Predictive preclinical models play an important role in the assessment of new treatment strategies and as avatar models for personalised medicine; however, reliable and timely model generation is challenging. We investigated the feasibility of establishing patient-derived xenograft (PDX) models of high-risk neuroblastoma from a range of tumour-bearing patient materials and assessed approaches to improve engraftment efficiency.PDX model development was attempted in NSG mice by using tumour materials from 12 patients, including primary and metastatic solid tumour samples, bone marrow, pleural fluid and residual cells from cytogenetic analysis. Subcutaneous, intramuscular and orthotopic engraftment were directly compared for three patients.PDX models were established for 44% (4/9) of patients at diagnosis and 100% (5/5) at relapse. In one case, attempted engraftment from pleural fluid resulted in an EBV-associated atypical lymphoid proliferation. Xenogeneic graft versus host disease was observed with attempted engraftment from lymph node and bone marrow tumour samples but could be prevented by T-cell depletion. Orthotopic engraftment was more efficient than subcutaneous or intramuscular engraftment.High-risk neuroblastoma PDX models can be reliably established from diverse sample types. Orthotopic implantation allows more rapid model development, increasing the likelihood of developing an avatar model within a clinically useful timeframe. However, limited engraftment success rates, lengthy model establishment time and high maintenance costs all pose major challenges to the routine use of PDX models for personalised therapy.18 Optimising the methodology to improve engraftment efficiency is critical for meeting urgent clinical needs in the era of personalised medicine.Patient-derived xenograft (PDX) models are generated from the implantation of patient tumour sample directly into immunodeficient mice, and the subsequent passage of tumour material from animal to animal.21 and has been described in more recent accounts.28 Herein, we demonstrate the feasibility of developing high-risk neuroblastoma PDX models at a high success rate from multiple sources of tumour-bearing patient materials at both diagnosis and relapse and from primary and metastatic tumour sites, and for the first time, residual tumour cells from cytogenetic analysis. We also show that the engraftment time is influenced not only by the number of tumour cells inoculated but also by the site of inoculation, with orthotopic  engraftment allowing more rapid model generation than subcutaneous engraftment. Finally, we also describe some of the challenges in developing PDX models—the development of xenogeneic graft versus host disease in recipient mice and the proliferation of Epstein–Barr virus (EBV)-infected cells rather than tumour cells—and provide strategies for obviating these hurdles.Establishment of neuroblastoma PDX models from primary tumour samples was first reported over 25 years agoFresh tumour specimens were obtained from patients at the Sydney Children’s Hospital Network (SCHN) under approval by the SCHN Human Research Ethics Committee . The primary purpose for developing the PDX models was to establish and improve xenografting approaches, to develop models to enable future preclinical testing, including in the context of personalised medicine. The relapse samples were from patients enrolled in a pilot feasibility study (TARGET) for a subsequent personalised medicine clinical trial , however without intention to return data to the treating clinician. Informed parental or guardian consent was obtained for each patient. The initial diagnosis of neuroblastoma was confirmed for each patient by a consultant paediatric pathologist. Samples were collected and transported in L-15 medium  at room temperature within 2–8 h after surgery. Solid tumour sample dissociation was performed enzymatically using a human tumour dissociation kit and a GentleMACS dissociator  into a single-cell suspension. Smaller samples were engrafted as 2–5-mm fragments. Bone marrow samples were collected in EDTA tubes and mononuclear cells isolated by Ficoll separation. Depletion of T lymphocytes was performed with human CD3 microbeads and an autoMACS Pro (Miltenyi Biotec) prior to assessment by flow cytometry and the CD3-negative population used for inoculation. Cells from pleural fluid were isolated by centrifugation and washed with red blood cell lysis buffer prior to inoculation. Cells from cytogenetics culture were obtained after completion of diagnostic analyses, harvested in EDTA-trypsin and washed in PBS prior to inoculation.2 with 2–6 mice/cage, and under 12:12 light/dark cycles. Bedding, enviro dry and igloos were provided for environmental enrichment. Irradiated rat and mouse breeder cubes and water were provided ad libitum. All animal procedures were performed inside a biosafety cabinet under sterile conditions during the animal’s light time cycle. Dissociated tumour cells, bone marrow, pleural fluid and cytogenetic cells (0.1–10 million cells) were mixed in RPMI (Life Technologies) and growth factor-reduced Matrigel  at equal volume ratio and implanted subcutaneously using a 27-gauge needle  into the flank of female NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice29 obtained from Australian Bioresources . For implantation of tumour fragments, mice were anaesthetised using a Stinger anaesthetic machine  with 2–2.5% isoflurane  and 800 mL/min oxygen. A 5-mm horizontal incision was made on the dorsum of the mice, 10 mm rostral to the base of the tail, to create a subcutaneous pocket. A 2–5-mm fragment of tumour dipped in growth factor-reduced Matrigel was inserted and the incision site was closed with a wound clip. Buprenorphine   was administered for analgesia. Tumour growth was measured by Vernier callipers at least twice a week. Mice were euthanised once tumour size reached 1000 mm3, calculated using the formula: Length × Width × Height/2, by CO2 overdose followed by cervical dislocation. Tumours were triaged for histological and molecular analyses, cryopreservation and expansion in secondary recipient mice. Failure to engraft was defined as no measurable tumour at maximum holding time (12 months) and was confirmed by necropsy.All animal studies were performed in accordance with the guidelines approved by the University of New South Wales Animal Care and Ethics Committee (ACEC 17/101B) and the requirements of the Australian Code for the Care and Use of Animals for Scientific Purposes. Animals were housed in a specific pathogen-free environment in Techniplast research animal cages with filter tops and floor area of 400 cm5, 1 × 105 and 1.35 × 105 cells per mouse for A6580, A6912 and A7560, respectively). Subcutaneous inoculation, as well as anaesthesia and analgesia for intramuscular and orthotopic engraftment were performed as described above. For intramuscular engraftment, a 10-mm midline dorsal skin incision was made, 15 mm rostral to the base of the tail.30 Cells suspended in a mixture of 10 μL of RPMI and 10 μL of Matrigel were injected into the paraspinal muscle and the incision closed with 9-mm wound clips . Orthotopic  engraftment was performed as previously described.31 Briefly, a 15-mm skin incision was made adjacent to the caudal border of the spleen and the superior tip of the left kidney, followed by a 10-mm incision into the abdominal wall to expose the left adrenal gland. Cells suspended in a mixture of 10 μL of RPMI and 10 μL of Matrigel were injected into the fat pad surrounding the left adrenal gland. The abdominal wall was closed with a polyglactin-coated Vilet absorbable suture  and the skin incision was closed with wound clips. Mice were monitored daily for 7 days post surgery, and wound clips removed on day 7 post surgery. Intramuscular engraftment was assessed as described above. Orthotopic tumours were assessed by weekly ultrasound imaging and with callipers upon killing to confirm imaging-based measurement. Mice were euthanised by CO2 overdose followed by cervical dislocation. Event-free survival (EFS) was determined as described previously.32For each of three patients, a single tumour sample was dissociated and inoculated simultaneously into randomised cohorts of NSG mice at subcutaneous, intramuscular or orthotopic sites. A fixed cell number per mouse was used for each inoculation based on sample availability (5 × 102) adjacent to the left adrenal gland and prewarmed Aquasonic Clear Ultrasound Transmission Gel  was applied to the area. Imaging was performed using VisualSonics Vevo 2100 . A linear-array transducer (MS-250) traversed the selected area, acquiring 2D images at ~100-μm intervals, which were reconstructed as a 3D image in a dynamic cube format and analysed using Vevo Lab 1.7.0 software . Tumour boundaries were manually outlined in parallel slices within the 3D image and the final tumour volume calculated as the sum of the outlined tumour boundary for each slice.Mice were depilated . Stains included haematoxylin and eosin (H&E), Mason trichrome, orcein elastic and Papanicolaou and Giemsa. Immunohistochemical (IHC) staining included NB84 , synaptophysin , CD56 , PHOX2B , CD45 , CD20  and CD3 . For EBV-encoded RNA in situ hybridisation (EBER-ISH), slides were stained using BOND EBER probe (Leica). Images were captured using an Olympus BX53 light microscope and CD73 camera with CellSens software.TMgDNA Tissue Miniprep System  or Allprep DNA/RNA/Protein Mini Kit . For copy-number analysis using single-nucleotide polymorphism (SNP) arrays, DNA samples were processed using the Illumina InfiniumOmni2.5–8 according to the manufacturer’s guidelines to interrogate ~2.5 M SNP loci . Standard clustering and genotyping were performed using GenomeStudio v2.0.3  using default settings to collect SNP calls. Regions of copy-number variations (CNV) were called using cnvPartition2.0 with default parameters. B-allele frequency (BAF), log R ratio (LRR) and CNV plots were generated using package karyoploteR within the R statistical software package. CNV was determined as a function of BAF and LRR as previously described.33 Patient CNV data were also validated against the clinical data obtained through routine diagnostic DNA microarrays (Affymetrix Cytoscan 750K). Short-tandem repeat (STR) profiling results were analysed using PowerPlexR 18D System at the Kinghorn Centre for Clinical Genomics .DNA was extracted from patient and PDX tumours using the ReliaPrepInfiltration of human lymphocytes into mouse liver and spleen was determined by staining with APC mouse anti-human CD45 and FITC rat anti-mouse CD45. T-lymphocyte-depleted samples were analysed using PE mouse anti-human CD3 and APC mouse anti-human CD45. B lymphocytes in PDX tumours were stained with PE mouse anti-human CD19. For each sample, red blood cells were lysed by BD FACS lysing solution. PE mouse anti-human GD2 staining was used to determine the human neuroblastoma cell content in PDX tumours. All reagents were from BD Biosciences . Data were acquired with the LSRFortessa (BD Biosciences) and analysed with FlowJo software.Dissociated patient and PDX-tumour cells were seeded on 384-well plates at 2000 cells/well in 25 μL of Iscove’s Modified Dulbecco’s Media, 20% foetal calf serum and 1× insulin–transferrin–selenium (Life Technologies). Human neuroblastoma cell content in the PDX tumour was determined by flow cytometry. After 72 h, cells were treated with a library of 165 anticancer drugs. After a further 72 h, Cell-titer glo 2D (Promega) was added to measure ATP as a surrogate for cell viability. Cell viability as a function of drug concentration was plotted with a three-parameter sigmoidal dose–response curve to determine area under the curve (AUC) values. Correlation of drug sensitivity  in patient tumour cells compared with that for PDX-tumour cells was assessed using Pearson’s correlation analysis.3 tumour was 107 days (range 33–210 days). For the remaining samples, there was no evidence of engraftment after 12 months. The established models represented a wide patient age range at the time of diagnosis (0.3–7.5 years) and were derived from both MYCN-amplified4 and non-amplified6 samples. Engraftment at diagnosis was associated with poor outcome  and patients at the point of relapse from either Sydney Children’s Hospital Randwick or Children’s Hospital Westmead (five patients) Table . For twoEach of the established PDX models was authenticated as being derived from the donor tumour specimen by STR profiling. In each case, the match was 100% across the 18 amplified loci. Morphologically, all ten established subcutaneous xenografts recapitulated the features of the patient tumours from which they were derived (Supplementary Table r = 0.84–0.99) than for diagnosis models (r = 0.035–0.82). The PDX models generally conserved the established prognostic molecular markers and recurrent aberrations observed in the original tumour, including chromosome 1p loss and 17q gain, MYCN and ALK amplification and loss of PHOX2B, TP53, NF1 and ATRX , despite confirmation of sample identity by STR profiling (100% match) and histologic features that were consistent with high-risk neuroblastoma. Interrogation of a previous SNP array (Affymetrix Cytoscan 750K array) conducted on this patient sample for clinical care revealed that MYCN amplification and the major chromosomal losses  and gains , were all present in a tumour population that comprised 50% of cells  with even fewer differences in the PDX from cytogenetics culture (r = 0.95)  and the second from cytogenetics culture of the fresh tumour material (CCI-NB06-RMC). Compared with the original patient tumour, minor differences of chromosome 1p loss and 18q gain were observed in the PDX derived from fresh tumour material (95) Fig. . The higDespite the high success rate for subcutaneous engraftment of relapse samples, the success rate for diagnosis samples was below 50%, and the relatively long engraftment time poses a barrier to the use of these models in personalised oncology. To determine whether engraftment at alternative anatomical sites could increase engraftment efficiency, tumour cells from three neuroblastoma patients  were simultaneously inoculated into three cohorts of mice, subcutaneously, intramuscularly or orthotopically , and monitored for engraftment and tumour growth. In each case, the tumour sample was already known to engraft (Table 3 tumour) after 230 days. Intramuscular engraftment was faster than subcutaneous engraftment in one model (A6580), but slower in another (A6912). To exclude the possibility of bias based on different tumour volume measurement approaches, tumour volumes for all orthotopic xenografts were also obtained at endpoint by using callipers and compared with ultrasound analysis. The two measurement approaches yielded equivalent tumour volumes  led to alopecia and reddening of skin around the ears and eyes in the recipient mice, consistent with xenogeneic graft versus host disease (GvHD). Postmortem histopathological analysis of the liver from mice inoculated with each sample was also consistent with GvHD Fig. . Mild efFlow cytometric analysis of mononuclear cells isolated from liver and spleen of the mice indicated 55% and 36% CD45-positive human leucocytes in the A7167 mouse liver and spleen, respectively, and 95% CD45-positive human leucocytes in the A5723 mouse liver Fig. . To precr = 0.71).An attempt to generate a xenograft model from a pleural fluid specimen (patient A7012) containing both metastatic neuroblastoma cells and inflammatory cells, including small lymphocytes and neutrophils Fig. , producer = 0.9018, P < 0.0001, Fig. To further assess the biological similarly of primary patient cells and those expanded via mouse engraftment, a high-throughput screen of 165 anticancer drugs was performed on dissociated cells from both the A6580 patient sample and the first passage of the cognate PDX (CCI-NB06-RMT) under identical conditions Fig. . Prior t27 These models are predictive of clinical outcomes and therefore are an excellent platform for preclinical drug testing and as avatars for personalised medicine.11 This study extends previous neuroblastoma PDX model establishment reports28 by demonstrating that PDX models can also be developed from diagnostic cytogenetic culture without additional loss of fidelity, that more rapid engraftment can be achieved with orthoptic implantation and that additional sample processing can circumvent loss of models by xenogeneic GvHD.PDX models are known to preserve many of the key biological properties of the tumours from which they were derived and remain stable across passages.26 in which neuroblastoma samples were engrafted para-adrenally with a success rate of 24% at diagnosis (9 of 39) and 33% at relapse (1 of 3). Several factors might contribute to the variation of engraftment rate between these studies, including the source of patient material, time to engraftment, number of cells inoculated and engraftment technique. In an earlier study,21 the subcutaneous engraftment rate of PDX from neuroblastoma patients at the time of diagnosis was 34% (20 of 58) for all patients attempted, and 45% (15 of 33) from INSS stage IV disease, which is similar to the current study. We found that histologic and molecular features are generally preserved in high-risk neuroblastoma PDX models commensurate with previous reports.28 The high success rate for samples from cytogenetics culture by comparison with samples from other patient sources that failed to engraft, strongly suggests that these materials should be accessed for model development where possible. While one PDX model (CCI-NB04-DML from A7167) developed from a lymph node metastasis via cytogenetics cells showed very poor correlation with the originating tumour material based on the CNV profile, further analysis of a clinical SNP array for these samples suggests clonal selection in the PDX, and good concordance with the expanded clone in the original patient material. More generally, evolution and copy-number alteration that occurs during development of PDX models might provide explanation for other differences.35Our subcutaneous engraftment success rates of 44% (4 of 9) for diagnosis patients and 100% (5 of 5) for relapse patients compare favourably with a recent reportscid mutant strains and do not develop spontaneous thymoma,36 which are important considerations when engraftment time is long, as is frequently the case for neuroblastoma models. While the absence of functional NK cells renders NSG mice highly susceptible to xenogeneic GvHD development,37 we show that this can be avoided by T-lymphocyte depletion of the patient sample.With further optimisation, other potential samples, such as pleural fluid and lymph node metastasis biopsy, can be used as valuable resources for PDX development, increasing engraftment success rate per patient, an essential advance for implementing personalised treatment in this childhood malignancy. Avoiding the loss of engrafted samples due to mouse pathologies is also critical for reliable model development and judicious choice of recipient mice and sample-processing approaches is essential. Highly immune-deficient NSG mice are advantageous for tumour engraftment, have significantly longer life spans than other 43 and the incidence rate in NSG mice has been reported to be as high as 32%.41 In healthy individuals, EBV infection is benign with reactivation and subsequent proliferation of EBV-infected lymphocytes suppressed by immune detection and response,44 whereas in an immunodeficient patient, EBV can transform B cells into the growth phase and trigger the lymphoproliferative disease.45 Similarly, the lack of immunosurveillance in recipient mice allows the proliferation of the EBV-infected lymphocytes contained in the patient.42 PCR-based detection of the EBV BamHI W region, a major internal repeat in EBV genome, in the patient specimen to be engrafted, has been reported to correlate with EBV-related lymphoid proliferation in the derivative PDX and may allow identification of samples requiring lymphocyte depletion.44 Alternatively, atypical lymphoid proliferations might be obviated by administration of rituximab, an anti-CD20 antibody, as the rate of lymphoma incidence in ovarian cancer PDX models has been reported to decline from 11.1 to 1.88% after the addition of rituximab.46 Aside from prevention, a robust approach to identify atypical lymphoid proliferation is essential to maintaining xenograft validity and mandatory prior to additional clinical assays, such as drug screening, being performed on the xenograft material.Development of EBV-associated atypical B-lymphoma proliferation or EBV-related B-cell lymphoma has been previously described after attempted xenografting of other human tumour types in immune-deficient mice30 or a greater capacity for angiogenesis.31 While enabling a shorter engraftment time, orthotopic inoculation is more technically challenging than subcutaneous inoculation and animal monitoring typically requires in vivo imaging,48 which can be particularly labour intensive for the frequent monitoring required in preclinical testing.48 Our observations that orthotopic, intramuscular and subcutaneous xenografts are equally representative of the donor tumour suggest that engraftment approaches can be selected based on utility.A major challenge for the use of high-risk neuroblastoma PDX models in personalised medicine is the development, validation and application of models in a timeframe that can still inform individual patient treatment. Approaches that shorten engraftment time or increase engraftment success rates without compromising model fidelity are essential to make this approach broadly applicable. Our observation that for each of three patient samples, orthotopic adrenal engraftment was substantially faster than either subcutaneous or intramuscular engraftment suggests one approach to reducing engraftment time. The reasons for more rapid adrenal engraftment are not clear; however, the adrenal gland is the most common site of origin for primary neuroblastoma. Alternatively, previous studies have suggested that non-subcutaneous sites may provide greater protection of the engrafted cells against hypothermic insult,49 This study demonstrated that PDX-derived cells were dependable alternatives for screening high-risk neuroblastoma, with ex vivo drug sensitivity of primary patient cells and PDX-expanded material being highly correlated.In addition to in vivo preclinical testing, limited patient material expanded as a PDX model can be used as a primary short-term culture for high-throughput ex vivo testing of drug sensitivity, including for combination chemotherapy.In summary, we identify dependable engraftment approaches that increase the likelihood of developing informative preclinical models for individual high-risk neuroblastoma patients within a clinically relevant timeframe and reliably expand limited patient material for ex vivo and in vivo drug testing.Supplementary materials


