This study was to investigate whether the previously proposed link between Alzheimer’s disease (AD) and decreased retinal nerve fiber layer thickness could be explained by the relationship between abnormal CSF profiles and optic nerve head characteristics, focusing on the influence of CSF tau protein on the lamina cribrosa (LC) thickness (LCT).-42 peptide, (Aβ1–42), total tau (T-tau) and tau phosphorylated at threonine 181 (P-tau181P) were measured from CSF samples of each subject. The relationship between the level of CSF proteins and the LCT was determined using linear regression and fractional polynomial analyses.A total of 44 eyes from 18 patients with AD and 26 healthy subjects were subjected to enhanced-depth-imaging volume scanning of the optic nerve using spectral-domain optical coherence tomography. The B-scan images were constructed three-dimensionally using maximum intensity projection (MIP), and the LCT was measured at three locations  using the thin-slab MIP images. CSF levels of amyloid β 1P = 0.004) and P-tau181P (P = 0.027), as well as a smaller central corneal thickness (P = 0.032), were significantly associated with a smaller LCT. Multivariate analysis indicated that only CSF T-tau (P = 0.041) was significantly associated with the LCT. The relationship was well explained by both linear regression  and fractional polynomial analysis . When we performed an assessment by linear regression with an indicator, the relationship was significant both in the healthy and AD groups, with a stronger correlation found in the healthy group .Univariate regression analysis revealed that higher CSF levels of T-tau (An increased CSF level of T-tau was significantly associated with a thinner LCT in both the healthy and AD groups. This result suggests that LCT could serve as a potential non-invasive indicator for increased CSF tau. The clinical meaning of the higher level of CSF T-tau in axonal degeneration of the optic nerve remains to be determined. Tau is a microtubule-associated protein that is most abundantly found in neurons and astrocytes within the central nervous system. Tau is known to play a pivotal role in various neurological pathways, interacting with signaling pathways that regulate microtubule stability and axonal transport –3. DysreThe optic nerve is a part of the central nervous system, and mainly comprises retinal ganglion cell (RGC) axons that project to the optic chiasm and optic tract and ultimately form synapses at the lateral geniculate nucleus. Therefore, the optic nerve could be affected by similar degenerative processes during neurodegeneration. It has been suggested that pathogenic mechanisms similar to those in neurodegenerative diseases may also occur in retinal degenerative diseases such as glaucoma , 7–13.Studies have suggested a possible role of tau protein in glaucomatous optic nerve damage. Yoneda et al.  demonstrGlaucoma is a progressive optic neuropathy characterized by the degeneration of RGC axons. Currently, the lamina cribrosa (LC) within the optic nerve head (ONH) is considered the primary site of axonal damage in glaucoma –21. It hConsidering the anatomical proximity between the ONH and retrobulbar CSF space, it is possible that the CSF protein affects the ONH. Given the implication of the LC in glaucoma pathogenesis and the possible influence of tau protein in glaucomatous axonal damage, it would be of interest to know how CSF tau protein is associated with the structural characteristics of the LC. The purpose of the present study was to determine the influence of CSF proteins on the ONH structure, focusing on the relationship between CSF tau and the lamina cribrosa thickness (LCT).This study is based on the Study for the Usefulness and Standardization of Cerebrospinal Fluid and Plasma Amyloid Biomarkers in Alzheimer’s Disease, which is an ongoing prospective study of patients with AD and healthy participants at the Neurocognitive Behavior Center in collaboration with the Glaucoma Clinic of Seoul National University Bundang Hospital. This study was approved by the Seoul National University Bundang Hospital Institutional Review Board. Informed written consent was obtained from all participants or their legally acceptable representatives in accordance with the Declaration of Helsinki.The subjects were enrolled by advertisement between May 2012 and April 2014. The participants included in the study were either normal or had been diagnosed with AD based on the National Institute on Aging–Alzheimer’s Association classification system ..Complete ophthalmic examinations were provided to all subjects, including a visual acuity assessment, Goldmann applanation tonometry, a refraction test, slit-lamp biomicroscopy, gonioscopy, a dilated stereoscopic examination of the optic disc, fundus photography , central corneal thickness measurements , corneal curvature measurements , axial length measurements , and enhanced depth imaging (EDI) spectral-domain optical coherence tomography (SD-OCT)  scanning of the optic disc and circumpapillary retinal nerve fiber layer (RNFL). The IOP was measured three times at 15-minute intervals, and the average of the three values was recorded as the baseline IOP. The subjects underwent cerebrospinal fluid (CSF) sampling with CSF pressure measurement on the same day as the ophthalmic examination (see ‘CSF sampling and storage’ section)3, uremia, hemophilia, or other impairment of the coagulation process), or severe cognitive impairment that resulted in low cooperability were also excluded. When both eyes were eligible, one eye was randomly selected from each subject.To be included in the present study, an eye had to have a best-corrected visual acuity ≥20/40 with a spherical refraction of –8.0 to +5.0 diopters (D), cylinder correction within ±3.0 D, and a normal appearing optic disc without glaucomatous optic neuropathy and pallor or swelling. Eyes with any ocular diseases other than cataracts or a history of intraocular surgery other than cataract extraction were excluded. Subjects with a neurological disease other than AD, history of intracranial surgery, lumbar puncture prior to enrollment in this study, a condition that could affect the intracranial pressure ), contraindication for lumbar puncture , thrombocytopenia <50,000/mmThe ONH was imaged using Spectralis OCT with the EDI technique. The details of the protocol for scanning the optic nerve using EDI SD-OCT to evaluate the LC are described elsewhere , 28. In The LCT was measured at three locations in each eye  using thin-slab maximum intensity projection (MIP) images. Thin-slab MIP images were used because they allow a straighter detection of the posterior LC border . The tecThe LCT was measured using the manual caliper tool of the Amira 5.2.2 software by two observers (EJL and DSL) who were masked to the clinical information for the subject. All measurements were repeated three times each by the two observers, and the average of the six values was used for the main analysis.The CSF was obtained using the fluoroscopy-guided lumbar puncture technique on the same day as the ophthalmic examination . In brie3, the samples were not included in the study (this criterion did not apply to any of the samples). The second fraction (10–25 ml) was used for biomarker analysis. This fraction was centrifuged immediately after sampling (10 minutes at 2000 x g), and the supernatant was distributed in different polypropylene vials and frozen in liquid nitrogen. The CSF samples were then stored at −80 °C until analysis.The first fraction (1 ml) was used for routine examination of the CSF, which included a cell count and total protein and glucose analysis. Whenever the CSF cell count revealed more than 5 white blood cells or more than 50 red blood cells per mmPrior to the CSF analysis, the samples were randomized to obtain a proportional distribution of samples from controls and AD patients on each microtiter plate (40 samples per plate). The laboratory technician was blinded to the expected test outcome in terms of clinical and definitive pathological diagnoses while performing and interpreting the tests.1–42), total tau (T-tau), and tau phosphorylated at threonine 181 (P-tau181P) were determined with INNO-BIA AlzBio3  using Luminex® 200™ . With each assay, the clinical samples together with a blank (sample diluent), the prepared calibrator solutions, and the appropriate controls were tested strictly following the test instructions provided in the kit inserts. All samples were run in duplicate.The CSF levels of amyloid-beta 1–42 peptide .If the duplicate results differed by more than 20 % or if the concentrations obtained were out of range, the samples were retested. The concentration ranges of the test kits are described in the package inserts , first with a univariate model and then with a multivariate model that included variables from the univariate model for which P <0.10. Statistical analyses were performed with Statistical Package for the Social Sciences software for Windows version 17.0 . For the factors that showed a significant relationship with the LCT, fractional polynomial analysis [P <0.05.To measure the interobserver reproducibility of measurement of the LCT, the intraclass correlation coefficients (ICC) and their 95 % confidence intervals (CIs) were calculated. The Shapiro–Wilk normality test was performed because of the relatively small number of subjects in this study. Comparison between groups for continuous variables was performed using the independent-samples analysis  was perfn = 3), a poor B-scan quality that did not allow delineation of the LC borders (n = 1), poor cooperation during the SD-OCT examination (n = 2), and failure to receive a CSF examination (n = 2), yielding a final sample of 18 AD patients and 26 healthy subjects.The study initially included 52 subjects . Of these, eight subjects were excluded because of a diagnosis of concurrent glaucoma  and had a lower total Mini-Mental Status Examination (MMSE) score  than the healthy subjects. There were no significant differences between the AD and healthy groups for any of the other baseline characteristics, including gender, spherical error, central corneal thickness, axial length, baseline RNFL thickness, body mass index, and the presence of diabetes or hypertension  were aged 66.0 ± 8.3 years (mean ± standard deviation) and had a spherical error of 0.5 ± 1.9 D. A comparison of the baseline clinical characteristics between the AD and healthy groups is presented in Table . 