These findings support a cell-type-specific role of miRNA-mediated gene expression in cortical expansion.Cortical expansion in primate brains relies on enlargement of germinal zones during a prolonged developmental period. Although most mammals have two cortical germinal zones, the ventricular zone (VZ) and subventricular zone (SVZ), gyrencephalic species display an additional germinal zone, the outer subventricular zone (oSVZ), which increases the number and diversity of neurons generated during corticogenesis. How the oSVZ emerged during evolution is poorly understood, but recent studies suggest a role for non-coding RNAs, which allow tight genetic program regulation during development. Here, using   •oSVZ-expressed microRNAs 137 and 122 promote superficial layer identity of neurons•miR-137 promotes basal progenitor proliferation and layer 2/3 neuron generation•miR-122 slows down neuronal differentiation pace Tomasello et al. demonstrate the role of two microRNAs (miRNAs) during cortical expansion. miR-137 induces proliferative behavior on basal progenitors in the subventricular zone (SVZ), and miR-122 slows down excitatory neuron differentiation progression. These distinct but complementary processes amplify the generation and molecular acquisition of layer 2/3 excitatory neurons. The human cortex has circumvolutions, but many species, including within primates, have a smooth cortex instead , 2009. AHow SVZ expansion occurred during evolution is poorly understood, but fine regulatory gene expression control over the proliferation and differentiation of progenitors and neurons may have played a role . Non-codGSE60687). This analysis identified 23 expressed microRNAs, with miR-137 and miR-122 being the most strongly expressed in the oSVZ (GSE63203). miR-137 is also expressed in the embryonic oSVZ of macaques  of the developing ferret cortex on postnatal day 2 (P2), when superficial layer (SL) neurons  are being born  on E14.5, when SL neuron generation is starting. We first performed a time course analysis 24, 48, and 72 h after electroporation  to assess progenitor proliferation, using KI67 as a marker for cycling cells. We found that miR-137 expanded progenitor numbers (KI67+ cells) 48 h after IUE . A scrambled (scram) version of miR-137 was used in control experiments. We distinguished APs, BPs, newborn (N0) neurons, and differentiating (N1) neurons by their transcriptional identity  or decrease (by Myt1l) in progenitor numbers and BP/AP ratio , confirming this effect . We used a control dataset of P7 neurons electroporated with scram on E14.0 (putative L4 neurons) and E15.5 (putative L2/3 neurons) to predict the molecular identities of miR-137-overexpressing neurons on P7 using a machine learning classifier (h current), an electrophysiological feature normally not found in L4 neurons and instead present in L2/3 neurons , confirming this effect , distinguishing APs, BPs, N0 neurons, and N1 neurons based on their transcriptional identity . Projection of human neurons in the mouse pseudo-maturation axis showed that they were even more immature than miR-122-overexpressing neurons , confirming this effect , supporting this possibility (GSE159596), which revealed that miR-122 neurons in the SL and DL displayed an increased ratio of L2/3-predicted to L4-predicted cells (GSE159596). Analysis of this dataset revealed that miR-122 neurons had a lower pseudo-maturation score than control neurons on P7, whatever their laminar position, with DL neurons being most immature  and white matter (WM), whereas the other half settled correctly in the SL A. Althousibility A. We nexed cells B, suggesed cells  results ed cells C. We neximmature D. In conimmature D, L4-locimmature F. Furtheimmature G. These immature E and S5Dimmature E, and inimmature F.Figure Cd63 and repression of Myt1l, a transcription factor promoting neuronal identity. On the other hand, miR-122 slows down neuronal maturation.Our results reveal that miR-137 and miR-122 expression in mice affects cortical development by acting on pre- and postmitotic cells, respectively. miR-137 mostly acts on BPs, maintaining their proliferative state, including through indirect upregulation of the extracellular matrix receptor We identify miR-137 and miR-122 as being expressed in the ferret oSVZ during SL neurogenesis. In the oSVZ, BPs highly proliferate to sustain cortical expansion and folding . Recent Cd63), which is known to promote proliferation of BPs  and gyrencephalic species is supported by essentially shared target genes and Gene Ontology families between mouse and human .Alexandre Dayer passed away during the submission process of this work. Further information and requests for resources should be directed to and fulfilled by his representative, Denis Jabaudon  0 and P21 for the postnatal experiments. Pregnant dams were kept in single cages and pups were kept with their mothers until P21, in the institutional animal facility under standard 12:12 h light / dark cycles.All experimental procedures were approved by the Geneva Cantonal Veterinary Authority and performed according to the Swiss law. Embryonic day (E) 0.5 was established as the day of vaginal plug. Wild-type CD1 mice were provided by Charles River Laboratories. Male and female embryos between E12.5 and E15.5 were used for the https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE60687 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63203.The miR expression data was obtained from HEK-T293 cells were maintained in culture flasks  with Dulbecco's Modified Eagle Medium  supplemented with 1% penicillin-streptomycin and 10% fetal calf serum  in an incubator .Timed pregnant CD1 mice were anaesthetized with isoflurane  and treated with the analgesic Temgesic . Embryos were injected unilaterally with 700 nL of DNA plasmid solution (diluted in endofree PBS buffer and 0.002% Fast Green FCF (Sigma)) into the lateral ventricle. Embryos were then electroporated by holding each head between circular tweezer-electrodes  across the uterine wall, while 5 electric pulses  were delivered with a square-wave electroporator .Injected plasmids were: pUB6-GFP and pUB6-TOM ; pSilencer-U6-scram, pSilencer-U6-miR-137 and pSilencer-U6-miR-122 (1 mg/mL); dUP-Cd63 and dUP-Myt1l ; DFRS, neo Vector  using the In-Fusion Kit (Clontech). The mir-137 and miR-122 sequence was thus under the control of the constitutive human RNA Polymerase III promoter U6.A vector backbone pSilencer 2.1 was used to clone the pSilencer-U6-miR-137 and the pSilencer-U6-miR-122. MiR-137 and miR-122 sequence is flanked by BamHI and HindIII excision sites, allowing the insertion into the pSilencer 2.1-U6 The Dual-Fluorescent-GFP-Reporter/mRFP-Sensor plasmid (DFRS) is able to detect strong and weak expressions of miRNAs at a single cell level . With ThThe DFRS137 and DFRS122 were the sensors of the level of miR-137 and miR-122 in the cell. For this, the 3′UTR of the RFP gene in the DFRS plasmid contained two perfect complementary sequence of miR-137 or miR-122 in tandem. Thus, these sequences were recognized and targeted by miR-137 or miR-122, which induced the degradation of the messenger RNA of the RFP. First, the primers for the complementary sequence of miR-137 or miR-122 were designed (miR-137 or miR-122 target cassette primers), flanked by PacI and FseI restriction sites. The cassette was then inserted by digestion and ligation into the pGEM shuttle plasmid. The complete cassette containing the 3′UTR information was amplified (pGEM 3′UTR cassette primers), digested by EcoRI and NotI and ligated to the DFRS empty plasmid, pre-amplified (DFRS empty plasmid primers) and pre-digested by the same enzymes . The miR-137 or miR-122 complementary sequences were thereby located on the 3′UTR of the RFP gene.CAG-loxP-mClover3-polyA-loxP-mScarlet-MCS-polyA). Upon plasmid delivery, only mClover3 is constitutively expressed. As this gene is flanked by loxP sites, subsequent delivery of Cre protein results in the excision of mClover3 sequence, allowing for expression of mScarlet and the gene of interest (cloned at the MCS). A vector backbone . Genes of interest were PCR amplified from a cDNA library from mouse embryonic brain RNA extracted at E14.5.A vector backbone (pUG001) was constructed to allow for temporal control of gene expression  was digested with BglII  and Gibson Assembly  was used to introduce Cd63 (pUG016) and Myt1L (pUG018) downstream of T2A site.For chronic administration, an osmotic pump  was filled with 10 mg/mL solution of EdU  was prepared in 1:1, DMSO: water and placed in the peritoneal cavity at the end of the surgery or at given gestational day . For sinAnesthetized pups were placed in a stereotaxic apparatus at postnatal day (P) 9 and injected with red Retrobeads (Rbeads) IX from Lumafluor in the primary somatosensory cortex (S1) .in utero electroporation and post-fixed overnight in 4% paraformaldehyde  at 4°C. Postnatal mice from P0 were perfused intracardially with 4% PFA and post-fixed overnight in 4% PFA at 4°C.Embryos were collected 24, 48 and 72 hrs following 80 μm coronal sections were performed using a vibratome . Sections were permeabilized in PBST  and incubated for two hrs at room temperature in blocking solution (10% Horse Serum Albumine in PBST), then