentially confound the accuracy of the Aβ1–42/Aβ1–40 ratio. We were curious what impact normalization of Aβ1–42 to Aβ1–40 would have on the Aβ1–42 recovery differences that emerge based on the timing of denaturation. We found that normalization to Aβ1–40 substantially reduced measured variability in recovery for all subjects, as did normalization to Aβ1–38 and Aβ1–37. The Aβ1–42/Aβ1–40 ratio demonstrated the lowest variability, possibly owing to the higher abundance of Aβ1–40 in CSF, and of the isoforms evaluated, it was closest in hydrophobicity to Aβ1–42. These results suggest that it may be possible compensate for inconsistent absolute Aβ1–42 recoveries by performing a simple normalization step.It has recently been reported that the CSF Aβ42 alone , 37–42.  results , 34. A s1–42, because improved recovery did not reduce the discrimination between patients with AD and control subjects. Maximum recovery of Aβ was observed when using 1.5-ml Eppendorf LoBind® tubes as assessed by immunoassay and a 2D-UPLC-MS/MS assay. Recovery can be further improved with denaturation of CSF using GuHCl early in the handling process. Collection of CSF directly into LoBind® tubes should also be explored as a strategy to resolve the adsorption issue. Additionally, our study further supports that the ratio of CSF Aβ1–42 to Aβ1–40 may be a more robust biomarker than CSF Aβ1–42 alone. These results contribute to our understanding of the key preanalytical factors impacting the measurement accuracy of Aβ peptides Aβ1–42, Aβ1–40, Aβ1–38, Aβ1–37, and Aβ1–34 and will contribute to emerging standardized operating procedures for handling CSF in clinical settings.This study confirms previous observations that CSF Aβ nonspecifically adsorbs to sample tubes, that disproportionate adsorption can occur as a function of sample volume, and that this can have a significant effect on measured peptide recovery. Interestingly, tube adsorption may not impact diagnostic performance of AβAdditional file 1:Table S1. Tube Types Table S2. MRM transitions, cone voltages, and collision energies for each peptide and internal standards Figure S1. Impact of aliquot size on CSF Aβ1-42 concentrations measured from low-retention tubes with the MSD Aβ1-42 assay. Matched 0.1mL and 0.5-mL aliquots of CSF from AD patients (n=15) were measured. Aβ1-42 concentrations from 0.5-mL aliquots were higher than 0.1-mL aliquots in 15/15 subjects. Figure S2. GuHCl denaturation increases Aβ1-42 recovery from the CSF of YNC and AD subjects. (A) Aβ1-42 concentrations recovered from aliquots of YNC and AD subjects when the samples were denatured with GuHCl before transfer from the original aliquot tube. (B) Aβ1-42 concentrations recovered from aliquots of YNC and AD subjects when the samples were denatured with GuHCl after transfer from the original aliquot tube. Figure S3. Receiver Operating Characteristic (ROC) Curves generated for CSF Aβ1-42. ROC curves generated from YNC and AD CSF denatured with GuHCl before transfer from the original aliquot tube (black) or from the same samples denatured with GuHCl treatment after transfer from the original aliquot tube (gray).Figure S4. CSF Aβ peptide recovery after GuHCl denaturation. Recovery of Aβ1-42, Aβ1-40, and Aβ1-38, Aβ1-37, and Aβ1-34 from healthy controls (black) and Dementia patients (red) after GuHCl denaturation at different times following collection. Condition 1- GuHCl denaturation immediately after CSF collection; Condition 2- GuHCl denaturation immediately before sub-aliquoting; Condition 3- GuHCl denaturation after sub-aliquoting, and immediately before analysis; Condition 4- sub-aliquot into Eppendorf LoBind® tubes immediately after collection, GuHCl denaturation immediately before analysis. Figure S5. Normalization of CSF Aβ1-42 to Aβ1-40, Aβ1-38, or Aβ1-37 reduces variability in CSF Aβ1-42 recovery from Eppendorf LoBind® tubes.Variation in CSF Aβ1-42 recovery due to the timing of GuHCl denaturation  was reduced in 10/10 Young Normal Controls  and 9/10 patients diagnosed with dementia (red circles) when the Aβ1-42 was normalized to Aβ1-40, Aβ1-38 or Aβ1-37 (****p≤ 0.0001).(DOCX 5078 kb)


Scientific Reports 10.1038/s41598-017-05294-0, published online 11 July 2017Correction to: This Article contains errors.In the Methods section, under the subheading ‘Quick CSF Analysis’,50.”“This re-sampling procedure automatically takes into account the covariance structure in the posterior distribution of the CSF parameters, and allows us to assess the precision of the estimated CSF curveshould read:30.”“This re-sampling procedure automatically takes into account the covariance structure in the posterior distribution of the CSF parameters, and allows us to assess the precision of the estimated CSF curveBehav Brain Res243, 102–8 (2013)’. The correct Reference 66 appears below as Reference1.In addition, Reference 66 is incorrectly listed as ‘Arena, Hutchinson, Shimozaki & Long Visual discrimination in noise: behavioural correlates of age-related cortical decline.


