Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower. Here, Droplet Digital PCR (ddPCR) and qPCR platforms were directly compared for gene expression analysis using low amounts of purified, synthetic DNA in well characterized samples under identical reaction conditions. We conclude that for sample/target combinations with low levels of nucleic acids (Cq ≥ 29) and/or variable amounts of chemical and protein contaminants, ddPCR technology will produce more precise, reproducible and statistically significant results required for publication quality data. A stepwise methodology is also described to choose between these complimentary technologies to obtain the best results for any experiment. Poorly optimized reactions can result in artifactual Cq values and misinterpreted data that are difficult or even impossible to reproduce3. For absolute quantification, data analysis is further complicated by the different sources of DNA from which the samples and standard curves are derived with unique backgrounds and contaminants that can variably affect the activity of Taq polymerase giving misleading results4. The Minimum Information for the Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and related articles published thereafter define a rigorous methodology for designing qPCR experiments that assures publication of reproducible and high quality data7. The consequence of ignoring MIQE-guided protocols has led to the retraction of multiple articles over the past several years and remains a major frustration in the scientific community8.Data from qPCR experiments are taken within each enzymatic reaction curve at the quantification cycle (Cq). Therefore, optimization is critical for each primer pair such that reaction efficiency is consistent between all samples and acceptable (between 90% to 110%) with sample contaminants diluted adequately to assure that all reactions and associated Cq values are within the efficient range of the respective standard curves10. As with qPCR, ddPCR technology utilizes Taq polymerase in a standard PCR reaction to amplify a target DNA fragment from a complex sample using pre-validated primer or primer/probe assays. However, there are two distinct differences: 1) the partitioning of the PCR reaction into thousands of individual reaction vessels prior to amplification and 2) the acquisition of data at reaction end point. These factors offer the advantage of direct and independent quantification of DNA without standard curves giving more precise and reproducible data versus qPCR especially in the presence of sample contaminants that can partially inhibit Taq polymerase and/or primer annealing13. In addition, end-point measurement enables nucleic acid quantitation independently of the reaction efficiency, resulting in a positive-negative call for every droplet and greater amenability to multiplexed detection of target molecules14. Thereby, ddPCR technology can be used for extremely low-target quantitation from variably contaminated samples where the sample dilution requirements to assure consistent and acceptable reaction efficiency, primer annealing and Cq values for qPCR would likely lead to undetectable target levels15.Droplet Digital PCR (ddPCR) is a recent technology that has become commercially available since 2011In this study, synthetic DNA samples were used to directly compare and contrast qPCR with ddPCR technologies under common experimental conditions that can generate variable results in typical gene expression studies. In samples with low concentrations of nucleic acids and variable amounts of Taq inhibitors, ddPCR technology was shown to convert uninterpretable results generated from qPCR to highly quantitative and reproducible data.13. Since qPCR is a sample interdependent technology where the relative quantity and normalized gene expression data rely on ΔCq values, the analysis from a single plate assures the best quality results by eliminating any bias from inter-plate variability17. Therefore, all reactions for the study were pipetted into a single 96-well plate each for ddPCR and qPCR technologies.Since the goal of the study was to directly compare the data quality between the qPCR and ddPCR platforms, care was taken to assure that the experimental design minimized all differences with the exception of the data acquisition platform (ie: qPCR versus ddPCR technology). A single reaction mix was therefore produced for each sample and for all experiments which was split (20 µL each) for data acquisition between platforms  as similarly designed in a previous studyFor qPCR, the primers gave good reaction efficiencies (between 90% and 110%) for low concentration samples diluted in water between 27 and 32 cycles Fig.  inset wiSince the most common contaminant in a qPCR reaction is the reverse transcription (RT) mix from which the cDNA is synthesized, reaction mixtures containing a 1/2 dilution series of low concentration synthetic DNA supplemented with either 4 µL or 5 µL of RT mix were split between the qPCR Fig.  and ddPCFor ddPCR technology, the number of interface droplets between the positive (blue) and negative (black) amplitudes increased with the amount of RT contamination ) or 280% contamination effect on the data , a subset of four samples per DNA dilution consisting of the first two replicates ie: Fig. . The ddPWhen the full set of three replicates from each DNA dilution and RT-mix background  were tabulated, the normalized relative expression values between the different levels of RT-mix were much tighter (varying by about 1.5-fold between the two levels of contaminant) than those from the non-normalized data which varied by about 4-fold  but this does not mean that reference genes are never required for this application. The protein and chemical contaminants that vary between RNA extracts from biological samples can not only partially inhibit the qPCR reaction but also the reverse transcriptase leading to variable levels of input cDNA in the ddPCR reactionAlways begin a study by testing each primer pair in a 1/10 diluted, pooled cDNA or gDNA sample  in duplicate with qPCR under optimized thermocycling conditions and a no template control for each target.Presuming good, sigmoidal amplification curves are obtained with single melt curve peaks for each primer pair, run the duplicate samples from the qPCR reactions on a gel to assess the molecular weight and identity of the amplicon and sequence if necessary.7.All targets must then be assessed for reaction efficiency with a standard curve using the same pooled gDNA or cDNA sample from point 1 serially diluted based on the Cq value obtained from point 2 starting from the concentrated sample plus seven dilutions13. Individual samples for any target that passes points 2 and 3 should be diluted appropriately according to the associated standard curves7 and quantified using qPCR but if the resulting data give high variability and poor p-values, these targets may also be good candidates for ddPCR technology but should also first be optimized13.Any target that passes point 2 but fails point 3 should be ported to ddPCR technology for optimization7.Primers that fail optimization using ddPCR technology should either be redesigned or the experimental design and samples should be re-assessed and undergo troubleshootingGiven the large diversity of samples used in life sciences, some general criteria can be applied to selecting the appropriate technology for gene expression analysis as follows:7. Thus quantification of low expressing/abundant targets using qPCR is challenging because sample dilution is not possible to adequately minimize the effect of contaminants while maintaining quantifiable levels of cDNA. ddPCR technology permits quantification of these samples with excellent precision.qPCR is an excellent tool for the detection of 2 fold or greater expression differences with good statistical significance above about 100 copies <30 Cqs) in applications such as gene expression and/or miRNA analysis and absolute DNA quantitation. Although reference gene normalization serves the important purpose of correcting for differential loading of cDNA between samples, it may not reduce contaminant dependent data variability observed with qPCR . Primers were diluted according to the kit instructions and template was diluted by approximately 400 fold to achieve the starting concentration used in each experiment. The iScript™ Reverse Transcription (RT) Supermix, 100 × 20 µl rxns (Bio-Rad: 170–8841) was diluted to 1X with corresponding volumes transferred to each reaction .50 µL reaction mixtures containing RT mix, primers, template and QX200™ ddPCR™ EvaGreen Supermix (Bio-Rad: 186–4034) were divided 20 µL each between ddPCR  System – Bio-Rad) and qPCR (The CFX96™ Touch System – Bio-Rad) platforms for quantification. For ddPCR technology, droplet generation and transfer of emulsified samples to PCR plates was performed according to manufacturer’s instructions .The cycling protocol was identical for both platforms with a 95 °C enzyme activation step for 5 minutes followed by 40 cycles of a two-step cycling protocol (95 °C for 30 seconds and 60 °C for 1 minute). For ddPCR technology, the ramp rate between these steps was slowed to 2 °C/second while for qPCR, the maximum ramp rate was employed. Post cycling, a standard melt curve protocol was applied for qPCR (a single step of 94 °C for 10 seconds followed by a melt curve from 65 °C to 95 °C with a plate read at 0.5 °C increments after a dwell time of 5 seconds at each temperature). For ddPCR technology the post-cycling protocol was in accordance with the kit instructions (Bio-Rad – 186–4034).Each reaction mixture (see previous section) was split between one plate each for ddPCR (ddPCR™ 96-Well Plates: Bio-Rad - 12001925) and qPCR (Hard-Shell® 96-Well PCR Plate: Bio-Rad - HSP9601). The qPCR plate was sealed  and run in qPCR. The ddPCR plate was sealed with a foil heat seal (Bio-Rad - 181–4040) and the PX1™ PCR Plate Sealer (Bio-Rad - 181–4000).http://www.analyse-it.com/; 2009).For qPCR, the relative quantity and normalized expression data were processed using CFX Manager (v.3.1). For ddPCR technology, the absolute quantity of DNA per sample (copies/µL) was processed using QuantaSoft (v.1.7.4). For both ddPCR and qPCR, the data were exported to Microsoft EXCEL for further statistical analysis using the Analyze IT plugin  Analyse-it Software, Ltd. 24, the log transformed normalized gene expression and relative quantity data from each biological replicate were also normality tested (Shapiro-Wilk) and then Student’s t tested for statistical significance.All statistically analyzed data conformed to a normal distribution as determined using a Shapiro-Wilk test. A Student’s t test was then used to assess the statistical significance between the two-fold dilutions of DNA for each experiment. The %CV (Standard Deviation/Mean *100) was also used to assess variability. Finally, since it is generally accepted that qPCR derived gene expression data are log transformed for statistical analysis


Herein, we examine this catalyst on a fundamental level using a 2-fold approach employing industrial-grade catalysts as well as surface science based model catalysts. In both cases we examine the atomic (HRTEM/XRD/LEED) and electronic (XPS) structure, as well as the adsorption properties (FTIR/PM-IRAS), with emphasis on in situ/operando studies under atmospheric pressure conditions. For technological Ni–ZrO2 the rather large Ni nanoparticles (about 20 nm diameter) were evenly distributed over the monoclinic zirconia support. In situ FTIR spectroscopy and ex situ XRD revealed that even upon H2 exposure at 673 K no full reduction of the nickel surface was achieved. CO adsorbed reversibly on metallic and oxidic Ni sites but no CO dissociation was observed at room temperature, most likely because the Ni particle edges/steps comprised Ni oxide. CO desorption temperatures were in line with single crystal data, due to the large size of the nanoparticles. During methane dry reforming at 873 K carbon species were deposited on the Ni surface within the first 3 h but the CH4 and CO2 conversion hardly changed even during 24 h. Post reaction TEM and TPO suggest the formation of graphitic and whisker-type carbon that do not significantly block the Ni surface but rather physically block the tube reactor. Reverse water gas shift decreased the H2/CO ratio. Operando studies of methane steam reforming, simultaneously recording FTIR and MS data, detected activated CH4 (CH3 and CH2), activated water (OH), as well as different bidentate (bi)carbonate species, with the latter being involved in the water gas shift side reaction. Surface science Ni–ZrO2 model catalysts were prepared by first growing an ultrathin “trilayer” (O–Zr–O) ZrO2 support on an Pd3Zr alloy substrate, and subsequently depositing Ni, with the process being monitored by XPS and LEED. Apart from the trilayer oxide, there is a small fraction of ZrO2 clusters with more bulk-like properties. When CO was adsorbed on the  Ni particles at pressures up to 100 mbar, both PM-IRAS and XPS indicated CO dissociation around room temperature and blocking of the Ni surface by carbon . The Ni nanoparticles were stable up to 550 K but annealing to higher temperatures induced Ni migration through the ultrathin ZrO2 support into the Pd3Zr alloy. Both approaches have their benefits and limitations but enable us to address specific questions on a molecular level.Ni