12.8 ± 2.6 mmHg, P = 0.062; Table 1–42 level , a higher T-tau level , and a higher P-tau181P level  than those of the healthy group (Table Table P = 0.004) and CSF P-tau181P (P = 0.027) and a thinner central corneal thickness (P = 0.032) on thinner LCT measurements. In the multivariate analysis, only the CSF T-tau level (P = 0.041) was significantly associated with the LCT ). This analysis showed that the association between CSF T-tau protein and the LCT was significant in both the healthy and AD groups, with a stronger association found in the healthy group   and a smaller subset of cellular components . The LC is thought to provide structural and metabolic support to the RGC axons passing through it.It has been suggested that eyes with thin LC are more susceptible to mechanical damage of the RGC axons. Thinning of the LC may expose the axon to a steep transition between the IOP and the retrolaminar tissue pressure (translaminar pressure gradient) and significantly influence the axoplasmic flow , 34–36, The present study suggests a potential mechanism of LC thinning associated with tau pathology; a higher level of CSF tau was associated with a thinner LC both in healthy individuals and in patients with AD. To the best of the authors’ knowledge, this is the first study to reveal a relationship between CSF proteins and the LC characteristics.Whether CSF tau is a causal factor for thinning of the LC or a consequence of LC thinning is unclear. Several possibilities may be considered for this issue. First, it is possible that the CSF tau is a causal factor in LC thinning. The cytoplasm of astrocytes contains networks of filamentous proteins, such as the actin cytoskeleton, intermediate filaments, and microtubules that maintain tissue and cell integrity, cell mobility, and cell differentiation . It has β = 10.417, P = 0.061). This may indicate that the LCT could also serve as a potential candidate marker representing cognitive function as well as the CSF tau level. A long-term follow-up study is required to verify whether these subjects are more susceptible to developing AD. Additionally, a study that includes subjects with mild cognitive impairment may help to answer this question.In the present study, more than half of the subjects were healthy individuals; thus, it is not known whether the non-AD subjects with a thin LC or an elevated CSF tau had subclinical AD. Although data were not presented because of the small number of samples, a subgroup analysis performed within the healthy group revealed a marginal but not significant linear association of a lower total MMSE score with thinner LCT . Instead, a smaller LCT was correlated with both higher CSF tau levels and a thinner RNFL. This finding suggests that CSF tau is more likely associated with glial tissues (LC) than with neural tissues (RGC axons), and the change in the neural tissues might be secondary to that in the glial tissues. This finding supports the assumption that glial cells are more directly associated with neurodegeneration than are neuronal cells –49.181P, was associated with the LCT. Although we do not have a clear reason for this result, it is possible that the level of P-tau181P was not sufficiently high to produce a statistical significance. We speculate that other forms of tau phosphorylation could be more highly associated with the LCT. However, the original study  did not analyze other types of CSF proteins besides Aβ1–42 and P-tau181P. Further study is expected to reveal the precise relationship between the phosphorylated tau and LC characteristics.In the present study, only the T-tau, not the P-tau1–42 level was not associated with the LCT. It is acknowledged that the concentration of Aβ1–42 in the CSF increases initially but then decreases decades before the expected onset of symptoms [1–42 and the LCT and the association between CSF tau and the LCT may suggest that the LCT could serve as a potential clinical indicator for neurodegeneration in patients with AD or those at risk of developing AD.In the present study, the Aβsymptoms –52, whersymptoms –52 and fsymptoms , 54. In This study is limited by the small sample size and its cross-sectional design. Hence, a causal relationship between CSF tau and the LCT and the pathogenic significance of this potential relationship could not be determined. A long-term follow-up study with a larger number of subjects, including subjects with RGC pathology, such as glaucoma, is needed to reinforce the findings of this study.The present study found that an increased CSF tau level is associated with a thin LC in healthy subjects and in patients with AD. Given the implications of LC thinning in RGC axonal degeneration, it is possible that tau protein contributes to a common pathogenesis of neurodegeneration in the brain and optic nerve. In addition, the LCT could serve as a non-invasive potential biomarker for estimating the level of CSF tau protein and for suspecting the presence of tauopathies. Although the study leaves many questions, its results may provide a better understanding of the mechanisms of tauopathy in neurodegenerative diseases and a basis for future studies investigating the pathogenesis of neurodegeneration in the brain and optic nerve.


Iron in asymptomatic plaque was present as hemosiderin/ferritin that stained positive with Prussian Blue, and was observed in association with CD68 positive macrophages. Iron in acutely symptomatic plaques is found within thrombus, in the presence of macrophages. The abundance of iron in symptomatic plaques is associated with the source patient’s LDL cholesterol. Within asymptomatic plaques, iron is found in association with macrophages, as hemosiderin/ferritin.Iron within atherosclerotic plaque has been implicated as a catalyst of oxidative stress that causes progression of plaque, and plaque rupture. Iron is believed to accumulate within plaque by incorporation of erythrocytes following plaque rupture and hemorrhage. There is only indirect evidence to support this hypothesis. Plaque specimens were obtained from ten symptomatic and fifteen asymptomatic patients undergoing carotid endarterectomy at a single institution. Plaques were sectioned for study using synchrotron radiation induced X-ray fluorescence the study the distribution of zinc, calcium and iron. Histologic staining was carried out with Prussian Blue, and immunohistochemical staining was done to localize macrophages with CD68. Data were compared against patient clinical variables. Ten symptomatic (15 ± 10 days between index symptoms and surgery) and fifteen asymptomatic carotid plaques were studied. Zinc and calcium co-localized in mineralized areas of symptomatic and asymptomatic plaque. Iron was identified away from zinc and calcium in both symptomatic and asymptomatic plaques. Within the symptomatic plaques, iron was found within the thrombus associated with plaque rupture and hemorrhage. It did not stain with Prussian Blue, but was found in association with CD68 positive macrophages. In symptomatic plaques, the abundance of iron showed an association with the source patient’s LDL cholesterol (R Atherosclerotic plaque at the carotid artery bifurcation is an important cause of stroke. This is the basis of plaque removal (carotid endarterectomy) as a useful stroke reduction strategy –7. PlaquOxidation of LDL cholesterol is important throughout the process of atherosclerotic plaque formation and destabilization . Iron isIt has been assumed that iron enters plaque macrophages through the endocytosis of heme rich erythrocytes following plaque rupture, hemorrhage, and healing. This assumption is based on previous work, in coronary arteries, demonstrating that the progression of atherosclerotic plaques is associated with histologic evidence of plaque rupture and healing, through the deposition of new connective tissue –21. SimiWe undertook the present work in order to seek further evidence that atherosclerotic plaques at the carotid bifurcation accumulate iron through rupture, hemorrhage and healing.Twenty-five patients undergoing carotid endarterectomy for stroke prevention were enrolled. Patients’ provided their written consent, which was then saved on file. The protocol for this study was reviewed by the Biomedical Research Ethics Board of the University of Saskatchewan and approved by the University of Saskatchewan, Regina Qu’Appelle Health Region, and the University of Regina through a harmonized ethics review process.All patients had carotid bifurcation atherosclerosis. Operative indications included either symptomatic or asymptomatic carotid artery plaque. Symptomatic carotid artery plaque was defined by the presence of focal neurologic symptoms attributable to the index carotid lesion, associated with > 50% stenosis of the internal carotid artery origin. Symptoms could include ipsilateral transient monocular vison loss (amaurosis fugax), contralateral sensory or motor loss, or aphasia for left sided carotid lesions. Symptoms could be transient in nature, or fixed, causing a permanent neurologic defect. The onset of symptoms must have occurred within 120 days prior to surgery, in accordance with NASCET definitions [After signed, informed consent to participate was provided, the following clinical data were collected regarding each subject: age, gender, side of the index carotid lesion, ethnicity of the patient, current medications, total cholesterol, LDL cholesterol, HDL cholesterol, serum triglyceride levels, blood pressure, presence or absence of diabetes, date of the last ipsilateral focal neurologic symptom, and the date of carotid endarterectomy. In addition, subjects were asked about their smoking status and were categorized as never having smoked, being current smokers, or being past smokers who quit. The duration of smoking cessation was recorded.Carotid endarterectomy was performed according to the standard practice of the subject’s surgeon with respect to the use of general or local anesthesia, use of intraoperative shunt, and patching. Once the carotid plaque was removed, it was placed in 10% neutral buffered formalin  and stored at 4 degrees C until further processing.The carotid plaques were then embedded in optimal cutting temperature embedding medium (OCT) in metallic cryomolds (25mm X 25mm X 15mm). Using a cryostat (MICROM HM 550) with a Teflon coated blade at -20 degrees C, 10μm thick sections of carotid artery tissue were sequentially obtained through the entire plaque. Tissue sections were alternately mounted on glass microscope slides and metal free Thermanox plastic cover slips . The tissue was air dried at room temperature for an hour then placed in a desiccator. The tissue sections were sequentially numbered and labelled.The preparation of specimens with alternating glass and Thermanox slides was necessary because glass microscope slides contain trace metals that preclude synchrotron imaging of the elements of interest in this study. Thermanox slides are depleted in the elements of interest, making them a good choice for synchrotron imaging. Alternating sections were mounted on glass microscope slides to facilitate immunohistochemical staining, for which Thermanox is unsuitable due to its autofluorescence in the 470–550 nm range.The Thermanox mounted tissue sections were visually examined and the samples which appeared to be from the most active part of each carotid plaque, based on plaque ulceration or degree of