Oropharyngeal squamous cell carcinoma (OPSCC) is often diagnosed at an advanced stage because the disease often causes minimal symptoms other than metastasis to neck lymph nodes. Better tools are required to assist with the early detection of OPSCC. MicroRNAs  are potential biomarkers for early head and neck squamous cell cancer diagnosis, prognosis, recurrence, and presence of metastatic disease. However, there is no widespread agreement on a panel of miRNAs with clinically meaningful utility for head and neck squamous cell cancers. This could be due to variations in the collection, storage, pre-processing, and isolation of RNA, but several reports have indicated that the selection and reproducibility of biomarkers has been widely affected by the methods used for data analysis. The primary analysis issues appear to be model overfitting and the incorrect application of statistical techniques. The purpose of this study was to develop a robust statistical approach to identify a miRNA signature that can distinguish controls and patients with inflammatory disease from patients with human papilloma virus positive (HPV +) OPSCC.Small extracellular vesicles were harvested from the serum of 20 control patients, 20 patients with gastroesophageal reflux disease (GORD), and 40 patients with locally advanced HPV + OPSCC. MicroRNAs were purified, and expression profiled on OpenArray™. A novel cross validation method, using lasso regression, was developed to stabilise selection of miRNAs for inclusion in a prediction model. The method, named StaVarSel (for Stable Variable Selection), was used to derive a diagnostic biomarker signature.A standard cross validation approach was unable to produce a biomarker signature with good cross validated predictive capacity. In contrast, StaVarSel produced a regression model containing 11 miRNA ratios with potential clinical utility. Sample permutations indicated that the estimated cross validated prediction accuracy of the 11-miR-ratio model was not due to chance alone.We developed a novel method, StaVarSel, that was able to identify a panel of miRNAs, present in small extracellular vesicles derived from blood serum, that robustly cross validated as a biomarker for the detection of HPV + OPSCC. This approach could be used to derive diagnostic biomarkers of other head and neck cancers. Head and neck cancer is the 6th most common cancer worldwide, with oropharyngeal squamous cell carcinoma (OPSCC) significantly increasing in incidence . HistoriBiomarkers are biological molecules found in blood, fluid or tissues that can signal either a normal or an abnormal process such as cancer. Serum biomarkers have emerged as potential tools to facilitate diagnosis in patients with head and neck cancer .MicroRNAs  have been identified as potential biomarkers for early head and neck squamous cell carcinoma diagnosis, prognosis, recurrence, and presence of metastatic disease , 6. miRNCirculating miRNAs obtained from blood have been described for head and neck cancer of several anatomical subsites including oral cavity, nasopharynx, larynx, salivary glands and cutaneous malignancies . HoweverSeveral reports have also indicated that the selection and reproducibility of biomarkers has been widely affected by the methods used for data analysis. Michiels et al.  reanalysit is essential to limit the false discovery of biomarkers so that the literature is not burdened with unreproducible findings”.Furthermore, in a critical review of microarray studies in cancer, Dupuy et al.  determinA key approach to improving medical biomarker studies is to validate findings in a separate set of samples. However, this approach alone does not maximise the information that can be derived from valuable samples, and for often necessarily small discovery studies it is prone to error resulting from biological variation. Cross validation is a more powerful method, but its implementation is not straightforward, and it is often used to compute an error estimate for a classifier that has itself been tuned using cross validation with the same data. This method of cross validation has been reported to give biased estimates of classification error . Cross vOur group has developed expertise in miRNA profiling for cancer biomarker identification using cross validation methodologies , 22. In Late diagnosis of OPSCC is a significant clinical problem. Primary care doctors and cancer specialists need improved methods for early diagnosis of OPSCC. miRNAs in tumor derived small extracellular vesicles, circulating in blood serum, have excellent potential for this purpose. Our aim was to develop a panel of serum small extracellular vesicle derived miRNAs which show robust cross validation as a diagnostic biomarker for OPSCC.Three patient cohorts were included in this study; a ‘control’ patient cohort and a cohort of patients with gastroesophageal reflux disease (GORD) and ulcerative esophagitis were included in the non-cancer group, and the cancer group were a cohort of patients with OPSCC. Blood specimens and related clinical data were accessed with appropriate ethical and governance approvals from the SA ENT Tissuebank , PROBE-NET  and Victorian Cancer Biobank from consenting participants. Specimens from cancer patients (n = 40) diagnosed with p16 positive advanced stage OPSCC (stage III or IV AJCC 7th Edition ) but no Diagnostic tissue blocks were accessed to determine the presence of HPV DNA utilising the method of Antonsson et al. , with mi® DNA Polymerase (0.125 µl), 2.5 µl of eluted DNA and water to make total volume 25 µl. PCR thermocycler conditions were 95°C 10 min, 50 cycles of 94 ℃ 90 s, 55 ℃ 90 s, 72 ℃ 2 min, followed by 72 ℃ 4 min and 20 ℃ 10 min.Primers for HPV detection and ß-globin were obtained from GeneWorks . DNA samples were analysed by PCR for the presence of HPV with the general mucosal HPV primers GP5 + (5′TTTGTTACTGTGGTAGATACTAC3′)/GP6 + (5′GAAAAATAAACTGTAAATCATATTC3′) , 25. PCRUltrapure water was used as a negative control. HeLa cells  were used as positive control. β-globin PCR with the primers PCO3 (5′CTTCTGACACAACTGTGTTCACTAGC3′) and PCO4 (5′TCACCACCAACTTCATCCACGTTCACC3′) was carried out on all samples to ensure they contained enough cells to detect human DNA  with the® (cat# 455078). All blood samples were left at room temperature for a period of 16–24 h before processing with a standardised protocol established in our laboratory [All pre-cancer treatment blood specimens were collected either at time of clinic consultation or at time of endoscopy/surgical procedure (before the administration of any medications). Blood was collected into 8 ml Z Serum Separator Clot Activator tubes Vacuetteboratory .g at 4 ℃ for 30 min to exclude larger microparticles. 250 µl supernatant from each sample was then processed with an ExoQuick™ kit  according to the manufacturer’s protocol. Samples were incubated with ExoQuick™ at 4 °C for 16 h. The pellet isolated from each sample was resuspended with 50 µl phosphate buffered saline (PBS). We have previously confirmed that pellets obtained from serum using ExoQuick™ contain particles consistent in size with exosomes (30–150 nm), using a Nanosight LM10 Nanoparticle Analysis System and Nanoparticle Tracking Analysis Software (Nanosight Ltd.) [For small extracellular vesicle isolation, 1 ml aliquots of serum were retrieved, quick thawed, and centrifuged at 16,000ht Ltd.) . We refeht Ltd.) . Extract® PCR custom made plates were used for miRNA profiling. These arrays were comprised of a panel of 112 miRNA probes  and the TaqMan® microRNA Reverse Transcription Kit (Life Technologies cat # 4366596). cDNA Pre-amplifications were carried out with a matching Custom OpenArray® PreAmp pool (Life Technologies cat # 4485255) and TaqMan PreAmp Master Mix (Life Technologies cat # 4488593) on 7.5 μl complementary DNA (cDNA)/sample for each pool. The pre-amplified products (4 μl per sample) were diluted at the recommended 1:40 dilution with 156 μl of RNase-free ultra pure water before mixing with TaqMan OpenArray Real-Time PCR Master Mix (Life Technologies cat # 4462164) and loading onto a 384-well TaqMan OpenArray loading plate. PCR runs were performed using a QuantStudio™ 12 K Flex Real-Time PCR System.High throughput QuantStudio™ 12 K Flex OpenArrayAnalyses were performed using R (version 3.4.3), and Microsoft Excel for Mac (version 16).https://cran.r-project.org/web/packages/qpcR/index.html). Only miRNAs with detectable Cts in at least 50% of samples in one group were considered for the expression analysis. The relative expression of each miRNA was calculated as 