nanoparticles supported on ZrO Steam reforming . Ho2/Pd3Zr model catalyst was prepared and the surface was saturated with CO at 90 K already in the preparation chamber (50 L CO exposure). Afterwards XP spectra of the C1 s and Ni2p regions were measured which revealed a peak at 284.9 eV characteristic of molecular CO . This clearly shows that CO dissociates on nickel upon annealing, a well-known property, thereby forming a carbon over-layer on the nickel surface, which prevents further CO from adsorbing. During this experiment the Ni2p signal remained unaffected, i.e. there was no Ni sintering (which would reduce the intensity) and no formation of Ni carbide .2 supported Pd nanoparticles. As expected, upon heating the Pd nanoparticles up to 550 K, CO desorption (~420 K) but no CO dissociation (carbon deposits) were detected by IRAS and XPS.Previous studies of mbar pressure CO adsorption on supported Pd nanoparticles  and smoo2 was investigated using XPS . Figure The thermal stability of Ni particles on ZrO2/Pt3Zr indicated that above 500 K metal atoms migrated through the oxide support into the alloy substrate . Such model systems are not only accessible by many surface sensitive methods but can also be modelled straightforward by theoretical methods, which are typically indispensable to interpret and thoroughly explain the experimental data , 91, 92.Apart from the materials or complexity gap, in many cases model studies are performed in UHV and at low temperature, leading to the “pressure gap”. However, there are a number of “(near) atmospheric pressure surface science methods” available that enable to study functioning catalysts or, at least, model catalysts at elevated gas pressure and elevated temperature. Among the most frequently applied are PM-IRAS and SFG (sum frequency generation), both working up to 1 bar, and NAP-XPS (up to a few mbar) . Neverth2 reforming catalysts, the Ni particles in the powder catalyst are rather large (~20 nm) and also the Ni islands on the model ZrO2 cover most of the surface. Clearly, the “real” ZrO2 is hydroxylated whereas the model oxide is not. Nevertheless, routines have been developed to create OH groups on UHV grown (ultra)thin oxides as well.In the current case of Ni–ZrO2 was reversible, i.e. CO could be adsorbed, desorbed at higher temperature, and readsorbed without significant loss of adsorption capacity. There were also no indications of CO dissociation. In contrast, the Ni nanoparticles grown in UHV dissociated adsorbed CO already around room temperature, which led to coking and irreversible CO adsorption, thus readsorbing CO on the poisoned surface was not possible. Taking into account the well-known fact that CO dissociation preferentially occurs on Ni edges/steps this difference may be explained by the partial oxidation of the technological Ni nanoparticles . Thus, it is likely that the edges of technological Ni particles are oxidized which prevents CO dissociation. Unfortunately, CO-FTIR was not able to differentiate step and terrace sites and also HRTEM was unable to demonstrate this (due to sample air exposure prior to microscopy).With respect to CO adsorption at mbar pressure, differences were observed. CO adsorption on the technological Ni–ZrO4 is the major carbon source and graphitic and whisker-type carbon were observed. Reverse water gas shift occurred in parallel and decreased the H2/CO ratio. For methane steam reforming operando (FTIR and MS) studies were performed, indicating activated CH4 (CH3 and CH2), activated water (OH), as well as different bidentate (bi)carbonate species. The latter originate from CO2 resulting from the water gas shift side reaction but it is unclear whether these are intermediates or spectator species.Under methane dry reforming conditions, when hydrogen is present at high temperature, the Ni particles (and their edges) are fully reduced and may also partly dissociate the reforming product CO. Clearly, CH2 model catalysts, prepared by surface science routes, turned out to be significantly more active for CO dissociation than their technological counterparts. When CO was adsorbed at pressures up to 100 mbar, both PM-IRAS and XPS revealed CO dissociation around room temperature and blocking of the Ni surface by carbon. This is most likely due to the fully metallic Ni particles whereas the technological particles were partially oxidized even after reduction at 673 K. CO adsorbed on the powder ZrO2 at room temperature, but it did not on the trilayer ZrO2 (CO desorption temperature of 155 K [Ni–ZrOof 155 K ).2/Pd3Zr the upper limit of stability is about 550 K. Around this temperature Ni atom migration through the ultrathin oxide (in)to the alloy substrate takes place and Ni is lost from the model catalyst surface. Such an effect does not occur for the technological catalyst and care must therefore be taken for model studies at higher temperature. Clearly, methane reforming cannot be studied in situ using the current model system.A major limitation of most model systems is their thermal stability, however. For Ni–ZrO2 reforming catalysts, employing both technological materials (powders) as well as single crystal based surface science model catalysts. In both cases the Ni nanoparticles were rather large (20 nm or larger) but exhibited different properties with respect to CO dissociation. Whereas the partially oxidized “technological” Ni nanoparticles did not dissociate CO around room temperature, the fully metallic “surface science” Ni particles did, with the carbon deposits blocking further CO adsorption. For the technological samples methane dry and steam reforming were performed, with both reactions affected by (reverse) water gas shift, modifying the H2/CO ratio and producing CO2, respectively. For dry reforming whisker-type carbon was produced, operando studies of steam reforming revealed CHx, OH, and different bidentate (bi)carbonate species. The limited thermal stability of model Ni–ZrO2 prevented in situ reforming studies. Nevertheless, the XPS binding energies of alloy supported ultrathin trilayer (O–Zr–O) ZrO2 as well as those of more bulk-like ZrO2 clusters have been determined which provide useful reference data.We have examined Ni–ZrO2 composition. Of course, “Ni–ZrO2” is a very crude description missing important features like metal nanoparticle size and morphology, type of surface facets/steps, oxidation state, supporting oxide (surface) structure, hydroxylation, defect density, nature of metal-oxide interface sites, etc. Answering all these questions would provide the molecular details of how catalytic processes work and is thus a continuing mission.Altogether the combined technological and surface science approach is rewarding and it is fascinating to observe marked differences despite nominally the same Ni–ZrO


To assess the neurological involvement and outcome, including school and professional performances, of adults and children with cryopyrin-associated periodic syndrome (CAPS).In this observational study, patients with genetically proven CAPS and followed in the national referral centre for autoinflammatory diseases at Bicêtre hospital were assessed. Neurological manifestations, CSF data and MRI results at diagnosis and during follow-up were analyzed.A439V mutation is frequently associated with a non-neurological phenotype while R260W mutation tends to be associated with neurological involvement. Eleven adult patients (61%) and 3 children (50%) underwent school difficulties.Twenty-four patients (15 adults and 9 children at diagnosis) with CAPS were included. The median age at disease onset was 0 year (birth) [range 0–14], the median age at diagnosis was 20 years [range 0–53] and the mean duration of follow-up was 10.4 ± 2 years. Neurological involvement at diagnosis, mostly headaches and hearing loss, was noted in 17 patients (71%). Two patients of the same family had abnormal brain MRI. Neurological involvement is frequent in patients with CAPS and the majority of patients presented difficulties in school performances with consequences in the professional outcome during adulthood. Further studies in larger cohorts of children with CAPS focusing in intellectual efficiency and school performances are necessary. NLRP3 gene , the median age at diagnosis was 20 years [range 0–53] and the mean duration of follow-up was 10,4 ± 2 years. Two patients had no family history of CAPS.Skin rash, musculo-skeletal involvement and fever were the most prevalent features as described in Table A439V mutation was frequently associated with no neurological signs and R260W tended to be associated with neurological symptoms  underwent a CSF analysis; the opening pressure of the CSF was measured for 3 of them: 2 patients had an aseptic meningitis and the CSF opening pressure of one patient was elevated at 36 mm HThe patients who had multiple sclerosis-like MRI lesions underwent after 8 months of imaging, a CSF analysis, which was normal.Twenty-one patients (87%) are treated with canakinumab (starting at a dose of 150 mg every 8 weeks for adults and 2 mg/kg for children administrated as a subcutaneous injection). Treatment was not started in two children (9 and 4 years old) as their symptoms had very low impact in their everyday life. One 42 year-old female patient still refuses the treatment.Fourteen (82%) of the treated patient described improvement of their headaches: a complete resolution of headaches in 12 patients (80%) and partial resolution in 2 (13%) was observed. One patient had no improvement of his migrainous headaches under treatment. The elevated opening CSF pressure of one patient, normalized two years after treatment onset.n = 3) or stabilization (n = 5) of their hearing loss proven by audiometry during follow up.Eight (80%) patients reported improvement  have learning difficulties needing school accommodations, and 3 (50%) have no school difficulties. The professional categories of adult patients (n = 18) are described in , neurological manifestations were observed in 40% and is mostly represented by headaches (70%), papilledema (52%), meningitis (26%), hydrocephalus (18%), mental retardation (16%), seizures (4%) [In the largest CAPS study based on Eurofever registry (res (4%) . Hearingres (4%) . In our The mechanism of headaches in patients with CAPS is not well known. In our study, 4 patients (27%) had migrainous headaches and 2 patients with non-migrainous headaches had an aseptic meningitis and one of them had a high opening CSF pressure showing an intracranial hypertension which can explain the headaches . Only 21Fifty nine percent of the patients with CAPS have a hearing loss. The physiopathology is not well known, it has been observed that some children with CINCA had a cochlear inflammation demonstrated on MRI, after IL-1 receptor antagonist treatment there was an improvement of cochlear enhancement . In our Before any anti- IL-1β treatment, one patient had white matter lesions with demyelination as it may be seen in multiple sclerosis without any neurological sign (except for chronic headaches) unlike the only other CAPS patient described in literature with demyelinating disease who had a hemiparesia . In MS, A439V mutation is frequently associated with a non-neurological phenotype as previously published [R260W mutation, tend to be associated with neurological involvement. Moreover 2 members of the same family with this mutation had brain MRI lesions suggesting that brain lesions in these patients may be linked to the disease. Larger studies are needed to detect any specific pattern of these lesions.Although we could not perform statistical test du to small numbers of included patients, we have observed that Our study described the neurological outcome under anti- IL-1β treatment with a mean duration of follow-up of 10,4 years. Canakinumab had shown its sustained efficacy on neurological symptoms: 82% improved their headaches under treatment, and 80% improved or stabilized their hearing loss. In our study the median age at onset is birth and the median age at diagnosis is 20 years. There is an important delay of diagnosis and therefore a delay for treatment, already noted in previous studies . This hiOur study focused on school performances and professional career during follow-up. Although there is only one objective mental retardation, school difficulties are frequent in children with CAPS. The cognitive difficulties may be under-estimated: only 4 patients underwent psychometric tests. The impact of CAPS in school and professional performances is multifactorial: chronic pains, school absences, sick leaves are also implicated. A recent study showed on 7 patients in a prospective monocentric study that canakinumab had not only a sustained effect on quality of life, but also allowed dramatic changes in social and work lives . The benWe provided a detailed description of the neurological involvement, which is frequent in adults and children with CAPS. Canakinumab seems to have changed the general outcome of these patients as well as the neurological outcome. Further studies in larger cohorts of children with CAPS focusing in intellectual efficiency and school performances are necessary. Because of the frequency of school difficulties shown in our study in children with CAPS, the role of the pediatric neurologist in the muldisciplinary follow-up of these patients is very important and regular neuropsychological evaluations and neurological following are advisable for children with CAPS.