stenosis, were selected for synchrotron imaging. Due to limitations on access to synchrotron beam time, the samples were imaged in batches. All symptomatic carotid plaques, as well as five asymptomatic plaques were imaged at Stanford Synchrotron Radiation Lightsource (SSRL) using X-ray fluorescence at an energy of 13.45 KeV, with a resolution of 40μm and a dwell time of 200ms per point. In addition to the elements of interest , fluorescence counts were obtained for copper, sulfur, potassium, titanium, manganese, nickel, and arsenic. Ten asymptomatic plaques, as well as four of the five asymptomatic plaques previously imaged at SSRL, were studied at the Canadian Light Source (CLS) synchrotron facility VESPERS beam line, using pink beam and X-ray fluorescence. Element maps were generated at 80μm resolution and a dwell time of 500ms per point. A subset of six of the asymptomatic plaques were restudied at CLS with 5μm resolution scans of localized iron rich areas. Digital data for each element were stored in a separate data channel.Data were collected in electronic files and processed using Sam’s Microprobe Analysis Kit . Fluorescence maps were produced for each element; each data channel was corrected for variations in incident beam strength and detector deadtime. Fluorescence channels for all elements were then combined into a single channel which was processed using inverse binary thresholding. Threshold levels were selected to maximize the signal from the plaque while reducing noise from empty areas around the plaque and within the plaque lumen. The threshold processed channels were used to create a digital mask. The mask was then applied to each data channel to eliminate noise from peripheral areas of the slide. Fluorescence levels for each element were quantified. Element correlation plots were obtained. Statistical analysis was done using Excel (Microsoft Corporation). Categorical variables were compared using Fisher’s exact test and continuous variables with Student’s t test. Linear regression was used to study correlations between continuous variables and ANOVA to study correlations with categorical variables. Ninety-five percent confidence intervals were determined, and p<0.05 was considered statistically significant.In order to further characterize the nature of iron deposits identified on synchrotron imaging, we performed staining with Prussian Blue (PB). PB stains hemosiderin and, to a lesser extent, ferritin deposits. The stain consists of equal quantities of 20% hydrochloric acid (v/v) and 10% potassium ferrocyanide . Glass mounted sections were stained with PB for 20 min, washed in running water for 1 min, counterstained with 0.5% aqueous nuclear fast red solution (Sigma) for 1 min. Subsequent dehydration steps used exact timings in order to maintain consistent counterstain intensity and distribution.The glass mounted carotid plaque section from the location adjacent to each synchrotron imaged specimen was stained with PB and surveyed in 100 μm segments. PB staining was scored from 0 (no blue present) to 4 (dark blue present).Sections mounted on glass slides, a maximum of 60 μm distant from sections imaged by synchrotron underwent immunohistological staining. Sections were re-hydrated with 1x PBS for 15 minutes. CD68  was added to a blocking solution consisting of 0.3% Glycine, 1x PBS, 1% BSA, 0.1% Triton X-100 and incubated with the specimen overnight at 4°C. The next day, sections were washed 3 x (5 min each) with PBS. Secondary antibody was added  for 45 minutes. After incubation period, sections were washed 3 x in PBS and flurogold mounting media was added. As a control, we omitted the primary antibody, to confirm secondary antibody specificity.Twenty-five patients were enrolled in the study: ten with symptomatic carotid stenosis and fifteen with asymptomatic stenosis. Subjects ranged in age from 45 to 81 years. (mean age 69.8 ± 3.5 years). The mean ages of the symptomatic and asymptomatic patients were not different (p = 0.46). Two of fifteen asymptomatic patients were female (13%), while four of ten symptomatic patients were female . Symptomatic subjects underwent carotid endarterectomy at a mean of 15 ± 10 days (range 5–56 days) following their last ipsilateral carotid territory neurologic event. Two asymptomatic patients had a history of remote, ipsilateral carotid territory symptoms 30 years, and 125 days, respectively and thirteen had no history of ipsilateral neurologic symptoms at any time in the past. Three of ten symptomatic patients were diabetic (30%) whilst seven of fifteen (47%) asymptomatic patients were diabetic (p = 0.68). The mean systolic and mean diastolic blood pressures were similar in the symptomatic and asymptomatic subjects . Of the symptomatic subjects, one had never smoked, one was a current smoker, and eight had quit smoking 1–26 years prior to surgery (mean quitting duration 11.9 years). Of the asymptomatic subjects, six had never smoked, two were current smokers, and seven had quit smoking between four months and 44 years prior to surgery .We noted only low levels of fluorescence from manganese, copper, nickel, arsenic and titanium in our samples. There was no correlation between the intensity of X-ray fluorescence from any of these elements and the symptomatic status of the subject.Plaques showed variable amounts of calcium and zinc fluorescence, depending on the degree of plaque mineralization. However, these elements consistently showed co-localization in areas of mineralized plaque , along w2 = 0.39; significance F = 0.05). This association was not observed in asymptomatic carotid plaques.Iron fluorescence was observed to occur away from zones of mineralization , in sub-Within the group of symptomatic carotid plaques, the areas identified by synchrotron imaging to have a strong iron signal were found to be associated with thrombotic material that did not take up PB stain . The PB In contrast to symptomatic carotid plaques, asymptomatic plaques did not contain thrombotic material. Areas of intense iron fluorescence were associated with PB stain uptake , consistImmunohistochemical staining for CD68, a macrophage specific antigen, was performed on glass slide mounted sections of plaque taken no further than 60µm from the corresponding sample imaged with synchrotron X-ray fluorescence mapping. There was close association of CD68 staining macrophages, both with PB stain uptake in asymptomatic plaques, and areas of iron fluorescence in both symptomatic and asymptomatic carotid plaques. The thrombotic material observed as the source of iron fluorescence in symptomatic plaques was found to be associated with intense localization of macrophages. Within the set of asymptomatic plaques, the iron fluorescence observed with synchrotron mapping was seen to localize with PB positive hemosiderin or ferritin, in the presence of CD68 staining macrophages.Iron has been presumed to accumulate within atherosclerotic plaques through a series of steps beginning with plaque rupture and hemorrhage. Macrophages are stimulated by hemoglobin exposure to differentiate into M2 polarized, mannose receptor positive macrophages that are specialized for phagocytosis ,26. EndoDirect confirmation that plaque iron accumulates through plaque rupture and hemorrhage has been lacking, partly due to the non-availability of an animal model in which to study this process . We presWe have used synchrotron radiation induced X-ray fluorescence to map the distribution of iron within carotid plaque specimens. Our findings are consistent with data reported by others ,28, demoWe observe, within the subset of asymptomatic plaques, that diabetes is associated with significantly greater levels of calcium fluorescence. This is consistent with other data demonstrating diabetes as a risk factor for calcification of plaque . This obIn symptomatic plaques, presenting with rupture and hemorrhage, we found iron within the thrombus. We also identified infiltration of the thrombus by macrophages, as early as five days after the onset of neurologic symptoms. Iron deposition in asymptomatic plaques is similarly associated with the presence of macrophages. This observation is consistent with macrophage mediated endocytosis of thrombus causing the iron to reside within the plaque after healing. The lack of PB staining of macrophage infiltrated thrombus in symptomatic plaques, and the presence of PB staining in asymptomatic plaques, is further consistent with transformation of heme iron to hemosiderin or ferritin within the macrophages. Interestingly, areas of PB staining are absent in symptomatic carotid plaques. We hypothesize that residual hemosiderin or ferritin within the plaque becomes obscured by fresh thrombus when the plaque ruptures. The lack of PB positive areas of plaque distant from the thrombus suggests that plaque rupture tends to occur in the location where previous iron deposits might have been located. This would be consistent with the hypothesis that iron in this area mediates the destabilization of the plaque.Our data demonstrate that the abundance of iron fluorescence in symptomatic plaque is associated with the source patient’s LDL cholesterol. Patients with higher LDL cholesterol appeared to have a more extensive plaque hemorrhage, as determined by iron content, than patients with lower LDL cholesterol. The nature of this association is not apparent from our study. The mechanism by which elevated LDL cholesterol might promote more extensive plaque hemorrhage is unclear and it is possible that elevated LDL cholesterol is the consequence of the inflammation that drives plaque instability, rather than a direct cause.The presence of subintimal iron deposits in the carotid plaques of patients lacking any history of prior ipsilateral neurologic symptoms, suggests that the process of plaque hemorrhage and healing can remain clinically silent when it occurs in the carotid arteries. This is consistent with findings in the coronary circulation, where plaques frequently undergo rupture without causing symptoms. It is possible that these carotid plaques do indeed cause ischemic injury, but no overt neurological symptoms, such as seen in silent strokes –38. DespZinc and calcium co-localize in areas of plaque mineralization. Calcium is more abundant in asymptomatic carotid plaques from diabetics than non-diabetics. Iron localizes to areas of plaque, away from calcium and zinc. Heme iron is found in thrombus of symptomatic plaques, and is infiltrated by macrophages within days of a heralding neurologic event. The abundance of iron in the symptomatic plaque is associated with the patient’s LDL cholesterol level. Asymptomatic plaques also contain subintimal deposits of iron, but these are in the form of hemosiderin or ferritin and are also associated with the presence of macrophages. It is hypothesized that these macrophages play a key role in the creation of plaque instability and is the basis of our present studies.