2(40−Ct). Relative expression values for each miRNA were used to derive per patient values for every possible permutation of miRNA ratios.The cycle threshold (Ct) value for each PCR assay was determined using the qpcR package v1.4 in R , using the cv.glmnet function (from the glmnet R-package v2.0-13) with the method set to “binomial” (i.e. logistic).We utilised leave-one-out cross validation in the outer loop to generate held-out test samples that would not be used in optimizing model parameters, and then utilized repeated (50 ×) tenfold cross validation in the inner loop (using the cv.glmnet function from the glmnet R-package v2.0-13) to optimise the regularisation parameter lambda for Lasso regression. Each of the 50 repeats of the tenfold cross validation consists of a random split of the samples into tenfolds, so this approach produces 50 lambda estimates from each of the outer loop training sets. These repeated lambda estimates were assessed for stability , and the average of the lambda estimates from the inner loop cross validations was used to build a Lasso regression model in each of the outer loop training sets, which was then used to predict each held-out test sample.In addition to optimizing the Lasso regression model regularization at the level that produced the minimum cross validated prediction error (lambda.min), we repeated the modelling using more stringent regularization to reduce model complexity .To stabilise variable selection, we extended the method utilised by Rosenburg et al.  for highOur novel variant of the Bach  method, We assessed the outer loop predictions using Receiver Operating Characteristic (ROC) curve analysis, with 2000 bootstrap samples to estimate 95% confidence intervals for the sensitivity and specificity at each threshold level .p > 0.1); (iii) they were not highly variable (coefficient of variation < 2 × standard deviation) and did not contain outliers (samples with levels not within fivefold of the mean); and (iv) they were correlated at r > 0.7 with the geometric mean of the house keeping genes. The values for these selection criteria for each of the 15 House Keeping Gene miRNAs, plus mature nucleic acid sequences and Accession numbers, are presented in Additional file For normalisation of the miRNAs we selected 15 miRNAs as House Keeping Genes using the following criteria: (i) they were expressed in all samples and at high levels (median Ct < 30); (ii) they were not statistically different in tissue comparisons , and were normalized using the geometric mean of the relative levels of the 15 House Keeping Genes.The relative levels of the miRNAs were determined using the formula 2The normalised miRNAs were pre-filtered using the following criteria: (1) at least 50% of samples amplified in one of the comparison groups, (2) the coefficient of variation was less than 200%, and (3) differential expression was greater than 1.3 fold. Mann–Whitney U tests were then used to determine which miRNAs were differentially expressed, and the False Discovery Rate was estimated using the method of Storey .Of the 80 RNA samples profiled on OpenArray™, one sample failed to amplify, and data import failed for one other sample. Therefore, the miRNA data available for biomarker discovery was derived from 19 controls, 20 patients with gastroesophageal reflux disease induced ulcerative oesophagitis, and 39 patients with p16 positive OPSCC  Table In order to discover miRNA ratios that can discriminate controls and patients with GORD and ulcerative oesophagitis from patients with OPSCC, we utilized lasso regression in a standard nested 2-stage cross validation. This standard approach produced a multi miR-ratio model with poor predictive capacity for the held-out samples Fig. , 6 with In order to determine how likely it was to obtain the observed classification performance of the 11-miR-ratio model by chance, we randomly permuted the sample labels 2000 times in order to estimate the empirical cumulative distribution of the cross validated classification error under the null hypothesis . The maxWe also investigated whether any of the miR-ratios in the model contained individual miRNAs that were significantly differentially expressed when normalised with house keeping gene miRNAs. For this differential expression analysis we estimated a false discovery rate of 18%. All 11 miR-ratios contained at least one differentially expressed house-keeping gene normalised miRNA  of the dataset, so there is the possibility that information about the data will be missed, which can result in bias. Furthermore, a single split of the data may not be able to generate an equitable distribution of all biological or clinical parameters . These iIn this current study increased penalisation of the lasso regression did not improve the cross validated predictive capacity of the model . A potenMany cancers are associated with a background of chronic inflammation . PatientThe main limitation of this study is the focus on the advanced stages (AJCC 7th edition) of HPV associated OPSCC. This is in part due to the later presentation of patients with OPSCC. Future studies need to test the ability of miRNA ratio model to detect early stage HPV associated OPSCC.Currently, there is no detection test available for primary care physicians to use for patients at risk of HPV associated OPSCC. Usually these patients have non-specific symptoms of a sore throat, or a lump in the throat or neck. These symptoms are not specific for cancer and may be mistakenly diagnosed as infectious or inflammatory. Consequently, some patients are not diagnosed as having HPV associated OPSCC until the cancer is at a more advanced stage. Therefore, a high specificity blood-based biomarker could provide a non-invasive test that could triage patients with HPV associated OPSCC in the primary care setting to receive prompt specialist care.The majority of studies examining the role of miRNAs in head and neck cancer have examined their potential role in pathogenesis or prognosis using tissue specimens . Examinid-glucose integrated with computed tomography (PET-CT), when available, is the preferred imaging modality for assessment of treatment response [Another potential area of benefit for a blood-based biomarker is as an adjunct test for the surveillance post treatment period and detection of cancer recurrences. Although HPV associated oropharyngeal cancers have a relatively good prognosis, 20–25% of patients develop recurrent disease within 5 years of treatment . Followiresponse , and is response , 50. Thiresponse , 52, whiThe 11-miR-ratio biomarker panel also has the potential to detect recurrences earlier than is currently possible. Currently there are no effective methods for detecting residual cancers within the first 6 to 12 weeks following treatment. In the most recent study investigating the use of PET/CTs for surveillance of HPV associated OPSCCs , the positive predictive value was only 13.4% . HoweverWhile the blood-based biomarker studies in HPV associated OPSCCs, including this current study, are relatively small, they have produced encouraging results, and should motivate the undertaking of larger studies. We have developed a stabilised biomarker selection approach, StaVarSel, using lasso regression, which enabled us to discover a panel of miRNA ratios in blood with levels of cross validated specificity and sensitivity that could potentially be useful for detecting HPV associated OPSCCs. The results of this study suggest that it will be worthwhile using this approach to discover molecular biomarkers for HPV negative OPSCCs, as well as other mucosal head and neck cancers.Additional file 1. Details of 112 miRNAs included on custom OpenArray™.Additional file 2. Further explanation of statistics and model derivation.Additional file 3. List of all lasso regression miR-ratios selected from the inner cross validation loop.Additional file 4. Details of selected House Keeping Genes.Additional file 5. Boxplots of the 11 miRNA ratios in the logistic regression model.Additional file 6. Details of the miRNAs included in the 11-miR-ratio logistic regression model.Additional file 7. Details of all differentially expressed house keeping gene normalized miRNAs (non-cancer vs cancer).Additional file 8. Details of non-differentially expressed miRNAs present in the 11 miRNA-ratios logistic regression model.Additional file 9. Boxplots of the differentially expressed miRNAs in the 11-miRNA-ratio logistic regression model.Additional file 10. Boxplots of the non-differentially expressed miRNAs in the 11-miRNA-ratio logistic regression model.