PCR for antigen receptor gene rearrangements (PARR) analysis is being increasingly used to assist diagnosis of canine lymphoma. In this study, PARR was carried out on consecutive samples received as part of routine diagnostic practice from 271 patients: 195 with lymphoid malignancies, 53 with reactive conditions and 23 with other neoplasms. Initially, published primer sets were used but later minor primer modifications were introduced and primers were rationalised to give a PARR panel that provides a good compromise between sensitivity and cost. Results were compared to diagnoses made by histology or cytology, coupled with immunophenotyping by flow cytometry or immunohistochemistry where possible. After exclusion of 11 poor quality samples, 230/260 (88%) gave a clear result with 162/163 (99%) of samples classified as clonal and 56/67 (84%) classified as polyclonal giving results concordant with the cytological/histological diagnosis. Among 30 samples with equivocal results, 21 had clonal peaks in a polyclonal background and nine showed little amplification. These were from patients with a range of neoplastic and non-neoplastic conditions emphasising the need to interpret such results carefully in concert with other diagnostic tests. The combination of primer sets used in this study resulted in a robust, highly specific and sensitive assay for detecting clonality. Several different primers sets for amplification of rearranged immunoglobulin heavy chain (IgH) and T-cell receptor gamma (TCRγ) genes have been described . Primer While assay sensitivity generally appears good, with detection of clonality in up to 98% of canine lymphomas , most PAThe aim of this study was to refine and rationalise previously published PARR primer sets in order to produce a panel of assays for routine use in the diagnostic setting. Initially, multiple published PARR primers were used to establish baseline sensitivity and specificity and, subsequently, modifications to both primer sequences and primer combinations were made. Assay conditions were also streamlined to facilitate use in a diagnostic setting. Diagnostic samples from 271 non-selected, suspected lymphoma patients were used in these analyses and results compared with morphological and, where possible, immunophenotypic diagnosis. Estimates of sensitivity and specificity were determined, and common difficulties in interpretation identified.22.1Clinical samples were collected from untreated patients presenting with suspected lymphoma to the University of Glasgow Small Animal Hospital, UK. Ethical approval was obtained from the Faculty of Veterinary Medicine Ethics and Welfare Committee, University of Glasgow. Samples consisted of excess material taken as part of routine evaluations at the time of diagnosis and were from 195 patients diagnosed with lymphoid malignancies , 53 with reactive or inflammatory conditions, and 23 with other neoplasms. Samples were either fresh , or fixed  biopsies or acetone-fixed, May-Grünwald-Giemsa-stained cytological smears). Most were from lymph node although a range of other tissues was sampled. Patient and sample characteristics are described in With the exception of 18 samples used in initial assay set-up , all samples were submitted as non-selected diagnostic samples; analysis and interpretation were carried out without knowledge of the diagnosis. Diagnoses were made by cytology or histopathology, with immunophenotyping by flow cytometry (FC) or immunohistochemistry (IHC) where possible. For FC, fresh samples were stained with the antibodies listed in Samples diagnosed as null-cell lymphoma (n = 6) were classified as lymphoma on morphological grounds but were negative for CD3 and CD79a/PAX5 by IHC, while one sample diagnosed as ‘double-labelled lymphoma’ was positive for both CD3 and PAX5. CD34-positive leukaemias (n = 6) were CD34 positive by FC and negative for other leukocyte antigens. Cases of lymphoma or leukaemia that were not subjected to immunophenotyping (n = 48) or where immunophenotyping by FC was inconclusive (n = 5) were designated ‘not otherwise specified’ (NOS). Plasma cell tumours (n = 3) were classified on the basis of morphology.2.22.2.1Primers were synthesised according to published sequences , and iniFour IgH V segment primers were retained in our PARR panel: two IgH framework region 1 (FWR1) primers (918 and 919) were included to help mitigate the problem of junctional mutation or somatic hypermutation disrupting the IgH framework region 3 (FWR3) primer binding site, and two IgH FWR3 primers (913 and 916) were retained as they will detect rearrangements in samples with fragmented DNA. Although some germline V segments have a perfect match to only one of these primers, most match more than one of the primers providing some redundancy and mitigating against false negative results. Two IgH J segment primers are required to bind to all rearrangements. A single J1-2 primer (914) was selected from the three tested, and a mismatch in the J3 primer corrected (915a).Two TCRγ V segment and three J segment primers were included in our final panel. Primer 928 should bind to TCRγ V2 and V6 segments. Following correction of four mismatches at the 5′ end of primer 925 (925a), this primer should bind to V3, V5 or V7 segments. Primer 930, which should bind the majority of TCRγ Jx-2 segments, was modified to include two mixed bases in the middle of the primer to allow for sequence variation between segments (930a). Primer 929 should bind well to Jx-1 segments, except for segment J5-1 where several mismatches occur at the 3′ end; primer 931 was therefore designed to match J5-1 more closely. The final PARR primer sequences are listed in The amplifiability of all DNA samples was assessed using two control PCRs. Initially Cμ (128 bp) and γ-actin (272 bp) primers were used, and later γ-actin (125 and 450 bp) primers; amplicon sizes correspond to those generated by the PARR primer sets .2.2.2DNA was extracted using QIAamp DNA Mini kits or DNA FFPE Tissue kits . Reactions were performed in a total volume of 25 μl, and contained 100 ng DNA (where possible), each primer at 250 nM  and 1 x HotStarTaq Plus Master Mix (Qiagen). Thermal cycling was carried out on a GeneAmp PCR System 9700 . Initially, all primer sets used identical thermal cycling conditions: 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s, with a final extension of 72 °C for 30 min to facilitate addition of terminal adenosine bases on each PCR product. Following optimisation, the annealing temperature for primer sets 9, 10, 13 and 14 only was reduced to 50 °C consistent with the low melting temperatures of primers 929 and 931.Fragment analysis was carried out using GeneScan methodology on an ABI 3130×l Genetic Analyser (Applied Biosystems) with a 36 cm capillary length loaded with POP-4 polymer. Products amplified with primer sets 1 and 4, 2 and 5, and 3 and 6, were combined prior to fragment analysis; 5′ primers in each combination were labelled with different fluorochromes so that reaction products could be distinguished .2.3For each individual PARR primer set, results were subjectively scored as clonal ; pBased on the overall pattern of results with all the PARR primer sets and the amplifiability controls, samples were then assigned to one of seven categories : ‘B-cell33.1Using the published primer sets in their original pairings (Tables S1 and S2), the majority of samples diagnosed as B-cell lymphoma had detectable clonal IgH rearrangements  and with TCRγ primer set 9 using the new thermal cycling conditions. Samples previously designated as ‘reactive’, ‘inadequate sample’, ‘no peak’, or ‘dominant peak only’ were assayed. Since further testing was not performed on samples that had a clear clonal result, each sample was not tested with every primer set. All samples submitted following primer review were tested with the standard panel of ten primer sets  samples. Fifteen of these samples had detectable clonal TCRγ rearrangements , and wer3.3Final results were then compared with the diagnosis reached on the basis of morphology. Results were clearly discordant in 12 samples (4.6%). There was one false positive result; a patient diagnosed with myeloid neoplasia had clonal peaks detected using two IgH primer sets. The clonal result was repeatable using the same sample (lymph node); however, fresh samples from both lymph node and bone marrow yielded polyclonal results. Concurrent cytology indicated that the lymph node contained a high proportion of myeloid precursors amidst a background of mature lymphoid cells. Eleven samples were diagnosed as lymphoid malignancy but had a polyclonal PARR result and could represent false negative results. These comprised two splenic B-cell lymphomas, four lymphoma NOS samples, two null-cell lymphomas, and three CD34-positive leukaemias. All four ‘diagnoses’ were over-represented in this group compared with the overall sample distribution. A further 12 samples diagnosed as lymphoid malignancy had equivocal PARR results: eight were ‘dominant peak only’ and four were categorised as ‘no peak’.Overall, 160 of 185 samples with a diagnosis of lymphoid malignancy produced a clonal result  with the standard panel of ten primer sets, or 162 of 185 samples when the additional primer sets were included. This gives sensitivities of 86.5% and 87.6%, respectively. Specificity was 98.7%, based on the absence of clonal IgH or TCR rearrangement in 74 of 75 samples with conditions other than lymphoid malignancy.3.4Conclusive immunophenotyping results were available for 139 of 195 lymphoid malignancy cases . Of thes3.5Six samples were classified as B- or T-cell lineage using PARR where there was no clearly defined B- or T-cell population following immunophenotyping. Of these, three were diagnosed as null-cell  lymphoma by IHC and PARR identified one as a B-cell and two as T-cell tumours. Similarly three samples classified as CD34-positive leukaemia with dim or negative staining for leukocyte antigens on FC were assigned to a T-cell lineage by PARR. Lineage was also assigned to the five samples previously designated as lymphoma NOS where FC was inconclusive.4The aim of this study was to develop a panel of PARR assays for routine use in the diagnostic setting. As a starting point we used PCR assays previously described in the literature but later modified some of the primer sequences, rationalised the use of highly similar primers, and adapted the thermal cycling parameters to allow use of a minimum number of different conditions. These changes improved the ability to detect clonal rearrangements, particularly in T-cell malignancies. We describe a ‘standard’ panel of ten primer pairs, recommended for routine use. These comprise six IgH and four TCR primer sets, which will pick up the vast majority of rearrangements. A further four additional primer pairs are described that are useful in certain circumstances.Comparison with morphological diagnoses suggested that the sensitivity of this PARR panel was 86.5%, which is within the range (80–97.9%) described elsewhere . HoweverAssay sensitivity improved as more primer sets were included, offset by increased cost of labour and reagents. We believe that the standard protocol described here is a good compromise between sensitivity and ease of use and has improved sensitivity over previous studies using fewer primer sets . RetainiThere are several plausible explanations for our failure to detect clonal rearrangements in samples with a morphological diagnosis of lymphoma. One obvious concern is that the primers used do not bind to the rearranged gene segments . AnalysiFalse positive