Bixa orellana, commonly known as “achiote” and “annatto” is a plant used for both human and animal foods and was thus identified for full chloroplast sequencing for the Center for Veterinary Medicine (CVM) Complete Chloroplast Animal Feed database. This work was conducted in collaboration with the Instituto de Medicina Tradicional (IMET) in Iquitos, Peru. There is a wide range of color variation in pods of Bixa orellana for which genetic loci that distinguish phenotypes have not yet been identified. Here we apply whole chloroplast genome sequencing of “red” and “yellow” individuals of Bixa orellana to provide high quality reference genomes to support kmer database development for use identifying this plant from complex mixtures using shotgun data. Additionally, we describe chloroplast gene content, synteny and phylogeny, and identify an indel and snp that may be associated with seed pod color.Full chloroplast genomes provide high resolution taxonomic discrimination between closely related plant species and are quickly replacing single and multi-locus barcoding regions as reference materials of choice for DNA based taxonomic annotation of plants. Bixa orellana accessions . Synteny and gene content was identical to the only other previously reported full chloroplast genome of Bixa orellana (NC_041550). We observed a 17 base pair deletion at position 58,399–58,415 in both accessions, relative to NC_041550 and a 6 bp deletion at position 75,531–75,526 and a snp at position 86,493 in red Bixa orellana.Fully assembled chloroplast genomes were produced for both red and yellow Bixa orellana to support kmer based identity markers for use with shotgun sequencing approaches for rapid, precise identification of Bixa orellana from complex mixtures. Kmer based phylogeny of full chloroplast genomes supports monophylly of Bixaceae consistent with alignment based approaches. A potentially discriminatory indel and snp were identified that may be correlated with the red phenotype.Our data provide high quality reference genomes of individuals of red and yellow  Bixa orellana, also known as “annatto” (English), ‘achiote’, ‘achiote amarrillo’, ‘achote’ (Spanish) ‘rocou’ (French), ‘urucu’(Brazilian Portuguese), ‘orlean’ (German), and Mzingifuri (Swahili)) . To. ToBixa Bixa orellana individuals with the only other existing published Bixa orellana full chloroplast accession, comparing gene count, organization and synteny. A phylogeny based on alignment free kmer distance metrics is compared to phylogenies using traditional alignment based methods with barcoding regions matK and rbcL [Bixa orellana (or any other plant) in modernized Next Generation Sequencing (NGS) approaches to identification and authentication of ingredients in complex mixtures such as natural products and animal feeds.Here, we compare the assembled chloroplast genomes of ‘red’ and ‘yellow’ and rbcL  and a fiBixa orellana accessions  by the Inverted Repeat in Bixa, as well as gain of rpl22, trnG-GCC in the LSC, and ycf15 in the Inverted Repeat. Bixa did not have the transfer-rna trnG-UCC, which was present in Theobroma, resulting in a net gain of 4 genes in Bixa relative to Theobroma cacao. The overall synteny of Theobroma and Bixa was continuous, and contrasted to Heritiera fomes, exhibited a much smaller inverted repeat  with an accompanying decrease in gene number (129 and 125 for Bixa and Theobroma versus 130 for Heritiera) due to duplication of genes in single copy portions of the genome. The representative Diperocarpaceae genome was more similar in gene structure and content to Bixa and Theobroma than was the Heritiera, with a similar size of the Inverted Repeat  gene number (130) and relative number of CDS, tRNA and rRNA   which places Thymelaeaceae basal to both Dipterocarpaceae and Bixaceae.The use of alignment free kmer distance metrics allows for the comparison of all DNA sequence data among distantly related chloroplast genomes, not just among genes which can be easily compared through sequence alignment. To demonstrate that organization using a kmer approach is consistent with traditional alignment based approaches, we contrast kmer phylograms with those of barcoding regions matK and rbcL and a 5 gene MLST. With the kmer approach, we were able to make direct comparisons among the full genomes of relatively divergent species  Fig. . The D2 Bixa chloroplast genomes with all the chloroplast genomes for Malvales found within the NCBI Refseq repository . We evaluated the accuracy of using whole chloroplast genomes by preparing three separate databases where all combinations of two Bixa chloroplasts genomes were included, and then used the whole genome shotgun/skim data from our two samples (SRR10320715 and SRR10320716) as well as data from available SRA accessions (SRR7941588 - SRR7941591) as input to test if the kmer software would correctly and unambiguously identify the raw data as that of Bixa orellana. The use of three databases allows us to avoid the circularity of testing the same data used in chloroplast assembly against its own assembled chloroplast genome (referred to as a ‘take-one-out’ strategy). We observed a 100% accuracy in assigning Bixa sequence data to Bixa reference chloroplast genomes  do not perform as well as full chloroplast genomes for closely related species , 6. Work species , 12. ThuBixa orellana from plant mixtures. The full genomes of Bixa orellana were sequenced, kmers were developed, and mixtures were queried against kmer formatted reference genome databases. Bixa orellana was consistently identified even when present at very small percentages of the total mixture with the lowest limit to exhibit reliable diagnostic capacity being 0.3%  has also been described [Bixa orellana is the part of the plant where bixin (the monomethyl ester of a dicarboxylic carotenoid (C25H30O4)), and norbixin ((C24H26O4), the saponified form of the same carotenoid) are produced [and rice . The ‘prand rice  and in pAmericas –17 and eescribed . The seed C25H30O), and nocis-bixin (~ 80%) with smaller amounts of other carotenoids. Cis-bixin is partially converted into the more stable trans isomer (which has the bright red color) upon heating which also produces a yellow byproduct. The quantity of trans isomer and yellow byproduct (red: yellow) balance is primarily influenced by temperature and duration of the heating processes [Annatto carotenoids are known as ‘E160(b)’ in the European Union (EU) and use of annatto is permitted in a variety of food products. In the United States, while there has been limited reporting of potential allergens , annattorocesses .Bixa orellana in food and textile industries continues to grow in popularity as the use of similar red and yellow colors provided by synthetic dyes  are linked with environmental toxicity [Use of natural color from toxicity –26. The toxicity . These dtoxicity –26, 28. Bixa orellana (NC_041550.1). Complete synteny was maintained, and the size and gene content of the inverted repeats was the same in all three accessions  can provide rapid and precise identification of Bixa orellana from mixtures of species for use with modernized Next Generation Sequencing (NGS) approaches to describe complex animal feeds and plant based food products with high accuracy.The structure of the two chloroplast genomes presented here was effectively identical to that of the previously published chloroplast genome of ana NC_04550.1. CoBixa orellana accessions in the display/research garden of the Instituto de Medicina Tradicional de ES SALUD (IMET),  and the germplasm collection of Bixa orellana in the Facultad de Agronomía - Universidad Nacional de la Amazonía Peruana (UNAP). DNA extraction from leaves was performed using DNeasy Plant Mini Kit from Qiagen® according to the manufacturers specifications. DNA was prepared for sequencing using the Nextera™ DNA Flex Library Prep Kit according to the manufacturer’s specifications. Libraries were loaded onto an Illumina MiSeq using a V2 cartridge with read lengths set for (2 × 250).Individuals were collected from Following DNA sequencing, data were trimmed to remove adapters and indexes using BCL2fastq. Sequences were then paired and quality trimmed using Usearch11 . QualityBixa orellana (NC_041550) to enrich reads for chloroplast specific sequences using Geneious R11 (https://www.geneious.com) Mapped reads were then denovo assembled using SPAdes [Paired, filtered reads were mapped to g SPAdes , with kmg SPAdes  in a refana NC_0450 to enrhttp://verdant.iplantcollaborative.org/plastidDB/). The existing reference for Bixa, NC_041550, was added to the Verdant database prior to upload and analysis of our sequences. The resulting gff files were downloaded and used to annotate the assembled Bixa chloroplast genomes , Vatica (Dipterocarpaceae) and Daphne (Thymelaeceae)). Genes were classified based on the type of gene  as well as their organization within the chromosome (Large Single Copy (LSC), Inverted Repeat (IR), Small Single Copy (SSC)). The order of all genes was established and changes in the structural order of genes along the chromosome for all 5 chloroplasts was compared. Gain or loss of genes was denoted, and movement of genes among subunits of the genome  based on expansion of the Inverted Repeat was recorded.Sequences were annotated with the Verdant online chloroplast assembly tool   and a 5 gene MLST schema comprised of rpoC1 (1–3116) rbcL (3117–4546) matK (4547–6154).Representative Chloroplast genome sequences for Malvales were downloaded from the GenBank RefSeq library for use in evaluation of the monophylly of the analysis  kmer disanalysis . The resanalysis  where AratpB 6155–7667) accD  to generate additional phylogenic trees based on universal alignment. NJ distance trees were computed and phylograms for all three trees were visualized in Fig Tree v1.4.4. To share distance values, phylogenetic trees are shared in Supplementary Materials Fig. 55–7667 aGenomes Oryza sativa (DRR228665), Zea mays (SRR10812881), Gossypium hirsutum (SRR10081030), Pseudomonas aeruginosa (DRR215956), Salmonella enterica (SRR11851888), and Escherichia coli (SRR11851885) were downloaded from NCBI. Bacteria were added as a “contaminant” to test potential interference). All sequences were trimmed to 125 kb, and then combined in the following proportions; Oryza sativa (DRR228665) 30%, Zea mays (SRR10812881) 30%, Gossypium hirsutum (SRR10081030) 30%, Pseudomonas aeruginosa (DRR215956) 3.3%, Salmonella enterica (SRR11851888)3.3%, and Escherichia coli (SRR11851885) 3.3%. Mixed data was subsequently combined with different proportions of Bixa orellana plastome data and analyzed using Genome2-ID portal, a metagenomic software for plant taxonomic classification using kmers . To evaluate limit of detection, random selection of Bixa at 5, 2.5, 1.25, 0.6, 0.3, 0.15, 0.07, 0.03, 0.01%, were added to the mixture. Genome2-ID algorithms were run on mixed “dilutions” to identify, based on this approach, the point at which there was until no longer statistical support for identification of Bixa from the mixed sample.Additional file 1: Table S1. Accession numbers, Family, Genus and species of all chloroplasts used for phylogenetic analyses.Additional file 2: Table S2. Genes and gene synteny across Malvales.Additional file 3: Table S3. Phylip formatted D2 distance matrix from kmer distances.Additional file 4: Supplementary Fig. 1. Phylogenies of Bixaceae and Malvales species using A) Kmer distance based and B) 5 gene MLST universal alignment approaches.