Populations of closely related microbial strains can be simultaneously present in bacterial communities such as the human gut microbiome. We recently developed a de novo genome assembly approach that uses read cloud sequencing to provide more complete microbial genome drafts, enabling precise differentiation and tracking of strain-level dynamics across metagenomic samples. In this case study, we present a proof-of-concept using read cloud sequencing to describe bacterial strain diversity in the gut microbiome of one hematopoietic cell transplantation patient over a 2-month time course and highlight temporal strain variation of gut microbes during therapy. The treatment was accompanied by diet changes and administration of multiple immunosuppressants and antimicrobials.We conducted short-read and read cloud metagenomic sequencing of DNA extracted from four longitudinal stool samples collected during the course of treatment of one hematopoietic cell transplantation (HCT) patient. After applying read cloud metagenomic assembly to discover strain-level sequence variants in these complex microbiome samples, we performed metatranscriptomic analysis to investigate differential expression of antibiotic resistance genes. Finally, we validated predictions from the genomic and metatranscriptomic findings through in vitro antibiotic susceptibility testing and whole genome sequencing of isolates derived from the patient stool samples.Bacteroides caccae. Comparative analysis of B. caccae genomes obtained using read cloud sequencing together with metagenomic RNA sequencing allowed us to identify differences in substrain populations over time. Based on this, we predicted that particular mobile element integrations likely resulted in increased antibiotic resistance, which we further supported using in vitro antibiotic susceptibility testing.During the 56-day longitudinal time course that was studied, the patient’s microbiome was profoundly disrupted and eventually dominated by We find read cloud assembly to be useful in identifying key structural genomic strain variants within a metagenomic sample. These strains have fluctuating relative abundance over relatively short time periods in human microbiomes. We also find specific structural genomic variations that are associated with increased antibiotic resistance over the course of clinical treatment. Microbial strains of the same species share “core” genes that encode conserved functions common to the species. However, strains of a given species can differ by single-nucleotide variants (SNV), insertions, and deletions as well as structural arrangement  and variGenetic variation within a population can either arise de novo as a consequence of new mutation or can be part of a reservoir of genetic variation in a community where multiple strains stably coexist . ExposurShotgun short-read sequencing methods facilitate study of the genomic content and strain-level architecture of complex microbial communities. Comparisons of microbial genomes obtained from both sequencing of isolates and single cells have shaped our understanding of strain-level genetic variability , 11, 12.Dedicated metagenomic assemblers –18 and bTo address these challenges, we recently developed a metagenomic shotgun sequencing and assembly method that provides complete microbial genomes from complex microbiome samples [24]. The molecular approach of linked-read (or “read cloud”) library preparation relies on partitioning long DNA fragments within more than a million nanoliter droplets . BarcodeIn this case study, we present the application of read cloud sequencing to longitudinal microbiome samples from a hematopoietic cell transplantation (HCT) patient to demonstrate the evolution of gut microbes during therapy. This experimental approach enables the discovery of strain-level sequence variants that confer selective advantages under differing environmental stresses in a clinical setting. We find that exposure of the patient to antibiotics is correlated with the predominance of strains with specific transposon integrations that result in differential transcription of antibiotic resistance genes. In vitro antibiotic susceptibility testing and whole genome sequencing of isolates derived from the patient stool samples support functional predictions made using our metagenomic approach.Stool samples were obtained from the study subject on an approximately biweekly basis, when available. Stool samples were placed at 4 °C immediately upon collection and processed for storage at − 80 °C the same day. Stool samples were aliquoted into 2-mL cryovial tubes with either no preservative or 700 μL of RNAlater and homogenized by brief vortexing. The aliquots were stored at − 80 °C until extraction.g for 20 min at 4 °C.Stool DNA was extracted for short-read libraries and Gemcode read cloud libraries with the QiAMP Stool Mini Kit  modified with an additional step after addition of ASL buffer consisting of 7 cycles of alternating 30-s periods of beating with 1.0 mm Zirconia/Silica beads in a Mini-Beadbeater  and chilling on ice. Stool DNA was extracted for Chromium read cloud libraries with the Gentra Puregene Yeast/Bacteria kit , modified with a chilling step at − 80 °C for 5 min, followed by ethanol DNA precipitation at 14,000Prior to read cloud sequencing library preparation, DNA was size-selected with the BluePippin instrument . A 5–50-kb size range was used for Gemcode libraries, and a 10–50-kb size range for Chromium libraries. Read cloud libraries were then prepared with either the Gemcode or Chromium instrument .DNA used for short-read library preparation was not size selected. Short-read libraries were prepared with the Truseq DNA HT library prep kit .All library fragment sizes were assessed with the 2100 Bioanalyzer instrument  using the High Sensitivity DNA chip and reagent kit. DNA and library concentration estimations were performed using fluorometric quantitation with the Qubit 3.0 fluorometer using the Qubit dsDNA HS kit .RNA was extracted with the Qiagen RNeasy Mini kit from stool samples stored in RNAlater at − 80 °C. Original total RNA concentration was assayed with the Qubit RNA HS kit. RNA was then ethanol precipitated and resuspended in nuclease-free water to concentrate, and then quantified again using both Qubit RNA HS and Qubit DNA HS kits to determine the degree of DNA contamination. Contaminating DNA was removed using the Baseline-ZERO DNase protocol  with 30 min incubation followed by a second ethanol precipitation. Ribosomal RNA was depleted with the Epicenter Ribo-Zero rRNA removal kit (Bacteria) and purified with another ethanol precipitation. The rRNA-depleted mRNA quality was assessed with the 2100 Bioanalyzer using the Agilent RNA6000 Pico kit and quantified with Quibit RNA HS assay. cDNA sequencing libraries were then prepared with the Illumina Truseq Stranded mRNA kit following the Truseq Stranded mRNA LT protocol. Resulting DNA libraries were quantified with Qubit DNA HS kit and their quality evaluated by 2100 Bioanalyzer instrument using the High Sensitivity DNA chip and reagent kit.Truseq libraries were sequenced with 2 × 101 bp reads on an Illumina HiSeq 4000 instrument, each library receiving 2–6 Gb sequence coverage with the exception of timepoint A, which was additionally sequenced with 34 Gb of coverage after the initial attempt produced insufficient coverage. 10X Gemcode libraries were sequenced with 2 × 148 bp reads on an Illumina NextSeq 500 instrument, with each library receiving 4–7 Gb sequence coverage. 10X Chromium libraries were sequenced with 2 × 151 bp reads on one lane of Illumina HiSeq 4000, each receiving 18–22 Gb of sequence coverage. RNAseq libraries were sequenced with 2 × 101 bp reads on an Illumina HiSeq 4000 instrument, each library receiving 8–12 Gb sequence coverage with the exception of timepoint A, for which a high-quality RNA sequencing library could not be obtained. Total reads and sequencing coverage for all metagenomic sequencing libraries before and after quality control can be found in Additional file Raw reads from all DNA libraries were first subjected to the same quality control and trimming as follows. Sequence data were trimmed using cutadapt  using a Trimmed reads from all libraries were then assembled using metaSPAdes 3.11.1  with defFor read cloud and short-read libraries, coverage was calculated for assembled contigs by aligning raw short reads with BWA v0.7.10 . MetabatIndividual contigs from all assemblies were assigned taxonomic classifications using k-mer-based classification with Kraken2  with a cBacteroides caccae genome drafts between timepoints , , and  using MUMMER [B. caccae genome within timepoint C, but were absent from the draft in timepoint B. The 17 kb and 57 kb islands were also found within the draft genomes of B. vulgatus and B. uniformis. Two genomic island incorporations of sizes 43 kb and 50 kb were observed in timepoint D with respect to timepoint C. Neither of these two islands were assembled into alternate genomic contexts in timepoint C.To discover large-scale genomic island incorporations, we first obtained pairwise sequence alignments of g MUMMER . We seark-mer sequences that were present in the B. caccae genome. We first obtained k-mer counts from the timepoint C read cloud draft using Jellyfish [k = 101. The vast majority of k-mers originate from single-copy portions of the genome, but we isolated the subset of these that occurred at a copy number of at least 10, and assembled them using SPAdes [B. caccae genome.To discover smaller-scale insertion sequences, we examined high-frequency ellyfish  with k =g SPAdes  with “--Illumina Truseq short-read data were aligned with BWA  to the iBacteroides fragilis group, including