overnight at 4°C with primary antibodies. Treatment with HCl 2N at 37°C for 30′ was performed before incubation with standard blocking solution for KI67 immunohistochemistry. Treatment with Na citrate pH 6 at 80°C for 40′ was performed before incubation with standard blocking solution for RORB immunohistochemistry.Sections were rinsed three times in PBST and incubated for 2 hrs at room temperature with corresponding secondary antibodies . Three washes in PBST were performed, followed by 10 min incubation with Hoechst staining solution  to label nuclei, before dry mounting on slides with Fluoromount (Sigma). For imaging, the putative primary somatosensory cortex (S1) was used as region of study for all the experiments. Images were acquired on Eclipse 90i epifluorescence microscope (Nikon) or on LSM 700 confocal laser scanning microscope (Carl Zeiss).Chicken anti-GFP ; Goat anti-TOM ; Rabbit anti-RFP ; Mouse anti-PAX6 ; Rat anti-EOMES ; Rat anti-EOMES ; Rabbit anti-KI67 ; Rabbit anti-NEUROD2 ; Mouse anti-RORB ; Rabbit anti-CUX1 , Rabbit anti-CASPASE3 , Mouse anti-BRN2 . All secondary antibodies were 488/555/647 conjugated .pSilencer scram, pSilencer miR-137 or pSilencer miR-122 along with pUb6-tdTOM (reporter) plasmids. Briefly, brains were dissected out in ice-cold HBSS (Gibco), embedded in 3% LM-agarose (Roth) and cut 250 μm-thick with a vibratome (Leica VT1000S). Slices were then transferred on Millicell inserts (Merck Millipore) on NBM medium in incubator at 37°C for at least 2h recovery before being placed in a Fluorodish (WPI) for imaging. In all the experiments, scramble and miR-electroporated cell were imaged simultaneously. Statistical significance was set at α < 0.05. All calculations were done using Excel (version 16.31) and statistics were done with GraphPad Prism software (version 8.1.2). Normality of the samples was assessed with D'Agostino-Pearson test and non-parametric tests used when criteria were not fulfilled.Acute slices were prepared at E17.5 from brains electroporated into the dorsal pallium at E14.5 with either 2 humidified at 95%. 50μm-thick z-stacks (3μm-stepped) were acquired with resonant laser scanning every 10min for 12h. In all the experiments, scramble and miR-electroporated cells were imaged simultaneously. At the end of the imaging session, stacks were piled into maximal intensity projections and time sequences corrected for drift with ImageJ (StackReg). To measure the multipolar to bipolar switch  equipped with long-working distance 20x or 40x objectives . The microscope chamber was kept at 37°C, with 25L/h continuous flux of 5% COr switch F, a box r switch F and S4Bn = 6 Scramble and n = 6 miR-137 embryos) and E17.5 (n = 6 Scramble and n = 6 miR-122 embryos) after E14.5 in utero electroporation extracted in ice-cold HBSS. 300μm-thick acute coronal brain sections were cut after embedding in 4% low melt-agar using a vibratome  under RNase-free conditions. The putative S1 was microdissected using a Dissecting Scope  and incubated in 0.05% trypsin at 37°C for 5 minutes. Following tissue digestion, cells were incubated in fetal bovine serum and manually dissociated via gentle pipetting. Cells were then centrifuged for 5 minutes at 500 rpm, resuspended in 1 mL of HBSS, filtered using a 70 μm-pored cell strainer  and incubated for 10 minutes at 37°C with Hoechst (0.1 mg/mL).Tissue collection was performed as in Tissue collection was performed as in . Brieflyn = 4 E14 GFP electroporated pups; n = 4 E15.5 GFP electroporated pups. P7: n = 4 E14 GFP electroporated pups; n = 4 E15.5 GFP electroporated pups; n = 4 E14.5 Scramble + GFP electroporated pups; n = 4 E14.5 miR-122 + GFP electroporated pups; n = 4 E14.5 miR-137 + GFP electroporated pups.P3: vs. L2/3 comparison at P3 and P7 (i.e. E14 vs. E15.5 GFP electroporated pups), layers were microdissected following the same procedure as described above with further separation of superficial (SL) and deep (DL) layers. 5000 to 10′000 SL and DL cells were next dissociated by incubating microdissected samples in 0.5 mg/mL Pronase  at 37°C for 10 minutes, followed by a 3 minute inactivation in 5% bovine serum albumin, washes in ACSF and manual trituration using pulled glass pipettes of decreasing diameters. Cells were then centrifuged for 10 minutes at 500 rpm, resuspended and filtrated using a 70 μm- cell strainer  and incubated for 10 minutes at 37°C with Hoechst (0.1 mg/mL).For L4 +/Hoechst+ embryonic and postnatal cells were sorted using a Beckman Coulter Moflo Astrios FAC-sorter according to their Forward and Slide scattering properties, and their negativity for Draq7™ . 5000 to 10′000 cells were FAC-sorted for each experiment. 3 μL of C1 Suspension Reagent (Fluidigm) was added to 10 μL of FACsorted cells, which were captured into 800 well- AutoPrep integrated fluidic circuit (IFC) designed for 10 to 17 μm diameter-cells  for embryonic cells, and for 10 to 17 μm diameter-cells  for postnatal cells, and imaged using the ImageXpress Micro Widefield High Content Screening System (Molecular Devices®). cDNA synthesis and preamplification was processed following the manufacturer's instructions . cDNA libraries were prepared using Nextera XT DNA library prep kit (Illumina), quality control was done using 2001 Bioanalyzer from Agilent and sequenced using HiSeq 2500 sequencer for E14.5 electroporated pups, and HiSeq 5000 sequencer for E14 and E15.5 electroporated pups.Singlet GFPin utero electroporated miR-122 pups at P7 (n = 3 pups). S1 was microdissected following the same procedure as described above with further separation of superficial (SL) and deep (DL) layers. 5000 to 10’000 SL and DL cells were dissociated and FAC-sorted as for single-cell RNA sequencing. RNA from each sample was extracted using Total RNA Isolation System kit (Promega SV) and quality control was done using 2001 Bioanalyzer from Agilent. cDNA libraries were obtained using SMARTseq v4 kit  and sequenced using HiSeq 2500 sequencer. All single cell RNA capture, library preparations and sequencing experiments were performed at the Genomics Core Facility of the University of Geneva.Brains were collected from E14.5 utero electroplated (at E14.5) CD1 mice from P20-P26 were used for electrophysiological recordings. Mice were anesthetized with isoflurane and decapitated to dissect out the brains and were immediately transferred into ice-cold sucrose cutting solution equilibrated with 95% O2 and 5% CO2 containing (in mM) Sucrose (75), NaCl (85), CaCl2 (0.5), MgCl2 (4), NaHCO3 (24), KCl (2.5), NaH2PO4 (1.25) and glucose (25). Three hundred μm thick coronal slices were cut using a vibratome (Leica VT 1200S).In For another set of experiments, embryonic slices were obtained at E17.5 from electroporated embryos (E14.5). Briefly, embryos were surgically dissected and transferred in ice-cold HBSS , embedded in 2% agarose solution, and four hundred μm thick coronal slices were cut in HBSS using a vibratome (Leica VT 1200S).2 (2.5), MgCl2 (1), NaHCO3 (26), KCl (2.5), NaH2PO4 (1.25) and glucose (25). Slices were kept at room temperature in a recovery chamber until recording. For recording, slices were transferred to a recording chamber continuously perfused with oxygenated ACSF that was maintained at 30 ± 0.1°C  using an in-line heating system . All the recordings were carried out into the somatosensory cortex; the barrels in L4 were used as a visual landmark for identification. Embryonic recordings were carried out in intermediate zone. Cortical layers 2/3 and 4 neurons were visualized by using an upright microscope and camera system , equipped with a 40x water-immersion objective, infrared/differential interference contrast (DIC) optics, and epifluorescence . Electroporated neurons were identified using GFP expression and were used for further recordings. Whole-cell recordings were obtained with recording pipettes of resistance between 3 and 5 MΩ. The recording pipettes were pulled using borosilicate glass capillaries  on Zeitz DMZ puller (Zeitz instrument). Pipettes were filled with an internal solution containing (in mM) CH3KO3S (140), MgCl2 (2), NaCl (4), creatine phosphate (5), Na2ATP (3), GTP (0.33), EGTA (0.2) and HEPES (10) adjusted to 295 mOsm and pH 7.3 with KOH.Slices were incubated at 35 °C for 20 min in a slice recovery chamber filled with artificial cerebrospinal fluid (ACSF) containing (in mM) NaCl (125), CaCls). Neurons with a stable Rs and stable resting membrane potential (RMP) negative than −60 mV were subjected to a battery of current injection protocol to study electrophysiological properties. Ih currents were recorded in voltage-clamp using a - 40 mV step (500 ms) and were calculated by the difference of current between the beginning and the end of the voltage step. Spontaneous excitatory postsynaptic currents (sEPSCs) were recorded at Vh = −70 mV 1 μM of SR95531 hydrobromide  was bath applied to block inhibitory neurotransmission. Firing characteristics were studied in the current-clamp mode by injecting incremental steps of depolarizing currents from+50 pA to 500 pA for 500 ms.Whole-cell voltage-clamp recordings were obtained using a Multiclamp 700B amplifier (Axon Instruments) filtered at 3 kHz and digitized at 20 kHz . Neurons were held at −70 mV in voltage-clamp mode and a pulse of −4 mV was