Central nervous system (CNS) inflammation is a mediator of brain injury in HIV infection. To study the natural course of CNS inflammation in the early phase of infection, we analyzed longitudinal levels of soluble and cellular markers of inflammation in cerebrospinal fluid (CSF) and blood, beginning with primary HIV-1 infection (PHI).-D-related (HLA-DR)) in blood and CSF.Antiretroviral-naïve subjects identified as having PHI (less than one year since HIV transmission) participated in phlebotomy and lumbar puncture at baseline and at variable intervals thereafter. Mixed-effects models were used to analyze longitudinal levels of CSF neopterin and percentages of activated cluster of differentiation (CD)4+ and CD8+ T-cells (co-expressing CD38 and human leukocyte antigenP <0.001), whereas it decreased by 6.7% in subjects with baseline levels of more than 21 nmol/L . In a subset with available flow cytometry data (n = 42), the percentages of activated CD4+ and CD8+ T-cells in CSF increased by 0.8 (P <0.001) and 0.73 (P = 0.02) per 10 weeks, respectively.A total of 81 subjects were enrolled at an average of 100 days after HIV transmission and had an average follow-up period of 321 days, with the number of visits ranging from one to 13. At baseline, the majority of subjects had CSF neopterin concentrations above the upper limit of normal. The baseline concentration was associated with the longitudinal trajectory of CSF neopterin. In subjects with baseline levels of less than 21 nmol/L, a cutoff value obtained from a mixed-effects model, CSF neopterin increased by 2.9% per 10 weeks (n = 33; Neopterin levels and the percentages of activated CD4+ and CD8+ T-cells in CSF progressively increase in most subjects without treatment during early HIV-1 infection, suggesting an accrual of intrathecal inflammation, a major contributor to neuropathology in HIV infection. With the increasing availability of combination antiretroviral therapy (cART) the prevalence of HIV-associated dementia ) decreased from between 10 and 15% to about 2% in the HIV-1-infected population in the United States . While pIn exploring explanations for persistent CNS injury, some studies have focused on the impact of early infection prior to initiation of cART, since it is known that HIV enters the CNS and induces immune activation soon after transmission ,12. EmerIn this study, we sought to examine the natural course of CNS inflammation starting in PHI. We hypothesized that CNS immune activation stimulated during initial infection would persist, and perhaps intensify, prior to initiation of ART, establishing a progressive inflammatory environment in the CNS. In a cohort of ART-naïve PHI subjects, we analyzed blood and CSF for longitudinal levels of soluble and cellular markers of inflammation. Neopterin was examined as a soluble surrogate biomarker of inflammation, and cluster of differentiation (CD)38 and human leukocyte antigen-D-related (HLA-DR) expressed together on CD4+ and CD8+ T-cells were examined as cellular markers of immune activation.ART-naïve HIV-1-infected subjects within one year of transmission were recruited in Gothenburg, Sweden n = 17) and San Francisco, United States (n = 64). As described previously , PHI in  and San Longitudinal paired samples of CSF and blood were obtained prior to cART. In San Francisco, visits were scheduled at baseline , six weeks, and every subsequent six months from baseline, while in Gothenburg, study intervals were variable. Opportunistic infections and major neurological diseases such as stroke, multiple sclerosis, and seizure disorders were ruled out at baseline. Blood samples were analyzed for HIV RNA, albumin, CD4+ lymphocytes and CD8+ lymphocytes, and CSF samples were analyzed for HIV RNA, albumin, and white blood cell (WBC) count, as previously described . ConcentAll statistical analyses were performed using SPSS version 19, IBM Corp., Armonk, USA. All descriptive statistics are presented as median and interquartile range (IQR) for continuous variables, and as frequency (%) for categorical variables. Fischer’s exact test, the Mann-Whitney U test, and Spearman’s rank correlation coefficient were used where appropriate.P = 0.31). We also included an interaction term (days post-transmission × baseline CSF neopterin) as a fixed effect to examine if the trajectory of CSF neopterin over days post-transmission depended on the baseline level. Since baseline CSF neopterin was used as a predictor, only CSF neopterin values from subsequent visits were used as observations of the dependent variable.To analyze the longitudinal trajectory of CSF neopterin, we used mixed-effects models, which can accommodate differences among subjects in the total number of visits and intervals between visits. CSF neopterin concentrations were log-transformed to approximate a normal distribution before formal modeling. The models included fixed effects of baseline CSF neopterin and days post-transmission, and the random effect of intercept. The quadratic time effect was excluded from the final model because of statistical non-significance (This model yielded a regression coefficient for slope (change in CSF neopterin per day post-transmission) equal to 0.000294 - 1.397E-5 × baseline CSF neopterin, suggesting that the slope of CSF neopterin trajectory was dependent on baseline CSF neopterin. Based on this regression coefficient, baseline CSF neopterin values greater than 21 nmol/L yielded a negative slope, whereas baseline CSF neopterin values less than 21 nmol/L yielded a positive slope. Therefore, a baseline CSF neopterin level of 21 nmol/L was a cutoff value that determined the increasing versus decreasing trajectory of CSF neopterin. These results were confirmed by another model that used baseline CSF neopterin as a dichotomous variable (above or below 21 nmol/L). Although only the group with baseline CSF neopterin below 21 nmol/L included subjects with visits beyond 150 weeks, sensitivity analysis constraining the time axis to 150 weeks yielded the same trajectories as above. Therefore the full analysis, which included these visits, is presented in this paper.Similarly, we used mixed-effects models to examine trends in the proportions of CD4+ and CD8+ cells co-expressing CD38 and HLA-DR in blood and CSF. In each of the four models, days post-transmission was a fixed effect, intercept was a random effect, and the percentage of cells co-expressing CD38 and HLA-DR was the outcome variable.The characteristics of participants are shown in Table s = 0.38, P = 0.02) and CD8+ cells  in blood, and the percentage of activated CD8+ cells in CSF . CSF neopterin correlated moderately with the percentage of activated CD4+ cells  and CD8+ cells  in blood and strongly correlated with the percentage of activated CD8+ cells in CSF .Consistent with a previous report on a subset of this cohort , the medP <0.001) being above or below 21 nmol/L, a cutoff value obtained from a mixed-effects model . As shown in Figure P <0.001), and decreased in the smaller high group by approximately 6.7% per 10 weeks (P = 0.001).To confirm the observation of 21 nmol/L as a cutoff value, we ran a similar mixed-effects model with baseline CSF neopterin as a categorical variable. This model yielded the average change in CSF neopterin level over time separately for the low and high groups, and showed that they were significantly different as evidenced by the group-by-time interaction (P <0.001), higher neopterin levels in plasma (P = 0.006) and CSF (P <0.001), and HIV RNA levels in both plasma (P = 0.02) and CSF (P = 0.006) that were greater by more than an order of magnitude. In both groups, the level of HIV RNA in CSF was lower than that found in plasma.When the groups with baseline CSF neopterin concentrations below and above 21 nmol/L were compared on baseline parameters (Table P <0.001), the two groups did not differ in the CSF-to-plasma HIV RNA ratio  or the CSF-to-plasma albumin ratio . The neopterin-to-HIV RNA ratio was higher in the high group in CSF (P <0.001) but not in plasma (P = 0.10).Despite a higher CSF-to-plasma neopterin ratio in the high group  and one out of 11 subjects in the high group (9.1%) had specific neurological seroconversion symptoms (excluding headache). The two neurosymptomatic patients in the low group had Guillain-Barré-like syndrome and meningitis, and the one patient in the high group had meningitis.P = 0.05, Figure P = 0.54, Figure P = 0.04). We noted an outlier value that had a sharp increase from 8 to 54% from day 26 to 51 post-transmission; re-analysis excluding this outlier yielded a rate of 0.8 per 10 weeks  showed an increasing trend in the percentage of activated CD4+ cells in blood at a rate of 0.23 per 10 weeks (In this study, we analyzed the longitudinal levels of CSF neopterin and percentages of activated CD4+ and CD8+ T-cells in blood and CSF, reporting for the first time the natural history of change in these measures of immune activation prior to cART beginning in PHI. HLA-DR and CD38 were used as markers of T-cell activation, and CSF neopterin was used as a soluble marker of CNS inflammation, as it is produced by activated macrophages and astrocytes mainly in response to interferon-gamma. It has been shown that 97.5% of neopterin in CSF is intrathecally produced , and it Our analysis showed two trajectories of CSF neopterin beginning in PHI that were dependent on the baseline concentration of CSF neopterin. In the majority of participants, a slow increase was observed, similar to trends in chronic infection ,27. PrevTo better characterize the groups, we compared them on multiple baseline parameters. In both groups, CSF-to-plasma HIV RNA ratio was lower than the 1 log reduction typically found during chronic infection, as has been previously reported in early HIV infection ,16. HoweInterestingly, despite a lower viral load in CSF compared to plasma in both groups, the concentration of neopterin was higher in CSF than in plasma in the high group, while the converse was true for the low group. Accordingly, a higher CSF-to-plasma neopterin ratio was observed in the high group even though the groups had similar CSF-to-plasma HIV RNA ratios. Further investigation revealed a higher neopterin-to-HIV RNA ratio only in the CSF of the high group, suggesting an amplified intrathecal immune response to the CSF viral load in this group.Additionally in this study, we analyzed changes in the percentages of activated CD4+ and CD8+ T-cells in blood and CSF, beginning in PHI. A significant trend was not observed for blood CD8+ CD38+ HLA-DR+ cells, a subset that has been associated with the rate of CD4+ cell decline and prognosis in chronic infection –30. HoweRegarding the percentage of activated CD4+ T-cells in blood, an upward trend was observed in agreement with a previous report of an increase in the density of CD38 on CD4+ cells in blood during early infection . A novelOur longitudinal analyses of both soluble and cellular measures of immune activation indicate that without treatment, CNS inflammation increases during primary HIV-1 infection in most individuals. In a minority of subjects with significantly higher levels of CSF neopterin at baseline, neopterin levels decrease, but only at a slow rate such that elevated levels persist throughout the follow-up period. These results are disconcerting because CNS inflammation and immune activation are considered to be major contributors to neurodegeneration in HIV infection ,34.et al. reported that the set-point of CSF neopterin in subjects on virologically suppressive cART is associated with the pre-ART level of CNS inflammation [cART dramatically reduces the level of immune activation in blood and CSF ,35–37, aammation . Therefo