Bacteroides caccae, were isolated directly from stool by streaking stool on solid BHI medium  under anaerobic conditions in a Bactron 300 anaerobic chamber . Individual colonies matching the described B. fragilis group morphology of circular, entire, convex, gray, translucent, shiny, and smooth [Members of the d smooth  were picd smooth  and incuWe extracted DNA using the Qiagen Gentra Puregene Bacteria DNA kit and prepared Illumina Nextera XT short-read sequencing libraries from all of the isolates that were cultured from the stool samples. The resulting 53 sequencing libraries were multiplexed and sequenced on a single Illumina HiSeq 4000 lane  and , we then searched for sets of k-mers that were exclusively present in the antecedent points, but not preceding ones. Each set of k-mers was then assembled using SPAdes [B. caccae drafts.In order to search for potential large genomic island sequences that are exclusively integrated into ellyfish  with k =g SPAdes  with “--B. caccae isolate strains were first selected for testing on the basis of their IS614 genotype determined by whole genome sequencing. One isolate from timepoint A (A2) was determined to contain both B. caccae and another Bacteroides species most closely related to Bacteroides uniformis. A2 was subsequently re-streaked on BHI plates; this allowed for isolation of the B. caccae strain, which was verified using PCR (described below).Susceptibility to ciprofloxacin and trimethoprim was assessed using a broth microdilution method for determining the minimum inhibitory concentration (MIC) adapted from CLSI  as follo600), and the culture were adjusted with TYG media to normalize the densities across all strains. The cultures were diluted 1:200 in fresh TYG media (OD600 ~ 0.1) and set up in the assay plates (100 μL per well) that were already prepared with the drug dilutions (100 μL per well) for a final total volume of 200 μL per well. Each selected B. caccae isolate was tested in at least two and up to four replicates. Assay plates were incubated for 48 h at 37 °C in the anaerobic chamber. The OD600 was then measured for each plate on a BioTek Epoch spectrophotometer . Prior to plate reading, wells were mixed by pipetting up and down to evenly mix the cultures as all B. caccae strains were observed to flocculate over the incubation period. The minimum inhibitory concentration (MIC) of each drug for each replicate of each isolate was determined by fitting the drug concentration vs. OD600 data to a Gompertz function [t-test with a significance threshold of p value ≤ 0.05.Clear 96-well flat-bottom assay plates were prepared with TYG media containing a twofold serial dilution of each drug over 11 concentrations in addition to no drug controls . Ciprofloxacin precipitated at concentrations higher than 512 μg/ml after incubation period; thus, these concentrations were excluded from susceptibility analysis. The turbidity of the overnight cultures was determined by measuring the optical density at 600 nm  with Phusion HF Buffer and NEB Deoxynucleotide Solution Mix. Primers were obtained from Elim Biopharm  with target melting temperature of 60 °C. Reactions consisted of 6 μL 5X Phusion HF buffer, 0.6 μL 10 mM dNTP, 0.3 μL Phusion, 2 μL 10 mM forward primer, 2 μL 10 mM reverse primer, 1 μL template DNA, and PCR clean water to 30 μL. Thermocycling was performed with 30 s of denaturation at 98 °C followed by 35 cycles of 5 s of denaturation at 98 °C, 10 s of annealing at 65 °C, and 30 s per kilobase extension at 72 °C. This was followed by a final extension at 72 °C for 5 min and an indefinite hold at 4 °C.The patient was a 46-year-old man who underwent HCT at the Stanford Hospital for treatment of an underlying myelodysplastic syndrome and myelofibrosis, which was refractory to treatment with azacitidine. The patient had been exposed to a course of therapy with azithromycin 1.5 months prior to the date of the first sample , pre-chemotherapy and pre-HCT; B day 13), post-chemotherapy and post-HCT; C (day 43), post broad-spectrum antibiotic exposure and onset of GI GVHD; D (day 56), following onset of GVHD and introduction of a new antibiotic regimen including trimethoprim , with the exception of one misassembly in the timepoint D draft around a 16S/23S ribosomal RNA gene operon  to our read cloud drafts using MUMMER [B. caccae genome contained 639 kb of novel sequence not represented in any reference isolate, compared to just 318 kb of novel sequence contained in the short-read draft. The median sequence identity for alignable bases between our B. caccae drafts and the reference isolates was 99.5% for both read clouds and short reads , we could not identify IS614 in any of the six available reference isolate genomes.We next searched the read cloud nce see “”. This pB. caccae, and 10 in B. vulgatus. We found 25 unique integrations of the unassigned IS, all of which were determined to be in B. caccae.We searched for integrations of IS614 and the candidate IS across all read cloud metagenome drafts in which at least 3 kb of flanking sequence was assembled on both sides of the integration. We found 28 unique integrations of IS614, 18 of which were determined to be in B. caccae, alignments of the short-read data to the Athena assembly confirmed the co-occurrence of both strains harboring the IS and strains with the pre-insertion “ancestral” sequence . Although a few strains with an IS614 integration upstream of thyA2 and dhfrIII are present in timepoint C (pre trimethoprim exposure), the majority of strains with this integration are present in timepoint D (post trimethoprim exposure). The observed shifts in abundance of the B. caccae strain subpopulations are consistent with the differing selective pressures applied from the patient’s drug regimen over the course of treatment. Notably, while clear subpopulations with varying IS614 positions appear to exist and have a selective fitness advantage at various timepoints, the larger genomic islands appear to be sporadically integrated amongst the individual strains. This suggests that none of the larger genomic islands were under selection between timepoints A and C or between timepoints C and D.All strains from timepoints C and D (post ciprofloxacin exposure) contain an IS614 integration upstream of B. caccae subpopulation to ciprofloxacin and trimethoprim. In total, we selected all three strains from timepoint A and four strains from each of the three subpopulations of timepoints C and D for this analysis   for members of the B. fragilis group have been ≤ 8 μg/mL for ciprofloxacin [50 values across all strain subpopulations were ≥ 64 μg/mL for both ciprofloxacin and trimethoprim. Nonetheless, we observed varying degrees of drug resistance in the isolated strains from the four subpopulations. Isolates from timepoints C and D, which we expected to be more resistant to ciprofloxacin using our metagenomic approach, showed a twofold to fourfold increase in the MICs relative to those from timepoint A . This suggests that the resistance phenotype to this drug appeared and was sustained over time. Trimethoprim resistance is consistently highest in the subpopulation with strains carrying the IS614 integration upstream of thyA2, although it is notable that some of the strains at timepoint C also have a high level of resistance in the absence of the IS614 integration upstream of thyA2. Differences in trimethoprim susceptibilities were statistically significant when comparing the MIC values of subpopulation A and subpopulation C/D-3 , and subpopulation C/D-2 and subpopulation C/D-3 . Collectively, the drug susceptibility results are generally consistent with the resistance phenotypes we predicted based on our metagenomics analysis. Whole genome sequencing of B. caccae isolate strains from timepoints A, C, and D together with antibiotic susceptibility testing support the presence of selection amongst strain populations of B. caccae with different IS614 integrations as predicted using our read cloud metagenomic approach.Grouping of replicates by subpopulation assignment enabled the examination of their collective antibiotic susceptibility. We found that the difference in ciprofloxacin susceptibility between strains from subpopulation A and each of the three C/D subpopulations  are shown in Fig. Additional file 3. Table S2. Species-level and genus-level classification of all the sequencing data for the four timepoints In this study. The relative abundance of the taxa in the samples was determined without removing human and viral reads from the data. Stacked bar graphs of these data are shown in Additional file Additional file 4. Table S3. Assembly statistics, completeness metrics, tRNA and rRNA loci counts, total annotated genes, and coverage depths for all annotated species draft genomes in short-read and read cloud libraries from each time point. Results are shown for the largest bin of each species.Additional file 5. Table S4. Assembly statistics, completeness metrics, tRNA and rRNA loci counts, total annotated genes, and coverage depths for bins created by merging all those annotated as B. caccae in short-read and read cloud libraries at each time point.Additional file 6. Table S8. Coding sequences with RNA sequencing read counts and fold-change between time points in the neighboring 10 kb around the five IS614 integration loci in B. caccae estimated to have large-scale ancestral strain shifts. Gene annotations were obtained using Prokka. The target gene downstream of the putative promoter as well as the upstream gene are highlighted .Additional file 7. Table S9. Total reads, sequencing coverage, assembled genome draft size and N50, and taxonomic annotation for all 53 isolates from stool samples of time points A, C, and D.