The high-sensitivity cardiac troponin T assay of Roche Diagnostics is known to have interference with high concentrations of biotin as this assay uses biotin-streptavidin binding as detection method. As studies so far have not shown if different biotin concentrations could have diverse influence on various troponin concentrations and whether interference could be removed by available protocol within corresponding turnaround time we aimed to investigate it.et al. was also tested.Plasma samples were spiked with different concentration of biotin solution. Troponin T concentrations were tested on a Roche Cobas® 8000 module 602 analyser. Final concentrations of biotin and troponin T were 50, 100, 500 and 1000 μg/L and 18, 59, 201 and 6423 ng/L, respectively. Impact of different incubation times following biotin neutralization protocol described by Piketty We observed a mean of negative biases of 24, 56, 97, and 98% of the troponin T expected value at biotin concentrations of 50, 100, 500, 1000 μg/L. Neutralization protocol was applied on the sample with initial concentration of TnT of 59 ng/L at a biotin concentration of 1000 μg/L. Same results across different incubation times from 60 to 0 minutes were obtained . We demonstrated that neutralization process had a dilution effect of the troponin concentration .Biotin interference is not dependent of initial troponin value. Interference could be successfully neutralized within a time frame compatible with emergency but results still should be carefully interpreted due to possible dilution effect. No significant effect on hs-cTnT was observed with bilirubin or turbidity. However, hs-cTnT is negatively influenced by haemolysis  and plasma samples were obtained by centrifugation . Following routine analysis residual samples with concentration close to the 99th percentile of healthy controls (14 ng/L), to the cut-off proposed by the European Society of Cardiology (52 ng/L) which has a positive predictive value for myocardial infarction of 75 to 80%, to a positive free value (200 ng/L) and with a very high concentration of troponin T (5000 ng/L) were selected, anonymized, pooled, aliquoted (900 µL) and used in further analysis , we demonstrated that proposed incubation time of 1 hour in biotin interference neutralization protocol is not necessary. Thus, turnaround time of troponin could be improved if neutralization protocol should be applied. We confirmed interference removal via neutralization method and absence of difference in case of suppression of the incubation time in three specimens from two patients with acute coronary syndrome who were treated for multiple sclerosis by high dose of biotin.Our study shows that hs-cTnT assay results are decreased by increasing biotin concentrations on various troponin concentrations. These interferences are not influenced by the initial troponin concentration. Indeed, we observed close negative biases at the same concentrations of biotin regardless of the initial value of troponin. High plasma concentrations of biotin from 500 to 1000 µg/L biotin, which are found in patients treated for multiple sclerosis, decreased the true value by more than 96% (et al. demonstrated, almost 10% and 20% negative bias at 15.6 µg/L and 31.3 µg/L of biotin, respectively (et al. for moderately high values of biotin. In fact, 45% of negative bias was observed on dose response curve at 60 µg/L of biotin (Trambas


Recent work suggests extensive adaptation of transposable elements (TEs) for host gene regulation. However, high numbers of integrations typical of TEs, coupled with sequence divergence within families, have made systematic interrogation of the regulatory contributions of TEs challenging. Here, we employ CARGO, our recent method for CRISPR gRNA multiplexing, to facilitate targeting of LTR5HS, an ape-specific class of HERVK (HML-2) LTRs that is active during early development and present in ~700 copies throughout the human genome. We combine CARGO with CRISPR activation or interference to, respectively, induce or silence LTR5HS en masse, and demonstrate that this system robustly targets the vast majority of LTR5HS insertions. Remarkably, activation/silencing of LTR5HS is associated with reciprocal up- and down-regulation of hundreds of human genes. These effects require the presence of retroviral sequences, but occur over long genomic distances, consistent with a pervasive function of LTR5HS elements as early embryonic enhancers in apes. Nearly half of the human genome is composed of transposable elements (TEs), which are increasingly being recognized not just as parasitic DNA, but as an important source of regulatory innovation for the host . In partDespite evidence suggesting the importance of LTRs and other TEs in rewiring gene regulatory networks, most current studies are either correlative or focus on the analysis of individual insertions, rather than on systematically perturbing specific TE classes, with one notable exception of a report utilizing transcription activator-like effector (TALE) fused to effector domains for functional perturbations of mouse LINE1 elements . This knHere, we couple CARGO with nuclease dead Cas9 (dCas9) fused to an activation or repression domain   to facilWe now demonstrate that a CARGO-based CRISPRa/CRISPRi strategy facilitates robust and specific targeting of dCas9 to ~90% of LTR5HS elements throughout the human genome for efficient activation or repression of HERVK transcripts