given at 0.1 Hz to monitor series resistance  downstream of the luciferase gene in the pmirGLO Dual-Luciferase miRNA target expression vector .2O). Firefly Luciferase and Renilla luminescence were measured using Dual-Glo Luciferase Assay System  on an Infinite M1000Pro (Tecan) instrument.HEK293T cells were seeded in 96-well plates  at a density of 35,000 cells in 50 uL culture medium  per well. Twenty-four hours later, cells were transiently transfected with pmiRGLO or pmiRGLO +3′UTR plasmids (40 ng/well) and miRNA delivering plasmids (60 ng/well) using Polyethylenimine (PEI) transfection . Where indicated, scram, miR-137 and miR-122 were analyzed together in order to perform more robust statistical analysis (One-way Anova in place of Unpaired t Test).Zen (Zeiss) and ImageJ softwares were used to analyze images. All results are shown as mean ± SEM, except when indicated otherwise. For statistical analyses, the following convention was used: n = 3) or pUB6-TOM + pSilencer-U6-miR-137  or pUB6-TOM + pSilencer-U6-miR-122  at E12.5 or E14.5, were used to quantify the number of KI67+, NEUROD2+, PAX6+ and EOMES+, cells among the fraction of TOM+ cells 36, 48 or 72hr after IUE. Three sections for each brain electroporated with pSilencer-U6-scram + dUP-Cd63  or pSilencer-U6-scram + dUP-Myt1l  at E14.5 were used to quantify the number of KI67+, NEUROD2+, PAX6+, EOMES+, cells among the fraction of GFP+ cells 48hr after IUE.+ in E16.5 scram: 47.91 ± 1.6, E16.5 miR-137: 75.11 ± 3.12, E16.5 miR-122: 50.37 ± 2.31. Percentage of KI67+ in VZ E16.5 scram: 56.81 ± 1.67, VZ E16.5 miR-137: 34.69 ± 2.3, VZ E16.5 miR-122: 53.68 ± 1.17, SVZ E16.5 scram: 43.18 ± 1.67, SVZ E16.5 miR-137: 65.3 ± 2.3, SVZ E16.5 miR-122: 46.31 ± 1.17.A one way-ANOVA with Dunnett's post hoc test was used when required.+ in E17.5 scram: 49.44 ± 1.24, E17.5 miR-137: 63.09 ± 1.4, E17.5 miR-122: 52.9 ± 0.67. A one way-ANOVA with Dunnett's post hoc test was used when required.+/EOMES+ in E16.5 scram: 1.03 ± 0.02, E16.5 miR-137: 3.76 ± 0.18, E16.5 miR-122 (not shown): 1.25 ± 0.07, E14 scram: 1.13 ± 0.18, E14 miR-137: 3.25 ± 0.33. A one way-ANOVA with Dunnett's post hoc test was used for E16.5. A student-T test was used for E14.+ in E16.5 scram: 47.91 ± 1.6, E16.5 miR-137: 75.11 ± 3.12, scram + Myt1l-gfp: 34.85 ± 1.02, scram + Cd63-gfp: 73.31 ± 1.79. Percentage of NEUROD2+ in E16.5 scram: 49.46 ± 1.17, E16.5 miR-137: 35.14 ± 1.75, scram + Myt1l-gfp: 71.5 ± 2.26, scram + Cd63-gfp: 36.31 ± 1.52, E16.5 miR-137: 3.76 ± 0.18. Ratio of PAX6+/EOMES+ in E16.5 scram: 1.03 ± 0.02, E16.5 miR-137: 3.76 ± 0.18, scram + Myt1l-gfp: 1.1 ± 0.1, scram + Cd63-gfp: 2.05 ± 0.07, E16.5 miR-137: 3.76 ± 0.18. The same scram, miR-137 and miR-122 brains were used for the n = 3) or pSilencer-U6-miR-137  were used to quantify the ratio of GFP+/RFP+ cells at E15.5. The analyses have been carried blindly. Ratio of GFP+/RFP+ in DFRS: 0.97 ± 0.08, DFRS137: 0.71 ± 0.03, DFRS137+pSil-miR-137: 0.25 ± 0.03, DFRS122: 0.42 ± 0.02, DFRS122+pSil-miR-122: 0.25 ± 0.002. A one way-ANOVA with Tukey's post hoc test was used when required.n = 4) or pSilencer-U6-miR-137 . Mean value for Y position in E14.5 control: 36.13 ± 0.29, miR-137: 34.36 ± 0.27.n = 4) or pSilencer-U6-miR-122 . Mean value for Y position in E14.5 control: 36.13 ± 0.29, miR-122: 66.67 ± 0.79. The same scram brains were used for the + cells in L2/3 at P7. Percentage of EdU/GFP+ scram: 10.74 ± 0.74, miR-137: 27.55 ± 2.09. A student-T test was used.n = 4) or pSilencer-U6-miR-137  or pSilencer-U6-miR-122  were used to quantify the number of RORB+ cells among the fraction of electroporated cells in L2/3 and L4 at P7. Ratio of RORB+ in E14.5 L2/3 control: 0.06 ± 0.01, L2/3 miR-137: 0.07 ± 0.008, L2/3 miR-122: 0.03 ± 0.004, L4 control: 7.29 ± 0.46, L4 miR-137: 2.53 ± 0.23, L4 miR-122: 0.62 ± 0.11. A two-way ANOVA with Bonferroni's post-hoc test was used.+ of the counted cells has been plotted to show the expression of the marker.n = 4) or pSilencer-U6-miR-137  and injected with retrobeads (Lumafluor) at P9, were used to quantify the number of retrobeads+ cells among the fraction of TOM+ cells at P11. Ratio of retrobeads+ in L2/3 scram: 1 ± 0.14, L4 scram: 0.03 ± 0.01, L4 miR-137: 0.23 ± 0.09. A two-way ANOVA with Tukey's post-hoc test was used when required.n = 3) or pSilencer-U6-miR-122  or pSilencer-U6-miR-137  were used to quantify the number of TOM+ or GFP+/SATB2+ cells in each cortical layer at E17.5. The analyses have been carried blindly.+ of the counted cells has been plotted to show the expression of the marker.n = 3) or pSilencer-U6-miR-122  were used to quantify the number of GFP+ cells in each cortical layer at P21. Analyses were carried blindly. Distribution of scram cells: , miR-122 . A two-way ANOVA with Sidak's post hoc test was used.n = 3) or pUB6-GFP with pSilencer-U6-miR-122  were used to quantify the number of TOM+/EdU+ or GFP+/EdU+ cells in each SL or DL + WM at P7. Analyses were carried blindly. Distribution of scram cells: , miR-122 . A two-way ANOVA with Sidak's post hoc test was used.n = 3) or pUB6-TOM/pUB6-GFP and pSilencer-U6-miR-122  were used to quantify the number of CASP3+ cells at P7. Analyses were carried blindly. Percentage of scram cells: (1.67 ± 0.98), miR-122 (0.51 ± 0.51). An Unpaired t Test was used.+ of the counted cells has been plotted to show the expression of the marker.+ of the counted cells has been plotted to show the expression of the marker.Reads were mapped on mouse genome GRCm38 following the same pipeline described in . Read 1,+ cell was observed were kept for further analyses  with convoluted shapes, multiple cells, or cell(s) with debris were excluded).For single-cell RNA sequencing, each transcriptome was further associated to a manual brightfield picture annotation, where the presence of a single cell in the wells of the Fluidigm HT800 chips was checked. Only wells where a single GFP10) gene expressions.All bioinformatics transcriptomics analyses were performed using R programming language and Bioconductor packages as described below on reads per million (RPM) normalized .Postnatal single cells: Cells expressing less than 1000 genes, or more than 15% mitochondrial genes were excluded from the analysis .Clustering analysis of embryonic single cells: Seurat bioinformatics pipeline (https://cran.r-project.org/web/packages/Seurat/citation.html) was used to determine the most 2000 variable genes using the FindVariableFeatures function (selection method = vst) and to perform t-Distributed Stochastic Neighbor Embedding dimensional reduction (tSNE) from the top 10 principal components. The FindClusters function (resolution = 0.5) next revealed 4 clusters, which were assigned to apical progenitor (AP), basal progenitor (BP), immature neuron (N0), and mature neuron (N1) identities based on their gene expression analyzed using the FindAllMarkers function. Expression of cell type-specific transcripts described in Telley et al., Science 2019 was used to further validate the identified clusters  to predict differentiation status of each cell. The linear models were built using the scramble cells, ranking the genes according to their ability to predict each cell category , and re-optimized on the best 100 genes (top 50 and bottom 50), before cross-validations using leave-one-out method. Pseudo-differentiation prediction scores were then calculated for miR or human embryonic neurons from  miR-122 D experimPredictions of SL vs. DL miR-122 identity  identity (vs. DL miR-122 cells (bulk RNA sequencing), (2) L2/3 (E15.5 in utero electroporated) vs. L4 (E14 in utero electroporated) P7 scram cells (single cell RNA sequencing), or (3) P3 vs. P7 L2/3 or L4 scram cells (single cell RNA sequencing). This implementation was provided by the hingeLoss function of the bmrm R package. This allowed to rank the genes based on their ability to predict the identity, and to re-train a new model on the best 100 genes (top- and bottom- 50 genes). All model performances were addressed by leave-one-out (1) or 20-fold  cross-validations on the subset of genes, which gave a prediction value to build receiver operating characteristic (ROC) curves. The layer/age prediction scores were then calculated for E14.5 in utero electroporated P7 miR vs. scram cells by the linear combination of the top gene expression. identity B, L2/3 videntity A or P3 videntity C: A logiDifferential gene expression analyses between scram and miR-137 (E15.5: bmrm R package). This allowed to rank the genes based on their ability to predict scram vs. miR identities, and to calculate an identity score based on the best 100 genes by 20-fold cross-validations. The areas under the ROC curve (AUC) were calculated based on the sensitivity/specificity of each model using the cross-validation values (roc.stat function).7 E15.5:  or scram7 E15.5:  cells weAll gene ontologies were performed using GSEA .s fluctuation of >20% during recording were excluded from the analysis. Offline analysis of electrophysiological data was carried out using Igor pro (Wave metric), several electrophysiological parameters were manually computed as illustrated in Neurons with Rh current. Mean of cells with Ih in L2/3 scram: 59.85 ± 11.2, L4 scram: 27.32 ± 4.69, L4 miR-137: 43.53 ± 6.25. A student-t test was used.