At high pressure, as intermolecular interactions increase significantly, the free rotation of H2 molecules is increasingly hindered, and consequently a modification of the coupling between molecular rotational properties and the nuclear spin system can be anticipated. To date, high-pressure experimental methods have not been able to observe nuclear spin states at pressures approaching 100 GPa  and consequently the effect of high pressure on the nuclear spin statistics could not be directly measured. Here, we present in-situ high-pressure nuclear magnetic resonance data on molecular hydrogen in its hexagonal phase I up to 123 GPa at room temperature. While our measurements confirm the presence of ortho-hydrogen at low pressures, above 70 GPa, we observe a crossover in the nuclear spin statistics from a spin-1 quadrupolar to a spin-1/2 dipolar system, evidencing the loss of spin isomer distinction. These observations represent a unique case of a nuclear spin crossover phenomenon in quantum solids.One of the most striking properties of molecular hydrogen is the coupling between molecular rotational properties and nuclear spin orientations, giving rise to the spin isomers  ortho and para spin isomers is lost.Solid hydrogen has increasingly hindered rotation under high pressure, but the effect on spin isomer populations had not been directly probed. Here the authors measure NMR spectra of solid hydrogen up to the megabar, and observe the crossover to a spin 1/2 dipolar system above 70 GPa where distinction between  It has direct applications for spintronics4 and enables the understanding of the stabilisation of magnetospheres of rocky (Earth-like) planets5 as well as gas- and ice-giants6. The degrees of freedom of the nuclei spins, however, are widely approximated as fixed within the analysis of experiments, due to large shielding by core electrons and the extremely short atomic distances necessary to induce such fundamental changes.Changes in electronic spin-statistics under changing thermodynamic conditions are an established physical crossover phenomenon29.Hydrogen, on the other hand, exhibits no core electrons and when bound contributes its electron to the molecular bond. Furthermore, due to the low mass of the hydrogen nuclei, quantum nuclear effects are considered to be significantly more pronounced compared to all other elements. The combination of both effects results in a number of fascinating physical phenomena in molecular H2 molecule and the resulting coexistence of the spin isomers ortho- (ortho-H2) and para-hydrogen (para-H2). Following Pauli’s exclusion principle, in order for the total H2 molecular wave function to be antisymmetric under exchange of atomic positions, demands for the rotational ground state J = 0, that the corresponding total nuclear wave function is antisymmetric . Analogously, for the J = 1 rotational state, the total nuclear wave function is required to be symmetric . Therefore, the spin allotropic isomerism of the H2 molecule originates in the coupling of both rotational state and nuclear spin. It has been argued11 that at high pressure (P) ortho- and para-hydrogen spin isomers remain stable up to the dissociative Wigner–Huntington transition at P > 400 GPa13.One property intrigued physicists in particular: the nature of the nuclear spin of the Hrn) are much shorter (≈0.7Å at ambient conditions) than next-nearest neighbour distances (rnn ≈3Å at ambient conditions), allowing for sufficient intramolecular wave function overlap  spectroscopy, detecting the linear response of the nuclear spin system upon radio-frequency excitation in a magnetic field B0. The spin-singlet state of para-hydrogen is NMR silent, whereas application of B0 lifts the three-fold degeneracy of the ortho states and allows for an excitation of nuclear spin transitions , intense 1H resonances of roughly 500 kHz width were detected. With increasing P, the resonance signals broadened significantly approaching 750 kHz at 68 GPa , the electric field V(r), defined by the local charge distribution based on the crystal structure of phase I, should influence the shape of the observed resonance lines. Calculated line shapes for an I = 1 spin system are shown in Fig. 19 and consequently treated as a first-order perturbation . Up to 68 GPa, the measured 1H-NMR spectra are well described by calculated line shapes broadened by first-order quadrupole interaction. The line shape is mainly controlled by two parameters: (i) the quadrupole coupling constant Cq describing the coupling between eQ and V(r) as well as (ii) the electric field gradient asymmetry parameter η accounting for the geometry of V(r).For the quadrupolar nature expected for Cq, which increase from 28.1(6) kHz at 20 GPa to 61.9(7) kHz at 58 GPa. This increase is likely originated in the high compressibility and rapidly reducing next-nearest neighbour distances between molecular H2 units, enhancing quadrupolar coupling. The asymmetry parameter η Å at 123 GPa, in excellent agreement with the computational estimates by Labet et al.15. Figure I = 1/2 line shapes between 71 and 123 GPa. The respective values derived for both nearest (top panel) and next-nearest (middle panel) neighbour distances can be found in Fig. 23 and diffraction data21, along with the unit cell volumes (blue dots) derived from rn and rnn extracted from the analysis of the NMR spectra are shown in the bottom panel of Fig. This pressure indicates a turning point in the behaviour of the 24 have been used to suppress quadrupolar and dipolar line broadening in order to resolve isotropic chemical shifts, δiso. Figure δiso: initially decreasing from 8.6 to 5.9 ppm between 20 and 59 GPa, δiso has an inflection point at ~60 GPa and raises under further compression to 22.7 ppm at 123 GPa. Comparison with Raman data25 suggests that the minimum in δiso coincides with the well-known turn-over in the Raman shift of the H2 vibron caused by a weakening of intramolecular and increased intermolecular interactions26.Homonuclear Lee–Goldburg decoupling sequencesP < 60 GPa, 1H-NMR data is characteristic for an I = 1 quadrupolar spin system as expected for ortho-H2. In this regime, individual nuclear spin angular momenta couple with their nearest neighbours (separated on average by rn), leading to a significant wave function overlap within the molecular units and a stabilisation of the spin isomers. The excellent agreement between NMR derived values for the electric field gradient asymmetry parameter η and values inferred from X-ray diffraction data21 strengthens this assessment. Following the theoretical study of Strzhemechny et al.27, this compression driven enhancement of the quadrupolar coupling constant Cq in this P-regime may be interpreted as experimental evidence for the mechanism of ortho–para conversion through electric quadrupole interaction.The presented data analysis leads to the following interpretation of the observed effects: At P > 60 GPa, quadrupolar coupling rapidly diminishes despite the absence of a structural rearrangement of the molecular H2 units. Starting from about 70 GPa, spectral features characteristic of homonuclear dipole–dipole coupling between nearest and next-nearest neighbours become apparent. Provided the good agreement between NMR data with DFT15 and experimentally21 derived intermolecular and interatomic distances, this shift in behaviour implies intramolecular coupling of nuclear spins to become increasingly perturbed. The inflection point in the isotropic chemical shift δiso strengthens this hypothesis as the increasing nuclear de-shielding above 60 GPa indicates a shift of electron density away from individual molecular centres towards intermolecular regions.At 14N-NMR on molecular nitrogen at P = 3 GPa (see “Methods” section) supports this argument, as the nitrogen spin system shows clear characteristics of a nuclear spin-triplet state anticipated within the non-pairing regime contrary to the quintuplet state stabilised in