results were rare (1 of 75), giving a specificity of 98.7%. Clonal IgH peaks were detected in a sample from a myeloid neoplasia. Although rearrangement of IgH has been documented in human and canine acute myeloid leukaemia , it is pDespite the use of routine diagnostic samples, only 11 of 271 (4%) samples were classified as inadequate. With one exception, these samples were extracted from FFPE material, which is likely to have contained more degraded DNA than the fresh tissue , or may Five of the nine samples placed in the ‘no peak’ category were from patients with non-lymphoid neoplasia, consistent with the PARR result. Two samples with clonal T-cell peaks were identified using only the new, modified primer sets and had previously been categorised as ‘no peak’. Results must be interpreted alongside findings using other diagnostic modalities and if this pattern is seen in samples where there is a high index of suspicion of lymphoma, repeat sampling and use of additional PARR primer sets are recommended.Perhaps the most problematic samples to interpret were those in the ‘dominant peak only’ category, with direct impact on overall assay sensitivity. Most previous reports either do not mention this phenomenon, or infer that all such results indicate clonality . Keller Dominant peaks were often observed in samples with a clear clonal peak detected with another primer set, suggesting that primer mismatches, most probably resulting from somatic hypermutation, can give rise to this pattern on electropherograms. This again emphasises the benefit of using multiple primer sets, including the additional primer sets when there is a high index of suspicion. A dominant peak may also indicate a small clonal population of cells within a background of reactive lymphocytes  either iEhrlichia species may generate clonal PARR results . Two of three discordant samples had clonal rearrangements of both IgH and TCRγ, which has been documented previously in canine and human lymphoid tumours . In humaIn this case series, PARR proved useful for assigning lineage where other methods were inconclusive. A previous study reported that FC more accurately determines lineage ; however5The combination of primer sets used in this protocol resulted in a robust, highly sensitive and specific assay. Although PARR provides diagnostic information unavailable from other tests and can help determine tumour lineage where other techniques have failed, interpretation of results must consider clinical presentation, cell morphology, immunophenotype and other ancillary tests. Knowledge of sample quality is essential, as samples with few cells or poor quality DNA will likely amplify poorly, giving an equivocal result. Dominant peaks, which may indicate a neoplastic population within a reactive background, but are also seen in non-lymphoma samples, pose a particular challenge. Better understanding of the nature of these peaks may come from longer term follow-up of cases and sequencing of products. Further investigation of these findings will improve the precision of this important diagnostic test.


Migrasomes are large vesicles that grow on the tips or intersections of retraction fibers at the rear of migrating cells. Following cell migration, the retraction fibers eventually break and the migrasomes become detached. The migrasomes and their contents, including cytosolic components and vesicles of unknown origin, are released into the extracellular space —— a process we named as migracytosis. We speculated that migracytosis may play important roles in cell-cell communication.Recently we reported the discovery of migrasome, a new organelle of migrating cells2. Different integrins bind to different ECM proteins2. For example, integrin α5β1 specifically binds to fibronectin, α3β1 to laminins, and α1β1 and α2β1 to collagens3.Integrins are a large family of transmembrane receptors that connect cells to the extracellular matrix (ECM). Integrins function as heterodimers composed of α and β subunits; there are 18 α and 8 β integrins in mammalsWhen cells migrate away, migrasomes adhere to the place where they are formed, which indicates that something holds the migrasome at its formation site. Mass spectrometry analysis revealed integrin α5β1 is enriched on migrasomes (unpublished data), suggesting this integrin is a possible candidate for the molecule that holds the migrasome on the ECM. To testify this hypothesis, we used MGC803 cells expressing TSPAN4-GFP that labels migrasomes, and stained the cells with antibodies against integrin α5 and β1. We found that both integrin α5 and β1 were enriched in migrasomes . MoreoveTo study the migrasomal integrin in more detail, we transfected integrin α5-GFP into normal rat kidney cells expressing TSPAN4-mCherry (NRK-TSPAN4-mCherry). We found that integrin α5-GFP was highly enriched in migrasomes, whereas there was relatively less integrin α5-GFP on retraction fibers . Time-laMigrasomes are spherical structures, so if integrin molecules adhere migrasomes to the ECM, they must be located at the bottom of the migrasome. To test this hypothesis, we checked the 3D distribution of integrin α5 and TSPAN4 on migrasomes. We found that integrin α5 was mainly enriched on the bottom side of migrasomes while TSPAN4 was on the upper side . Similar4. We wondered whether the integrin-enriched regions on migrasomes are FAs. To test this idea, we co-transfected cells with various FA markers 5 and TSPAN4. We found that none of these FA markers was localized on migrasomes  are the sites that cells are linked to the ECM. Integrins are highly enriched in FAsgrasomes . Moreovegrasomes . These dITGA5 that encodes α5 impaired the formation of migrasomes on cells cultured on fibronectin, but not on other ECMs  on different ECM proteins. We found that NRK-TSPAN4-GFP cells produced many more migrasomes on cover glasses coated with fibronectin than with laminin 511 or collagen I, whereas very few migrasomes formed on non-coated cover glasses . Next, wher ECMs . Thus, mIn NRK-TSPAN4-GFP cells, the expression level of integrin α3 is very low. We overexpressed integrin α3-mCherry in NRK-TSPAN4-GFP cells, and found that it significantly enhanced migrasome formation on cover glasses coated with laminin 511, but not on cover glasses covered with other ECM proteins . Due to 1. Formation of retraction fibers requires adherence of at least a portion of the retraction fiber to the cell surface to provide a tethering point, which enables the retraction fiber to be pulled from the trailing end of the cell as the cell migrates. The fact that integrins are enriched on the bottom of migrasomes, and that migrasomes are largely stationary while retraction fibers are often swaying around (Migrasome formation depends on cell migrationg around  indicateIn this report, we found that activated integrin is highly enriched on the bottom of migrasomes, and we further demonstrated that the expression level of integrin affects migrasome formation. Finally, we showed that the correct pairing of integrin with its specific ECM partner protein is a determinant for migrasome formation.2, and each ECM protein has a specific spatial and temporal distribution pattern in a given organism. Thus, our finding that pairing of integrins with their specific ECM partner proteins is a determinant for migrasome formation may emerge as an important principle for determining where and when migrasomes can be generated invivo.There are 18 α and 8 β integrins in mammals and their expression is subject to various regulatory mechanisms. Different integrins bind different ECM proteinsin vivo.So far, the detection of migrasomes has depended on TSPAN4, a marker we identified in our original paper. In this regard, integrins may serve as more specific markers for migrasomes . While b


In this work the role of p62 in energy metabolism was studied in fibroblasts from FTD patients carrying two independent pathogenic mutations in the p62 gene, and in a p62-knock-down (p62 KD) human dopaminergic neuroblastoma cell line (SH-SY5Y). We found that p62 deficiency is associated with inhibited complex I mitochondrial respiration due to lack of NADH for the electron transport chain. This deficiency was also associated with increased levels of NADPH reflecting a higher activation of pentose phosphate pathway as this is accompanied with higher cytosolic reduced glutathione (GSH) levels. Complex I inhibition resulted in lower mitochondrial membrane potential and higher cytosolic ROS production. Pharmacological activation of transcription factor Nrf2 increased mitochondrial NADH levels and restored mitochondrial membrane potential in p62-deficient cells. Our results suggest that the phenotype is caused by a loss-of-function effect, because similar alterations were found both in the mutant fibroblasts and the p62 KD model. These findings highlight the implication of energy metabolism in pathophysiological events associated with p62 deficiency.Abnormal mitochondrial function has been found in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Mutations in the  Mutations in the p62 gene have been found to cause both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). FTD and ALS are sometimes associated in patients or within families showing ALS and FTD as a pathological continuum (ALS/FTD) as they share common pathological features1. Supporting this idea, mutations in the same disease-causing genes in both disorders have been reported. These include VCP2, p623, OPTN4, UBQLN25 and especially the hexanucleotide repeat expansion in C9orf726. It is worth mentioning that mutations in OPTN, VCP and p62 also cause Paget disease of the bone (PDB)1. p62 has been related to neurodegenerative phenotypes and it has been linked to the ubiquitin-proteasome system and autophagy, main protein degradation mechanisms in cells. Protein aggregates containing p62 have been also found in many disorders, including ALS and FTD8, in some cases colocalizing with the transactive response DNA-binding protein 43 (TDP-43)9 and also in ubiquitinated inclusions along with FUS protein and TDP4310.p62, also known as sequestosome 1, is a scaffold or an adaptor protein involved in multiple cellular activities and is encoded by the 15. Studies with cell models showed the implication of p62 in mitophagy which is disrupted in some forms of Parkinson’s disease15. The silencing of the p62 orthologue in drosophila ref(2)P, resulted in mitochondrial dysfunction, and mitochondrial DNA accumulation and this was linked to the observed locomotor deficits in the flies13. In 2014, Seibenhener and colleagues further showed the link between p62 and mitochondrial protein turnover using p62 silenced mouse embryonic fibroblasts (MEFs)14. Mitochondria are essential organelles especially for neurons as they are the main source of ATP due to limited glycolysis. The p62 knockout mouse model as well as p62 silenced MEFs showed impaired mitochondrial function resulting in reduced ATP production17.The link between p62 and neurodegeneration has been further investigated using animal and cell models. Most of them have focused on autophagy, the ubiquitin-proteasome degradation pathway and particularly, the mitochondrial quality control process known as mitophagy21. This has been demonstrated using different animal and cell models. In particular, we recently demonstrated that mutations in VCP cause mitochondrial uncoupling leading to decreased mitochondrial membrane potential and a significant reduction of cellular ATP production highlighting the pathophysiological events that may occur in FTD and ALS22. In the present study we aimed to