and proteins. Moreover, perturbation of LTR5HS function by recruitment of an activator or a repressor leads to the reciprocal up- and down-regulation of nearly 300 human genes, along with widespread effects on the chromatin landscape surrounding the promoters of these genes and LTR5HS insertions. Remarkably, these effects on host gene expression occur over long genomic ranges, indicating that LTR5HS elements function as distal enhancers for a substantial number of genes. In agreement, deletion of select LTR5HS elements confirms their strong contribution to host gene transcription. These LTR5HS-regulated genes are preferentially expressed in naïve relative to primed hESCs and their transcripts are also elevated in developing human blastocysts as compared to those of rhesus macaque, a primate species that does not contain LTR5HS insertions. These observations suggest that recent HERVK endogenization has contributed to the establishment of unique gene expression patterns in preimplantation embryos of humans and other apes. Altogether, our work provides a novel and broadly applicable strategy for functional manipulation of specific TE classes across the genome and supports a pervasive role of LTRs as embryonic gene enhancers.To investigate the role of HERVK LTR5HS insertions in the regulation of embryonic gene expression and, more broadly, to establish a proof of principle for using CARGO to simultaneously target hundreds of repetitive elements interspersed across the genome, we designed a CARGO array with 12 distinct gRNA transcriptional units, altogether predicted to recognize ~91% (635/697) of LTR5HS integrations in the human genome (hg38 assembly) when allowing zero mismatches between gRNA sequences and LTR5HS sequences . We compAlthough our custom scoring algorithm penalized potential gRNAs that target genomic regions other than LTR5HS, we ‘masked’ the highly related (~88% sequence similarity) HERVK LTR5A and LTR5B sequences to exclude them from negatively affecting candidate gRNA scores. Consequently, our CARGO array is computationally predicted to exhibit some binding to LTR5A and LTR5B, but should not target other classes of LTRs or other TEs , includiStreptococcus pyogenes gRNA scaffold  or the Staphylococcus aureus gRNA scaffold (LTR5HS Sa). As a non-targeting control, we also assembled a CARGO array with gRNAs that should not pair anywhere in the human genome, with the S. pyogenes gRNA scaffold (nontarget Sp). To couple CARGO with CRISPRa/CRISPRi approaches for systematic perturbation of function, we used the human embryonal carcinoma NCCIT model to generate six transgenic cell lines, each expressing one of the three aforementioned CARGO arrays and a doxycycline-inducible S. pyogenes dCas9 fused to either the strong transactivation domain VPR  or to a repressive KRAB domain  , thereby serving as a control for the presence of RNA-loaded dCas9 complexes (We assembled CARGO LTR5HS-targeting arrays using either the CRISPRi)   . Only ceCRISPRi) . By contomplexes .env, gag, and pro, as well as protein levels of Env. We found that although HERVK is already highly expressed in NCCIT cells, levels of both HERVK proviral transcripts and LTR5HS-derived transcripts further increase between 10- and 15-fold in the dCas9-VPR activating lines in the recruitment condition LTR5HS Sp, as compared to the control conditions LTR5HS Sa and nontarget Sp  to characterize the prevalence and specificity of dCas9 targeting to individual LTR5HS instances across the genome. We derived NCCIT lines stably expressing doxycycline-inducible dCas9 fused to EGFP (dCas9-GFP), and one of the three CARGO arrays: the recruitment (LTR5HS Sp) array or the two control (LTR5HS Sa or nontarget Sp) arrays. For each CARGO array condition, we performed ChIP-seq using three antibodies: one against Cas9, and two against GFP . We also performed ChIP-seq for dCas9 using the Cas9 antibody described above, in the same three cell populations. As expected, in WT NCCIT cells, which do not express a dCas9 fusion, we did not detect any enrichment of dCas9 signal. We also found that in these cells, a large subset of LTR5HS elements is marked by H3K27ac and H3K4me3, with H3K4me3 showing the expected asymmetric distribution consistent with the direction of LTR-driven transcription . Thus, the function of LTR5HS as alternative promoters cannot broadly explain the observed regulatory effects on host gene transcription.For these 275 LTR5HS-regulated transcripts, we found that the nearest LTR5HS insertion is upstream of the promoter in 150 cases, and downstream in 125 cases. This finding suggests that even downstream LTR5HS insertions can have a transcriptional effect on the gene, meaning that these insertions do not serve as alternative promoters. Furthermore, since LTR sequences do have a natural orientation, we also examined the relative orientation of the nearest LTR5HS for each of these genes. In the 150 cases in which the nearest LTR5HS insertion is upstream of the promoter, the LTR5HS has the same orientation as the gene (both on Watson strand or both on Crick strand) 83 times, compared to 67 in the opposite orientation. In the 125 cases in which the nearest LTR5HS insertion is downstream of the promoter, the LTR5HS has the same orientation as the gene 48 times, compared to 77 in the opposite orientation. Together, these findings suggest that neither the relative position of the LTR5HS insertion to the promoter, nor its orientation, determines