Whether FEOBV PET can be used to assess cholinergic activity in the heart is not known. Hence, this study aimed to evaluate the properties of FEOBV for cardiac PET imaging and cholinergic activity mapping. PET data were collected for 40 minutes after injection of 230 ± 50 MBq of FEOBV in four healthy participants . Dynamic LV time activity curves were fitted with Logan graphical, 1-tissue compartment, and 2-tissue compartment models, yielding similar distribution volume estimates for each participant. Our initial data show that FEOBV PET has favorable tracer kinetics for quantification of cholinergic activity and is a promising new method for assessing parasympathetic function in the heart.In contrast to cardiac sympathetic activity which can be assessed with established PET tracers, there are currently no suitable radioligands to measure cardiac parasympathetic (cholinergic) activity. A radioligand able to measure cardiac cholinergic activity would be an invaluable clinical and research tool since cholinergic dysfunction has been associated with a wide array of pathologies . [ Sympathetic and parasympathetic systems in the heart are critical for adequate cardiac function. While sympathetic innervation of the heart is assessed routinely with positron emission tomography (PET) radiopharmaceuticals mimicking norepinephrine,18F]fluoroethoxybenzovesamicol ([18F]FEOBV) is the most promising radioligand.18F]FEOBV has also been extensively validated in the central nervous systems of animals and humans as a marker of cholinergic innervation density.18F]FEOBV possesses desirable kinetics, high affinity for VAT (19.6 nM), low affinity for sigma-1 receptors and low sigma-1/VAT affinity ratio; is sensitive to physiological and pathological VAT density fluctuations; and releases relatively low quantities of free [18F] fluoride.18F]FEOBV a potentially better cholinergic tracer than non-vesamicol alternatives. The most prominent of which is [11C]Donepezil,18F]FEOBV has negligible affinity for these receptors.18F]FEOBV PET could be used to assess cardiac cholinergic activity is not known. Thus, the objective of this study was to test if [18F]FEOBV could be a viable tracer for cardiac PET imaging and cholinergic activity mapping.Cholinergic neurotransmission is dependent upon vesicular storage of acetylcholine by the vesicular acetylcholine transporter (VAT), thus making an ideal target for radioligand aimed at assessing cholinergic activity. In the brain, the quantity of VAT determines the capacity to release acetylcholine. Vesamicol and several of its derivatives bind preferentially to the VAT and act as a non-competitive inhibitor, resulting in the decrease of stimulated acetylcholine release.for VAT 1.6 nM, lofor VAT 1.6 nM, lo18F]FEOBV was injected into a left antecubital vein as a bolus injection over 1 minute. PET data were collected for 40 minutes (first participant scanned for 30 minutes only) starting at injection using a Siemens Biograph mMR.This investigation was approved by Health Canada and the The Royal Ottawa Health Care Group Research Ethics Board. Data are available from the authors upon request. Four healthy participants  were recruited. Before entering the study, each participant signed a written informed consent form. All participants were non-smokers, did not have any known past or present heart disorders and gave a negative urine drug screen before the PET scan. An average dose of 230 ± 50 MBq of FEOBV PET is a promising radioligand to assess cholinergic activity in the heart and may become a useful non-invasive method to study cardiac parasympathetic function and improve risk assessment and prognosis in cardiac patients with conditions associated with autonomic imbalance or impaired parasympathetic activity.[


Dear Editor,1. While therapies such as high-dose melphalan followed by autologous stem cell transplantation (ASCT) can result in deep and durable responses2, only approximately a quarter of newly diagnosed patients are deemed transplant eligible3. Even with successful initial therapy, relapses occur, and not infrequently the response is of insufficient depth to protect against further amyloid deposits in vital organs. Alternative therapeutic options are required for such patients.Systemic light chain (AL) amyloidosis is a clonal plasma cell disorder characterized by the deposition of immunoglobulin light chain-associated amyloid material in various organs4, with encouraging activity in multiple myeloma (MM), particularly in patients whose clonal plasma cells harbor t and/or overexpress BCL-2 (refs. 6). In preclinical models, venetoclax demonstrates synergy in combination with bortezomib against MM cell lines7. Approximately 50% of patients with AL amyloidosis have t, making venetoclax a suitable agent to treat this rare disorder10. Venetoclax could induce a complete response (CR) in a patient with AL amyloidosis whose disease markers plateaued on bortezomib, cyclophosphamide, and dexamethasone (VCD)11. A case series presented in an abstract form, involved seven patients with relapsed/refractory AL amyloidosis (RRAL) with cardiac involvement who were treated with venetoclax12. Two of four patients who were treated with at least two cycles of venetoclax achieved a CR and had t. We report a larger cohort demonstrating efficacy and safety of venetoclax in RRAL.Venetoclax is an oral, small-molecule B cell lymphoma 2 (BCL-2) inhibitor that induces cellular apoptosis14. Patients with normal light chain levels at time of initiation of venetoclax were considered inevaluable for hematologic response. The patients were risk stratified according to the 2012 Mayo staging system15.We conducted a review of records of all patients with RRAL, treated with venetoclax between January 2017 and May 2019 at Mayo Clinic. Organ involvement and response was assessed according to the Consensus Criterian = 9) were males. The median number of prior lines of therapy was 2 (range 1–4). Previous therapy exposures included proteasome inhibitors (100%), oral alkylators (92%), or high-dose melphalan with autologous stem cell rescue (25%), immunomodulatory drugs (25%) and anti-CD 38 antibodies, daratumumab (33%). Most common organs involved were renal (75%) followed by heart (50%), neurological (25%), and gastrointestinal (17%). The t signature was detected on interphase fluorescence in situ hybridization (FISH) studies of the marrow in 11 of 12 patients. Of these 11 patients with t, BCL2 expression by immunohistochemistry was assessed in 5 patients, all of whom exhibited strong expression. Venetoclax was used alone or in combination with dexamethasone in seven (58%) patients. The remainder received venetoclax in a triplet or quadruplet combination that invariably included bortezomib and dexamethasone. The quadruplets administered to two patients incorporated lenalidomide and cyclophosphamide, respectively, to venetoclax, bortezomib, and dexamethasone backbone.We identified 12 patients who were treated with venetoclax for RRAL amyloidosis, with patient characteristics listed in Table The dose of venetoclax used was 800 mg daily in 7 (58%) and 400 mg daily in 5 (42%) patients Fig. . A dose n = 1), dyspnea (n = 1), failure to respond (n = 1), and attainment of desired response (n = 2). The two patients who had stopped therapy after attaining a response did so at 19 months  and 5 months (hematologic CR), respectively.None of the patients experienced tumor lysis syndrome and five have discontinued therapy. The reasons for the discontinuation included cytopenias . One patient developed an upper respiratory tract infection (URTI) 3 weeks after starting venetoclax that only required supportive management and oral levofloxacin. Another patient developed an URTI a month after starting treatment and also pneumonia nearly a year later, which was uncomplicated and resolved with oral cefuroxime. At last follow-up, two patients have progressed at 4 and 5 months post initiation of venetoclax therapy, respectively, and none have died.Our study shows that venetoclax is a generally well-tolerated and efficacious agent in patients with RRAL amyloidosis. It can effectively induce both hematologic and organ responses as a single agent or in combination with other agents. In our cohort, four patients have received venetoclax therapy for >12 months, of whom three are continuing on the drug, suggesting durability of response and tolerability of the agent. The patient who discontinued therapy did so after achieving a renal response and remains in remission. Notably, our study was almost exclusively in AL amyloidosis patients with presence of t. The single patient in whom the t status was not available did not respond to therapy. Although venetoclax was used as a single agent or in combination with dexamethasone in the majority of our cohort, some patients did receive venetoclax as a component of a triplet or quadruplet.6. However, in an interim analysis of an ongoing phase III study of venetoclax in combination with bortezomib and dexamethasone for relapsed or refractory myeloma , safety concerns were raised as a result of increased number of deaths from both infectious and non-infectious causes in the venetoclax plus bortezomib and dexamethasone arm compared to the control arm of bortezomib and dexamethasone, despite a remarkable improvement in the progression-free survival. Despite these surprising findings, the subset of patients with t MM, interestingly, demonstrated a trend towards improved survival in this trial. Patients with AL amyloidosis often have significant organ involvement (cardiac and kidney) and therefore these safety signals need to be carefully considered as prospective trials using venetoclax in this patient population are conducted (NCT03000660). Notably, in our cohort no deaths have been observed so far. Albeit a case series, with a small sample size, our study suggests high efficacy and good tolerability of venetoclax monotherapy and combination therapy in patients with RRAL amyloidosis who harbor t.Responses were seen in both subsets of patients receiving venetoclax as a single/doublet and those receiving it in other combinations. Given the small sample size and the retrospective nature of the current study, it is not possible to accurately ascertain the impact of single versus combination venetoclax-based therapies. Data on efficacy of venetoclax in early clinical trials (phase I/II) in myeloma were encouraging, with higher response rates seen in patients with t