the molecular spin-pairing regime.I = 1/2 value. Crossovers of the nuclear spin statistics of a quantum solid such as hydrogen have so far not been observed and given the large compressibility of hydrogen in conjunction with strong nuclear quantum effects, this crossover phenomenon might only be experimentally observable in molecular H2. Nuclear spin statistics of similar diatomic molecules (e.g. N2) are likely to be best described as non-pairing nuclear spins due to enhanced atomic masses as well as reduced compressibilities due to the presence of core electrons.In this work, in-situ high-pressure nuclear magnetic resonance was used to investigate the nature of the nuclear spin statistics of molecular hydrogen up to 123 GPa in Phase I at room temperature. It was found that even at moderately high pressures (<100 GPa) intramolecular nuclear spin coupling broke down and the hydrogen spin system adopted an average dipolar 2.This nuclear spin-crossover may have far-reaching consequences for understanding different phenomena such as the stabilisation of magnetospheres of gas and ice giant planets containing large quantities of molecular HTwo diamond anvil cells, equipped with pairs of diamond anvils with a culets size of 250 and 100 µm, were prepared. Rhenium gaskets were pre-indented to 25 and 10 µm, respectively, and 80 and 40 µm diameter holes were laser drilled in the centre of the indentation to form the sample cavities, resulting in sample volumes of about 125 and 13 pl, respectively.28. Double29 (in the case of the 250 µm diamonds) and triple30 (for the 100 µm diamonds) stage Lenz-lens radio-frequency resonators were produced by using focused ion beam milling. To ensure electrical insulation and avoid hydrogen diffusion into the rhenium, the gaskets were coated by 500-nm-thick layers of Al2O3. Radio-frequency excitation coils were made from 100 µm thick, Teflon insulated, copper wire and arranged such that a Helmholtz coil pair is formed.The diamond anvils were coated with a 1-µm-thick layer of copper using physical vapour deposition32. Comparison of the vibron frequencies of the H2 samples at elevated pressures shows a slight systematic offset of less than 5 GPa at the highest pressures where Raman data was collected33.Hydrogen loading was conducted at the ESRF and pressure was increased at cryogenic temperatures to avoid rapid hydrogen diffusion into the diamond anvils. The pressure was calibrated by means of the diamond edge Raman scale1H-NMR signals were recorded at frequencies of 45.26 MHz, corresponding to an external magnetic field strength of about 1063 mT. Using nutation experiments, we found optimal excitation pulses between 1 and 1.2 µs for both cells, in reasonable agreement with earlier experiments34.All NMR experiments were conducted using a solid-state NMR spectrometer from Tecmag Inc. (Redstone) equipped with a 100 W pulse amplifier. To polarise the nuclear spin system, we used a sweepable electromagnet with an average magnetic field of 1 T and sufficiently high homogeneity. Intense 1H-NMR spectra recorded by this method. 25,000 scans were accumulated for each spectrum  and ri the average distance between interacting hydrogen nuclei, rn or rnn, respectively. η describes the asymmetry of the electric field gradient tensor (Vij) in the principal axis system as:Using the standard expressions for the resonance frequency distribution ω for both first-order quadrupole interaction as well as homonuclear dipole–dipole interaction:P(ω), for quadrupolar spin interactions, is given by:β denotes the inverse function of Eq. , is given byβ denotes the inverse function of Eq.  is optimised by varying Cq and η for quadrupolar coupling and rn and rnn for dipolar coupling. The corresponding Python scripts are available from the authors upon request. Table Cut-off frequencies of the resulting spectral line functions were chosen according to the Heaberlein convention for NMR shift tensorsη of the electric field gradient tensor in the spin-pairing regime, we used the second derivative of the electric potential, V(r), defined by the molecular centre of gravity positions from diffraction measurements14:In order to calculate the asymmetry parameter Vij in the principal axis system allows the computation of η from crystallographic data.Using Eq.  under co14N nuclei. As 14N nuclei have a nuclear spin of I = 1, one can expect a spin-pairing scenario similar to molecular D2: the para-N2 states consist of a quintuplet subsystem with I = 2, whereas the ortho-N2 states are a triplet subsystem.Molecular nitrogen was measured using natural isotopic composition, where the majority of molecules can be expected to be pairs of η was estimated according to diffraction data14 to be around 0.23. Recorded 14N-NMR spectra (Supplementary Fig. I = 2 quadrupolar powder pattern in absence of m−2→−1 and m1→2 transitions. In fact, the spin system is well described by an I = 1 spin system using the estimated value for η (Supplementary Fig. The electric field gradient asymmetry parameter 38, rn can be estimated to be around 1.2 Å at this pressure; four times longer than the thermal de-Broglie wavelength of a single 14N atom. Therefore, the wave function overlap should be negligible in molecular nitrogen at these pressures and nuclear spins considered unpaired.According to structural dataSupplementary Information


SHANK3 mutation. We observed a reduction in cell soma size of selective neuronal categories and in axonal projections at 30 days post-transplantation. In contrast to previous in vitro studies, we did not observe any alteration in spinogenesis at this early age. The humanized chimeric mouse models offer the means to analyze ASD-associated mutations further and provide the opportunity to visualize phenotypes in vivo.Using human induced pluripotent stem cells (iPSC), recent studies have shown that the events underlying autism spectrum disorders (ASD) can occur during neonatal development. We previously analyzed the iPSC-derived pyramidal cortical neurons of a subset of patients with ASD carrying de novo heterozygous mutations in postsynaptic SHANK3 protein, in culture. We reported altered spinogenesis of those neurons. The transplantation of human iPSC-derived neuronal precursors into mouse brain represents a novel option for in vivo analysis of mutations affecting the human brain. In this study, we transplanted the neuronal precursor cells (NPC) into the cortex of newborn mice to analyze their integration and maturation at early stages of development and studied axonal projections of transplanted human neurons into adult mouse brain. We then co-transplanted NPC from a control individual and from a patient carrying a de novo heterozygous  SHANK gene family located on chromosome 22 in humans which contains multiple structural domains as we previously demonstrated by homology modeling1. Mutations in SHANK3 represent some of the best-known genetic causes for ASD and account for the severe intellectual deficiency (ID) and language deficits observed in 1–2% of patients with ASD3. SHANK3 haploinsufficiency also contributes to the clinical symptoms of patients with Phelan-McDermid syndrome which presents a deletion of chromosome 22q13 that includes SHANK3 in the large majority of cases5. SHANK3 is a critical partner of a major signaling complex expressed at postsynaptic densities (PSD) of glutamatergic synapses which involves cytoskeletal networks at both the soma and the neurites of neuronal cells6. SHANK3 therefore plays a crucial role in synapse formation and dendritic spine maturation. The fundamental role of SHANK3 at such excitatory synapses was initially investigated by using genetically modified mouse models9, or by overexpressing mutated SHANK3 protein in transfected rat neurons in culture10. Human induced pluripotent stem cells (iPSC)-derived neurons represent a valuable model for in vitro analysis of SHANK3 deficiencies in humans15. Accordingly, we recently selected four independent