its ability to effect a transcriptional change on the gene in question under CRISPRa or CRISPR, consistent with the putative enhancer function. Furthermore, when we analyzed the RNA-seq data for the presence of chimeric transcripts between LTR5HS and the LTR5HS-regulated genes, we detected an appreciable level (i.e. transcripts per million [TPM] > 1) of chimeric transcription at only four of the 275 genes  are also upregulated in naïve as compared to primed hESCs (−21) are expressed in the human preimplantation epiblast . Interestingly, however, even though our experiments were performed in NCCIT embryonal carcinoma cells, we analyzed previously published RNA-seq data and observed statistically significant relationships between LTR5HS-regulated transcripts and differentially expressed genes in these public datasets. Specifically, we found that 138 of the 275 LTR5HS-regulated transcripts  were among the 275 LTR5HS-regulated genes with reciprocal effects on expression upon CRISPRa/CRISPRi, (ii) contained no more than two LTR5HS within 1 Mb of the TSS, (iii) spanned a large range of promoter distances from LTR5HS , and (iv) represented all potential combinations of position relative to the promoter as well as orientation of LTR5HS-driven transcription with respect to the gene. We deleted the nearest LTR5HS element at each selected locus via CRISPR/Cas9 genome editing using WT NCCIT cells as a parental cell line.We next sought to test whether the presence of LTR5HS DNA sequences is required for both the deposition of enhancer marks in the vicinity of the LTR5HS and for the observed long-range effects on host gene expression. To this end, we selected six genes, CACNA2D2, EPHA7, and ALPPL2. We found that upon deletion of the LTR5HS, both H3K27ac and H3K4me1 were significantly reduced in the regions directly flanking the LTR5HS insertion, consistent with the idea that the presence of the LTR5HS sequence is required for the deposition of these marks (EPHA7 and ALPPL2) upon deletion of the LTR5HS . For ALPPL2, we measured an average of ~3.3-fold decrease in expression with deletion of the nearest LTR5HS, which is also human specific, located ~16 kb downstream of the TSS, and transcribed in the same orientation as the gene (both are on the Watson strand) . We similarly observed an average of ~6.9-fold decrease in expression after deleting the LTR5HS element nearest to NFKB2, ~2.1 kb upstream of the TSS and transcribed in the same orientation as the gene (both are on the Watson strand) . We found an average of ~7.0-fold loss of expression of SERPINB9 upon deletion of the nearest LTR5HS element, ~5.8 kb upstream of the TSS and transcribed in a divergent orientation with respect to the gene . Finally, upon deletion of the LTR5HS closest to GDPD1, ~69 kb downstream of the TSS and transcribed in a convergent orientation towards the gene, we found an average of ~3.2-fold loss of expression of the gene  contain a consensus motif and are bound by the pluripotent stem cell/primordial germ cell/reprogramming factor and master regulator OCT4, which may have contributed both to their endogenization and cooption for enhancer function . Indeed,It is intriguing to consider whether regulatory repurposing of LTR5HS elements for enhancer function may have contributed to human-specific transcriptome divergence and endowed the early developmental stages of the human embryo with species-specific attributes. All LTR5HS insertions are unique to apes, and a subset is human-specific or even human-polymorphic . We founNCCIT cells were obtained from ATCC. NCCIT cells were grown in RPMI-1640 , supplemented with 10% FBS , 1x Glutamax (Thermo Fisher Scientific), 1x non-essential amino acids (Thermo Fisher Scientific), and 1x antibiotic/antimycotic (Thermo Fisher Scientific). Cell lines were tested for mycoplasma contamination using MycoAlert Detection Kit . All cell lines tested negative for mycoplasma contamination.All unique SpCas9 16 nt seed sequences derived from known instances of LTR5HS were aligned against hg38 human genome with bowtie  using ‘-For analysis shown in CARGO arrays containing twelve guides were assembled as described previously . The 12 For CRISPRa/CRISPRi, dCas9-VPR, dCas9-KRAB, and dCas9-GFP fusions were inserted into a PiggyBac transposon containing a puromycin-selectable cassette. For CRISPR/Cas9 deletion of LTR5HS, a guide upstream of a targeted LTR5HS insertion was cloned into px458 (pSpCas9(BB)−2A-GFP, a gift from Feng Zhang, Addgene plasmid #48138) , which eNCCIT cells were transfected with PiggyBac plasmids containing a dox-inducible dCas9 fusion, along with PiggyBac transposase, and selected using puromycin . These lines were then transfected with PiggyBac CARGO plasmids, along with PiggyBac transposase, and selected using G418 (Thermo Fisher Scientific). Cells were re-selected with puromycin (Invivogen) to ensure that dCas9-fusions were not lost during second transposition event. For all dCas9 fusion experiments, expression of fusion proteins was induced for four days with 2 ug/mL doxycycline Cells for RT-qPCR and RNA-seq were homogenized in Trizol (Thermo Fisher Scientific), then RNA was extracted using Direct-zol RNA columns , with DNase treatment on-column, and eluted in water.Reverse transcription for RT-qPCR was performed using SensiFAST cDNA synthesis kit , according to the manufacturer’s instructions with input from the RNA extraction described above.qPCR was performed using