Despite the utility of neuroimaging in the diagnostic and therapeutic management of patients with acute ischemic stroke (AIS), imaging characteristics in patients with preceding direct oral anticoagulants (DOAC) compared to vitamin K antagonists (VKA) have hardly been described. We aimed to determine presence of large vessel occlusion (LVO), thrombus length, infarction diameter, and occurrence of hemorrhagic transformation in AIS patients with preceding DOAC as compared to VKA therapy.Using a prospectively collected cohort of AIS patients, we performed univariate and multivariable regression analyses regarding imaging outcomes. Additionally, we provide a sensitivity analysis for the subgroup of patients with confirmed therapeutic anticoagulation.N = 75) and VKA (N = 61) therapy, median age 79 (IQR 70–83), 39% female. Presence of any LVO between DOAC and VKA patients , and target LVO for endovascular therapy  was equal with a similar occlusion pattern. DOAC as compared to VKA were associated with a similar rate of target LVO for EVT . The presence of multiple lesions and characteristics of the thrombus were similar in DOAC and VKA patients. Acute ischemic lesion diameter in real world patients was equal in patients taking DOAC as compared to VKA. Lesion diameter in VKA patients , but not DOAC patients was smaller in the setting of confirmed therapeutic VKA. The frequency of radiological hemorrhagic transformation and symptomatic intracranial hemorrhage in OAC patients was low. Sensitivity analysis considering only patients with confirmed therapeutic anticoagulation did not change any of the results.We included AIS in patients with preceding DOAC (Preceding DOAC treatment showed equal rates of LVO and infarct size as compared to VKA in AIS patients. This study adds to the knowledge of imaging findings in AIS patients with preceding anticoagulation. Patients with atrial fibrillation (AF) have a five-fold increased risk of acute ischemic stroke (AIS). Therefore, oral anticoagulation (OAC) is recommended after risk stratification to prevent thromboembolic events . Due to Initial imaging in AIS is crucial to rapidly obtain an accurate diagnosis, select patients for acute recanalization treatments, predict outcome, guide etiological work-up and initiate early secondary prevention –6. NeverHence, we aimed to explore the frequency of any LVO or target LVO for endovascular stroke treatment and other imaging characteristics of AIS in a prospectively collected registry of patients admitted to our comprehensive university stroke center prescribed with OAC. Additionally, we present a sensitivity analysis considering only patients with confirmed therapeutic DOAC and VKA activity on admission.Inclusion criteria for this study were a diagnosis of AIS and preceding OAC prescription at the time point of symptom onset (either DOAC or VKA). We selected a random sample of patients fulfilling the inclusion criteria from our prospectively consecutive local stroke registry from February 2015 to August 2017. We chose random sampling because of lack of capacity to analyze the images of all DOAC and VKA patients of the registry. For this purpose, a random number between 0 and 1 was generated using the excel function = RAND for each patient. By sorting the random number ascendingly, the first 80 patients were included in the study. We analyzed only patients with MRI on admission, which represents the preferred imaging modality in our center (about 60–70%). We excluded patients with transient ischemic attacks, stroke mimics, non-cerebral ischemic events, refusal of further use of biological data and missing information on OAC medication from this analysis. Antiplatelet prescription did not affect group assignment. For the sensitivity analysis, we further defined confirmed therapeutic OAC as specific drug activity > 50 ng/ml in patients taking DOAC –15 and IWe assessed the following information from the registry and in case of missing items in the medical records: demographic variables ), cardiovascular risk factors, clinical parameters , onset type, TOAST-etiology, antithrombotic, antihypertensive and lipid lowering medication before admission, laboratory parameters  and acute recanalization treatment such as intravenous thrombolysis (IVT) and endovascular therapy (EVT).We acquired MRI scans on the day of admission, either on a 1.5 T scanner  or a 3 T system . Our MRI protocol includes axial diffusion-weighted imaging (DWI) with apparent diffusion coefficient (ADC) (5 mm slice thickness), Fluid-attenuated inversion recovery (FLAIR) (5 mm slice thickness), susceptibility-weighted imaging (SWI) (1.6 mm slice thickness), and a time of flight (TOF) angiography (0.5 mm slice thickness). After application of intravenous Gadobutrol  in an antecubital vein with a 5 ml/s injection rate, we acquired a standard dynamic susceptibility contrast (DSC) MRI perfusion  as well as a contrast enhanced T1-weighted sequence (slice thickness 5 mm). Finally, a contrast enhanced magnetic resonance angiography (CE MRA) of the head and neck vessels was acquired after injection of a second bolus of Gadobutrol with a 3 ml/s injection rate. Post-processing of DSC was performed using Olea sphere v.2.3 , using deconvolution method with oscillant singular value decomposition. Follow-up imaging was performed using the same scanners and same MRI protocol.Two raters (EA and SF) assessed the imaging outcomes after a standardized training and blinded to patients’ OAC status. All ratings were controlled by a board-certified neuroradiologist (AH) with more than 10 years’ experience in stroke imaging. The primary outcome was the presence of any LVO defined as occlusion of an artery supplying the brain visible on TOF or contrast enhanced MR-angiography. Secondary outcomes included: target LVO for EVT defined as LVO until or proximal to A1/M2-segment of the anterior circulation or P1-segment of the posterior circulation; presence and length of thrombus as determined by SWI; size of hypoperfusion; degree of DWI infarction core and hypoperfused area mismatch defined as small mismatch (infarction core ≥2/3 of the hypoperfused region), moderate mismatch (infarction core > 1/3 and < 2/3 of the hypoperfused region) and large mismatch (infarction core ≤1/3 of the hypoperfused region) by visual judgment; maximal diameter in mm of the acute ischemic lesion as determined in DWI (axis 1), maximal diameter of a perpendicular axis of the acute ischemic lesion (axis 2). In case of multiple lesions, the largest lesion was analyzed for the primary analysis. Other outcomes included size of ischemia demarcation in FLAIR sequence at 24 h; presence of cerebral microbleeds (CMB) according to established criteria at baseline ; presencWe compared the two OAC groups (DOAC versus VKA), and patients with confirmed and infra-therapeutic OAC using appropriate statistical measures . We determined published predictors of stroke severity as pre-specified covariates and factors for the multivariate analysis avoiding collinearity. For the primary analysis, the association of OAC type (DOAC versus VKA) with any LVO or target LVO for EVT was assessed using binary logistic regression adjusting for the following confounders: age (continuous), sex , admission glucose  per mmol/L increase), arterial hypertension  and atrial fibrillation . For the secondary analysis, the same confounders were included in a multivariable linear regression analysis. Patients with missing data items were excluded from the multivariate analysis. For the sensitivity analysis, the same model was used considering only patients with confirmed therapeutic OAC activity on admission. We used a level of significance of 0.05.The random sample of OAC patients with AIS fulfilling the in- and exclusion criteria included 75 DOAC patients  and 61 VKA patients. Either documented information on compliance or a reliable assessment of DOAC activity was available in 45/75 (60%) of patients taking DOAC. INR was available in all patients taking VKA. OAC activity was confirmed therapeutic in 35/75 (46.7%) of DOAC patients and 45/61 (73.8%) of VKA patients. Table P = 0.361) on univariate analysis. There was also no difference in target LVO for EVT between DOAC and VKA patients , but the size of the initial hypoperfusion was significantly larger in DOAC patients . The occlusion pattern was similar in patients with DOAC and VKA  of DOAC patients  and 57% of VKA patients .P = 0.607, Table P = 0.705) as compared to DOAC. Both analyses remained unchanged when only considering patients with confirmed therapeutic OAC activity. There was no difference in the maximal diameter of the perpendicular axis of the acute ischemic lesion. In VKA patients, acute DWI lesion diameter was smaller in the case of confirmed therapeutic OAC activity. For DOAC patients, this effect could not be seen  was equal to VKA  on univariate analysis (P = 0.409). The rate of multiple lesions was equal between groups.Size of ischemia lesion diameter at 24 h (FLAIR sequence) was equal in DOAC patients  and VKA patients  in the DOAC group (Heidelberg class 3) and one patient (1.6%) in the VKA group (Heidelberg class 2) suffered symptomatic intracranial hemorrhage at 24 h. 5/75 (6.7%) of DOAC patients and 10/61 (11.5%) of VKA patients showed asymptomatic hemorrhagic transformation of the ischemic lesion at 24 h.The comparison of MRI findings in patients with AIS and preceding DOAC versus VKA prescription in our real world university dataset shows the following main findings:P = 0.361), and target LVO for endovascular therapy  were equal with a similar occlusion pattern. (2) Also, the presence of multiple lesions and thrombus characteristics were similar in DOAC and VKA patients. (3) Ischemic lesion diameter in real world patients is equal in patients taking DOAC and VKA and this finding holds true in case of confirmed therapeutic OAC activity. (4) Lesion size in VKA patients was smaller in the setting of confirmed therapeutic VKA. (5) The frequency of radiological hemorrhagic transformation and symptomatic intracranial hemorrhage in OAC patients was low.(1) Between DOAC and VKA patients, the frequencies of any LVO  . FurtherThe low rate of radiological as well as clinically symptomatic intracranial hemorrhage is reassuring; however recanalization strategies were very infrequently used in this cohort.The data was collected prospectively, but our study has the inherent limitations of a single-center retrospective analysis. We reliably differentiated between therapeutic and non-therapeutic OAC in patients prescribed VKA and DOAC. For assessment of lesion size, we only measured the diameter and did not use three-dimensional segmentation tools, which does not accurately reflect true ischemic lesion size. Nevertheless, the correlation between maximal diameter and volume seems acceptable  and our In conclusion, we found that about 1 in 4 patients with preceding OAC has a target LVO for EVT. The ischemic lesion size was equal in patients taking DOAC and VKA, also in the setting of confirmed therapeutic OAC activity. Since imaging features are similar, the mechanisms associated with AIS despite preceding DOAC or VKA treatment seem alike.