analyze mitochondrial function and pathophysiology using fibroblasts from FTD diagnosed patients carrying two independent p62 mutations and the p62-knock-down (p62 KD) human dopaminergic neuroblastoma cell line (SH-SY5Y) model. We found that p62 deficiency induces inhibition of mitochondrial respiration due to a lack of substrate delivery. This inhibition results in higher cytosolic ROS production inducing the cells to increase the pentose phosphate pathway activity (PPP) in order to enhance the reduced glutathione (GSH) levels. We also demonstrated that the p62 mutations cause a loss-of-function effect as the p62 KD shows the same phenotype as the p62 mutations from patients.Accumulating evidence from recent studies suggests that mitochondrial dysfunction plays a significant role in both the FTD and ALS etiopathogenesism). The implication of p62 in the mitochondrial function was assessed by transient silencing of the p62 gene in the SH-SY5Y human neuroblastoma cell line using siRNA (p62 KD)  as a fluorescent indicator of ΔΨm in the p62 KD cells and in mutant fibroblasts from patients carrying independent p62 mutations  compared to either untransfected  or cells transfected with scrambled (SCR) siRNA control   compared to control fibroblasts  Fig. . The ΔΨm 4) Fig. . Equivalols Fig. . The TMRsts Fig. .Figure 1p62 KD SH-SY5Y cells and p62 mutant fibroblasts showed a depolarization in response to the F0-F1-ATP synthase (ATPase or complex V) inhibitor oligomycin (2 µg/ml), suggesting a reverse mode function for the ATPase  compared to either untransfected  or SCR-transfected  cells  compared to untransfected  and SCR  cells  compared to untransfected  and SCR  cells  compared to untransfected  and SCR  confirming an inhibition in complex I and reduced substrate availability  and the rate of substrate supply can be estimated by measurement of mitochondrial NADH and FAD autofluorescence as previously shownlls Fig. . Then, tlls Fig. . Equivallls Fig. . The analls Fig.  was usedlls Fig.  which walls Fig. . Increaslls Fig.  and in tlls Fig.  indicatiity Fig. . Furtherity Fig. . It is pity Fig. .Figure 2p62 KD cells showed higher Het rates  compared to the untransfected  or SCR cells  . Cytosolic ROS production was analyzed in the p62 deficient cells using dihydroethidium (Het). Figure  5) Fig. . Equival 5) Fig. . Togethe25. It was reported that p62 and Nrf2 form a positive feed-forward regulatory loop, whereby p62 activates Nrf2 by competing for binding with the negative regulator Kelch-like ECH associated protein 1 (Keap1), and Nrf2 activates the transcription of p6226. These observations, together with the fact that both models of p62 deficiency show similar mitochondrial alterations to the ones we had previously shown in Nrf2-deficient cells24 prompted us to evaluate the role of Nrf2 activation on mitochondrial function in p62-deficient cells. To this end we used three different Nrf2 activators, the synthetic acetylenic tricyclic bis(cyanoenone) (TBE-31), the naturally occurring isothiocyanate sulforaphane (SFN) and the synthetic triterpenoid RTA-408 (RTA). Each of the three pharmacological Nrf2 activators rescued the respiration deficiencies in the fibroblasts carrying the p62 mutations -related factor 2 (Nrf2) directly regulates cellular energy metabolism by modulating the availability of substrates for mitochondrial respirationons Fig. . The NADons Fig.  and the ons Fig.   were significantly increased compared to untransfected  and SCR-transfected  SH-SY5Y cells indicating an increased activity in the PPP due to p62 deficiency , which is one of the major endogenous antioxidants in the cell. During an oxidative insult, neurons divert part of their glucose pool towards the PPP, thereby increasing the production of NAD(P)H, a necessary cofactor in the regeneration of GSH. PPP activity can be estimated by analysing the basal levels of NAD(P)H autofluorescence after addition of 1 μM FCCP, which maximises respiration Fig. . This bancy Fig. . Similarncy Fig. . We founncy Fig. . Finallyncy Fig.  we foundncy Fig.  but not ncy Fig.  GSH levency Fig. . These d27. Previous works presented p62 protein as one of the main regulators of mitochondrial function17. In this study, the mitochondrial bioenergetics in p62 deficient cells and fibroblasts from FTD patients carrying independent p62 mutations was investigated. Reduced p62 levels are associated with reduced ΔΨm in both p62 SH-SY5Y deficient cells and mutant fibroblasts. These cells also exhibit inhibited mitochondrial respiration through complex I due to the lack of substrates for the ETC complexes and higher superoxide production. The boost in the cytosolic ROS levels leads to activation of the pentose phosphate pathway, thereby increasing the GSH levels in order to counteract the oxidative stress environment.Mitochondria are the main source of energy in neurons due to their limited glycolytic capacity. Harnessing the proton gradient generated in the respiration through complexes I to IV, neurons rely on the oxidative phosphorylation process to generate ATP through the ATP synthase in the mitochondria. Mutations in FTD and ALS have been found to induce impairments in crucial mitochondrial metabolic processes causing a fatal fate for cells, organs and patients30 but not much is known about this topic in FTD. Recent studies found mitochondrial alterations in FTD cases together with ALS supporting the idea of the FTD/ALS continuum34. Other works showing mitochondrial alterations were carried out in different cohorts of patients with variations in the CHCH10 gene, a common cause of both FTD and ALS35. A number of studies have demonstrated that mitochondrial activity is compromised in FTD and ALS using animal models and cells from patients36. In this study, a reduction in the ΔΨm is observed in the p62 deficient cell models. The ΔΨm reflects mitochondrial health and function and mitochondrial viability and it is defined as the proton gradient generated in the intermembrane space during respiration. This proton gradient is harnessed by the ATPase to synthesise ATP through the oxidative phosphorylation (OXPHOS) process. Here it is also shown that ATP hydrolysis by the ATPase maintains the ΔΨm in the p62 deficient cells since these cells exhibited depolarised mitochondria when oligomycin, the inhibitor of the F1-F0 ATPase, was added. Under normal conditions the process of respiration maintains the ΔΨm but mitochondria can also hydrolyze the ATP forcing the ATPase to work in a reverse mode in order to maintain the ΔΨm37. This has been thoroughly described in other neurodegenerative models in which an inhibition of respiration hampers the normal ΔΨm maintenance forcing the reversal ATPase40.Mitochondrial dysfunction in ALS has been thoroughly demonstratedm an inhibition in the respiration process was detected in p62 deficient cells. This was reflected by both the elevated NADH redox index and the decreased FAD redox index, pointing to the inhibition of complex I. This inhibition is a consequence of lack of substrates as demonstrated by the lower NADH pool observed in the p62 deficient cells as well as the lower FAD pool. The inhibition of the respiration through the complex I induces an increase in ROS production in the cells. Damaged mitochondrial respiration including reduced complex I and II activities were previously found in mitochondria from p62 KO mice brains16. These authors also found an exacerbated oxidative stress due to inhibition of respiration. On the other hand, in 2012 Kwon and colleagues showed mitochondrial impairments in a p62 KO model reflected in reduced mitochondrial membrane potential and reduced oxygen consumption together with increased cellular oxidant levels17. Increased ROS generation has been found to be related to inhibited mitochondrial respiration in neurodegenerative disorders43. In AD, it has been found that an inhibition in mitochondrial respiration through complex I increased ROS generation44. Increased ROS production is also a demonstrated feature in some forms of PD. This has been related to inhibition in the complex I dependent respiration both in cells and PD animal models46.In combination with the reduced ΔΨ47. More recently, new roles regarding the maintenance of mitochondrial function and bioenergetics have been attributed to Nrf249. It has been shown that cells from Nrf2-deficient mice have impaired mitochondrial function. Specifically, it was demonstrated that Nrf2 is a main regulator in the substrates supply to both complex I and complex II as the rate of NADH and FADH2 production was much lower in the Nrf2 KO cells compared to controls24. Nrf2 and p62 were shown to intersect through the direct interaction between p62 and Keap126. Under normal conditions Nrf2 is ubiquitinated and degraded by the proteasome as it is bound to the E3-ubiquitin ligase adaptor protein Keap1. When Keap1 is challenged with ROS or electrophiles, Nrf2 degradation is blocked, activating the antioxidant response elements (ARE)-mediated transcription. In the noncanonical pathway of Nrf2 regulation, Keap1 is sequestered by p62 and can no longer bind Nrf2, leading to increased Nrf2 signalling with the subsequent activation of the ARE-mediated transcription. Our findings point to a disruption in the Keap1 sequestration by p62, leading to enhanced degradation of Nrf2. The consequence would be inhibition in respiration due to low Nrf2 levels, which could be restored by Nrf2 pharmacological activators.The results presented herein show that Nrf2 activators restore the inhibition of the respiration and the NADH pool in the p62 deficient cells. Nrf2 is a transcription factor implicated in the activation of antioxidant genes, providing cytoprotection against oxidative stress and inflammation50. The expression levels of the rate limiting enzyme responsible for GSH biosynthesis, the glutamate-cysteine ligase, are very low in neurons. This fact makes neurons more vulnerable to oxidative stress as the GSH-dependent antioxidant system is very weak in these cells. Neurons can compensate their oxidative stress vulnerability by diverting part of the glucose pool to the PPP, which generates NAD(P)H and increases the GSH levels51. It is possible that p62 deficient cells divert part of their glucose to the PPP to increase GSH levels after an oxidative stress stimulus, as has been shown in neurons52.Finally, it is shown that p62 deficiency is associated to an increased activity in the pentose phosphate pathway as demonstrated by the elevated levels of NAD(P)H and reduced glutathione. It has been demonstrated that elevated ROS levels in cells induce the activation of the PPP to generate the antioxidant GSHIn summary, our results show that p62 deficiency induces mitochondrial respiration inhibition by deficient delivery of substrates to the mitochondrial complexes, which subsequently results in elevated cytosolic oxidative stress. The rise in the oxidative stress could partially trigger glucose diversion to the PPP in order to increase the GSH levels.p62 KD model, suggesting a loss-of-function effect upon p62 deficiency. This point makes the results applicable not only to FTD patients carrying p62 mutations but also to ALS patients with mutations in p62, and therefore supports the idea of FTD/ALS continuum. The data presented here along with recent works showing functional mitochondrial impairments linked to mutations causing both FTD and ALS provides significant basis to support the hypothesis that mitochondrial dysfunction is involved in the underlying pathogenic mechanisms within the FTD/ALS spectrum. Additionally, the pharmacological