patients with heterozygous truncating de novo SHANK3 point mutations who had previously been characterized in our laboratory3. We generated the corresponding human iPSC for their selective reprogramming into cortical neurons16. We then examined the effects of SHANK3 haploinsufficiency on the levels of SHANK3 mRNA and on the spine morphogenesis to demonstrate the correlation between spinogenesis defects and SHANK3 levels1. In accordance with earlier reported results, our data from these studies with cultured human neurons confirmed the existence of early synaptic deficits that may occur in the brain of ASD patients who carry SHANK3 mutations. Together with local synaptic deficits, a defective short-range cortico-cortical wiring was recently reported in a mouse model presenting a homozygous loss of the Shank3B isoform17. The human cortex is an important region that undergoes multi-developmental processes. Recent findings from animal models suggest that pathological modifications that lead to ASD start before birth at early stages of cortical development18. Another recent study with iPSC, derived from ASD patients, has shown a dysregulation of specific neurodevelopmental gene modules which occurs at stages of neuronal precursors19. The neuronal connections that are observed in two-dimensional cultures cannot fully reflect the in vivo conditions, and this represents an important issue for interpretation of data. Recent alternative protocols including the transplantation of human cells, derived either from human embryonic stem cells (ESC) or from human iPSC, offer the possibility to analyze the projection patterns of grafted human cortical neurons within the adult mouse brain20. These transplantation protocols have also been used to repair brain lesions by employing mouse ESC-derived cortical neurons22. In the present study, we used a human neuronal-chimeric mouse by transplanting human NPC into the brain of immune-deficient newborn mice. We analyzed the in vivo integration and maturation of cortical cells from a control individual in different regions of mouse brain. We then co-transplanted, for the first time, the neurons from the control individual and from an ASD patient with a heterozygous truncated SHANK3 mutation to study at early stages of neuronal maturation some key morphological parameters of mutated neurons, under in vivo conditions.The use of human-derived cellular models to study neurodevelopmental disorders such as autism spectrum disorders (ASD) is an appropriate approach to link causal genetic alterations to molecular mechanisms that may occur in humans during pre- and postnatal brain development. The “SH3 and multiple ankyrin repeat domain 3” (SHANK3) is a member of the 23.The experimental design of the study as well as the illustration of spine remodeling is depicted in Fig. We transplanted the NPC, without initial differentiation in culture, into the cortex of newborn mice and followed their maturation in vivo. We compared the morphological parameters of dendritic spines in a pool of neurons from three control individuals with the selected control neurons (CTR) which were either kept in vitro for 40–50 days or transplanted into the cortex of newborn mice for analysis at 50-day-old. Our data show a significant reduction in the density of spines as well as a decrease in the percentage of stubby spines in transplanted neurons, as compared to neurons which were kept in vitro. We also observed a significant increase in spine straightness and length in transplanted neurons, as compared to in vitro conditions Fig. d. Other 23.The detailed mapping of axonal projections from ventricular transplants is presented in Fig. 2 , it is reasonable to assume that in addition to its synaptic role, it may play other earlier functional roles after its cellular expression. Schafer et al. have shown an aberrant neurodevelopmental growth dynamic in maturing neurons derived from the iPSC of several patients with ASD, triggered by an altered chromatin accessibility19. In a second set of experiments, we analyzed the role of a SHANK3 mutation in vivo to determine if transplanted neuronal precursors mutated in SHANK3 respond to endogenous guidance cues from host brain, and if they behave differently as they do in vitro in culture. We selected a patient with severe autistic traits from our previous study, who presented a frameshift mutation in SHANK3 which leads to a premature STOP codon1. For an accurate comparison of phenotypes within cortical transplants, we co-transplanted NPC derived from a CTR individual and those from the patient with ASD. We had initially checked for identical levels of fluorescence between GFP-positive (CTR) and mCherry-positive (CTR) cells. We then quantified the densities of cells with either green or red fluorescence within each transplant and found no significant difference. CTR and ASD neurons displayed a mixed homogeneous distribution within the transplant with no visible boundaries with host cortical cell layers. We analyzed the development and maturation of transplants at 30 days post-injection which is reported to be a sufficient period for the development of cortical and subcortical projections21 and a stage at which the early events can be observed. Several morphometric studies have shown a reduction in neuronal size in the neocortex of patients with ASD27. We used the approach which has been developed by Lingley et al. to specifically map neuronal soma size to identify the differences between neuronal cell populations28. By applying the same color code and by ranging neurons within the same size categories, we were able to evaluate the distribution of the soma size of human grafted CTR and ASD neurons within the host cortex. Our results show a reduction in soma areas in grafted ASD neurons as compared to CTR neurons. This phenotype was specifically observed for somas with areas that were smaller than 40 μm2. In agreement with our results, other authors have recently reported a reduction in cell soma size in cultured iPSC-derived neurons under SHANK3 knockdown conditions and during the early stages of maturation30. We did not observe any difference in cell morphology between human ASD cells transplanted alone or with the control cells. This indicates that the presence of CTR cells does not modify the survival of ASD cells under our experimental conditions. These data are in accordance with those we obtained using separate cultures of CTR and ASD cells1. We did not detect any increase in caspase-3 labeling in ASD neurons as compared with CTR neurons. These data suggest that at early stages of neuronal development and maturation, the reduction in cell soma size is neither a consequence of apoptotic events with neuronal death, nor a consequence of the existence of deleterious experimental conditions. In addition, it has been reported that vascularization of transplanted human cerebral organoids reduces cell apoptosis31. We have also reported a strong vascularization of transplants from NPC by two-photon imaging23. This suggests that vascularization may have a strong protective effect as well. We also observed a decrease in the densities of cortical axonal projections from ASD neurons as compared to CTR neurons, as illustrated for the infra-limbic and piriform cortical areas. Our data are in agreement with those of Pagani et al. who recently reported a defective short-range cortico-cortical wiring in a mouse model presenting a homozygous loss of the Shank3B isoform17. In contrast to our previous in vitro results, we did not observe any significant difference in morphological parameters of dendrites and spines from transplanted CTR and ASD neurons. Since SHANK3 is known to play a crucial role in both synaptic development and function, our results suggest that compensatory changes may occur under in vivo conditions, as was recently observed in Shank3 knock-out mice having partial deletions of exons32. Other authors also reported unexpected levels