Paraneoplastic syndrome generally results from tumor-derived hormones or peptides that cause metabolic derangements. Common metabolic conditions include hyponatremia, hypercalcemia, hypoglycemia, and Cushing’s syndrome. Herein, we report a very rare case of tongue carcinoma presenting with leukocytosis and hypercalcemia.A 57-year-old man was admitted to our hospital with tongue squamous cell carcinoma . He underwent radical resection following preoperative chemoradiotherapy, but locoregional recurrence was detected 2 months after surgery. He presented with marked leukocytosis and hypercalcemia with elevated serum levels of granulocyte colony-stimulating factor (G-CSF) and parathyroid hormone-related protein (PTHrP). He was therefore managed with intravenous fluids, furosemide, prednisolone, elcatonin, and pamidronate. However, the patient died 1 month later of carcinomatous pleuritis following distant metastasis to the lung. Immunohistochemical analyses of the resected specimens revealed positive staining for PTHrP and G-CSF in the cancer cells.In this case, it was considered that tumor-derived G-CSF and PTHrP caused leukocytosis and hypercalcemia. Malignant tumors occasionally secrete hormonal factors that can cause some types of paraneoplastic symptoms –3. Hyper3/μL). The serum levels of PTHrP and G-CSF increased in parallel with progression of his hypercalcemia and leukocytosis  presenting with both hypercalcemia and leukocytosis among 225 oral malignancies , though Hypercalcemia can be categorized as humoral hypercalcemia of malignancy (HHM) or local osteolytic hypercalcemia (LOH) . HHM is Numerous previous studies have shown that G-CSF is responsible for paraneoplastic leukocytosis , 23–25. FDG-PET, F-18 fluorodeoxyglucose positron emission tomography; G-CSF, granulocyte colony-stimulating factor; HHM, humoral hypercalcemia of malignancy; IL-6, interleukin 6; LOH, local osteolytic hypercalcemia; PTHrP, parathyroid hormone-related protein; SCC, squamous cell carcinoma


Quantum dots patterned by atomically precise placement of phosphorus donors in single crystal silicon have long spin lifetimes, advantages in addressability, large exchange tunability, and are readily available few-electron systems. To be utilized as quantum bits, it is important to non-invasively characterise these donor quantum dots post fabrication and extract the number of bound electron and nuclear spins as well as their locations. Here, we propose a metrology technique based on electron spin resonance (ESR) measurements with the on-chip circuitry already needed for qubit manipulation to obtain atomic scale information about donor quantum dots and their spin configurations. Using atomistic tight-binding technique and Hartree self-consistent field approximation, we show that the ESR transition frequencies are directly related to the number of donors, electrons, and their locations through the electron-nuclear hyperfine interaction. Silicon is an excellent host for spin qubits as it is a spin-free environment with the added advantage of weak spin-orbit coupling. The coherence time of single electron spins bound to phosphorus donors in enriched silicon-28 was measured to be 0.6 s with Hahn echo in bulk564The popular Kane qubit proposal of single phosphorus based quantum computerAlthough transport spectroscopy can also be used to determine the number of electrons bound to a donor dot3B0) is applied to the system to polarize the electron spins bound to the dot. The metal wire (the blue bar) deposited on the SiO2 layer carries an alternating current that can generate an electromagnetic field (Bac represented by the blue dashed ellipses) to rotate the qubit electron spin with microwave frequency. Spin readout can be performed by spin dependent tunneling to a single-electron-transistor using the protocol described in refs First, we will discuss how this metrology works theoretically on different device samples. 14m nuclear spins and n electron spins localized in a silicon crystal lattice. In spin qubit experiments, a static external magnetic field To accurately calculate the ESR frequencies of a Si:P QD, it is crucial to capture its electronic structure. Here we obtain the ESR frequencies by solving the effective spin Hamiltonian based on the atomistic tight-binding approach. A donor-dot consists of HZeeman, asThe external B-field is included in the Zeeman term, ith electron and the jth nucleus respectively. ge and gn are the electron and nuclear g-factors respectively. μB is the Bohr magneton and μn is the nuclear magneton. The second term, Hhyperfine, is the hyperfine interaction between electron spins bound to the donor-dot and the nuclear spins of the donors, and is given as,where Aij represents the Fermi contact hyperfine coupling of the ith electron and the jth nuclei, which is proportional to the electronic probability density at the jth donor site. Aij and the components of where μ0 is the vacuum permeability. Ψi is the wavefunction of the ith electron, jth nucleus, and rk,l =  are the electron position vector components. Although we consider both contact and the anisotropic hyperfine interactions in the simulations, we find that the Aij terms, analogous to the case of Si29 in silicon21Aij terms. This helps in the direct mapping of ESR frequencies to Aij and eventually to the physical properties of the donor dot, as needed for the metrology. In obtaining the Aij coupling from the tight-binding wavefunction, we adopt the same technique as ref. where 3d5s* spin-resolved atomic orbital basis with nearest neighbor interaction and spin-orbital coupling. The resulting eigenvalue problem is solved by a parallel block Lanczos algorithm for about 1.4 million atoms using the nanoelectronic modeling tool (NEMO-3D)To obtain the electron wavefunctions, the tight-binding Hamiltonian of the silicon and the donor atoms is represented by an spHSi represents the Hamiltonian of the silicon lattice, HD denotes the central-cell corrected Coulomb potential energy given by the positive charges of donor cores, and He−e is the mean-field electron-electron interaction energy present for multi-electron occupation of the donor dot. The term HSi contains the full bandstructure of siliconHD is represented by a Coulomb potential screened by the dielectric constant of silicon along with a central-cell correction term which assumes a constant potential U0 at the donor site− stateT1 times in donor dotswhere Si which leads to no contribution to the hyperfine term As for multi-electron occupation, for qubit applications, we are mostly interested in effective 1/2 spin configurations of a donor dot, where there is an odd numbered electron in the dot. As indicated by B0 = 1.5 T is applied in the [001] direction.In the results presented in the following, the simulation domain is set to 30 nm × 30 nm × 30 nm of a regular silicon crystal lattice (~1.4 million atoms) in NEMO-3D, so that the Coulombic potential of the donors approach almost zero near the boundaries of the domain. This ensures that the donor-dot bound wavefunctions are not affected by the artificial hard-wall confinement of the box of silicon. The P-donor dots are placed in the middle of the box in the (001) atomic plane. A static magnetic field First, we show how we can infer the number of donors in a donor-dot using ESR measurements. T1 timesNot only are the number of ESR transitions important, but their locations and spacings in the frequency axis can also provide information about the locations of the P donors and the number of bound electrons in the quantum dot. In the next section, we discuss the effect of the donor locations on ESR frequencies. The hyperfine couplings between the electron and the nuclear spins, which are proportional to electron probability density at each donor site, are quite sensitive to the relative donor locations within the quantum dot. The quantum confinement and hence, the on-site wavefunction concentrations strongly depend both on the radial and angular separation of the donors due to the crystal symmetry, even for ~1–2 nm variations in quantum dot sizes. It is critical to obtain information about the extent of the electron wavefunction for the design of two-qubit gatesAij) of one of the two donors, in which the other shares the same value because of symmetry. In a STM patterned Si:P quantum dots, a lithographic patch defines a region where donors can be incorporated into the silicon crystal. The donors in reality can occupy different energetically favourable atomic sites within the patch, which provides some variations in hyperfine couplings between dots with the same number of donors. Here, we use 2P donor dots as an example to demonstrate how the hyperfine couplings and hence the ESR frequencies are correlated with donor separations within a 2P donor quantum dot. Aij as a function of inter-donor separation along the [100] (circles) and the [110] direction (crosses). In the [100] direction, Aij first decreases from 287.4 MHz to 57.1 MHz from a separation of ~0.54 nm to ~3.8 nm, and smoothly levels off when the separation is larger than 4 nm. A very close separation distance increases the quantum confinement of the electron wavefunction as the potential of the two donors enhance each other significantly near the donor cores. Here the electron density from the outskirts of the dot is redistributed to the central region forming a strongly hybridized molecular state, thereby enhancing the hyperfine coupling.Effects such as dopant diffusion and segregation can often cause the precisely placed donors to occupy lattice sites outside the lithographic patch. To account for this scenario, we explore the hyperfine interaction of a P donor pair for larger radial separations along two high symmetry directions in the plane. Aij converging to about half of the bulk value of 117.53 MHz, as the two donors still share one electron. This is in contrast to the case of two far separated donors with one electron each where Aij = 117.53 MHz. In the [110] direction, Aij can be seen to oscillate as a function of separation due to the interference between the wavefunctions of the six conduction band valleys of silicon, similar to the predicted exchange oscillationsAij of a P-donor pair in silicon with one bound electron can vary from ~366.0 MHz to ~48.9 MHz within a 5 nm separation range.When the separation is even larger (>~5 nm), the two P donors approach the regime of two isolated bulk-like donors with Next, we evaluate the ESR frequencies of the donor-based quantum dots for multi-electron occupation. As discussed earlier, the resultant hyperfine coupling in a donor dot originates from the unpaired electronic spin of the outermost electron. In such a case, the unpaired electron is screened from the positively charged donor cores by the spin-paired electrons of the inner orbitals, and the wavefunction spreads out more in space causing a reduction in the density at the nuclear sites in the central region. This causes a sharp decrease in the Fermi contact hyperfine coupling as more electrons are loaded. Note that, by referring to spin-paired inner electrons, we are still referring to the extra electrons in the donor molecules that do not take part in the covalent bonds with the nearest silicon atoms as these bonds remain intact in the donor-dot.nd figure in In We have proposed a metrology technique to characterize spin qubits hosted by Si:P quantum dots in silicon. The metrology can help extract information about the number of donors, donor locations and the number of electrons based on ESR measurements, and is potentially more accurate than charging energy based metrology. An atomistic tight-binding method is coupled with a Hartree self-consistent field approach to obtain the orbital wavefunctions of multi-donor quantum dots in silicon. Solving a multi-spin Hamiltonian with parameters computed from the wavefunctions enables us to predict ESR transition frequencies for the Si:P quantum dots. We found that the number of ESR frequencies increases with the number of donors, while the locations and the spacings of the ESR peaks on the frequency axis can help extract information about the spatial extent of the donors within the quantum dot and the number of bound electrons. The extracted information is useful for engineering single and multi-qubit operations in a system with very long coherence times. The method proposed here may also be applied to obtain information about other How to cite this article: Wang, Y. et al. Characterizing Si:P quantum dot qubits with spin resonance techniques. Sci. Rep.6, 31830; doi: 10.1038/srep31830 (2016).