activation of Nrf2 needs to be further explored as therapeutic tool for the p62-associated ALS/FTD prevention and treatment.Even though our results were obtained using fibroblasts from FTD patients, they have been also validated using the SH-SY5Y All methods were performed following the relevant guidelines and regulations approved by the local ethical review committee from the National Hospital for Neurology and Neurosurgery and the Institute of Neurology.p62 mutations and two non-related healthy donors used as controls were generated from a 4-mm skin punch biopsy taken under local anesthetic following informed consent. Biopsies were dissected into ~1-mm pieces and cultured in 5-cm2 petri dishes in DMEM, 10% FBS, 1% L-Glutamine until fibroblasts were seen to grow out from the explants. When fibroblasts reached confluency, they were detached from culture dishes using TrypleE from Thermo Fisher Scientific  and transferred to larger culture vessels for further expansion and cryopreservation. Age, sex, age of onset, clinical features, clinical diagnosis, and p62 mutations carried by the patients as well as healthy donors characteristics are given in Table Written informed consent was obtained from the donors for publication of their individual details. The consent form is held by the authors’ institution within the patients’ clinical notes and is available for review by the Editor-in-Chief. Donors gave written consent and the project was approved by the local ethical review committee from the National Hospital for Neurology and Neurosurgery and the Institute of Neurology . Fibroblasts from two patients carrying independent 53. Unless otherwise stated, SH-SY5Y cells and fibroblasts were seeded at a density of 4 × 104 cells/cm2, grown to 75–80% confluence and maintained at 37 °C and 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% (v/v) foetal bovine serum (FBS), 2 mM L-glutamine and 1% (v/v) penicillin/streptomycin.Human neuroblastoma (SH-SY5Y) cells were purchased from the European Collection of Cell Cultures  and maintained as previously describedCells lysates were obtained after 2 minutes of incubation with the lysis buffer NP-40  and the lysates were immediately frozen. 10 μg of denatured protein previously estimated  from each sample were loaded in a precast 4–20% Tris-Glycine SDS-PAGE . After electrophoresis, proteins were transferred to 0.45 μm PVDF membranes  and identified by the appropriate primary and secondary antibodies and visualised using Enhanced Chemiluminescence  with the ImageQuant LAS4000 . Anti-human p62 rabbit monoclonal antibody (1:20000) and anti-human glucose 6 phosphate dehydrogenase (1:1000) were obtained from Abcam . Anti-human β-Actin rabbit polyclonal antibody (1:5000) was obtained from Sigma-Aldrich .The anti-rabbit secondary antibodies (1:5000) coupled to horseradish peroxidase and bovine immunoglobulins (IgG) were from Bio-Rad .2.The non-targeting scramble siRNA, the targeted siRNA (siGenome SMARTpool) against human p62 and the Dharmafect transfection reagent were purchased from Dharmacon, Thermo Fisher Scientific . The siRNA transfection was performed following the manufacturer’s instructions once the plated cells reached 60% confluence. Cells were ready for subsequent experiments after 48 h post transfection 37 °C and 5% CO54, the triterpenoid RTA-408 (RTA)55, and the naturally occurring isothiocyanate sulforaphane (SFN)56. The concentration of each compound was optimized based on the potency in inducing NAD(P)H:quinone oxidoreductase 1 (NQO1), a prototypic Nrf2 target gene, without causing any cytotoxicity, using a quantitative bioassay57. The compounds were prepared as stock solutions in acetonitrile and diluted (1:1000) in the cell culture medium, such that the final concentration of acetonitrile was maintained at 0.1% (v/v). Cells were exposed to the Nrf2 activators for 24 h.Three different activators for the transcription factor Nrf2 were used: the synthetic acetylenic tricyclic bis(cyanoenone) TBE-31m was measured as was described previously22. Briefly, cells were plated on 25 mm coverslips and loaded with 40 nM tetramethyl-rhodamine methyl ester (TMRM) in a HEPES-buffered salt solution (HBSS)  for 40 minutes at room temperature and keeping the dye present in the chamber at the time of recording. TMRM is a cell-permeant fluorescent dye used in the redistribution mode to assess ΔΨm, and therefore a reduction in TMRM fluorescence represents ΔΨm depolarization. Confocal images were obtained using a Zeiss 710 VIS CLSM  equipped with a META detection system and a × 40 oil immersion objective. TMRM was excited using the 560 nm laser line and fluorescence was measured above 580 nm. The Z-stack images were analyzed using the Volocity software  and TMRM values for control cases were set to 100% and the p62 deficient cells values were expressed relative to controls. For analysis of response to mitochondrial toxins, images were recorded in a time course-dependent manner from a single focal plane and analyzed using ZEN Zeiss software (Zeiss).ΔΨNADH autofluorescence was measured using an epifluorescence inverted microscope equipped with a 20× fluorite objective. Excitation light at a wavelength of 350 nm was provided by a Xenon arc lamp, with the beam passing through a monochromator . Emitted fluorescence light was reflected through a 455 nm long-pass filter to a cooled CCD camera  and digitised to 12 bit resolution. Imaging data were collected and analyzed using software from Andor . FAD autofluorescence was monitored using a Zeiss 710 VIS CLSM equipped with a META detection system and a × 40 oil immersion objective. Excitation was measured using the 454 nm Argon laser line and fluorescence was measured from 505 to 550 nm. Illumination intensity was kept to a minimum (at 0.1–0.2% of laser output) to avoid phototoxicity and the pinhole set to give an optical slice of ~2 μm.+, coupled to the 1:1 reduction of the reporter dye of which product concentration was proportional to the increase in absorbance at 450 nm, measured over time using a spectrophotometer. Complex I activity was expressed as the rate of increase in absorbance per amount of sample loaded in the well.The NADH dehydrogenase activity of isolated complex I was measured using the complex I enzyme activity microplate assay kit . Cell lysis was carried out using the lysis buffer provided by the manufacturer and left on ice for 20 min to allow protein extraction. Samples were then centrifuged at 12, 000 × g for 20 min at 4 °C and total protein concentration in the supernatant was estimated using the Pierce BCA Protein Assay Kit . Samples were then diluted in Incubation Solution (provided by the manufacturer) to reach a final protein concentration of 0.5 μg/μl. 200 μl of each sample were transferred to each well of the microplate containing immobilised anti-complex I antibody bound to the wells and incubated for 3 hr at RT. After the incubation period, the wells were rinsed twice in 300 μl of Buffer, provided by the manufacturer, and 200 μl of Assay Solution containing NADH and a reporter dye were added to each well. NADH dehydrogenase activity was determined by measuring the oxidation of exogenous NADH to NADCellular ROS generation was measured using dihydroethidium  from Life Technologies . All imaging was performed in HBSS and the dye was present in the solution during the experiment. No preincubation (“loading”) was used for Het to limit the intracellular accumulation of oxidized products. Fluorescence images were collected with a 12-bit resolution cooled CCD camera coupled to an epifluorescence inverted microscope equipped with a 20× fluorite objective (Leica Microsystems). The excitation wavelength for the oxidised form (ethidium) was 530 nm collected at 605 nm while excitation to measure changes in the reduced form (hydroethidium) was 380 nm collecting the emission at 405–470 nm. Ratiometric Het fluorescence was recorded with excitation light at 380 and 530 nm. All imaging data were collected and analyzed using the Metamorph software .2PO4, 1 mM MgCl2, 5 mM EGTA and 1.86 mM CaCl2 at pH 7.1) including digitonin (40μM) was added to permeabilise the cells allowing us to measure the mitochondrial GSH. The fluorescence images of the MCB-GSH adduct were acquired using the cooled CCD imaging system as described above using excitation at 380 nm and emission at >400 nm. The fluorescence was then quantified using the Metamorph analysis software . Additionally, quantitative GSH analysis was performed in live cells in a modified protocol as was previously described58. Briefly, cells were plated in 96 well plates. Once they reached 70–80% of confluence they were loaded for 30 minutes with 2 mM MCB allowing the formation of the MCB-GSH fluorescent adduct inside cells. The fluorescence was then quantified using the Varioskan plate reader  with excitation wavelength at 380 nm and emission at 470 nm. Fluorescence values were then related to the protein content .The reduced glutathione (GSH) levels were determined in live cells after incubation with 50 μM monochlorobimane (MCB) for 40 minutes at room temperature in HBSS until a steady state was reached. MCB is a nonfluorescent probe until conjugated with GSH. Once the cytosolic GSH was analyzed, HBSS was removed and a hypotonic medium . For all graphs, error bars represent mean ± SEM.Data were generated from a minimum of three independent replicate per experiment (n ≥ 3) performed in different days. Each replicate consisted of at least 1 coverslip per condition where a number of 15–30 cells per coverslip were analyzed. Statistical analysis and exponential curve fitting were performed using GraphPad Prism 6.01  software. Statistical significance for multiple comparisons was performed by one-way ANOVA followed by Fisher’s LSD correction. All results are related to healthy control fibroblasts or untransfected cells accordingly and expressed as percentage. In all cases, Primary fibroblast lines were generated from skin punch biopsies. Donors gave written consent, and the project was approved by the local ethical review committee from the National Hospital for Neurology and Neurosurgery and the Institute of Neurology.Supplementary information