SensiFAST SYBR No-Rox kit (Bioline) in a LightCycler 480II , using technical duplicates or triplicates for each sample. Each condition was also analyzed with at least two independent biological replicates. Figure legends indicate transcript normalization for RT-qPCR.Whole cell nuclear extracts were prepared by lysing cells for 30 min at 4° C with overhead vertical rotation in protein extraction buffer , then clearing by centrifugation and recovery of the supernatant. Total protein concentration was quantified by Bradford assay . Equal amounts of protein were denatured in LDS buffer (Thermo Fisher Scientific) supplemented with 2-mercaptoethanol (Sigma-Aldrich), then loaded in 3-fold serial dilutions onto tris-glycine 4–20% SDS-PAGE denaturing gradient gels (Thermo Fisher Scientific), then transferred onto nitrocellulose membrane. Chemiluminescence was assayed using Lumi-light Plus (Roche) or Amersham  and visualized on autoradiography film.7 NCCIT cells were fixed in 1% formaldehyde for 10 min at room temperature in PBS, then quenched with glycine to a final concentration of 0.125 M for 10 min. Chromatin was sonicated to 0.5–2.0 kb using Bioruptor , cleared by centrifugation, divided into separate aliquots for each antibody, and incubated with 5 μg of antibody overnight at 4° C. Subsequently, 100 μL of Dynabeads protein G (Thermo Fisher Scientific) were added to the ChIP reactions and incubated for 4–6 hr at 4° C. Magnetic beads were washed and chromatin was eluted, followed by reversal of crosslinks overnight at 65° C, proteinase K and RNase A treatment, and DNA purification by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. ChIP DNA was resuspended in water.ChIP assays were performed as described previously . BrieflyFor ChIP-seq data presented in Total RNA (10 ug) from two independent biological replicates was subjected to oligo-dT purification using Dynabeads oligo(dT) (Thermo Fisher Scientific), then fragmented with 10x fragmentation buffer (Thermo Fisher Scientific). Fragmented RNA was used for first strand cDNA synthesis with Superscript II (Thermo Fisher Scientific) and random hexamer primers (Thermo Fisher Scientific). Second strand cDNA synthesis was performed using RNase H (Thermo Fisher scientific) and DNA polymerase I (New England Biolabs). The resulting double-stranded cDNA was used for Illumina library preparation as described for ChIP-seq experiments, but was size-selected on acrylamide gels, and pooled and sequenced on a NextSeq 500 (Illumina) at the Stanford Functional Genomics Facility, using 2 × 150 sequencing with index read.gRNAs upstream and downstream of individual LTR5HS insertions with low potential off-targets were identified using CRISPOR . For eacQuality of FASTQ files was assessed using FASTQC software. Reads were aligned to hg38 genome using bowtie2 , with ‘bFor analysis shown in Quality of FASTQ files was assessed using FASTQC software. Reads from were trimmed of Illumina adapter sequences using cutadapt. For analysis of human non-repeat transcripts, trimmed reads were aligned using hisat2  to the hAll possible 150 bp paired-end reads for fragments in size range 150–400 bp within −400 bp to +400 bp of known LTR5HS were generated from hg38 reference sequence with bedtools getfasta and aligned to hg38 assembly with bowtie2 (--end-to-end --no-mixed --no-discordant). MAPQ score for each pair was extracted and assigned to LTR5HS instance. Plot of fraction of uniquely mappable (MAPQ > 20) reads was generated in R.All antibodies, primers, and gRNAs used in this study are listed in Sequencing data have been deposited in GEO under accession code GSE111337. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Detlef Weigel as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Deborah Bourchis (Reviewer #1); Cédric Feschotte (Reviewer #2).Thank you for submitting your article "Systematic perturbation of retroviral LTRs reveals widespread long-range effects on human gene regulation" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this study, the question of the extent to which Transposable Elements and their relics in a genome have been adapted to influence "host" gene regulation is addressed. By adapting their recently published gRNA multiplexing CARGO method  the authors target dCAS9-fusion activator (VPR) or repressor (KRAB) proteins to the ~700 copies of the HERVK (HML-2), LTR5HS elements of the human genome (known to be expressed early on in human development). The authors examine the effects of this TE modulation on nearby gene expression in human embryonal carcinoma cells derived from a germ-line tumor (NCCIT). The authors report that activation/silencing of LTR5HS is associated with reciprocal up- and down-regulation of nearly 300 human genes, although the VPR activation shows more striking effects than KRAB repression. They go on to look at the range of impact that these LTR5HS elements have is up to 160kb, suggesting that they might be acting as potential enhancers. The authors also specifically delete three individual LTR5HS elements, and examine effects on nearby genes by qRT-PCR which points to their potential role in gene regulation.This study is of a great interest for the field as it serves as an important demonstration that expression of hundreds of copies of a specific family of LTR retrotransposons can be efficiently modulated using dCas9 effectors and the recently developed CARGO system and it allows the impact of a whole LTR family on gene