Hif1a gene that the oxygen-labile transcription factor HIF-1α is essential for HSC mobilization in response to G-CSF and Plerixafor. Conversely, pharmacological stabilization of HIF-1α with the 4-prolyl hydroxylase inhibitor FG-4497 synergizes with G-CSF and Plerixafor increasing mobilization of reconstituting HSCs 20-fold compared with G-CSF plus Plerixafor, currently the most potent mobilizing combination used in the clinic.Many patients with hematological neoplasms fail to mobilize sufficient numbers of hematopoietic stem cells (HSCs) in response to granulocyte colony-stimulating factor (G-CSF) precluding subsequent autologous HSC transplantation. Plerixafor, a specific antagonist of the chemokine receptor CXCR4, can rescue some but not all patients who failed to mobilize with G-CSF alone. These refractory poor mobilizers cannot currently benefit from autologous transplantation. To discover alternative targetable pathways to enhance HSC mobilization, we studied the role of hypoxia-inducible factor-1α (HIF-1α) and the effect of HIF-1α pharmacological stabilization on HSC mobilization in mice. We demonstrate in mice with HSC-specific conditional deletion of the  However in the autologous setting, 20–60% of chemotherapy-treated patients fail to reach this minimal threshold in response to G-CSF, precluding transplantation1 particularly in patients with prior radiotherapy and chemotherapy.2 The chemotactic interaction between the chemokine CXCL12 and its receptor CXCR4 is pivotal to HSPC retention within the BM4 and is weakened by G-CSF treatment causing mobilization.7 Additional inhibition of the CXCL12:CXCR4 interaction with specific small synthetic inhibitors such as Plerixafor  synergistically enhances HSPC mobilization in response to G-CSF in humans and mice.8 The synergistic effect of Plerixafor has been confirmed in large clinical trials with multiple myeloma and non-Hodgkin lymphoma patients eligible for autologous HSC transplantation who previously failed to mobilize in response to G-CSF alone. Plerixafor injected daily 1 h before blood aphaeresis from day 4 of G-CSF administration enables approximately 70–80% patients who previously failed to mobilize in response to G-CSF alone to reach 2 × 106 CD34+ cells/kg threshold.10 However, the remaining 20–30% of patients who failed to mobilize with G-CSF alone still fail to mobilize with G-CSF and Plerixafor.10 It is therefore important to further understand the mechanisms of HSC mobilization to identify novel therapeutics to overcome mobilization failure in a larger proportion of patients.The cytokine granulocyte colony-stimulating factor (G-CSF) is the main agent to mobilize hematopoietic stem cells (HSCs) for transplantation. Administered daily at doses of 10 μg/kg, G-CSF mobilizes hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) into the circulation. In most healthy allogeneic donors, CD3411 Extensive myeloid progenitor proliferation in response to G-CSF is thought to enhance O2 consumption increasing overall BM hypoxia, which in turn stabilizes the O2-labile protein hypoxia-inducible factor-1α (HIF-1α) in mobilized BM.11 HIF-1α is a transcription factor that regulates metabolic adaptation and cell responses to hypoxia.14 Considering the critical role of HIF-1α in regulating HSC proliferation, self-renewal and homing to the BM,18 we investigated its role in HSPC mobilization in mice.The hypoxia sensing pathway is activated in the BM of mice mobilized with G-CSF.Hif1atm3Rsjo/J (Hif1aflox/flox) mice from the Jackson Laboratory . Scl-CreER transgenic mice expressing a tamoxifen-inducible Cre recombinase driven by a HSC-specific Scl gene enhancer,19 B6-Gt(ROSA)26Sortm1(EYFP)Cos/J (abbreviated as R26RYFP) with a loxP-flanked STOP sequence preceding an enhanced yellow fluorescent protein (YFP) reporter gene inserted into the genetrap ROSA26 locus20 were backcrossed at least 10 times in C57BL/6 background. Mouse genotyping is described in Supplementary Methods.C57BL/6 mice were purchased from the Australian Resource Centre, Perth, Australia; B6.129-Scl-CreER mice were gavaged daily with tamoxifen-free base diluted in peanut oil containing 10% ethanol for 3 days to induce the Scl-CreER transgene. For mobilization experiments with G-CSF, G-CSF was administered for 3 days beginning on the final day of tamoxifen gavaging unless otherwise stated. For mobilization experiments with Plerixafor, Plerixafor was administered intraperitoneally 1 week following the final day of tamoxifen induction, 1 h before harvest. Following treatments, mice were then euthanized by cervical dislocation and BM and bones harvested. For migration assay and CXCR4 staining, mice were harvested 1 week following the final day of tamoxifen induction.21 Tissues were harvested 1 h after Plerixafor administration. FG-4497 was injected daily intraperitoneally at 20 mg/kg. Control mice were injected with an equivalent volume of saline. Tissues were harvested on the morning following the last G-CSF injection, or 1 h following Plerixafor injection.All experiments were performed on 9- to 12-week-old male mice. Recombinant human G-CSF was injected twice daily subcutaneously at 125 μg/kg and Plerixafor (AMD3100 octohydrochloride) intraperitoneally at 10 mg/kg as described.For studies with additional 4-prolyl hydroxylase domain enzyme (PHD) inhibitors, G-CSF was administered twice daily subcutaneously for 4 consecutive days. PHI-1 and PHI-2 were administered daily by oral gavage at 20 mg/kg for the last 3 days of the mobilization regimen.22 Spleens were harvested, weighed, dissociated in 10 ml phosphate-buffered saline with 2% FCS. RNA from the endosteum was isolated from femurs as previously described.23Mice were anesthetized with isofluorane and approximately 1 ml of blood collected into heparinized tubes by cardiac puncture before cervical dislocation. Femoral BM was flushed and spleens dissociated between the frosted ends of two glass slides in phosphate-buffered saline containing 2% fetal calf serum (FCS) for further analyses. For flow-cytometry analyses, red cells were lysed from blood samples as previously described.M phenylmethylsulfonyl fluoride.16 Quantitative western blots with rabbit anti-mouse HIF-1α antibody were performed using the Odyssey Infra-Red Imaging System  as described previously.16Following treatment of mice with Saline, G-CSF or FG-4497, BM cells from one femur were flushed with 1 ml of urea cell lysis buffer containing complete protease inhibitors  supplemented with 200 μ24 When results are reported as proportion of mobilized cells versus whole BM content, we considered that one femoral BM represents 5.6% of total mouse BM and that the total volume of blood is 0.08 ml per gram of body weight.26In all, 10 μl whole blood or splenocytes from 1/1000 of a whole spleen were seeded in 35-mm petri dishes and covered with 1 ml IMDM supplemented with 1.6% methylcellulose, 35% FCS and optimal concentrations of mouse IL-3, IL-6 and soluble kit ligand as described previously.Antibody staining for expression of α4 and α5 integrins, PSGL1, CXCR4, binding of P- and E-selectins and for HSPC cell cycling are described in Hif1a gene deletion in HSPC and measure Cxcr4, Cxcr7 and Cxcl12 mRNA is described in Quantitative real-time reversed transcribed PCR (qRT-PCR) to confirm 24 Briefly, lethally irradiated recipient congenic B6.SJL CD45.1+ female mice were transplanted with 200 000 competitive whole BM cells from untreated B6.SJL CD45.1+ mixed with 20 μl blood aliquots from 6 pooled mobilized CD45.2+ C57BL/6 donor mice. CD45.2/CD45.1 chimerism was measured in blood at 8, 12 and 16 weeks post transplant in myeloid, B and T lineages by flow cytometry. Content in repopulating units was calculated as previously described.27The content of mobilized blood samples in competitive repopulating HSC was determined in competitive repopulation assays as previously described.YFP/YFPHif1aWT/WT or SclCreER R26RYFP/YFPHif1afl/fl mice were plated in each 5 μM pore transwell inserts  in IMDM supplemented with 10% FCS in duplicate. CXCL12 was added to wells at a concentration of 250 ng/ml. After 4 h at 37 °C, the percentage of YFP+ and YFP− Lin−Sca1+Kit+ cells that had transmigrated to the lower chamber was determined by flow cytometry after addition of 1000 fluorescent counting beads  in both insert and input cells. For chemotaxis assays with BM cells from wild-type mice treated with FG-4497, assays were set up as described above except that 40 μM FG-4497 was added in both top and bottom chambers containing BM cells from FG-4497-treated mice, whereas an equivalent volume of DMSO  was added in both chambers containing BM cells from saline-treated mice.Five million whole BM cells from SclCreER R26Rt-test, paired t-test, ANOVA with Bonferroni correction or non-parametric Mann–Whitney test or Dunn's multiple comparison test depending on distribution normality. A value of P<0.05 was considered as significant. Data are presented as mean±standard deviation.Differences between treatment groups were calculated using a two-tailed Hif1a gene alleles are specifically deleted and YFP reporter induced in a Cre-dependent manner20 in HSCs following treatment with tamoxifen  to the blood  and YFP+ (Hif1a deleted) Lin−Sca1+Kit+ HSPCs mobilized to blood or spleen compared with those remaining within the BM. Within each individual mouse, the proportion of YFP+Hif1a-deleted HSPCs that left the BM to mobilize into blood or spleen was 12-fold and 5-fold lower, respectively, than the proportion of YFP-Hif1a-intact HSPCs mobilized to blood and spleen  and YFP+ (Hif1a deleted) Lin−Sca1+Kit+ HSPCs mobilized to blood versus the number of Hif1a-intact or Hif1a-deleted HSPCs remaining within the BM. Within each individual mouse, the proportion of YFP+Hif1a-deleted HSPCs that mobilized into blood or spleen significantly reduced compared with the proportion of YFP+Hif1a-intact HSPCs that mobilized to the blood  and YFP+ (Hif1a deleted) Lin−Sca1+Kit+ HSPCs that migrated toward CXCL12. YFP+ (Hif1a deleted) HSPCs exhibited significantly reduced migration toward CXCL12 compared with YFP- (Hif1a gene intact) HSPCs  HSPCs . In contifferent , suggestCxcr4 mRNA by qRT-PCR on Lin−Sca1−Kit+ YFP+ HSPCs from SclCreER R26RYFP/YFPHif1afl/fl mice and SclCreER R26RYFP/YFPHif1aWT/WT mice 4 days after a 3-day induction with tamoxifen. Cxcr4 expression was however not altered by Hif1a deletion. We also tested for the expression of CXCR7, a related chemokine receptor previously shown to bind CXCL12 and dimerize and co-signal with CXCR4 on human CD34+ HSPCs.29 Using an anti-CXCR7 monoclonal antibody cross-reactive for human and mouse CXCR7, as well as TaqMan primer probe set for mouse Cxcr7 mRNA, we could not detect CXCR7 protein or mRNA in mouse BM HSPCs (data not shown), in agreement with expression array reported on www.biogps.org showing lack of expression in adult mouse HSCs.As cell surface expression of CXCR4 is very low, we quantified 2-labile, may enhance mobilization in response to G-CSF and Plerixafor. We injected wild-type mice with the PHD inhibitor FG-4497 to block HIF-1α proline hydroxylation in oxidative conditions and downstream recruitment of the ubiquitin ligase VHL and O2-dependant proteasomal degradation of HIF-1α.31 HIF-1α protein was very low in BM cell lysates from saline-injected animals and significantly increased following G-CSF treatment  incorporation over 3 days and Ki67 antigen