Few prospective studies have examined the relationship between a patient’s expectation of pain improvement and the pain prognosis. The aim of this prospective study was to investigate whether patients’ expectation to pain reduction was associated with pain intensity after morphine treatment in opioid treatment-naïve patients with various types of cancerThe subjects were patients scheduled for cancer pain treatment with morphine who were taking nonsteroidal anti-inflammatory drugs daily. Morphine treatment was performed according to the standard method, including titration . Simple regression analysis was performed between pain intensity numerical rating scale (NRS) (day 8) as the dependent variable, expectation of pain decrease NRS (day 1), tumor types, and the following covariates as independent variables: patients’ characteristics such as age, gender, PS (day 1), genotype of catechol-O-methyltransferase, total scores of Hospital Anxiety and Depression Scale (day 1), and pain intensity NRS (day 1). Multiple regression analysis was performed using forced entry methods with pain intensity NRS (day 8) as the dependent variable, and expectation of pain decrease NRS (day 1) and the covariates as independent variables that had a p value <0.05 in the simple regression models.A total of 100 patients with baseline data were included, and 97 patients  met the inclusion criteria. Patients with a high expectation of pain decrease NRS had a significantly lower pain intensity NRS (day 8) (p = 0.001).Non-pharmacological factors such as expectations for pain treatment could also be important factors to treat cancer pain, which might be associated with communication skills in physicians. Pain is a common symptom in cancer patients , 2 that There is a need for a multifaceted approach to non-cancer chronic pain that includes the effects of catastrophizing (exaggeration of pain)  and expeFurthermore, almost all guidelines have mentioned the importance not only the use of pharmacological therapy  but also the non-pharmacological therapy including reduction of pain catastrophizing and rise of expectation of patients in chronic non-cancer pain because safe and effective chronic opioid therapy for chronic non-cancer pain requires clinical skills and knowledge in both the principles of opioid prescribing and on the assessment and management of risks associated with opioid abuse, addiction, and diversion , 19.On the other hand, in treating cancer patients, the main approaches to cancer pain  are drug therapies, such as non-steroidal anti-inflammatory drugs (NSAIDs) and opioids. Since the report is less about the importance of non-drug therapies , which are often frequently used in chronic pain models of non-cancer, results and guidelines of non-cancer patients study , 19 mighHowever, there are few studies on expectation of symptom improvement before treatment in cancer patients , and theIn this prospective study, we investigated whether patients’ expectation to pain reduction was associated with pain intensity after morphine treatment in a cohort of opioid treatment-naïve patients with various types of cancer.This prospective study was conducted from 2009 to 2012 at the Kindai University Faculty of Medicine and Sakai Hospital. A total of 100 patients with opioid treatment-naïve and histologically confirmed malignant neoplasms who were scheduled to undergo opioid treatment were evaluated in the study. They were recruited and selected by non-probabilistic convenience sampling by their physician’s selections from the outpatient and inpatient service of Department of Medical Oncology. Of these patients, 97 met inclusion criteria of ≥18 years of age, a verified cancer diagnosis, cancer pain suitable for morphine treatment , daily use of non-steroidal anti-inflammatory drugs, and ability to give signed informed consent. Three patients were ineligible, 1 patient did not meet inclusion criteria, 1 patient was due to medical event, and 1 patient withdrew consent. All 97 patients underwent gene expression analysis, but only 91 patients were evaluated in genotype analysis because the DNA samples were insufficient in 6 cases. The required dose of morphine (day 8) was evaluated in 85 patients, excluding 12 who could not continuously receive morphine due to adverse effects or death  performance status (PS), and types of primary malignant neoplasm were recorded. Morphine treatment was performed according to the standard method, including titration  by specialized palliative care doctors. On this step, they explained standardized information about potential benefits and adverse effects to their patients. Assessments were conducted pre-treatment (day 1) and 1 week after treatment (day 8).This study was approved by the Regional Committee for Medical Research Ethics, Kindai University Faculty of Medicine and informed consent was obtained from all individual participants included in the study.Pain score was assessed with 0–10 Numerical Rating Scale  (pain intensity NRS) on day 1 (pre-treatment) and on day 8 (1 week after treatment) by asking patients by the following question; “How intense was your average pain for the past 24 hours?”Patients’ expectation of a decrease in pain determined via a questionnaire with a 0–10 NRS (0 = no improvement to 10 = full improvement) on day 1 (pre-treatment) (expectation of pain decrease NRS (day 1)) and on day 8 (1 week after treatment). Nurses who were not related to this study handed questionnaires to patients. The questionnaire had the following question; “How much do you think your pain will get better?” The questionnaire was self-administered.To assess correlations with pain intensity NRS (day 1 and day 8), anxiety/depression before treatment (day 1) and on day 8 was evaluated using the Hospital Anxiety and Depression Scale (HADS). In accordance with a systematic review by Cosco et al. , we use O-methyltransferase (COMT) 472G→A  also modulate the genetic response to opioid medications [OPRM1 118A→G  and COMT 472G→A . The treatment outcome of morphine was examined based on the plasma concentration of morphine and the required dose according to genotype on days 1, 2, and 8 [The single nucleotide polymorphisms (SNPs) of catechol-ications . We perf2, and 8 . On day COMT forward, 5′-GAT TCA GGA GCA CCA GCC CTC C-3′ and reverse (intronic), 5′-CAC TGA GGG GCC TGG TGA TAG TG-3′. PCR was performed in a 20-μl volume containing 20 ng of template, 0.5 μM of each primer, Ampdirect Plus , and 0.5 units of NovaTaq™ DNA Polymerase . Amplification was performed for 35 cycles . The resulting PCR fragments consisting of 210 bp (COMT) were directly sequenced with the corresponding forward and reverse primers, respectively [Genomic DNA isolated from blood samples using a QIAamp® DNA Blood Mini Kit (Qiagen) was amplified with the following primers: ectively .p value <0.05 in simple linear regression analysis, which means that the required level for p values was <0.00625 with Bonferroni correction for multiple tests in eight simple regression models. A two-sided significance level of 0.05 was used for multiple regression analysis. All statistical analyses were performed using SPSS software .All data are expressed as mean ± standard deviation. We first undertook a bivariate relationship using linear regression analysis between pain intensity NRS (day 8) as the dependent variable and expectation of pain decrease NRS (day 1) and the following covariates as an independent variable: patients’ characteristics such as age, gender, PS (day 1), genotype of COMT, pain intensity NRS (day 1), and total scores of HADS (day 1). We also tested the association between pain intensity NRS (day 8) and cancer types (pancreatic cancer or not) because pancreatic cancer patients had significantly lower expectation of pain decrease NRS (day1) than the other cancer patients. Then, multiple linear regression analysis was performed using forced entry methods with pain intensity NRS (day 8) as the dependent variable and expectation of pain decrease NRS (day 1) and the covariates as independent variables that had a The characteristics of the 97 patients  are shown in Table p value  and leverage (0.11).With regard to simple regression analysis with pain intensity NRS (day 8) as the dependent variable, only expectation of pain decrease (day 1) and PS (day 1) reached the set ue Table . Then, mp = 0.001, partial correlation coefficient = 0.36). Those with a low PS also had a significantly lower pain intensity NRS (day 8) (p = 0.001) (Table Patients with a high expectation of pain decrease NRS on day 1 had a significantly lower pain intensity NRS (day 8) (1) Table . The norr = 0.199).Pain intensity NRS (day 8) showed almost no correlation with HADS total score on day 8 (The results of this study show that patients with an expectation for pain decrease NRS (day 1) had a significantly lower pain intensity NRS (day 8). To our knowledge, this is the first prospective study to examine the effect of an expectation of a change in pain before morphine treatment on pain score 1 week later in opioid-naïve patients with various cancers and PS. The results are consistent with findings in non-cancer patients  that pain score is decreased in patients who expect pain improvement , 13.To explain how a health expectation influences the outcome, research on the mechanisms of placebo effects, of which expectancy is generally considered to be the core mechanism, provides more well-grounded suggestions for the causal influence of expectations on pain and other outcomes and for suggestions to improve pain management by modulating expectations .According to the response expectancy theory of Kirsch et al., response expectancy is defined as the anticipation of automatic, subjective, and behavioral responses to particular situational cues, and studies of the effects of placebos reveal that response expectancies can produce lasting changes in pain .g = 0.15) in clinical analgesic trials (studies used only placebo as a control condition) as compared to studies of placebo analgesia mechanisms (Hedges’ g = 0.95). Further comparison between acute and chronic pain indicated that magnitudes of placebo analgesic effects have been smaller (Hedges’ g = 0.33) in chronic pain than acute procedural pain (Hedges’ g = 0.67) in the recent meta-analyses by Peerdeman et al. [An investigation of the effects of the expectation of pain decrease NRS (day 1) indicated some effects on pain intensity NRS (day 8)  in the present study. According to the meta-analyses on placebo and expectancy effects by Vase et al. , it was n et al. . Althougn et al. .Our results suggest that pain relief in cancer patients can be improved by increasing the patient’s expectation of pain improvement. However, these findings are from a prospective observational study, and an interventional study is required for further evaluation.p = 0.001), which may be because most of low PS patients were inpatients who received improved pain control due to more frequent contact with medical staff.Patients with low PS also had lower pain intensity NRS (day 8) (This study has several limitations. First, 20–40% of cancer patients have mental disorders that require therapy . Scores Within these limitations, we conclude that cancer patients with pain who expect to have relief from pain before pain control treatment are more likely to have an actual improvement of pain. For control of cancer pain, it may be helpful for oncologists and palliative care doctors to develop their communication skills. By improving the communication skills to improve the doctor-patient relationship, the amount of required dose of analgesics can be reduced by the rise of “expectations” of a patient and the reduction of pain. It may be possible to treat cancer patients of chemical coping –22, whic