of complexity and modularity of SHANK3 functions in vivo using mouse models33. Our findings confirm that a heterozygous mutation in SHANK3 associated with a reduced SHANK3 expression1 may significantly dysregulate some initial properties of neuronal development and maturation, at least with respect to cortical neurons. The fact that we did not observe any modification in morphological parameters of dendritic spines under in vivo situations as compared to in vitro models, supports the hypothesis of diverse layers of complexity in the function of SHANK3, as well as its isoforms and the other members of SHANK family. These observations are also in line with the diversity of phenotypes, including the lack of behavioral effects that are observed in heterozygous Shank3 mouse models which have been developed35.The second objective of the present study was to take a further step towards understanding the consequences that are associated with In conclusion, as compared to in vitro studies, the humanized chimeric mouse models offer the possibility to better understand the role of human mutations that are associated with ASD and provide the opportunity to visualize the neuronal phenotypes in more details.1.We confirm that all methods were carried out in accordance with relevant guidelines and regulations. We also confirm that the experimental protocols were approved by the named institutions. Informed consents were obtained from all subjects as detailed in our previous study16. For the control iPSC lines, we used GM04603 and GM01869 from Coriell Biorepository . We also used the line PB12, which we described in our previous study. The NPC obtained from the three control individuals for this study were referred to as “C1 + C2 + C3” in the present study. Briefly, 6-well culture plates with glass coverslips were coated under the flow hood with 0.01% poly-ornithine solution diluted to 1/6 in DPBS. After an overnight exposure at 37 °C, the coverslips were washed three times with DPBS. Then we added the laminin stock solution of 1 mg/ml, which was diluted 500 times in DPBS and incubated the coverslips for at least 4 h. at 37 °C. The NPC were then plated at the density of 50,000 cells/cm2 in the 6-well culture plates with the glass coverslips in 3 ml of culture medium. The culture medium consisted of 500 ml of DMEM/F-12 mixed with 500 ml of Neurobasal medium and supplemented with 2 vials (5 ml each) of N2 supplement, 2 vials (10 ml each) of B27 supplement, 10 ml of Penicillin-Streptomycin , 1 ml of 50 mM 2-mercaptoethanol Stock solution and laminin (1/500), as described previously1. After removal of culture medium, fresh N2B27 medium containing 2 μg/ml of fresh laminin solution was added to keep the neurons attached on the glass coverslips and to avoid clumping. Medium was changed every 3 days.The production of iPSC, their characterization, commitment to the neural lineage and derivation of stable NPC were performed according to Boissart et al.1. The patient for this study carried a de novo frameshift mutation which leads to a premature STOP codon. The pedigree of the family carrying the L1142Vfs*153 mutation as well as the clinical features of the patient are presented elsewhere3.For the in vivo transplantation into newborn mice, we used NPC derived from the GM04603 control individual (CTR) and from the “AUN291_3/L1142Vfs*153” ASD patient as representative samples from our previous study1. Lentiviral vectors were prepared according to a published protocol36. The map of the CMV vector is detailed in Fig. −3 μg/ml), epidermal growth factor (10 × 10−3 μg/ml) and fibroblast growth factor (10 × 10−3 μg/ml), then aliquoted in cryotubes using FBS with DMSO (10%) and frozen in liquid nitrogen. Eight to 10 days before transplantation, labeled NPC were thawed, plated and amplified using the same supplemented culture medium described above. Culture steps of NPC are detailed in Figure NPC were labeled either by transduction with a CMV-GFP-lentivirus (green fluorescence) or with a CMV-mCherry-lentivirus (red fluorescence) as described in Gouder et al.5 cells/µl) before transplantation according to D’Alessio et al.23. GFP-labeled cells were transplanted into either the motor cortex or into the lateral ventricle of immunodeficient NOD/SCID mice (Charles Rivers Laboratory), at postnatal day 1–day 2. Pups were first locally anesthetized using a lidocaine gel. The head of newborn pups was gently held horizontally between the operator’s fingers. Bregma was used as a visible landmark to localize the injection site. We used the published coordinates Dorso-Lateral = + 1 mm, Antero-Posterior = − 1 mm, depth = − 1 mm to target the cortex, or depth = − 2 mm to target the ventricle23. Two µl of resuspended cells were injected slowly using a Hamilton syringe  unilaterally. The syringe needle was set for injecting at 1- or 2-mm depth and was kept in the pups’ brain for 30 s following the injection before it was removed. The injection itself was done over 1 minute. This protocol was approved by the Institut Pasteur Ethics Committee and the French “Ministère de l’Education Nationale, de la Recherche et de l’Innovation” under reference APAFiS #20686. The accuracy of injection was monitored by immunofluorescence labeling of GFP-labeled transplants, after brain slicing at different periods post-injection. All pups per litter were injected and their survival was evaluated at 60%. Only the mice which presented well-defined GFP-labeled transplants within the same cortical areas were kept for further analysis. The co-transplantation of equal numbers of CTR and ASD cells was monitored by initial cell counting and immunofluorescence labeling of the distinct cell populations. Brains with small number of labeled transplanted cells were excluded from our analysis, even though they presented similar phenotypes.Cultured NPC were dissociated and resuspended in DPBS  and Xylazine , and were transcardially perfused with 15 ml of DPBS, followed by 50 ml ice-cold paraformaldehyde (PFA 4%) in 0.1 M phosphate buffer (pH 7.4). Brains were dissected and post-fixed overnight at 4 °C in 4% PFA. Fifty μm coronal sections were obtained using a vibratome , collected in 24-well-culture plates, and stored in DPBS at 4 °C until use. Briefly, the free-floating sections were rinsed twice in PBS (pH 7.4) and then incubated in DPBS solution containing 0.5% Triton X-100 and 10% serum at room temperature (RT) for 30 min to block nonspecific binding. Primary antibodies, diluted in blocking solution, were added overnight at 4 °C. The list of primary and secondary antibodies with their respective dilutions is presented in Table Immunofluorescence analysis on brain sections was performed at 30 and 50-days post-transplantation according to our previously described protocol with slight modifications37. Briefly, confocal imaging was performed using a confocal laser-scanning microscope with a 40X oil NA = 1.3 objective, a 488 nm laser for GFP excitation, and a 555 nm laser for mCherry excitation. A Z spacing ranging from 150 to 300 nm was used for Z-stack acquisitions. A semi-automatic tracing of dendrites and automated segmentation of spines was performed using the Filament Tracer module of Imaris 9.5 software . Spine categories were defined by their morphology as follows: Stubby: length < 1 μm; Mushroom: Length (spine) < 3 μm and Max width (head) > mean width (neck) × 2; Long thin: Mean width (head) ≥ Mean width (neck); Filopodia-like: length ≤ 4 μm (no head). For the quantification of cell soma areas of transplanted cells, Z-stacks were acquired under the same microscopic parameters. Soma size areas were measured using Fiji software.We have previously reported a stepwise protocol for imaging of dendritic spines after immunofluorescence in a video journalStatistical analysis was performed using GraphPad Prism Version 6 software . Statistical analyses are reported in each figure legend. 95% confidence levels have been used.Supplementary Information.