expression in BM HSPC from mice treated with saline, FG-4497 for 3 days, G-CSF for 2 days or G-CSF for 2 days together with FG-4497 for 3 days . Therefore, this short FG-4497 treatment did not increase HSPC mobilization by altering their cycling. We also measured the effect of FG-4497 on HSPC apoptosis. In overnight cultures of BM cells with 10 ng/ml Kit ligand, addition of 40 μM FG-4997, a concentration that stabilizes HIF-1α in cultured HSPC,32 did not alter the proportion of live non-apoptotic annexin V-negative, 7-aminoactinomycin D-negative HSPCs . This may explain why FG-4497 alone does not mobilize HSPC Cxcl12 mRNA was further decreased in the G-CSF plus FG-4497 combination group compared with G-CSF alone, suggesting that FG-4497 further reduce expression of CXCL12 by BM stromal cells in response to G-CSF  from chemically distinct structural classes (US patents number 7 696 223 and 7 928 120). Although representing different chemical series, FG-4497, PHI-1 and PHI-2 are all structural mimetics of the PHD substrate 2-oxoglutarate and inhibitors of the HIF PHD enzymes. animals .Since FG-4497 synergized with either G-CSF or Plerixafor, we tested whether FG-4497 could further enhance the mobilizing effect of the combination of G-CSF with Plerixafor. Mice were treated with G-CSF for 2 or 4 days, with or without Plerixafor 1 h before harvest or with or without FG-4497 for 3 days prior harvest . The com+ C57BL/6 mice in competition with 2 × 105 BM cells from CD45.1+ B6.SJL congenic donors. CD45.2+ donor versus CD45.1+ competitor chimerism 16 weeks post transplantation confirmed that the tri-combination G-CSF plus Plerixafor plus FG-4497 mobilized competitive repopulating HSCs more than the dual combination G-CSF plus Plerixafor at both time points of G-CSF treatment P<0.01  quantitatively increasing the number of HSCs mobilized into the blood and (2) increasing their subsequent homing and engraftment efficiency once transplanted.Using conditional deletion of the Hif1a gene deletion inhibited HSPC mobilization in response to the CXCR4 inhibitor Plerixafor, and reduced their migration toward CXCL12 gradient without altering Cxcr4 mRNA expression. However, due to the extremely low levels of CXCR4 protein at the surface of mouse HSPCs in the BM, we could not detect significant changes in CXCR4 protein at the surface of HSPCs following Hif1a gene deletion. Because of this we could not detect changes in cell surface CXCR4 protein on HPSCs from mice treated with FG-4497. However, treatment of wild-type mice with FG-4497 alone or in combination with G-CSF, which further stabilizes HIF-1α protein, significantly reduced intracellular CXCR4 protein in HSPCs compared with mice treated with saline or with G-CSF alone. However, FG-4497 treatment did not alter Cxcr4 mRNA expression in HSPCs. This is consistent with previous findings showing that deletion of Hif1a gene in all hematopoietic cells and in stromal cells in Mx1-Cre Hif1afl/fl mice did not alter Cxcr4 mRNA or CXCR4 protein surface expression on HSPCs.17 In addition, we found that in vitro chemotaxis of BM HSPCs isolated from mice treated with G-CSF plus FG-4497 was also decreased compared with HSPCs from mice treated with G-CSF alone. This is in contrast with a recent report that ex vivo treatment of BM HSPCs with dimethyl prostaglandin E2, which also increases HIF-1α protein, enhanced HSPC transmigration toward CXCL12.18 A possible reason for this discrepancy is that dimethyl prostaglandin E2 was used in ex vivo once HSCs removed from their physiological niches while in our experiments, FG-4497 was administered in vivo. Also, dimethyl prostaglandin E2 is likely to activate via its four receptors EP1-4 many other pathways in addition to HIF-1α.38 Finally, we found that the combination of FG-4497 plus G-CSF further exacerbated the reduction of Cxcl12 mRNA expression in the BM stroma in response to G-CSF. Therefore, the effect of FG-4497 is likely mediated in part by perturbation of CXCR4 signaling (HSPC autonomous effect) and CXCL12 expression (extrinsic effect) in G-CSF-treated mice, which may ultimately lead to enhanced mobilization. In summary, our data suggest that in G-CSF-treated mice, HIF-1α stabilization with FG-4497 qualitatively alters or quantitatively decreases CXCR4 chemotactic signaling. However, as HIF-1α activates the transcription of hundreds of genes,39 it is likely that HIF-1α enhances HSPC mobilization via several parallel mechanisms. Therefore, the effect of HIF-1α stabilization on HSPC mobilization is likely to be complex resulting from both HSC autonomous mechanisms and stroma mediated-mechanisms. Ongoing studies in our laboratory to profile the transcriptome of HSPCs from mice treated with FG-4497 will provide insights about its additional mechanisms of action on HSPC mobilization.Mechanistically, we show that 37 Therefore, PHD inhibitors may have the potential to (1) reduce the high cost of remobilization41 and make it more accessible to patients requiring autologous HSC transplant and (2) be used upfront with G-CSF to increase the number of HSCs harvested in both the allogeneic and autologous setting as transplantation of higher doses of HSCs (>5 × 106 CD34+ cells/kg) further reduce the period of cytopenia, risks of potentially fatal febrile neutropenia, and transfusion costs associated with prolonged anemia and thrombocytopenia.If corroborated in humans, then our current study suggests that PHD inhibitors such as FG-4497 could advantageously replace Plerixafor or be added to G-CSF and Plerixafor to form an even more potent mobilizing cocktail that could further reduce the numbers of patients who fail to mobilize sufficiently to proceed with autologous HSC transplantation. Indeed, PHD inhibitors are safe in humans and currently tested in several clinical trials to treat anemia associated with chronic renal diseases.In conclusion, our work demonstrates that HIF-1α is necessary to HSPC mobilization in mice and opens the perspective of using PHD inhibitors in combination with G-CSF to boost HSC mobilization for transplantation in humans.


KD values broadly agree with published biochemical measurements.We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100’s to 1000’s of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway.  With increasing knowledge of intracellular signalling networks, it becomes more evident that molecules can be involved in processes occurring in multiple pathways. Understanding the complex interconnections between different pathways requires comprehensive identification of specific binding partners, and therefore it is important to develop higher throughput techniques to search for new interactions. Currently, biochemical methods are most often used to this end and provide high sensitivity. However, they require long separation procedures, during which the active molecules are isolated from their native environment and may present different reaction kinetics than in live cells where molecular crowding and high compartmentalisation could have an impact. Fluorescence microscopy – particularly exploiting genetically expressed fluorescent proteins – can be applied directly to map and quantify protein interactions in live or fixed cells and preserve information concerning the inhomogeneous cellular distribution of molecules, with typical spatial resolution below 0.5 μm. With the advent of superresolution microscopy, the prospect of sub-50 nm resolution could permit the study of the organisation and dynamics of molecules within organelles and large interacting complexes1A widely used fluorescence technique to study bi-molecular interactions within cells is FRET (Förster resonant energy transfer), which utilises the non-radiative (dipole-dipole) energy transfer from a fluorescent donor to an acceptor that can take place only when the two fluorophores are situated at distances <~10 nm. In the case of two proteins labelled with donor and acceptor tags, this implies that FRET occurs only if and when the two proteins interact with each other. FRET has therefore been widely exploited to study protein interactions using fluorescence microscopes. However, its application for high content analysis in automated multiwell plate readers is much more limited. FRET can be read out using a wide range of techniquesOne approach to detect FRET is to measure the fluorescence intensity ratio of the acceptor and the donor fluorophores observing the increase of the fluorescence intensity in the acceptor channel with the simultaneous decrease of the intensity in the donor channel. This spectral ratiometric imaging acquisition is fast but requires additional control samples to correct for spectral cross-talk between the fluorophores and to calibrate the spectral response of the specific optical set-up (instrument and sample corrections), making comparison between different samples difficult. Quantitation can be degraded by unknown variations in donor-acceptor stoichiometry and quantitative readouts of FRET efficiency and population fraction of FRETing donors are not possible without additional measurements of reference FRET constructs5It is also possible to utilise the depolarisation of the acceptor fluorescence as a FRET readout Polarisation-based measurements can achieve similar acquisition speeds as spectral ratiometric readouts and are highly sensitive to detect the occurrence of FRET, but it is again difficult to quantify FRET efficiencies and population fractions of interacting donors7Fluorescence lifetime imaging (FLIM)13KD. In order to achieve the fast FLIM acquisition required for reading 96-well plates, we utilise wide-field time-gated imaging to realise a FLIM microscope that is able to automatically acquire wide-field or optically sectioned fluorescence lifetime images with a typical mean acquisition time of 10 seconds per field of view for cells expressing fluorescent proteins, including the time required to move from the previous field of view and to automatically focus the microscope. For the first time to our knowledge, we report the application of automated FLIM FRET to screen for protein binding partners within cells – here shown to identify interactions between the Ras-association domain family (RASSF) and mammalian sterile 20-like kinases (MST) – and the estimation of the KD for these interactions.In this paper we report the application of a prototype high content assay platform providing unsupervised FLIM FRET of multiwell plate arrays that can identify protein binding partners in their cellular context and quantify the dissociation constant, The RASSF family consists of ten members, RASSF1-10, which share a common Ras association domain. The role of this domain is not yet fully understood161718192021Salvador/RASSF/HippoDrosophila melanogaster.The C-terminal domains differ between the classical RASSF1-6 and the N-terminal RASSF7-10. The classical RASSF members have a common α-helical SARAH domain that is absent in the N-terminal RASSF members, which instead are predicted to have coiled-coil motifs at various positions towards their C-terminal regionRecent studies have shown that the SARAH domain is able to dimerise in solution26271529301718The SARAH dimer is formed via a head-to-tail interaction of the two helices in an antiparallel arrangement. Although different coiled-coil motifs have been described to form oligomers33MST1) were labelled with mCherry at the N-terminus