expression to be addressed in a systematic way. Although previous studies  have targeted whole TE families, the present study is one of the first to describe an in depth, genome-wide analysis of what the functional contribution of LTR elements might be for gene expression control across the human genome. The manuscript is well written, easy to read and the experiments are of high quality. There are however some important issues that the authors will need to address.Essential revisions:1) The authors claim that the LTR5HS sequences they have affected are acting as enhancers. However, they need to prove this enhancer potential at the endogenous locations. As the study stands they cannot rule out that the elements are acting as alternative promoters, particularly as the three LTR5HS elements that they deleted were in close proximity (a few kb) to the associated genes. Several approaches can address this, as outlined in the reviewers' comments. These include demonstrating enhancer orientation independence for the LTRs; presenting their analyses in Figure 4 using stranded information, and directionality of the LTR as well as clarifying distances from TSS; demonstrating that chimeric transcripts are not produced between the LTR5HS elements and the up-regulated genes in the CRISPRa approach using 5'RACE or CAGE RNAseq; performing ChIP-PCR to examine enhancer marks such as H3K27ac, H3K122ac and H3K4me1 to see if their distributions change in wt versus LTR-deleted condition across at least two of the loci studied ; performing deletion experiments for LTRs that are more distant to the misregulated genes identified; examining the effects both upstream and downstream of the genes.2) The authors should provide more information about the transcription factor and chromatin landscape of the LTR sequences. This could thus assess whether there is a pre-existing regulatory activity of individual LTR5HS elements in NCCIT cells that could influence the efficiency and outcomes of the CRISPRi/a assays.NFKB2 and SERPINB9 upon deletion of the unique LTR is not shown.3) The repressive effects on neighbouring genes upon KRAB targeting is not clear . There are no statistics and the number of genes falling into different categories are not given; the effects on basal transciprition of CACNA2D2, 4) Statistics need to be checked and included in several of the figures including Figure 4 above. The distance analyses lack statistics: how many genes per 'distance' block were analysed? Are the changes in transcription statistically significant? How would this look with a negative control (e.g. another repeat)? Essential revisions:1) The authors claim that the LTR5HS sequences they have affected are acting as enhancers. However, they need to prove this enhancer potential at the endogenous locations. As the study stands they cannot rule out that the elements are acting as alternative promoters, particularly as the three LTR5HS elements that they deleted were in close proximity (a few kb) to the associated genes. Several approaches can address this, as outlined in the reviewers' comments. These include demonstrating enhancer orientation independence for the LTRs; presenting their analyses in Figure 4 using stranded information, and directionality of the LTR as well as clarifying distances from TSS; demonstrating that chimeric transcripts are not produced between the LTR5HS elements and the up-regulated genes in the CRISPRa approach using 5'RACE or CAGE RNAseq; performing ChIP-PCR to examine enhancer marks such as H3K27ac, H3K122ac and H3K4me1 to see if their distributions change in wt versus LTR-deleted condition across at least two of the loci studied ; performing deletion experiments for LTRs that are more distant to the misregulated genes identified; examining the effects both upstream and downstream of the genes.We thank the reviewers for this comment and agree that the claim that LTR5HS elements function as distal enhancers is central to the thesis of our manuscript, and thus should be further strengthened. We followed the reviewers’ suggestions and took a multi-pronged approach to addressing this comment, as outlined below:Demonstrating orientation independence for the LTR5HS effect on gene expression: We reanalyzed the data concerning the LTR5HS-regulated genes  and their nearest LTR5HS insertion (the putative enhancer). We found that for these 275 genes, the nearest LTR5HS is upstream of the promoter in 150 cases, and downstream in 125 cases. This finding suggests that even downstream LTR5HS insertions can have a transcriptional effect on the gene, a finding not compatible with their function as alternative promoters. Importantly, we deleted three of those downstream LTR5HS insertions and confirmed that they indeed significantly affect expression of the candidate target gene (see next section for details). As LTR sequences do have a natural orientation, we also examined the relative orientation of the nearest LTR5HS for each of these genes. In the 150 cases where the LTR5HS insertion is upstream of the promoter, the LTR5HS insertion has the same orientation (both on the Watson strand or both on the Crick strand) 83 times, compared to 67 in the opposite orientation. In the 125 cases where the LTR5HS insertion is downstream, the insertion has the same orientation as the gene 48 times, compared to 77 in the opposite orientation. These findings together suggest that neither the relative position of the LTR5HS to the