Monocyte-derived Dendritic cells (DCs) have successfully been employed to induce immune responses against tumor-associated antigens in patients with various cancer entities. However, objective clinical responses have only been achieved in a minority of patients. Additionally, generation of GMP-compliant DCs requires time- and labor-intensive cell differentiation. In contrast, Blood DCs (BDCs) require only minimal ex vivo handling, as differentiation occurs in vivo resulting in potentially better functional capacities and survival. We aimed to identify a protocol for optimal in vitro activation of BDCs including the three subsets pDCs, cDC1s, and cDC2s. We evaluated several TLR ligand combinations and demonstrated that polyinosinic:polycytidylic acid [poly(I:C)] and R848, ligands for TLR3 and TLR7/8, respectively, constituted the optimal combination for inducing a positive co-stimulatory profile in all BDC subsets. In addition, TLR3 and TLR7/8 activation led to high secretion of IFN-α and IL-12p70. Simultaneous as opposed to separate tailored activation of pDCs and cDCs increased immunostimulatory capacities, suggesting that BDC subsets engage in synergistic cross-talk during activation. Stimulation of BDCs with this protocol resulted in enhanced migration, high NK-cell activation, and potent antigen-specific T-cell induction.We conclude that simultaneous activation of all BDC subsets with a combination of R848 + poly(I:C) generates highly immunostimulatory DCs. These results support further investigation and clinical testing, as standalone or in conjunction with other immunotherapeutic strategies including adoptive T-cell transfer and checkpoint inhibition.The online version contains supplementary material available at 10.1007/s00262-021-03109-w. Generating, enhancing, and maintaining tumor-specific immune responses is a major challenge in the search for cures for cancer. Dendritic cells (DCs) orchestrate innate and adaptive immunity, thereby inducing tailored, strong, and durable immune responses. They have therefore been extensively evaluated as an immunotherapeutic tool, including various in vivo targeting approaches , 2. HoweNumerous clinical trials have evaluated monocyte-derived DCs (moDCs) against different cancer entities, including multiple myeloma and Acute myeloid leukemia (AML). Their overall safety and induction of tumor-specific immune responses have been demonstrated –8. Howev+ CD141+ CLEC9A+ conventional DCs (cDC1s), CD11c+ CD1c+ cDC2s, and CD11c− CD303+ CD123+ plasmacytoid DCs (pDCs)  and R848 (Resiquimod), respectively. Our data suggest this to be the optimal combination for inducing a positive co-stimulatory profile in all BDC subsets, high secretion of IFN-α, and maximal secretion of IL-12p70. Activating all BDC subpopulations together increased immunostimulatory capacities compared to separately activating pDCs and cDCs with tailored protocols, indicating a synergistic cross-talk between BDC subsets during activation. Moreover, we demonstrate that activation of BDCs with this protocol results in enhanced migration, high NK-cell activation, and potent antigen-specific T-cell induction.Heparinized peripheral blood was collected from healthy donors after informed consent in accordance with the Declaration of Helsinki and approval by the Institutional Review Board of the Ludwig-Maximilians-Universität .BDCs were isolated from peripheral blood mononuclear cells (PBMCs) using the human Blood Dendritic Cell Isolation Kit II  and cultured in DC medium .Unless otherwise indicated, BDCs were activated for 20 h with 0.5 µM CpG , 5 µg/mL R848, 25 µg/mL poly(I:C)-LMV , 5 ng/mL IFN-γ (Peprotech), 2 µg/mL CD40L , 5 µg/mL protamine-RNA or combinations thereof.Protamine-RNA complexes were formed as previously described  using mMExpression of immune checkpoint molecules on BDCs was assessed using Aqua-LIVE/DEAD  and the antibodies and isotype controls listed in Table Cytokine concentrations were quantified using the human MACSPlex Cytokine 12 Kit (Miltenyi Biotec).Flow cytometry measurements were performed on a CytoFLEX S . Data were analyzed using FlowJo v10.7  and Prism v9.0.1 . All stated values are reported as mean ± SEM.pDCs were isolated from PBMCs by using the human CD304 MicroBead Kit, followed by isolation of cDCs from the flow-through by using the human Myeloid Dendritic Cell Isolation Kit (both Miltenyi Biotec). Alternatively, cDCs were isolated from whole PBMCs. pDCs were cultured for 16 h at 37 °C in DC medium, followed by stimulation with CpG for 3 h. cDCs were resuspended in DC medium and stimulated for 20 h with R848 and poly(I:C) immediately after isolation.Activated BDCs, cDCs, pDCs, or a 1:1 mixture of cDCs and pDCs were pulsed with a cytomegalovirus (CMV), Epstein–Barr virus (EBV), influenza, and tetanus (CEFT) peptide pool  for 2 h. T cells were isolated from PBMCs using human CD3 MicroBeads (Miltenyi Biotec) and stained using CellTrace Far Red (Life Technologies). DCs and autologous T cells were co-cultured at a ratio of 1:10 in R10 medium  for 5 days and subsequently stained for CD3 , CD4 , and CD8  .The lower part of a 96-transwell plate  was filled with 200 µL X-Vivo15 medium supplemented with 2% HS and either 200 ng/mL CCL19 (R&D Systems), 200 ng/mL CCL21 (Biolegend) or no chemokine. BDCs were seeded in the upper chamber in technical duplicates. Cells were harvested after 3 h incubation at 37 °C from both chambers, stained for CD11c and CD123, and analyzed by flow cytometry together with Precision Count Beads (Biolegend).NK cells were isolated from fresh PBMCs using the human NK Cell Isolation Kit (Miltenyi Biotec). BDCs were co-cultured with autologous NK cells for 24 h at a ratio of 1:10 in R10 medium. NK-cell activation was assessed by staining for CD69  or mIgG1 isotype  on cells positive for CD16  and CD56 .+ T cells were isolated using CD8 T Cell Isolation Kit (Miltenyi Biotec) and cultured in T-cell medium  supplemented with 5 ng/mL IL-7 (Peprotech) for 20 h. Autologous BDCs, activated with R848 + poly(I:C), were pulsed for 2 h with 1 µg/mL/peptide of PepTivator CEF MHC Class I Plus, WT1, or SARS-CoV-2 Prot_M, Prot_N and Prot_S . T cells and pulsed BDCs were co-cultured at a T cell/BDC ratio of 4:1 in T-cell medium containing 30 ng/mL IL-21 (Peprotech). On days 3, 5, and 7, co-cultures were expanded 1:1 with medium containing 10 ng/mL IL-7 and IL-15 (Peprotech). Antigen-specific T-cell expansion was monitored after 10 days by restimulation for 4 h with equal numbers of autologous PBMCs and 1 µg/mL peptides in medium containing 25 µM monensin and 10 µg/mL brefeldin A (both Sigma–Aldrich). Cells were stained for CD3  and CD8 . The percentage of cytokine-secreting CD8+ T cells was analyzed by intracellular staining for IFN-γ  and TNF-α .CD8BDCs were isolated from PBMCs with a median yield of 0.69% Fig. a. The exAnalysis by flow cytometry and calculation of MFI ratios revealed that all BDC subsets expressed HLA-DR and several positive co-stimulatory immune checkpoint molecules. All subsets expressed high levels of CD80 , whereas CD86 was only weakly expressed on cDC1s and cDC2s, and absent on pDCs. ICOSL was highly expressed by pDCs (24.4 ± 4.8) and cDC1s (53.1 ± 17.2), whereas CD40 was present predominantly on cDC1s (16.3 ± 2.7). Furthermore, BDCs expressed no or only minimal levels of the immune checkpoint molecules CD70, CD137L, GITRL, and OX40L. We detected no expression of co-inhibitory markers, including PD-L1, PD-L2, Gal-9, B7-H3, B7-H4, and B7-H5. All BDC subsets expressed high levels of HVEM . The migratory receptor CCR7 was weakly expressed on all subsets, with its highest expression on pDCs (7.0 ± 1.6)  led to the highest expression of PD-L1 on all BDC subsets , whereas R848 + poly(I:C) caused only intermediate levels of expression  . Using poly(I:C) ± CpG caused intermediate IL-12 levels . Interestingly, adding CpG led to significantly reduced IL-12 levels , similar to activating with R848 + poly(I:C) (1.8 × 103 ± 4.4 × 102 pg/mL). Poly(I:C) induced only negligible IFN-α secretion. Activation with CpG, R848, or protamine-RNA caused notable IFN-α secretion . Addition of IFN-γ or CD40L to the combination of R848 and poly(I:C) did not change the IFN-α response .By contrast, we observed the highest IFN-α concentrations using poly(I:C) + CpG Fig. f. AddingSince R848 + poly(I:C) caused an overall positive co-stimulatory expression on BDCs, high IL-12 secretion, and intermediate levels of IFN-α secretion, we focused on this combination for subsequent experiments.p = 0.018) if BDCs were activated with R848 + poly(I:C)   Fig. g. SimilaTo determine when cytokines are secreted by BDCs, we analyzed cytokine secretion 1, 3, 6, and 20 h after TLR activation. Owing to time constraints, we introduced an additional resting step of 16 h between BDC isolation and activation. As a control, we also evaluated a 20-h activation without the resting step.3 ± 1.1 × 103 pg/mL)  . In contrast, when we added the R848 + poly(I:C) directly after cell isolation, we observed strong IL-12 secretion for the same donors (2.1 × 10p < 0.001). "Resting" versus "no resting" prior to 20-h activation did not result in a significant difference (p = 0.781) , compared to BDCs rested for 16 h . No significant differences in IFN-γ secretion between BDCs activated for 1, 3, 6, or 20 h after the resting step was observed  for 1, 3, 6, and 20 h after 16 h "resting" or for 20 h directly after cell isolation. We analyzed T-cell proliferation and IFN-γ secretion after five and four days, respectively. BDCs activated for 20 h induced a T-cell proliferation of 59.0 ± 6.0%. This was significantly higher compared to shorter activation periods of between 1 and 6 h . Activation with CpG + poly(I:C) resulted in strong secretion of IFN-α by pDCs (4.9 × 103 ± 9.2 × 102 pg/mL), while observed cytokine concentrations for R848 + poly(I:C)-activated BDCs were lower (2.9 × 103 ± 8.1 × 102 pg/mL). We did not observe any cytokine secretion in controls without TLR ligands  for 20 h immediately after isolation. By contrast, pDCs were rested for 16 h before exposing them to CpG + poly(I:C) for 3 h, as described for optimal IFN-α secretion above. Secretion of IL-12 was higher by BDCs than by cDCs (3.5 × 10p = 0.044) , whereas the mixture of pDCs and cDCs exhibited a 