While atomic scale structural and dynamic information are hallmarks of nuclear magnetic resonance (NMR) methodologies, sensitivity is a fundamental limitation in NMR studies. Fully exploiting NMR capabilities to study membrane proteins is further hampered by their dilution within biological membranes. Recent developments in dynamic nuclear polarization (DNP), which can transfer the relatively high polarization of unpaired electrons to nuclear spins, show promise for overcoming the sensitivity bottleneck and enabling NMR characterization of membrane proteins under native-like conditions. Here we discuss fundamental aspects of DNP-enhanced solid-state NMR spectroscopy, experimental details relevant to the study of lipid assemblies and incorporated proteins, and sensitivity gains which can be realized in biomembrane-based samples. We also present unique insights which can be gained from DNP measurements and prospects for further development of the technique for elucidating structures and orientations of membrane proteins in native lipid environments. Solid-state NMR spectroscopy (ssNMR) is distinctly capable of determining membrane protein structure and dynamics at high resolution within native lipid environments ,2,3,4; iTo increase signal strength, dynamic nuclear polarization (DNP) can transfer the relatively high polarization of unpaired electrons to nuclear spins A, theore17O  L-Leucine  was purchased and modified for Fmoc solid phase peptide synthesis [+] = 2469.83. Pure KL4 was dissolved in methanol to yield a 1 mM monomer peptide solution, with concentration and purity confirmed via analytical RP-HPLC. DPPC, POPG, and DPPC-d62 were purchased as chloroform solutions from Avanti Polar Lipids  and quantified by phosphate analysis . Cholesterol was also purchased from Avanti Polar Lipids as a powder and dissolved in chloroform at 25 mg/mL. Following lipid quantitation, stock lipid mixtures were prepared by combining individual lipid chloroform solutions. KL4 in methanol was added to lipid chloroform mixtures to achieve 2.0 mol% peptide relative to total lipids. Lipid/peptide samples were dried under N2 gas, resuspended in warm cyclohexane (>45 °C), frozen in liquid nitrogen, and lyophilized overnight to remove residual solvent. Lyophilized lipid/peptide powders (~20 mg) were first hydrated in 1.0 mL of 10X Universal Buffer (UB), pH 7.4, made with 90/10 (v/v) 9:1 D2O:H2O, and equilibrated with 15 freeze/thaw cycles. For AMUPol-containing samples, liposomes were subsequently ultracentrifuged to obtain phase separated proteoliposomes and buffer layers. The buffer was carefully removed with a syringe, and 50 v/v) DMSO-d6 was added to the liposome pellet and an additional 15 freeze/thaw cycles were performed. The lipid and DNP matrix suspensions were then vortexed to create a uniform suspension and pelleted into 3.2 mm sapphire NMR rotors and sealed with silicone plugs. The final NMR samples consisted of ~10 mg of proteoliposomes in a 20–25 μL volume.KLynthesis . Crude p4 whereas sample 1 does not contain peptide. For samples containing peptide, we observed a slightly different AMUPol spectrum than for the sample that did not contain peptide. One potential explanation for this difference is that the PEG-modification in AMUPol leads to partial biradical partitioning at the lipid interface, which would decrease AMUPol motion, and that the peptide mediates this partitioning.CW EPR spectra were collected on a Bruker X-Band EMX Nano benchtop spectrometer . DNP sample rotors were placed in 5 mm quartz sample tubes and placed into the cavity with its associated adaptor. Spectra were collected at room temperature (298 K) and at 100 K by using cold nitrogen gas to cool the cavity. Spectra recorded at both RT and 100 K allow assessment of PA concentration and distribution at cryogenic temperatures prior to performing DNP experiments . In our 1H/13C/15N MAS-DNP probe cooled to ~95 K. Microwaves were delivered with a gyrotron source operating at 395 GHz and output optimized for ~12 W at the probe base. Microwaves on/off spectra were collected with a MAS frequency of 10 kHz and a 13C CP-echo pulse sequence with a 1H CP ramp from 40 to 70 kHz, a 13C RF field of 50 kHz, 100 kHz SPINAL-64 1H decoupling and a recycle delay of 5 s. An initial comparison of spectra for MW on vs. MW off, shown in MAS-DNP NMR spectra were collected at 600 MHz (14.1 T) using a Avance III DNP system (Bruker Biospin) with a Bruker 3.2 mm, 4 containing samples show significantly less enhancement of the lipid moieties relative to the sample made without peptide. This again suggests the peptide affects partitioning of the PA within the samples. Nonetheless, enhancements of 8–58 are seen across the different samples and resonances, showing a substantial increase in sensitivity from DNP. Between the two peptide-containing samples we note a difference in enhancements even though they were prepared in parallel using the same level of PA. Differences in overall PA levels were not detected by EPR or as sample bleaching, suggesting the primary cause of differences in enhancement is the distribution of the PA within the MLVs.Signal intensities in microwave on and microwave off spectra, normalized for the number of scans, were used to calculate DNP enhancements  effects observed at ambient temperature indicate preferential localization of AMUPol and TOTAPOL biradicals in the lipid membrane . The gly1n, TB reports on the efficiency of polarization transfer from electrons to nearby protons and subsequent diffusion. It is an indirect probe of biradical distribution within a sample. At 10 mM PA concentration and 600 MHz/395 GHz DNP conditions, typical TB times of ~3–5 s are expected for samples with uniformly-dispersed PA based on measurements made with model samples [B times suggest that the peptide, which is fully protonated and contains many methyl groups, induces T1n relaxation changes which are observed as reduced enhancements at the lipid moieties. This enhanced relaxation plays against the DNP process and prevents observed resonances from reaching high polarization levels, thus leading to lower To gain more insight into the underlying causes of differences in polarization between samples, we also compared DNP build up times . For sim samples . This wa13C T2 relaxation times in the samples with well-dispersed PA  less efficient DNP polarization due to increased longitudinal nuclear relaxation in peptide containing samples, and (b) sample-to-sample variability in equilibrating PA throughout the MLVs. The former source of variability may be overcome with deuteration of peptide residues at methyl positions and the latter can likely be overcome by increasing the number of freeze/thaw cycles standardly used while the former source of variation will be dependent on the lipids and the membrane proteins or peptides in a given sample. The use of 10% DMSO and freeze/thaw equilibration of PA within MLVs is likely to produce superior reproducibility of PA dispersion and sample integrity in general for membrane protein samples compared to previously employed sample preparations utilizing “DNP juice” with 60% glycerol as the DNP matrix and an “add on top” approach where PA dissolved in DNP matrix is added to a membrane protein sample with minimal hydration. This is due to better matching of viscosity between the MLVs and the DNP matrix made with 10% DMSO, less dehydration at the membrane interface by DMSO, and better control of PA concentration within the samples by using a slight excess of matrix with a known PA concentration. Importantly, organic acid-based buffers, which exhibit minimal pH changes with temperature and freezing, should be utilized in sample preparations.Comparison of DNP enhancements for MLV sample preparations containing the surfactant peptide KLEvaluation of DNP enhancements and DNP buildup times can quickly provide valuable assessments of PA distribution within samples and enable optimization of sample conditions for maximal DNP gains. Variability in DNP enhancements and buildup times throughout lipid membranes due to partitioning of water-soluble PA within the aqueous phase or at the membrane interface can provide valuable guideposts for evaluating membrane proteins samples with limited prior information regarding the localization of protein domains relative to the membrane environment.13C spin T2 relaxation times are slightly shortened by addition of PA but spin coherence lifetimes are comparable to those measured by ssNMR at ambient temperatures. This suggests most ssNMR pulse sequences can be employed in MAS-DNP NMR experiments without any detrimental loss of signal in multidimensional NMR experiments. We note that inhomogeneous broadening of resonances for the samples we studied was a factor of 1.5–2-times worse than for similar samples frozen and studied above the protein glass transition via ssNMR spectroscopy.Under DNP conditions, 17O and 43Ca. This will enable fundamental studies of chemical mechanisms within membrane proteins.The added sensitivity of DNP, combined with robust sample preparation methodologies for membrane proteins, will likely lead to routine use of MAS-DNP NMR approaches to garner one to two orders of magnitude improvements in sensitivity relative to conventional ssNMR. This will fundamentally alter the application of NMR spectroscopy in membrane protein structural biology. In particular, with improved PAs , and very low temperature MAS-DNP (He recirculating systems), the improved sensitivity of NMR will open new possibilities. Such technology will indeed extend biomolecular NMR methodologies to nuclei which are typically not observed by ssNMR due to sensitivity limitations, such as