The controlled creation of defect centre—nanocavity systems is one of the outstanding challenges for efficiently interfacing spin quantum memories with photons for photon-based entanglement operations in a quantum network. Here we demonstrate direct, maskless creation of atom-like single silicon vacancy (SiV) centres in diamond nanostructures via focused ion beam implantation with ∼32 nm lateral precision and <50 nm positioning accuracy relative to a nanocavity. We determine the Si+ ion to SiV centre conversion yield to be ∼2.5% and observe a 10-fold conversion yield increase by additional electron irradiation. Low-temperature spectroscopy reveals inhomogeneously broadened ensemble emission linewidths of ∼51 GHz and close to lifetime-limited single-emitter transition linewidths down to 126±13 MHz corresponding to ∼1.4 times the natural linewidth. This method for the targeted generation of nearly transform-limited quantum emitters should facilitate the development of scalable solid-state quantum information processors. Interfacing spin quantum memories with photons requires the controlled creation of defect centre—nanocavity systems. Here the authors demonstrate direct, maskless creation of single silicon vacancy centres in diamond nanostructures, and report linewidths comparable to naturally occurring centres A central goal in semiconductor quantum optics is to devise efficient interfaces between photons and atom-like quantum emitters for applications including quantum memories, single-photon sources and nonlinearities at the level of single quanta. Many approaches have been investigated for positioning emitters relative to the mode-maximum of nanophotonic devices with the necessary subwavelength-scale precision, including fabrication of nanostructures around pre-localized or site-controlled semiconductor quantum dots12347Unlike quantum emitters such as molecules or quantum dots, diamond defect centres can be created through ion implantation and subsequent annealing911121314157The silicon vacancy (SiV) belongs to a group of colour centres in diamond that has emerged as promising single-photon emitters and spin-based quantum memories. Among the many diamond-based fluorescent defects that have been investigated202122252529304 sites per second, which allows creation of millions of emitters across a wafer-scale sample. We also show that additional post-implantation electron irradiation and annealing creates an order of magnitude enhancement in Si to SiV conversion yield. By repeated cycles of Si implantation and optical characterization, this approach promises nanostructures with precisely one SiV emitter per desired location. Finally, we demonstrate and evaluate the site-targeted creation of SiVs in pre-fabricated diamond photonic crystal nanocavities. The ability to implant quantum emitters with high spatial resolution and yield opens the door to the reliable fabrication of efficient light–matter interfaces based on semiconductor defects coupled to nanophotonic devices.Here we introduce a method for positioning emitters relative to the mode-maximum of nanophotonic devices: direct FIB implantation of Si ions into diamond photonic structures. This post-fabrication approach to quantum emitter generation achieves nanometre-scale positioning accuracy and creates SiV centres with optical transition linewidths comparable to the best ‘naturally' growth-incorporated SiV reportedAs outlined in 2 write field, created via a single-point exposure from the Si beam. Room temperature spectral measurements in a dense region containing many centres  of 1.3) objective into a single-mode fibre under 20 mW of 532 nm pump power. The red line in We characterized the resulting SiV arrays at room temperature through confocal fluorescence microscopy in a home-built set-up (Methods). χ-distribution with a mean separation in R of σ=40±20 nm and underlying lateral  distributions with zero mean and s.d.'s of 32 nm. These measured values agree well with the expected precision of 26 nm calculated by the addition in quadrature of the uncertainties arising from the nominal 40 nm FHWM beam size and 19 nm lateral implantation straggle.To determine the spatial precision of creating SiV with our method, we analysed their distribution relative to the implantation lattice grid. 12 to 1014 Si cm−2, and the implantation energy linearly from 10 to 100 keV. The dose and energy determine the number and depth of vacancies created during the implantation process, with increased energy resulting in more vacancies at increased depth. The vacancy density affects the probability that a Si defect captures a diffusing vacancy and converts to SiV during annealing, which is a proposed mechanism for SiV formation2 region of constant implantation dose and energy, and normalized to the average single-emitter intensity and implanted ion number. 12 cm−2. These measurements indicated a decreasing yield as a function of dose  that indeed only the SiV typical peak at 737 nm was detected. In the second measurement, we observed increased fluorescence for all four spots by a factor of ∼10 −1=94 MHz for a typical fluorescence lifetime of 1.7 ns . The inhomogeneous distribution of SiV transitions at 18 K is plotted in  ns ref. , and equx direction and a main offset of 48 nm in the y direction.Finally, we demonstrated the targeted implantation and subsequent creation of SiV centres inside diamond nanostructures. We first fabricated two-dimensional (2D) photonic crystal nanocavities into a ∼200-nm-thick diamond membrane through oxygen reactive ion etching41η, prevents the generation of exactly one emitter with high yieldWhile we have demonstrated targeted creation of high-quality SiVs through FIB, there are several avenues for improvement. The stochastic nature of the SiV creation process, characterized by a mean yield of 12 cm−2, indicating that high densities of implanted SiVs are not detrimental for their optical properties. Although we found that FIB-implanted SiVs are similar in homogeneous transition linewidth to ‘natural', as-grown centres, the inhomogeneous linewidth of ∼51 GHz  is still slightly broader than the ∼15 GHz demonstrated for a similar SiV creation method with annealing temperatures around 1,200 °C  diameter, corresponding to an implantation dose of ∼10 °C ref. . PotentiIn summary, we demonstrated SiV creation with high spatial accuracy by FIB implantation of Si ions into bulk and nanostructured diamond. The SiV-positioning accuracy relative to the targeted nanocavity mode-maximum was 48±21 nm, which is sufficiently precise to locate the SiV within ∼90% of the mode-field intensity maximum of nanocavities or waveguides. We also demonstrated that the SiV creation yield can be increased after implantation by a factor of 10, up to 20%. The targeted implantation technique demonstrated here likely applies to other quantum emitters like the germanium defect centre in diamondThe ZPLs of SiVs created by our method have optical linewidths within a factor of 1.4 of the lifetime limit, making them as narrow as naturally occurring SiVs described to date. Considering both this narrow linewidth and the narrow inhomogeneous distribution of implanted SiV of only ∼51 GHz, this fabrication method represents a significant step towards the high-yield generation of thousands to millions of efficiently waveguide-coupled indistinguishable single-photon sources. Such arrays of atom-like quantum emitters would be of great utility for a range of proposed quantum technologies, including quantum networks and modular quantum computing485052Focused ion implantation was performed at the Ion Beam Laboratory at Sandia National Laboratories using the nanoImplanter (nI). The nI is a 100 kV FIB machine (A&D FIB100nI) making use of a three-lens system designed for high mass resolution, using an ExB filter, and single ion implantation, using fast beam blanking. The ExB mass-filter (M/ΔM of ∼61) separates out different ionic species and charge states from liquid metal alloy ion sources, providing the capability for implantation of ∼1/3 the periodic table over a range of energies from 10 to 200 keV. For the Si implantation discussed here, we used an AuSbSi liquid metal alloy ion source with typical Si beam currents ranging from 0.4 to 1 pA. Fast beam blanking allows direct control over the number of implanted ions. We determine the number of implanted ions by measuring the beam current and setting the pulse length to target a given number of ions per pulse. The nI is a direct write lithography platform that uses electrostatic draw deflectors, controlled by a Raith Elphy Plus pattern generator, to position the beam. Single ion positioning is limited by the beam spot size on target. With typical spot sizes ranging from 10 to 50 nm, we have measured the targeting accuracy to be <35 nm for 200 keV Si++ beam using a series of ion beam-induced charge measurements.For targeting into nanostructures, we align the ion beam relative to the sample by registering a secondary-electron image of the alignment markers generated using the ion beam to scan the sample. Shift, rotation and magnification corrections are calculated and applied in the pattern generator control package. This allows for any location within the write field to be individually targeted for implantation.The lithography pattern is the original design file that was used to pattern the diamond thin film via electron beam lithography and reactive ion etching. Errors resulting from inaccuracy during electron beam lithography were not taken into account.To create a single SiV per cavity with high probability, we implanted ∼20 Si ions per cavity mode-maximum, yielding ∼1.8 SiVs per cavity on average according to an extrapolated conversion efficiency of ∼3% under Poisson statistics for 160 keVSi ions  that tar−6 mbar at maximum temperature) for 2 h to form SiV centres and eliminate other vacancy-related defects. Finally, we clean the sample surface through boiling tri-acid treatment (1:1:1 nitric:perchloric:sulfuric) and subsequent dry oxidation in a 30% oxygen atmosphere at 450 °C for 4 h.We annealed the sample at 1,050 °C under high vacuum (<10We used a modified fluorescence microscope (Zeiss Axio Observer), customized to allow confocal illumination at 532 nm (Coherent Verdi) and single-mode fibre fluorescence collection. Collected fluorescence is spectrally filtered (Thorlabs FEL0650) and detected on avalanche photodiodes with single-photon resolution (Excelitas) or spectrally resolved on a grating spectrometer .These measurements were performed at 18 K in a closed cycle helium cryostat (Janis). A home-built confocal microscope collects the fluorescence with a high NA objective (Olympus UMplanfl 100 × 0.95 NA) and directs the emission to either the input of a single-mode fibre connected to an avalanche photodiode or to a free-space spectrometer with a resolution of ∼61 pm (∼34 GHz) at 737 nm .Photoluminescence excitation measurements were performed using a modified helium flow probe-station (Desert Cryogenics model PS-100) with a 0.95 NA microscope objective (Nikon CFI LU Plan Apo Epi 100 × ) inside the vacuum chamber. Details of this set-up are described in ref. χ-distribution with two degrees of freedom (Rayleigh distribution), which describes the distribution of the distance R=X and Y are independent zero mean normal random variables with identical variance (χ-distrbution corresponding to the mean separation in R (40 nm), and the error is the square root of the variance (20 nm). The mean separation in the X and Y directions is 0 nm with a s.d. of 32 nm.To determine the spatial precision of the SiV implantation, we created and imaged a square array of SiV colour centres following the procedures in Methods A and D. We then fitted each SiV site with a 2D Gaussian to determine the location of the SiV centres below the diffraction limit, and considered only SiV sites with fluorescence intensities consistent with single emitters. Using these locations, we fitted a 2D grid allowing for affine transformation and found the distance between each SiV site and its nearest grid point. Finally, we binned the distances and fitted to a central variance . The repTo determine the positioning accuracy of the cavity-targeted SiV creation, we performed a spectrally resolved photoluminescence confocal scan at room temperature. At each pixel of a 2D 532 nm laser scan we recorded a spectrum and determined the intensity for different spectral positions. For each wavelength, we then plotted its 2D-intensity map as in The data that support the findings of this study are available from the corresponding author.How to cite this article: Schröder, T. et al. Scalable focused ion beam creation of nearly lifetime-limited single quantum emitters in diamond nanostructures. Nat. Commun.8, 15376 doi: 10.1038/ncomms15376 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.