promoter, nor its orientation, determines its ability to effect a transcriptional change on the gene in question under CRISPRa or CRISPRi, consistent with the proposed enhancer function. This information has now been incorporated in the text of the revised manuscript under the header “Reciprocal effects of LTR5HS CRISPRa/CRISPRi on host gene expression.”Additional deletion experiments for LTRs that are more distant to the misregulated genes identified and located downstream from the TSS:“in close proximity” to the associated genes and thus may not be enhancers. First, we would like to clarify that since these LTRs were respectively ~17 kb, ~2 kb and ~6 kb upstream from the promoter, they all fall under the definition of distal regulatory elements, typically defined as those that do not overlap with promoter sequences . Furthermore, two of the genes (SERPINB9 and CACNA2D2) are transcribed in opposite orientation relative to the LTR5HS, making their LTR5HS-originating chimeric transcription unlikely. Nonetheless, we agree that since all deleted elements were upstream from the target genes, additional deletions were needed to further exclude a possibility that the LTR5HS elements function as alternative promoters. In the revised manuscript, we present analysis of clonal lines with homozygous deletions of three LTR5HS elements located downstream from the candidate target genes: (i) ~16 kb downstream of the ALPPL2 gene TSS, (ii) ~245 kb downstream of the EPHA7 gene TSS  and (iii) ~69 kb downstream of the GDPD1 gene TSS. In all three cases, we observed downregulation of the candidate target gene . Together with our previous results, we now show examples of deletions of LTR5HS elements located within a wide distance range from the target gene promoter , positioned either upstream or downstream from the gene TSS and transcribed in either direction with respect to the gene. In all cases, we observed downregulation of the candidate target gene , providing definitive evidence that LTR5HS elements indeed function as enhancers.The reviewers suggested that the three LTR5HS elements deleted in our original manuscript were all We also want to clarify , that these deletion experiments were all performed in WT NCCIT cells that do not express any dCas9 fusions. We believe that the confusion might have been caused by our inclusion of the genome browser images with dCas9 binding at these selected LTRs in the figure, and thus we eliminated those browser images and reorganized the figure to incorporate new data.ChIP-PCR analysis of enhancer marks in the LTR5HS deletion lines:CACNA2D2, ALPPL2, and EPHA7. In each case, we observe loss of both H3K27ac and H3K4me1 from the LTR5HS flanking regions in the deletion lines , demonstrating that deposition of these enhancer marks is dependent on the presence of the LTR sequence. Furthermore, analysis of the H3K27ac at the target gene promoters revealed downregulation of promoter acetylation levels in LTR5HS deletion lines, though we note that this downregulation did not reach statistical significance at one of the genes .We performed ChIP-qPCR for the histone modifications H3K27ac and H3K4me1 in homozygous deletion and wild type clones for three separate LTR5HS insertions: those near NBPF12, SLC4A8, FA2H and TIMM50). Notably, of the six LTR5HS elements that we deleted, all showed effect on candidate target gene expression, but none had detectable levels of chimeric transcription between the LTR5HS and the regulated gene. We therefore conclude that while such chimeric transcripts may indeed arise at some loci, they cannot explain the regulatory effects observed in our study. This information has been incorporated into the manuscript under the header, “Reciprocal effects of LTR5HS CRISPRa/CRISPRi on host gene expression.”Analysis of chimeric transcripts between the LTR5HS elements and the upregulated genes: We analyzed deeply sequenced (~500 million 150 bp paired-end reads) RNA-seq data for the presence of the chimeric transcripts between LTR5HS and 275 LTR5HS-regulated genes identified in our study. We detected an appreciable level  of chimeric transcription at only four of the 275 genes  The authors should provide more information about the transcription factor and chromatin landscape of the LTR sequences. This could thus assess whether there is a pre-existing regulatory activity of individual LTR5HS elements in NCCIT cells that could influence the efficiency and outcomes of the CRISPRi/a assays.S. pyogenes CARGO array (i.e. under targeting conditions). This experimental design allows us to not only examine the landscape in unperturbed NCCITs, but also to measure the effects of VPR activation and KRAB repression on the chromatin states. Results from these experiments are presented in a new figure .To investigate the chromatin landscape of the LTR sequences, we performed ChIP-seq for H3K27ac, H3K4me3, and H3K9me3. In addition to doing these experiments in the parental WT NCCIT line, we also performed these ChIPs in lines expressing dCas9-VPR and dCas9-KRAB along with the LTR5HS Briefly, our results can be summarized as follows:H3K27ac, H3K4me3 and H3K9me3 patternsA subset of LTR5HS elements is marked by H3K27ac and H3K4me3 in WT cells in the absence of perturbation, with H3K4me3 showing asymmetric distribution consistent with the direction of the LTR-driven transcription, as has previously been observed at promoters and highly transcribed enhancers. Under CRISPRa conditions, most (over 90%) LTR5HS elements gain high level of H3K27 acetylation, but interestingly, H3K4me3 levels remain relatively unaffected. Strong gains of H3K27ac occur even at those LTR5HS elements that have low/no endogenous acetylation, which may indicate that ectopic enhancer activation is relatively common and efficient with dCas9-VPR system. Conversely, under CRISPRi conditions, endogenous H3K27ac and H3K4me3 are suppressed, and most LTR5HS elements become decorated with high levels of H3K9me3, as would be expected, given that KRAB repression is mediated by the H3K9me3 deposition . Notably, in WT NCCIT cells, LTR5HS elements typically lack H3K9me3, regardless of the presence or absence of the active marks, suggesting that in these cells LTR5HS escapes KRAB-mediated repression, a major mechanism of endogenous retrovirus silencing.In addition to examining histone modifications at LTR5HS insertions, we also assessed H3K27ac, H3K4me3, and H3K9me3 patterns surrounding the promoters of the 275 LTR5HSregulated transcripts . We found that most of these promoters have at least some H3K27ac and H3K4me3 in WT cells, and most gain or lose, respectively, H3K27 acetylation under CRISPRa or CRISPRi conditions . Notably, these changes occur in the absence of direct dCas9 binding to the promoters, suggesting that they result from the long-range effects we describe. Furthermore, although some gains of H3K9me3 can be observed in the vicinity of the promoters under CRISPRi conditions, most of the TSS remain unmethylated at H3K9 and, unlike at the LTRs, their H3K4me3 levels are relatively unaffected, suggesting that direct silencing of promoters via H3K9me3 spreading from a nearby LTR5HS is not likely to explain the transcriptional effects we examine in this study. As a control, we performed these same analyses on a set of 275 randomly selected promoters, and we detected no changes in any histone mark under CRISPRa or CRISPRi conditions .dCas9 binding patternsWe also performed ChIP-seq for dCas9-VPR and dCas9-KRAB fusions , as suggested in minor point 9. This experiment shows no dCas9 signal in WT (non-dCas9-expressing cells), but widespread binding to over 90% of LTR5HS in dCas9-VPR- and dCas9-KRAB-expressing cells . We further observed that the dCas9-VPR levels at LTR5HS were higher than dCas9-KRAB levels. This is likely attributable to the fact that VPR, a strong activation domain, recruits coactivators that promote nucleosomal depletion, whereas KRAB-mediated H3K9me3 facilitates chromatin compaction, which may in turn provide, respectively, positive or negative feedback for dCas9 fusion binding. Nonetheless, dCas9-KRAB still occupies and mediates H3K9me3 deposition at the vast majority of LTR5HS elements .3) The repressive effects on neighbouring genes upon KRAB targeting is not clear . There are no statistics and the number of genes falling into different categories are not given; the effects on basal transciprition of CACNA2D2, NFKB2 and SERPINB9 upon deletion of the unique LTR is not shown.CACNA2D2, NFKB2, and SERPINB9 upon deletion of the nearest LTR5HS, this result was shown in the initial manuscript in Figure 5C . Again, perhaps due to our inclusion of the genome browser images with dCas9 binding at these selected LTRs, it was not clear to the reviewers that these deletion experiments were all performed in WT NCCIT cells that do not express any dCas9 fusions. In any case, as discussed above, we now extended the analysis to the additional three elements and show the effect of deletion of six different LTR5HS on the basal transcript levels of their candidate target genes .A discussion of statistics in Figure 4B  follows in point 4. As for the effects on basal transcription of 4) Statistics need to be checked and included in several of the figures including Figure 4 above. The distance analyses lack statistics: how many genes per 'distance' block were analysed? Are the changes in transcription statistically significant? How would this look with a negative control (e.g. another repeat)?We analyzed a total of 26517 genes. These are broken down into 11 bins . Each bin contains genes whose TSS is within +/- 20 kb of the bin description. Therefore, bin “0” contains genes whose TSS are between -20 and +20 kb from the nearest LTR5HS; bin “40” contains genes whose TSS are between +20 and +60 kb from the nearest LTR5HS; and so on. The number of genes per bin is as follows:As for statistical significance, we have now performed one-sample Wilcoxon signed rank tests of the null hypothesis that the distribution of log2 fold change at each bin is symmetric around 0, which would describe no change in gene expression. Those values are indicated in the table above. We would like to point out that all bins we show reach statistical significance, including the +/-220-180 kb bins. However, if we perform significance testing on further bins, we show that log2FoldChange figures do fail to disprove the null hypothesis of the Wilcoxon signed rank test at bins further away. See below for results:These results have now been incorporated to the revised manuscript as Supplemental file 1. To further address the reviewer’s point, as a negative control we generated similar plots of fold change under CRISPRa and CRISPRi, using an unrelated LTR, in this case LTR2 of the HERVE family, which has a similar number (~900) of insertions as LTR5HS, but is not active in NCCIT cells. It is clear that there is no effect at any distance .