1.8-fold reduction (4.4 × 102 ± 1.5 × 102 pg/mL)   significantly increased IL-12 secretion  or SARS-CoV-2, or peptides derived from the tumor oncogene WT1 . Similarly, expansion of T cells specific for SARS-CoV-2 from donors that had been previously infected by the virus was higher by R848 + poly(I:C)-activated BDCs compared to nonactivated BDCs (9.0 ± 2.1 vs. 8.2 ± 1.2). For WT1-specific T cells, R848 + poly(I:C)-activated BDCs similarly caused an improved expansion of antigen-specific T cells compared to non-activated BDCs (2.9 ± 1.1 vs. 1.6 ± 0.5).R848 + poly(I:C)-activated BDCs demonstrated enhanced expansion of CEF-specific CD8p < 0.001) at very high levels  . Addition of R848 + poly(I:C)-activated BDCs led to CD69 expression by almost all NK cells  Fig. e.p = 0.031) upon TLR activation. Similar results were obtained for CCL-21  increased the specific migration of DCs toward the chemokines CCL-19 and CCL-21. pDCs migrated toward CCL-19 only upon activation with R848 + poly(I:C) (52.3 ± 5.4%). For cDCs, TLR-activation increased migration from 44.9 ± 4.8% to 83.5 ± 3.6% , IL-12 secretion was maximal with R848 + poly(I:C). In contrast, single TLR stimulation-induced generally lower BDC responses. This is in line with previous results, describing TLR synergy between TLR3 or TLR4 and TLR8 in human moDCs and cDC2s resulting in potent IL-12p70 induction . AdditioWe decided to focus on R848 + poly(I:C) to optimize Th1 differentiation, which is induced by IL-12. Similar to our results, R848 + poly(I:C) has been reported to upregulate co-stimulatory immune checkpoints in BDC subsets and to induce cytokine and chemokine secretion  in a humanized mouse model . AdditioBy analyzing the kinetics of cytokine secretion, we found that IFN-α production by BDCs starts only a few hours after stimulation with R848 + poly(I:C), whereas the onset of IL-12 secretion appears to be later. This is consistent with results from a humanized mouse model . UnexpecEvaluation of tailored activation protocols for BDC subsets allowed us to elucidate the individual contributions of pDCs and cDCs to induce immune responses. cDCs induced stronger T-cell responses than pDCs, correlating with IL-12 secretion. The role assigned to pDCs in shaping the immune response in oncological malignancies remains controversial, with contradicting correlations between pDC infiltration and disease prognosis , 33. RecDC vaccines aim to elicit and maintain T-cell responses against tumor-specific antigens. WT1 is a universal tumor target antigen due to its expression pattern in multiple different cancer entities, thus having great potential for developing immunotherapies . HoweverDC vaccination trials to date have mainly focused on induction of T-cell responses, however, NK-cell activation by DCs constitutes an interesting upside for immunotherapies. Not only do NK cells recruit and activate further DCs, promoting T-cell responses, they can also eliminate tumor cells directly. Thus, tumor-cell material is released that can be processed by further DCs and be presented to T cells . As actiMigration of DCs to draining lymph nodes relies on the migratory receptor CCR7 and is central to induce adaptive immune responses. CCL-19 and CCL-21 promote the migration of DCs to the lymph nodes themselves and to the T-cell zone within the lymph nodes, enabling T-cell activation by DCs . Only a Our investigations show R848 + poly(I:C) to be an optimized cocktail for ex vivo activation of all BDC subsets. Our findings support the further investigation and usage in early clinical trials – as standalone or in conjunction with other immunotherapeutic strategies including adoptive T-cell transfer and checkpoint inhibition.Supplementary file1 (PDF 877 KB)Below is the link to the electronic supplementary material.


Igh) locus. The role of interleukin-7 receptor (IL-7R) signaling in Igh recombination has been difficult to partition from its role in B cell survival and proliferation. With a detailed description of the Igh repertoire in murine IL-7Rα−/− bone marrow B cells, we demonstrate that IL-7R signaling profoundly influences VH gene selection during VH-to-DJH recombination. We find skewing toward 3′ VH genes during de novo VH-to-DJH recombination more severe than the fetal liver (FL) repertoire and uncover a role for IL-7R signaling in DH-to-JH recombination. Transcriptome and accessibility analyses suggest reduced expression of B lineage transcription factors (TFs) and targets and loss of DH and VH antisense transcription in IL-7Rα−/− B cells. Thus, in addition to its roles in survival and proliferation, IL-7R signaling shapes the Igh repertoire by activating underpinning mechanisms.Generation of the primary antibody repertoire requires V(D)J recombination of hundreds of gene segments in the immunoglobulin heavy chain J recombination of the Igh, causing a severely restricted repertoire. Defects include reduced Igh antisense transcription and diminished expression of B lineage transcription factors.Baizan-Edge et al. show that the interleukin-7 receptor drives antibody repertoire diversity. Deletion of the IL-7R impairs usage of most V Il7r), which also pairs with the thymic stromal lymphopoietin receptor (TSLPR), important in fetal liver (FL) B cell survival (Igh recombination (Interleukin-7 (IL-7) is a critical cytokine for B and T lymphocyte development. It is bound by the IL-7 receptor (IL-7R), composed of the common gamma chain (γc), shared with the IL-2R, and the IL-7-specific IL-7Rα chain  stage, resulting in reduced progenitors and impaired B-lineage potential  undergo Igh VH-DJH recombination J recombination.Although IL-7Rαbination , their uin vitro . ConversH and DH genes. Large-scale processes, including non-coding transcription and Ig locus contraction, are thought to facilitate accessibility of distal 5′ VH genes to the recombination center over the DJ region, where the recombination activating gene (RAG) 1 and 2 bind  enabling more comprehensive analysis of /− mouse  to addre/− mouse , a DNA-b−/− BM B cells are not derived from the FL. Junctions between VH, DH, and JH gene segments in IL-7Rα−/− BM B cells are much more variable than FL B cell sequences, indicating that IL-7Rα−/− BM pro-B cells undergo de novo V(D)J recombination. Furthermore, they have a much more severe reduction in repertoire diversity than FL. Transcriptome analysis reveals loss of large-scale antisense intergenic transcripts in both DH and VH regions and reduced expression of key transcription factors required for Igh recombination, including EBF1 and Pax5. Thus, IL-7R signaling promotes Igh repertoire diversity in BM pro-B cells by activating mechanisms that enable large-scale access to VH and DH genes.Importantly, IL-7Rα−/− BM and two wild-type (WT) FL pro-B cell samples and compared with two WT BM datasets we previously generated  relative to WT pro-B cells (128 genes). All but three were within the WT group .To visualize Vgenotype A. A muchpertoire B. NotablH genes segregate into 16 families in 3 clans  were significantly less frequently recombined relative to the architectural state VH genes  and on several middle region families in the architectural state. This distribution also applies to the clans: loss of IL-7R reduces recombination of clan 1  as well as the middle genes from clans 2 and 3. Importantly, this suggests that the IL-7R does not influence either clans or local chromatin states selectively but rather linear positioning in the Igh V region, i.e., loss of IL-7R impairs recombination of middle and 5′ V genes in the Igh locus.The 195 V 3 clans . Their d do not recombine in IL-7Rα−/− pro-B cells  incorporations within VDH and DJH junctions. Terminal deoxynucleotide transferase (TdT), which inserts N-nucleotides, is absent in FL and first expressed in pro-B cells in the BM J recombination de novo in the BM, but loss of the IL-7Rα severely restricts usage of VH and DH genes in the formation of the primary Igh repertoire.To distinguish whether VDJn the BM . Accordin the BM D and 2E.dditions D and 2E.H genes in IL-7Rα−/− BM, together with normal DJ/VDJ ratios, suggests that signaling through IL-7R is specifically required for 5′ VH gene recombination. We first asked whether altered Recombination Signal Sequence (RSS) accessibility could account for this reduced recombination, because the local enhancer chromatin state is predominantly associated with 5′ VH genes  with IL-7Rα−/− × Rag2−/− (referred to as IL-7Rα/Rag2−/−) BM. Duplicate libraries for both genotypes were highly correlated, indicating the data are highly reproducible J recombination. Reduction in recombination of 5′ VVH genes . We perfVH genes . We perfoducible .−/− pro-B cells, VH RSSs coincided with a peak of accessibility  RSSs were used as a negative control because the Igκ locus does not become fully activated until the pre-B stage  and DJH regions  were also analyzed. Although the RNA-seq libraries were not strand specific, these known transcripts are easily distinguished from the much less frequently transcribed genic transcripts in Rag2−/− pro-B cells  or the CH (Iμ) transcript regions was relatively unchanged , downregulated in EBF1-deficient cells (Smarca4 (encoding BRG1) and Lef1, were also decreased. Conversely, FLT3R (Flt3), downregulated in pro-B cells by PAX5 , Myb, and Pdcd1 (activated and repressed by EBF1) showed no significant transcriptional changes in IL-7Rα/Rag2−/− cells. Indeed, Irf8 and Ikzf3 (Aiolos), activated by both, were more highly expressed in IL-7Rα/Rag2−/− cells. Together, these results suggest that EBF1 and PAX5 function is mis-regulated in IL-7Rα−/− cells.We next examined genome-wide alterations in gene expression in the absence of the IL-7R . Gene se/− cells B–S4D. Co-B cells . Important cells . PAX5-ac by PAX5 , was upr-B cells E. Howeve−/− than Rag2−/− cells. We used hypergeometric optimization of motif enrichment (HOMER) to identify TF motifs within these sites. This allowed us to infer TFs that bind less often (less accessible sites) in IL-7Rα/Rag2−/− relative to Rag2−/− and vice versa. EBF1 bound less often in IL-7Rα/Rag2−/− cells, correlating with reduction in its expression and that of its target genes. The PAX5 motif was not found in HOMER. We infer that the PAX8 motif, with a similar binding pattern, is PAX5, because