Shallow feed-forward networks are incapable of addressing complex tasks such as natural language processing that require learning of temporal signals. To address these requirements, we need deep neuromorphic architectures with recurrent connections such as deep recurrent neural networks. However, the training of such networks demand very high precision of weights, excellent conductance linearity and low write-noise- not satisfied by current memristive implementations. Inspired from optogenetics, here we report a neuromorphic computing platform comprised of photo-excitable neuristors capable of in-memory computations across 980 addressable states with a high signal-to-noise ratio of 77. The large linear dynamic range, low write noise and selective excitability allows high fidelity opto-electronic transfer of weights with a two-shot write scheme, while electrical in-memory inference provides energy efficiency. This method enables implementing a memristive deep recurrent neural network with twelve trainable layers with more than a million parameters to recognize spoken commands with >90% accuracy. Accomplishing complex cognitive tasks such as speech recognition calls for artificial intelligence hardware with high computing precision. John et al. propose deep recurrent neural networks based on optoelectronic transition metal dichalcogenide memristors with high weight precision for in-memory computing. Despite the impressive progress already demonstrated with conventional CMOS-based programmable architectures3, innovative neuromorphic hardware approaches are required to emulate the scale, connectivity and energy efficiency of biological neural networks5. The first wave of neuromorphic hardware solutions based on electrical memristors have demonstrated advantages in parallel summing and update operations, scalability, cost, and power consumption8. However, the switching dynamics of conventional memristors produces abrupt transitions and write non-linearity, resulting in asymmetric weight updates and limited number of accessible states9. Such limited precision weights can only be used for shallow feed-forward networks trained to classify simple datasets and are incapable of addressing applications like speech recognition and natural language processing (NLP) that require learning of temporal signals. To address these requirements, we therefore need neuromorphic architectures with recurrent connections and deeper architectures (>10 hidden layers) such as deep recurrent neural networks (DRNNs)11. However, the efficient training of DRNNs calls for fully-parallel write/read operations, which demands non-volatile memory elements with multiple linearly distributed conductance states addressable via blind updates- not satisfied by current electrical implementations. A potential way to accelerate the training process of DRNN is to exploit the increased speed offered by optical processing platforms. However, such all-photonics-based computing platforms require meticulous integration of numerous optical components with a large footprint12 and are incapable of updating weights in a precise, linear manner, necessary for training DRNNs via parallel updates13. Present electrical and optical approaches therefore do not sufficiently address the effective bit precision and accuracy required for energy-efficient computation-in-memory implementation of DRNNs.The success of deep learning in diverse fields such as image classification14. Optogenetics- a photo-stimulated neuromodulation technique, utilises optical pulses to monitor and manipulate neuronal activities by controlling ionic currents in biological tissues16. With higher speed and spatiotemporal precision over its electrical counterparts, optogenetics has facilitated precise probing of neuronal circuits14, unravelling underpinnings of cognition and memory. Beyond probing the neural pathways, optogenetics is now extensively employed to stimulate and silence neural activity to manipulate locomotion17 and rewire neural pathways to cure disorders18. This strategy of utilising light to tune the learning and memory behaviours20 can be adopted to selectively activate artificial neurons and synapses in hardware circuitry to address the drawbacks of limited number of accessible states, non-linearity and asymmetric weight updates. Inspired from optogenetics, we propose an optoelectronic neuromorphic computing platform comprising of photo-excitable neuristors (PENs) to selectively control the excitability of artificial neurons and update the weights of synapses with high linearity to obtain precise weights necessary for DRNNs.This motivates us to re-examine neuroscience, specifically those based on optogenetics, for a second wave of neuromorphic devices for advanced AI applications2) active channels -based synapses on atomically-thin low-bandgap PENs with rhenium disulfide  of incident light. This opens up the possibility of accessing linearly distributed weights with high precision utilizing optical blind updates, essential for DRNNs.While conventional optogenetics targets specific neurons for excitation or inhibition, our approach of using PENs gives us the flexibility to selectively activate either neurons or synapses in an artificial neural network Fig. . We firs2 and 20 insensitive indium tungsten oxide (IWO) synapses was assembled as shown in Fig. 25. All synapses were initially programmed to a common low conductance state (5 nS). Upon illumination with red light , the channel conductance of the ReS2 PENs depicted a non-volatile increment (5–6.5 nS) due to its low bandgap (1.5–1.8 eV)26. On the other hand, the channel conductance of IWO synapses remained constant (5 nS) due to their large bandgap (3.6 eV)27. A spatial conductance map readout of the array resembled the image of “N” with the light-sensitive ReS2 PENs spatially arranged to resemble this alphabet as shown in Fig. Spatiotemporally selective perturbation is another key factor in optogenetics, enabling probing of a specific group of neurons with high precision. To demonstrate selective perturbation, a 6 × 6 mini-array of PENs was selectively subjected to sinusoidal global clocks of red and blue wavelengths Fig. . The PEN28. Even for transferring offline learnt weights with high bit precision to a neuromorphic hardware necessary for speech recognition or NLP, blind weight updates without iterative read-write procedures are necessary to avoid prohibitively long write times for large-scale DRNNs29. Update linearity and write noise have been identified as the prime analogue device properties which degrade the accuracy of neural networks30. However, most memristive systems depict a nonlinear and asymmetric response due to their abrupt filamentary switching physics, failing to sufficiently address the bit precision and accuracy required for in-memory computations in DRNNs. We propose to exploit the exquisite write linearity and low write noise afforded by the optical gating to get very accurate weight transfer with a two-shot write scheme.From the algorithmic point of view, an ideal synapse should typically satisfy linear weight updates for high classification accuracies using online blind programming schemesλ = 623 nm, 65 mWcm−2 intensity), the conductance updates exhibited excellent linearity, non-volatility and retention characteristics. Real-time monitoring of the conductance changes revealed a precise stepped linear increase in conductance from 1.25 to 180 nS with a step size of ~0.18 nS, equivalent to 980 distinct conductance states (~10-bit) , while Supplementary Note To demonstrate the extremely high density and linearity of non-volatile states available for analogue computation, the PENs were subjected to an input optical pulse train of constant pulse width and interval. Upon illumination (Gavg) over the entire range of conductance (G) to enable a high dynamic range of weights with extremely accurate weight updates31. A test of write endurance (1600 switching cycles – 800 write and 800 erase steps between the 1st 16 conductance states) revealed high endurance and low write noise in the investigated devices for all wavelengths of optical stimulation  of 39 (potentiation)/30 (depression), 77 (potentiation)/24 (depression) and 75 (potentiation)/53 (depression) for red, green and blue wavelength optical write and their corresponding electrical erase operations  generated in the channel increase the conductance (potentiation) and the photogenerated holes are localised/trapped in states within the semiconductor bulk or/and at the semiconductor-dielectric interface resulting in a delayed recombination. This photogating effect33 results in a permanent change in the channel’s carrier concentration, resulting in a non-volatile weight update. Carriers generated during the subsequent illumination adds to the existing carrier concentration with every input pulse, updating weights in a linear manner. While optical pulses enable linear incremental non-volatile write steps, electrical gating is used to erase via defect-assisted recombination. During erase, the electrical gating raises the Fermi level to induce electron accumulation in the channel and this accelerates the recombination with the holes in the trap sites reducing the photoconductance34 (depression) as shown in Fig. The linear increase/decrease of weights in a material system depend on a number of factors including the nature of traps and its temporal response, the kinetics of photo-carrier generation and their recombination. The programming scheme we adopt caters for many of these factors (Supplementary Note 35) within memory 39 that achieve state of the art results in speech classification, require much more precise weights40. Earlier work42 on memristive devices have shown their ability to classify handwritten digits from the MNIST dataset43 using fully connected networks (FCN) with online learning. However, such online learning schemes using stochastic gradient descent are unsuited for training DRNNs on large-scale datasets; instead sophisticated momentum based learning rules such as ADAM44 are preferred for offline training which enables gradient averaging across mini-batches. Hence, in this work we use offline learning of weights followed by optically-assisted weight transfer to the PEN crossbar which can then perform the inference operation in electrical mode with extremely low energy dissipation  operations  obtained by dividing the linear range of weights  by the standard deviation of write noise  due to the recurrent layers40 Fig. . Programise Fig. . Howeverise Fig. . The lin48  that is almost indistinguishable from its software full precision counterpart (99.2%) and at par with the state-of-the-arters Fig. , where ters Fig. . Withouters Fig. .52, a Vgs that keeps the trap states empty, a Vds that ensures the leakage currents do not interfere with the weight readouts, a low initial background carrier concentration to prevent screening effects and a light intensity sufficient to fill the traps ensures the better linear update of weights in our devices  and subsequent lift-off process. Height profiling with atomic force microscopy (AFM) was conducted to determine the thickness of the sample. Raman characterisation was performed to confirm the purity of the sample.A scotch-tape method was used to exfoliate ReSFor all the optoelectronic characterisations in the manuscript, the PENs were subjected to input optical pulse trains of constant pulse width and interval using an LED light source (ThorLabs SOLIS-445C) equipped with a DC2200 driver. The device conductance was measured using a parameter analyser (Keithley 4200SCS) in sync with the LED light source.Supplementary InformationPeer Review File