to provide the acceptor for the FRET assays. To evaluate the effect of possible non-specific interactions on the FRET readouts , two negative controls were employed: the free fluorescent protein mCherry was expressed (without being linked to the MST1 or SARAHMST1 domain) and, as a more biologically relevant control, the kinase domain of the MST1 with a deletion of the SARAH domain (MST1ΔSARAH) was tagged with mCherry at its N-terminus.FLIMfithttp://www.openmicroscopy.org/site/products/partner/flimfit), based on the variable projection method and providing tools for segmentation of cells containing both donor and acceptor, monoexponential and global analysis of EGFP lifetime using convolution and background correction, as well as analysis of fluorescence intensity images.To obtain reliable statistics, we automatically acquired FLIM images from 10 fields of view (FOV) per well, using 5 time gates to sample the fluorescence decay profiles with exposure times around 1 s per gate for the donor (EGFP) images. Intensity images of the acceptor (mCherry) were also obtained with direct excitation for the same fields of view. Approximately 800 FLIM images were thus acquired for each multiwell plate. Such large FRET data volumes require rapid automated analysis, for which we have developed an open source program called i) EGFP-RASSF + mCherry-SARAHMST1 (interaction partner) and (ii) EGFP-RASSF + mCherry-MST1ΔSARAH as the negative control (τ) per cell that can be used for qualitative readouts of the occurrence or absence of the protein-protein interaction.For these assays, COS7 cells were transfected with donor only plasmids (EGFP-RASSF) and two different conditions of donor plus acceptor constructs:  are compared with those transfected with the donor only, the average of the mean lifetime differences for each RASSF family member was 6 ± 8 ps, with a maximum change in mean fluorescence lifetime of 16 ps for RASSF3. These results represent the biological noise in our measurement. Therefore, to be conservative, we considered that a lifetime shift of at least 32 ps – i.e. twice the maximum difference observed between donor only and donor plus negative control – should be required for it to be considered significant.MST1 co-transfection, the average EGFP lifetime is reduced by 130–310 ps for RASSF1-6 . However, the reduction in mean EGFP lifetime in individual cells expressing the mutants is smaller than that observed with the wild type RASSF5C, suggesting that the fraction of the bound molecules is reduced in the case of RASSF5C mutants compared to the wild-type protein, which could be due to a reduction in binding affinity.In the case of the three RASSF5C mutants, we observe a reduction in the average EGFP lifetimes compared to the negative control based on the box plots in These FRET screening data are also supported by biochemical assays, represented in The different degrees of disruption to heterodimerisation caused by the SARAH mutations in RASSF1 and RASSF5 observed both in our FRET and biochemical data , 6, 7, 8MST1 domain compared to when they dimerise with the full length MST1. This is observed for all RASSF1-6, and is supported by the 2D plots of EGFP lifetimes vs. donor/acceptor ratios . r ratios  even thor ratios .These data could be explained by a larger mean donor-acceptor FRET distance for the interaction of full length MST1 with RASSF1-6 compared to the interaction with the isolated SARAH domain, e.g. due to steric constraints. This would reduce the FRET efficiency, due to its dependence to the sixth power of the donor-acceptor distance. Alternatively, the fraction of bound molecules, e.g. due to a different binding affinity of RASSF1-6, could be different for the two interactions.FLIMfitTo understand more about the interactions producing the observed differences in the readout based on fitting to the monoexponential decay model, the data underlying τD) and from RASSF molecules that are bound either to the isolated SARAH domain or to the full length MST1 kinase . The unquenched donor lifetime was determined using data from the cells transfected only with RASSF1-6 and this component was fixed during the global fitting. The donor lifetime quenched by FRET was allowed to vary, but was constrained to be spatially invariant across all cells within a given experimental condition. Thus, the fraction of interacting molecules (β term in equation 2) could be estimated and these results are presented in the box plots shown in MST1 domain.The two lifetime components contributing to the EGFP (donor) decay profiles arise from non-interacting RASSF molecules , where D is the donor-labelled partner, A is the acceptor-labelled partner and DA is the complex formed by their association:Dissociation constants KD is then given by equation 4, which relates the concentrations of the binding partners to the complex:Dtotal) and acceptor  concentrations per cell, while the FRET fraction β obtained from the global analysis provides an estimate of the concentration of the DA complex via the bound fraction of the donor.Using the fluorophore concentration calibration we can determine the total donor  to produce constructs with an N-terminus EGFP tag as described in ref. Full length RASSF1-10 were cloned into the GatewayMutagenesis was performed using the QuickChange® Site-Directed Mutagenesis Kit (Stratagene) following the manufacturer’s instructions. All mutant constructs were sequence verified.Co-immunoprecipitation was performed using the Anti-c-Myc Immunoprecipitation kit (Sigma) using co-transfected cell lysates as described in ref. Antibodies used for identification are anti-GFP (B2) (Santa Cruz), anti-GAPDH (Santa Cruz), anti-myc (in-house). Bands from Western blotting were quantified using ImageJ. The relative intensity of the WT control was set at 1 for each experiment and used as a reference point. The error bars are the standard deviations, p-values were calculated using the Student’s t-test and indicated as follows: p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***).http://hexserver.loria.fr/index.php) and ClusPro (http://nrc.bu.edu/cluster) were used for rigid body docking and to run simulations of the heterodimers consisting of the monomer structures of MST1 (PDB: 2JO8) and the RASSF SARAH homology models from ref. http://www.bioinf.manchester.ac.uk/naccess/).Docking programs Hex  bovine serumalbumin (pH = 7.4), filtered through a 0.2 μm membrane for sterilisation). 4.5 μg of plasmids in different combinations (indicated in the figures) were added to this suspension. Electroporation was performed using an Amaxa NucleofectorTM II  using the manufacturer’s program for COS7 cells. Cells were then seeded in a 96-well plate (Greiner Bio-One) at a density of 30 000 cells/well and fixed the following day using 4% paraformaldehyde for 20 minutes at room temperature, washed 3 times in phosphate buffer saline (PBS) and imaged in PBS.COS7 cells (ECACC) were grown in DMEM supplemented with 10% fetal calf serum, 2 mM glutamine, 1 mM sodium pyruvate and 1% penicillin-streptomycin and were used for all experiments. All fluorescent constructs were transfected via electroporation. Typically, 1–1.5 × 10The instrument shown in For wide-field imaging the excitation is directed via a single-mode optical fibre to the back illumination port of the microscope after passing through a rotating diffuser wheel and relayed to the focal plane of the microscope to realise Köhler illumination. The samples arrayed in a 96-well plate were mounted on a motorised x-y stage  and imaged using a 20 × objective (Olympus UPlanFl 20×/0.5). Appropriate dichroic mirrors and emission filters in the motorised filter cube wheel  provided automated selection of spectral channels. The emitted fluorescence light was imaged via the left-hand port of the microscope to a gated optical intensifier (GOI)  and the resulting gated images at the phosphorus screen were imaged to a cooled CCD camera . The GOI gating voltage signal is synchronised and delayed with respect to the laser excitation pulses under computer control. For the work reported here, the GOI gate width was set to 1 ns and typically time-gated images of EGFP fluorescence were acquired at 5 different delays after excitation while only one time gated image (at the beginning of the decay) was acquired for the mCherry emission. The integration time of the CCD camera was set to 1–2 s per gate delay for EGFP and 5–6 s for the mCherry image acquisition such that the dynamic range of the CCD was utilised.KD calculations, the instrument was configured to incorporate a spinning Nipkow disk unit , as described in refs For the optically sectioned FLIM acquisitions used to provide the data for the In addition to the time-gated FLIM FRET data, a FLIM acquisition of a scattering sample detected at the excitation wavelength was acquired to provide an instrument response function (IRF) for the data analysis. Time-gated FLIM was also applied to a well containing only PBS in order to determine the time varying background.FLIMfit, described in detail in ref. www.openmicroscopy.org/site/products/partner/flimfit. For the work reported here we utilised the following capabilities of FLIMfit: cell segmentation based on donor and/or acceptor intensity; calculation of average fluorescence intensity of donor and acceptor per cell; fitting the donor fluorescence intensity decays to monoexponential and to double exponential decay models (including instrument response function (IRF) and time-varying background correction); global fitting of donor fluorescence intensity decays across multiple fields of view and wells; visualisation of FLIM data (including rendering of plate maps showing mean EGFP decay times per well and images of one field of view per well). To utilise the relatively small changes in donor lifetime that we have obtained in the FLIM FRET assays reported here from fits to double exponential decay models, it is critical to minimise fluctuations in the IRF and to account for any residual variation. This was realised by acquiring FLIM data of a reference dye solution (rhodamine 6G) in some of the plate wells and fitting the measured decay data to a monoexponential model in order to precisely determine the relative excitation time , for each plate. This information was combined with the measurement of a scattering sample to construct the IRF that is convolved with the exponential decay model to provide the function to which the experimental FLIM data is fitted. Graphs of lifetime and intensity ratio parameters were plotted in Origin 8 .The instrument is controlled using a programme written in LabVIEW . This controls the automatic movement of the stage, the autofocusing of each field of view, the automatic change of the excitation filters, of the filters and dichroics in the filter cube wheel, the objective lens change, the GOI gating and the CCD camera acquisition. A “prefind” scan was implemented to image the well plate using fluorescence intensity to identify and localise cells and to acquire donor and acceptor intensity images. Specific fields of view in various wells were selected for subsequent FLIM after applying an intensity threshold. FLIM data analysis was performed using the custom written open source software, How to cite this article: Margineanu, A. et al. Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM). Sci. Rep.6, 28186; doi: 10.1038/srep28186 (2016).