PAX8 is not expressed in B cells. Again, we found reduced representation of this motif in ATAC-seq-accessible sites in IL-7Rα/Rag2−/− cells  caller  function/− cells A.Figure Igh locus, we examined MACS peaks in published PAX5 (GSM932924) and EBF1 (GSM876622 and GSM876623) chromatin immunoprecipitation (ChIP) data in Rag−/− pro-B cells. We quantified ATAC-seq reads over these sites to infer TF binding differences between IL-7Rα/Rag2−/− and Rag2−/− cells. Of 24 accessible sites in the Igh locus (not counting RSSs), all overlapped with one of the 34 PAX5 binding sites. Five showed a marked decrease in accessibility in IL-7Rα/Rag2−/− cells  and early B cell priming TFs, including PU.1, MYB, and RUNX  and PAX5 J recombination progressed with normal dynamics but severely restricted participation of both VH and DH genes. Previous models of a block in B cell development in IL-7Rα−/− BM J recombination.A previous study showing that IL-7Rαsurvival , suggestelopment , indicatH genes. It must be noted that we performed RNA- and ATAC-seq on a Rag2−/− background. Although an established model for studying mechanisms underpinning recombination because the Igh locus remains in an intact, poised state, it has the caveat that DH to JH recombination, which normally affects locus structure and V region accessibility, has not occurred. Nevertheless, several marks of V region accessibility are acquired, against which we measured the effect of IL-7R loss. There were no significant differences in local chromatin accessibility over VH gene RSSs in IL-7Rα/Rag2−/− cells. Importantly, low accessibility at Vκ gene RSSs suggests that surviving IL-7Rα−/− B cells have not “rushed through” to the IL-7R-independent pre-B cell stage where increased Vκ access occurs. IL-7R signaling must be downregulated to enable Igκ recombination  locus recombination in vivo , althougbination . These abination . Here, wbination  suggestsbination .−/− cells, is essential for Igh locus contraction .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Anne Corcoran  C57BL/6 mice were maintained in accordance with Babraham Institute Animal Welfare and Ethical Review Body and Home Office rules under Project License 80/2529. Recommended ARRIVE reporting guidelines were followed. Mice were bred and maintained in the Babraham Institute Biological Services Unit under Specific Opportunistic Pathogen Free (SOPF) conditions. After weaning, mice were maintained in individually ventilated cages (2–5 mice per cage). Mice were fed CRM (P) VP diet  ad libitum, and millet, sunflower or poppy seeds at cage-cleaning as environmental enrichment. Health status was monitored closely and any mouse with clinical signs of ill-health or distress persisting for more than three days was culled. Treatment with antibiotics was not permitted to avoid interference with immune function. Thus, all mice remained ‘sub-threshold’ under UK Home Office severity categorization. 6-8-week-old IL-7Rα−/− and IL-7Rα−/− x RAG2−/− mice , and 10-12 week old RAG2−/− mice  were used. Although wild-type (WT) comparison data were from 12 week old mice, IL-7Rα−/− animals were taken before 10 weeks because they produce fewer BM B cells as they age, with very few produced after 10 weeks , Ly6G , Ly6C , Ter119  and CD3e , incubated on ice for 30 mins, followed by streptavidin MACs beads (10 μl/107 cells in 100 μl) (Miltenyi) at 4°C for 15 mins. MACS LS columns were equilibrated and the flow through collected for flow sorting. MACS depletion for FL was carried out as for BM, using TER119-biotin at a higher concentration (1:200). Cells were stained for 45 mins on ice in the dark with the following sorting antibodies from BD Bioscience: BV421-anti B220,1:200; PerCP-Cy5.5-antiCD19, 1:400; FITC-anti CD43, 1:200) Thereafter, pro-B cells from IL-7Rα−/− BM and WT FL were flow-sorted for forward and side scatter and cell surface markers as B220+ CD19+, while IL-7Rα−/− x RAG2−/− and RAG2−/− BM B pro-B cells were sorted as a B220+ CD19+ CD43+ population on a BD FACSAria in the Babraham Institute Flow Cytometry facility.Following COGenomic DNA was isolated from mouse B cells using the DNeasy kit (QIAGEN) according to the manufacturer’s instructions, except for the incubation step, which was changed to 30 mins. DNA was eluted in nuclease-free water and quantified by Nanodrop.Total RNA was extracted from ~200,000 cells for each replicate using the RNeasy Plus kit (QIAGEN). cDNA preparation was performed using the Ovation RNaseq System V2 kit (NuGen) protocol, and 200 ng of cDNA (made up to 130 μL with nuclease-free water) was carried through to generate 50bp paired-end RNA-seq libraries for Illumina sequencing. cDNA was sonicated using a Covaris E220 to fragment lengths between 200-700 bp . End repair was carried out by adding 16 μL of 10x T4 Ligase Buffer (NEB), 4 μL of 10 mM dNTP mix, 3 μL T4 DNA polymerase , 1 μL Klenow , 1 μL T4 polynucleotide kinase  and 3 μL nuclease-free water, and incubating the reaction for 30 min at 20°C. Samples were purified again using QIAquick columns and eluted in 43 μL of nuclease-free water. A-tailing was then performed by adding 5 μL of 10x Klenow Buffer (NEB), 1 μL of dATP (10 mM) and 1 μL of exo minus Klenow ; the reaction was incubated at 37°C for 30 min and purified using MinElute PCR purification columns (QIAGEN) and cDNA eluted in 10 μl. To sequence multiple libraries in one lane, Illumina TruSeq adaptors (6 bp index) (TruSeq RNA Library Prep Kit v2) were ligated by adding 15 μL 2x Rapid Ligation Buffer (Enzymatics), 4 μL of Rapid T4 DNA Ligase  and 1 μL of adaptor mix  to the 10 μL of eluted cDNA. This reaction was incubated at 23°C for 30 min and 15°C for 30 min. Agencourt AMPure XP Beads (Beckman Coulter) were used to select library fragments between 200-700 bp by performing double-sided SPRI bead selection. Amplification was performed as follows: a 50 μL reaction was set up by adding 5ul of 10x Pfx amplification buffer (Invitrogen), 0.8 μL Pfx Platinum (2.5 U/μl), 2 μL of dNTPs (10 mM each), 2 μL MgSO4 (50 mM) and 1 μL of each Illumina paired-end primer  to the 38.2 μL library. Program: 94°C for 2 min, 8-11 cycles of 94°C for 15 s, 62°C for 30 s and 72°C for 30 s, and a final 10 min at 72°C. The library was purified by single-sided SPRI selection as above , 2 μL dNTP (10 mM each) and 1 μL Vent exo- . Incubation: 95°C- 4 min, 55°C- 5 min, 72°C- 15 min. Reactions for each sample were pooled and purified using QIAquick columns. Unrecobined JH sequences were removed from the samples using streptavidin magnetic beads  and samples were purified using QIAquick columns. To enrich for recombined sequences, primer extension was carried out as above  using biotinylated reverse primers for each of the four JH genes. Recombined sequences were recovered using streptavidin beads as above. Beads were washed twice using 100 μL of washing buffer and once with EB buffer, and resuspended in 42 μL of EB buffer. To amplify the library, a PE1 primer (Illumina) and a mix of four JH primers which included a PE2 adaptor sequence , were used. The resuspended beads were split into four aliquots (10.5 μL each) and 12.5 μL Pwo master mix (Sigma-Aldrich), 1 μL PE1 primer (10 μM) and 1 μL JH reverse primer mix (10 μM) were added to make a 25 μL reaction. Incubation: 94°C- 2 min, 15 cycles of 94°C- 15 s, 61°C-30 s, 72°C- 45 s, followed by a final 72°C- 5 min. The sample was pooled again, and beads were washed using 30 μL EB (QIAGEN), which was added to the supernatant containing the amplified library. These were put through a double-sided selection using Ampure XP beads (Bekman Coulter) for 200-700 bp. The library was further amplified to incorporate flowcell-binding indexes. PE1 and PE2 (including the index) primers (Illumina) were added to a PCR reaction as described above but with only 5 cycles and an annealing temperature of 55°C, rather than 61°C. PCR products were combined and purified using SPRI beads, eluting in 20 μl.VDJ-seq was carried out as previously described in −/− BM, VDJ-seq libraries were generated with approximately 6-fold less starting material, resulting in reduced numbers of sequences relative to the WT BM VDJ-seq libraries (Table S7). Nevertheless, this amount of starting material does not compromise detection of the wide dynamic range of frequency of VDJ and DJ recombined sequences . Due to the low number of cells in IL-7Rαequences .cf. for 10 min at 4°C, resuspended in 50 μl 1xTD buffer containing 2.5 μl TDE1 transposase (Illumina Nextera DNA Sample Preparation Kit), and incubated for 30 min at 37°C. Samples were purified using MiniElute columns (QIAGEN) according to the manufacturer’s instructions and eluted in 21 μl RSB buffer . PCR amplification and index incorporation were performed in a 50 μl reaction containing 5 μl of forward and reverse index primers (Illumina Nextera Index Kit), 15 μl NPM, 5 μl PPC (Illumina Nextera DNA Sample Preparation Kit) and 20 μl DNA. Libraries were purified using QIAquick PCR clean-up columns (QIAGEN) and sequenced on an Illumina HiSeq (6 libraries/lane).ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) was performed as previously described , 2015 onhttps://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). Differential expression analysis was performed using DESeq2  was performed using GSEA 4.1  and the sequence and position of the VH gene  recombined alleles. To overcome this issue, modified PE1 adaptors containing random barcodes were used to generate the IL-7Rα−/− and FL libraries, allowing PCR duplicates, biological duplicates and Illumina sequencing errors to be distinguished  and EBF1 Rag−/− pro-B .ChIP reads were mapped using Bowtie and peaks called using MACS2 (in the narrow peak mode): PAX5 RagFor calculating statistical significance between groups for the above datasets, a two-tailed ANOVA (type III) together with a pairwise t test (adjusted by Benjamini and Hochberg method) were used to calculate significance and p values (significant when > 0.05) when data were normal. When data failed the normality test a Kruskal-Wallis test (followed by pairwise Wilcox test (adjusted by Benjamini and Hochberg method) was performed. All results for specific tests are explained in the figure legends, including the statistical test used, value and meaning of n, and confidence intervals.