Alligator mississippiensis) over two consecutive breeding seasons, we observed that 80% of nests exhibit both male- and female-promoting thermal cues during the thermosensitive period, and of these nests, all exhibited both male- and female-promoting temperatures within the span of a single day. These observations raise a critical question—how are opposing environmental cues integrated into sexually dimorphic transcriptional programs across short temporal scales? To address this question, alligator embryos were exposed to fluctuating temperatures based on nest thermal profiles and sampled over the course of a daily thermal fluctuation. We examined the expression dynamics of upstream genes in the temperature-sensing pathway and find that post-transcriptional alternative splicing and transcript abundance of epigenetic modifier genes JARID2 and KDM6B respond rapidly to thermal fluctuations while transcriptional changes of downstream effector genes, SOX9 and DMRT1, occur on a delayed timescale. Our findings reveal how the basic mechanisms of TSD operate in an ecologically relevant context. We present a hypothetical hierarchical model based on our findings as well as previous studies, in which temperature-sensitive alternative splicing incrementally influences the epigenetic landscape to affect the transcriptional activity of key sex-determining genes.An organism’s ability to integrate transient environmental cues experienced during development into molecular and physiological responses forms the basis for adaptive shifts in phenotypic trajectories. During temperature-dependent sex determination (TSD), thermal cues during discrete periods in development coordinate molecular changes that ultimately dictate sexual fate and contribute to patterns of inter- and intra-sexual variation. How these mechanisms interface with dynamic thermal environments in nature remain largely unknown. By deploying thermal loggers in wild nests of the American alligator ( Embryos often experience dynamic environmental conditions during development which can exert lasting influences on organismal phenotypes. This is especially true for species with temperature-dependent sex determination (TSD) in which the sex of offspring is determined by temperature cues experienced during discrete periods in development. Occurring in a diverse range of taxa, including all crocodilians, many turtles, and some fish , TSD proTrachemys scripta, temperature fluctuations appear to play critical roles in influencing population sex ratios. Mean temperatures of T. scripta nests in the field rarely exceed those necessary to produce females under constant laboratory conditions, yet mark-recapture studies indicate females are frequently recruited into these populations. This apparent discordance is reconciled by the observation that female development can be triggered in T. scripta by as few as 5 days of exposure to female-promoting temperatures , are among the earliest genes displaying temperature-sensitive differential expression during TSD in both T. scripta and Alligator mississippiensis (JARID2 is a component of the master Polycomb repressive complex 2 (PRC2) which plays a key role in localizing the PRC2 complex to its target loci for subsequent transcriptional silencing via histone 3 lysine 27 trimethylation (H3K27me3)  and is associated with increased H3K27me3 at the promoter of DMRT1, a transcription factor involved in male development  . KDM6B eget loci . Intriguelopment . TogetheA. mississippiensis, T. scripta, and Pogona vitticeps, exhibit temperature dependent alternative splicing of the epigenetic modifier genes, JARID2 and KDM6B . Following transport to the Savannah River Ecology Laboratory , a representative embryo was examined to determine the developmental stage of each clutch . Eggs wen = 10), intermediate increasing temperature , maximum temperature , and intermediate decreasing temperature  of the FLUX. Embryos exposed to a constant FPT , and constant MPT  were sampled at the same time points to assess daily variability in gene expression unrelated to temperature and to yield a baseline to which the FLUX group could be compared.At Stage 22, the middle of the TSP, alligator embryos was sampled at four time points spanning a daily thermal fluctuation in the fluctuating temperature treatment FLUX ; . The samJARID2 and KDM6B were selectively targeted for quantification via specially designed primer sets. In particular, we targeted transcripts retaining the same introns reported to be differentially retained in previous studies (11th intron in JARID2 and 19th intron in KDM6B [ACTB) and ribosomal protein L8 (RPL8), respectively, which served as internal controls and were also derived from standard curves. There were no significant effects of temperature, sampling timepoint, or their interaction on the absolute expression levels of either of these housekeeping genes  and resulting concentrations were quantified via spectrophotometry . Synthesis of cDNA was carried out using the iScript cDNA synthesis kit  according to the manufacturer protocol and using 903.4 and 999 ng of input RNA from the gonad and brain, respectively. Gene expression was assessed via quantitative real-time PCR and reactions were performed in triplicate using an SYBR green reaction mix. Intron-retaining transcripts of in KDM6B ). Informng genes .JARID2 , KDM6B , and SOX9. A square-root transformation was applied to values for DMRT1. Transformations were not sufficient to alleviate non-normality for the JARID2 intron–retention ratio in both the gonad and brain. We examined the influence of temperature treatment, sampling timepoint, and their interaction on relative expression of each gene and isoform using linear mixed effects models with clutch identity included as a random effect. These models were fit using the “lme4” package in R. We obtained P-values from the models using likelihood ratio tests. Due to the persistent non-normality of the JARID2 intron–retention ratio following transformation, we also assessed the effect of temperature and sampling timepoint in the gonad and brain by conducting non-parametric Kruskal–Wallis tests. In both cases, results of the Kruskal–Wallis test conformed to those of the linear mixed effects model. To further examine potential relationships among the responses of genes in the gonad to thermal regime, we assessed pairwise Pearson correlations between each of the isoforms of JARID2 and KDM6B, as well as DMRT1 and SOX9.Statistical analyses were conducted using R statistical software version 3.6.1. Distributions of relative expression values for each gene were tested for normality and homoscedasticity using a Shapiro–Wilk test and Levene’s test, respectively. If necessary, a transformation was applied to relative expression values to achieve normality. A log-transformation was applied to values for Thermal profiles of 20 alligator nest cavities were monitored between 2015 and 2016 at the YWC . The overall mean temperature during the TSP for all nests was 32.02°C, a temperature predicted to produce 69.8% males . The cooJARID2 intron-retaining transcripts to all transcripts  = 49.86, P < 0.001; JARID2 intron–retention ratio supported this result . While embryos exposed to the constant FPT exhibited consistently higher intron–retention in JARID2 compared to embryos exposed to the constant MPT, embryos exposed to the FLUX exhibited variable levels of intron–retention. In particular, intron–retention of JARID2 peaked in FLUX embryos at the timepoint in which these embryos were experiencing a FPT (T2), consistent with a rapid response of intron–retention to temperature . The response of JARID2 transcript abundance to temperature in the brain was less clear, but there was a significant interactive effect of temperature treatment and sampling timepoint  but not sampling timepoint or their interaction on the ratio of KDM6B intron-retaining transcripts to all transcripts in the gonad  but not sampling timepoint or their interaction on KDM6B transcript abundance in the gonad (KDM6B transcript abundance appeared highest in the FLUX embryos at the timepoint after embryos received an FPT cue (T3), this variation was not statistically significant. In contrast to JARID2, there was no significant effect of the temperature-by-timepoint interaction on KDM6B transcript abundance  (Levels of intron–retention in he gonad . There whe gonad . While gbundance . In the = 0.025) .SOX9 relative expression . Consistent with its role in promoting testicular differentiation, SOX9 relative expression was higher in embryos incubated at MPT compared to those incubated at FPT  on DMRT1 relative expression in the gonad. Similar to SOX9, relative expression of DMRT1 in FLUX embryos was highest at timepoint T3  and thus occupy upstream positions in the sex-determining transcriptional cascade. Despite extensive research efforts, the thermo-sensory mechanism governing TSD has remained elusive. Several candidates have been hypothesized to initially translate incubation temperature into a biological response during TSD including transient receptor potential channels JARID2 anchannels  though echannels . Based oted here . This, hted here . A recend JARID2 , lendingd JARID2 , proteind JARID2 , and/or d JARID2  during TJARID2. Counterintuitively, the intronic sequence retained in mature JARID2 transcripts contains premature stop codons and intron-retaining transcripts are predicted to produce truncated products with altered or ameliorated function if they avoid nonsense-mediated decay and are translated , this observation is, in part, a result of our sampling scheme and may not reflect the true timescale of this response. In order to better resolve the temporal scale on which alternative splicing, transcription, and translation respond to fluctuating thermal cues during TSD, future experiments must sample embryos across a higher resolution time-series and across multiple days. Furthermore, the thermal regime implemented here represents a simplification of the thermal fluctuations occurring in wild nests. Nests frequently exhibit thermal fluctuations of smaller magnitudes and daily fluctuations often vary over the course of the TSP. It remains unknown how characteristics such as the magnitude and repeatability of fluctuations impact molecular responses to these thermal cues and, ultimately, how these complex thermal profiles shape sex ratio outcomes in species with TSD.While this study revealed intriguing patterns in the responses of upstream epigenetic regulators and downstream effector genes of sex determination to a thermal fluctuation between MPT and FPTs, there are limitations to the current experimental design that leave several important questions unanswered. In particular, while transcript abundance of Many questions regarding the epigenetic mechanisms of TSD await further exploration and studies like the one presented here underscore the importance of implementing thermal regimes that accurately reflect the dynamic environment in which TSD evolved and examining these processes across multiple temporal scales in future experimental investigations of this phenomenon.obaa033_Supplementary_DataClick here for additional data file.