Differential networks (DN) are important tools for modeling the changes in conditional dependencies between multiple samples. A Bayesian approach for estimating DNs, from the classical viewpoint, is introduced with a computationally efficient threshold selection for graphical model determination. The algorithm separately estimates the precision matrices of the DN using the Bayesian adaptive graphical lasso procedure. Synthetic experiments illustrate that the Bayesian DN performs exceptionally well in numerical accuracy and graphical structure determination in comparison to state of the art methods. The proposed method is applied to South African COVID-19 data to investigate the change in DN structure between various phases of the pandemic. Therefore, correctly estimating the precision matrices with sparse structures is essential to adequately gauge the conditional dependency relations between variables. The task to estimate the precision matrix for both required –36. A porequired . An alterequired . In partation by  claim thη in η, in the range of 0.2 and 0.6 in increments of 0.02. The absolute sparsity error is computed for each graph structure for each Bayesian sparsity criterion in Eqs p = 10. The optimal threshold plots for p = 30 and p = 100 are available in the η*, for the Bayesian DN is, in most cases, in the neighborhood of the minimum absolute sparsity error and in the region of η* ∈ {0.2 − 0.4}. Both Bayesian sparsity criterion candidates perform comparably well noting, however, that The original recommendation for Np and Np respectively. The true DN isr = 10−2 and s = 10−6, see ij for i < j and λii = 1 for i = 1, …, p. The iterative shrinkage-thresholding approach jointly estimates the precision matrices for The synthetic experiment is designed to test the parameter estimation and graphical structure determination of the DN estimation for both the novel Bayesian approach (referred to as ‘B-net’) and the iterative shrinkage-thresholding estimator (referred to as ‘D-net’) from . The itestructure 1: An AR(1) model.Component 1: θij = 0.7i−j||.Component 2: θij = 0.75i−j||.structure 2: An AR(2) model.Component 1: θii = 0.1, θi,i−1 = θi−1,i = 0.05 and θi,i−2 = θi−2,i = 0.025.Component 2: θii = 1,θi,i−1 = θi−1,i = 0.5 and θi,i−2 = θi−2, i = 0.25.structure 3: A sparse random model where both components have approximately up to 80% off-diagonal elements set to zero.structure 4: A moderately sparse random model where both components have approximately up to 40% off-diagonal elements set to zero.structure 5: A scale-free model where the second component is a scalar multiple of the first.structure 6: A band or diagonal model.Component 1: θii = 1, θij = 0.2 for 1 ≤ i ≠ j ≤ p/2, θij = 0.5 for p/2 + 1 ≤ i ≠ j ≤ p and θij = 0 otherwise.Component 2: θii = 1, θij = 0.7 for 1 ≤ i ≠ j ≤ p/2, θij = 0.9 for p/2 + 1 ≤ i ≠ j ≤ p and θij = 0 otherwise.structure 7: A cluster model containing two disjoint groups.Component 1: θii = 1, θij = 0.5 for 1 ≤ i ≠ j ≤ p/2, θij = 0.5 for p/2 + 1 ≤ i ≠ j ≤ p and θij = 0 otherwise.Component 2: θii = 1, θij = 0.9 for 1 ≤ i ≠ j ≤ p/2, θij = 0.9 for p/2 + 1 ≤ i ≠ j ≤ p and θij = 0 otherwise.structure 8: A star model with every node connected to the first node.Component 1: θii = 1, θi1, = θi,1 = 0.1 and θi,j = 0. otherwise.Component 2: θii = 1, θi1, = θi,1 = 2.1 and θi,j = 0. otherwise.structure 9: A circular model.Component 1: θii = 2, θi,i−1 = θi−1,i = 1 and θp1, = θp,1 = 0.45.Component 2: θii = 4, θi,i−1 = θi−1,i = 2 and θp1, = θp,1 = 0.95.n1 = n2 ∈ {50, 100, 200} and p1 = p2 ∈ {10, 30, 100}, respectively. The Bayesian estimates are based on 10000 Monte Carlo iterations after 5000 burn-in iterations. To assess the performance of DN matrix estimation, six loss functions are considered and defined in p denotes the dimension and γi the ith eigenvalue, respectively. Notice that some loss functions utilise the true DN matrix and its estimates, while others utilise the eigenvalues and their respective estimates. p = 10, 30, 100 in structures 1−9 based on 40 replications. For each scenario, the best performing measure is boldfaced.The sample sizes and dimensions for each model are The eigenvalue based loss functions are designed to investigate the extremes of the eigenvalue spectrum. In particular, the MAXEL1 loss function highlights which estimator is favourable in a principal component setting, . A couplη, associated with the MCC values in To assess the performance on graphical structure determination, the specificity, sensitivity, false negative rate, f1 score and the MCCs are computed and defined in p = 100. p = 10; higher dimensions are available in the p = 10. Both Figs The B-net estimator generally outperforms the D-net estimator across all models and all dimensions according to the MCC, f1-score, sensitivity and false negative rate, with the exception of the star case for https://archive.ics.uci.edu/ml/datasets/spambase to investigate changes in DN structure between spam and non-spam data. In addition, the B-net estimator is applied to South African COVID-19 data, obtained from https://www.nicd.ac.za/diseases-a-z-index/disease-index-covid-19/surveillance-reports/, https://ourworldindata.org/coronavirus/country/south-africa and https://mediahack.co.za/datastories/coronavirus to investigate the change in DN structure between various phases of the pandemic.This section focuses on applying the novel Bayesian DN estimator, B-net, as well as the terative shrinkage-thresholding estimator, D-net, to the spambase dataset, available at The objective here is to compare the B-net and D-net graphical model estimates of the spam and non-spam emails. The dataset consists of 1813 spam emails and 2788 non-spam emails. The attributes include, amongst others, the average length of uninterrupted sequences of capital letters; total number of capital letters in the e-mail; an indicator denoting whether the e-mail was considered spam or not, in this study.Following the approach of , the datth of February 2020 to the 27th of April 2021. The daily metrics include, deaths; performed tests; positive test rate; active cases; tests per active case; recoveries; hospital admissions; hospital discharges; ICU admissions and the number of ventilated patients. It should be noted that no sensitive patient information is used, however, the interested reader is referred to [The 2019 novel coronavirus (COVID-19) has affected more than 180 countries around the world, including South Africa. The current body of knowledge boasts a wealth of statistical literature that aims at empowering researchers to study and alleviate the impact of the disease, see for example . Understerred to  for a deThe Bayesian differential network estimator is the first of its kind which utilises the excellent graphical structure determination and matrix estimation of the Bayesian graphical lasso . In compThe graphical structure learning is a crucial component of the Bayesian differential network estimator. The ad hoc approach provided in S1 Filep = 30 and p = 100.Contains a block Gibbs sampler, as well as, additional optimal threshold; adjacency heatmaps and graphical network figures for dimensions (PDF)Click here for additional data file.


The crystal structures of anhydrous and hydrated ceftibuten are reported. Both crystallize as zwitterions. 15H14N4O6S2, with the systematic name -7-{[(Z)-2--4-carboxybut-2-enoyl]amino}-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, is a third generation, orally administered cephalosporin antibiotic with broad antimicrobial activity and stability against extended spectrum β-lactamases. Ceftibuten can exist in various hydration states and to better understand the location of the water molecules of crystallization and their effect on the structure, the crystal structures of anhydrous (I) and hydrated (II) ceftibuten were determined and both occur as zwitterions with proton transfer from the carboxylate group adjacent to the β-lactam ring to the N atom of the thiazole ring. The β-lactam ring in (I) is almost planar but the equivalent grouping in (II) is slightly buckled. In the extended structure of (I), O—H⋯O and N—H⋯O hydrogen bonds link the molecules into a three-dimensional network. In (II), O—H⋯Oc, N—H⋯Oc, O—H⋯Ow, N—H⋯Ow and Ow—H⋯Ow  hydrogen bonds link the components into a three-dimensional network. A large void space is present within the anhydrous crystal structure that can accommodate between two and three molecules of water.Ceftibuten, C The asymmetric unit of (I)R. This is reflected in the N13—C12—C8—S7 torsion angle of 5.0 (10)°. The C24—C25—O26—O27 atoms were treated as disordered over two adjacent sets of sites with a population ratio of 0.841 (11): 0.159 (11). The β-lactam ring is almost planar with the C8/C12/C10/N9 atoms in the ring having a calculated r.m.s. deviation of 0.032 Å. Based on the refined bond distances of C3—O1 = 1.258 (9) Å and C3—O2 = 1.254 (9) Å, we have assigned the O1—O2—C3 group as a carboxylate and the N22 atom of the thiazole ring as protonated based on peaks in the residual electron density map, i.e., the molecule exists as a zwitterion in the solid state.The anhydrous compound (I)15H14N4O6S2·2.7H2O and crystallizes in the orthorhombic space group P212121 with similar unit-cell parameters to (I)R and N13—C12—C8—S7 = 17.2 (4)°. The β-lactam ring is slightly buckled in (II)The hydrated compound (II)3 each , which account for 9.2% of the unit-cell volume, as calculated in PLATON Compound (II)et al., 2016A Cambridge Structural Database search for compounds containing a β-lactam ring resulted in 1381 hits [CSD version 5.42 (December 2020), ConQuest version 2020.3.0; Groom et al., 2007et al. . Dehydration occurs following exposure to an atmosphere below 30% relative humidity at 298 K, and the material was confirmed to be anhydrous following receipt at the University of South Florida X-Ray Facility. A crystal in the form of a colorless needle was selected directly from the bulk sample (I)For rehydration, ceftibuten powder was placed in an uncapped scintillation vial within a container of pure water. The sealed container was stored at room temperature for four weeks, and a sufficiently large crystal (a colorless needle) was selected for analysis.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989022002110/hb8008sup1.cifCrystal structure: contains datablock(s) I, II. DOI: 10.1107/S2056989022002110/hb8008Isup3.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989022002110/hb8008IIsup2.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989022002110/hb8008Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989022002110/hb8008IIsup5.cmlSupporting information file. DOI: 2154016, 2154015CCDC references:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


In the crystal, N—H⋯N hydrogen bonds between neighboring benzimidazole rings form chains along the  13H13N3O, the isoxazole ring is inclined to the benzimidazole ring at a dihedral angle of 69.28 (14)°. In the crystal, N—H⋯N hydrogen bonds between neighboring benzimidazole rings form chains along the a-axis direction. Hirshfeld surface analysis indicates that the most important contributions to the crystal packing are from H⋯H (48.8%), H⋯C/C⋯H (20.9%) and H⋯N/N⋯H (19.3%) interactions. The optimized structure calculated using density functional theory at the B3LYP/6–311 G level is compared with the experimentally determined structure in the solid state. The calculated highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy gap is 4.9266 eV.In the title molecule, C Parallel chains interact through C4—H4⋯N2ii hydrogen bonds between neighboring isoxazole rings  resulting in the three-dimensional structure. The crystal packing contains no voids.The crystal packing is characterized by N—H⋯N and C—H⋯N interactions Fig. 2. Chains CrystalExplorer program . The positive electrostatic potential (blue region) over the surface indicates hydrogen-donor potential, whereas the hydrogen-bond acceptors are represented by negative electrostatic potential (red region). The shape-index . The most important interaction is H⋯H, contributing 48.8% to the overall crystal packing, which is reflected in Fig. 5b as widely scattered points of high density due to the large hydrogen content of the molecule, with the tip at de = di = 1.28 Å. In the presence of C—H interactions, the pair of characteristic wings in the fingerprint plot delineated into H⋯C/C⋯H contacts (20.9% contribution to the HS), Fig. 5c, has the tips at de + di = 2.69 Å. The pair of scattered points of spikes in the fingerprint plot delineated into H⋯N/N⋯H, Fig. 5d (19.3%), have the tips at de + di = 1.81 Å. The H⋯O/O⋯H contacts, Fig. 5e (9.6%), have the tips at de + di = 2.65 Å. The C⋯C contacts, Fig. 5f, contribute 0.9% to the HS and appear as a pair of scattered points of spikes with the tips at de + di = 3.60 Å. Finally, the C⋯N/N⋯C contacts, Fig. 5g, make only a 0.5% contribution to the HS and have a low-density distribution of points.The overall two-dimensional fingerprint plot  isoxazole, (IV) , hardness (η), ionization potential (I), dipole moment (μ), electron affinity (A), electrophilicity (ω) and softness (σ) are collated in Table 3H-benzimidazole system. The energy band gap [ΔE = ELUMO - EHOMO] of the molecule is 4.9266 eV, and the frontier molecular orbital energies, EHOMO and ELUMO, are −5.8170 and −0.8904 eV, respectively.The structure in the gas phase of the title compound was optimized by means of density functional theory. The density functional theory calculation was performed by the hybrid B3LYP method and the 6–311 G basis-set, which is based on Becke’s model ]: γNH = 3416; γCH = 3012–3263; γC=N–C=C= 1525–1672; 1H NMR : 2.32 ; 2.57 ; 4.23 ; 6.22 ; 7.00–7.60 ; 5.0 . 13C NMR : 13.2 (CH3 isoxazole); 24.3 (CH3 benzimidazole); 26.7 (CH2); 101.8 (CH isoxazole); 115.2–125.8 (CH aryl); 132.7–169.6 (C quaternary).(Z)-7-Methyl-4-(2-oxopropylidene)-1,5-benzodiazepin-2-one  and hydroxylamine hydrochloride  were brought to reflux in 40 ml of methanol for 2 h. After neutralization with NaHCOUiso(H) = 1.2Ueq(C) (1.5 for methyl groups). At the end of the refinement, the final difference Fourier map showed no residual peaks of chemical significance.Crystal data, data collection and structure details refinement are given in Table 410.1107/S2056989021002723/tx2037sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989021002723/tx2037Isup2.hklStructure factors: contains datablock(s) I. DOI: 2048487CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Furthermore, we show that βarr1 in complex with a synthetic phosphopeptide mimicking the human V2 vasopressin receptor tail that binds and functionally activates βarrs also allosterically activates C-Raf. We reveal that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for C-Raf activation. Interestingly, the interaction between βarr1 and C-Raf was considerably reduced in the presence of excess activated H-Ras, a small GTPase known to activate C-Raf, suggesting that H-Ras and βarr1 bind to the same region on C-Raf. Furthermore, we found that βarr1 interacts with the Ras-binding domain of C-Raf. Taken together, these data suggest that in addition to canonical scaffolding functions, GPCR–βarr complexes directly allosterically activate C-Raf by binding to its amino terminus. This work provides novel insights into how βarrs regulate effector molecules to activate downstream signaling pathways.G protein–coupled receptors (GPCRs) convert external stimuli into cellular signals through heterotrimeric guanine nucleotide-binding proteins (G-proteins) and β-arrestins (βarrs). In a βarr-dependent signaling pathway, βarrs link GPCRs to various downstream signaling partners, such as the Raf–mitogen-activated protein kinase extracellular signal–regulated kinase–extracellular signal-regulated kinase cascade. Agonist-stimulated GPCR–βarr complexes have been shown to interact with C-Raf and are thought to initiate the mitogen-activated protein kinase pathway through simple tethering of these signaling partners. However, recent evidence shows that in addition to canonical scaffolding functions, βarrs can allosterically activate downstream targets, such as the nonreceptor tyrosine kinase Src. Here, we demonstrate the direct allosteric activation of C-Raf by GPCR–βarr1 complexes  G protein–coupled receptors (GPCRs), also called 7-transmembrane receptors, are the largest group of membrane proteins that regulate a multitude of physiological processes , 2. ApprA). CR1, on the amino terminus, contains a Ras-binding domain (RBD) and a cysteine-rich domain (CRD); CR2 corresponds to a region rich in serine/threonine residues, whereas CR3 includes the catalytic domain on the carboxyl terminus , binds C-Raf and allosterically activates it. Therefore, GPCR–βarr complexes not only function just as typical scaffold proteins for the C-Raf/MEK/ERK module but also play an active role in regulating MAPK signaling.GPCR–βarr complexes interact with C-Raf to initiate MAPK cascade signaling , 14. BotS-transferase (GST) pull-down assays using GST-C-Raf fusion proteins and βarr1 (A). βarr1 coeluted with the GST-C-Raf amino terminus, and no βarr1 was detected for GST-C-Raf carboxyl terminus (B). These results show that GST does not interact with βarr1 and, therefore, βarr1 and C-Raf amino terminus interact specifically. Both free and active βarr1 bind C-Raf amino terminus; however, the binding was significantly enhanced in the presence of V2Rpp and the stabilizing antibody Fab30. The amino terminus of C-Raf encompasses both the RBD and CRD. To investigate the roles of both domains in the C-Raf–βarr1 interaction, GST-C-Raf-RBD, GST-C-Raf-CRD, and GST-C-Raf-CR1 (which contains the RBD and CRD) were used as bait proteins to perform the pull down with βarr1. Whereas GST-C-Raf-CRD pulled down no βarr1, GST-C-Raf-CR1 binds more βarr1 than GST-C-Raf-RBD (C). These results indicate that the RBD domain of C-Raf directly interacts with βarr1. In contrast, the presence of the CRD domain might be essential for optimal folding and stability of the RBD domain, thus contributing to the enhanced binding of βarr1. Interestingly, H-Ras, a small GTPase known to activate C-Raf, also interacts with the same domains of C-Raf (D), consistent with competitive binding of both βarr1 and H-Ras to the same region of C-Raf. Therefore, these data suggest that βarr1 binds to the amino terminus of C-Raf similarly as H-Ras.To identify the βarr1-binding elements within C-Raf, we performed glutathione-nd βarr1 A. βarr1 terminus B. These -Raf-RBD C. These of C-Raf . To inveof C-Raf D, consisA). To exclude the scaffolding function of βarr1, we measured the phosphorylation of myelin basic protein (MBP) instead of the physiological substrate MEK, by C-Raf. We used a chimeric M2 muscarinic receptor with a phosphorylated C-tail of the vasopressin 2 receptor (M2V2R) that confers tight binding to βarr1. M2V2R was reconstituted in ∼12-nm membrane scaffold protein (MSP) MSPD1E3 nanodiscs as this environment closely mimics a native membrane, and therefore, it represents a fully engaged M2V2R–βarr1 complex based on the previous cryo-EM study  and as calculated initial rates  of C-Raf activity. The empty nanodisc and Fab30 or M2V2R alone failed to increase the C-Raf activity. To further ascertain whether the M2V2R–βarr1 complex activates C-Raf, we utilized a complementary in vitro kinase assay using γ-32P labeled ATP to assess the phosphorylation of MBP by C-Raf in the absence or the presence of M2V2R–βarr1. We found that the M2V2R–βarr1 complex could increase the C-Raf activity in vitro, consistent with our enzyme-coupled fluorescence-based assay . Therefore, our findings reveal that the M2V2R–βarr1 complex allosterically activates kinase C-Raf. Next, we used an excess of the C-Raf amino terminus to test its ability to competitively inhibit the allosteric activation of C-Raf by M2V2R–βarr1. The allosteric activation of C-Raf is blocked by excess C-Raf amino terminus . These results reveal that the M2V2R–βarr1 complex allosterically activates C-Raf by interacting with its amino terminus. These data also demonstrate that βarr1 binds to the amino terminus of C-Raf.GPCR–βarr complexes have been demonstrated to allosterically activate signaling partners such as the tyrosine kinase Src , 23. To Fig. 2EM study . GPCR–βaA) . Both M2V2R–βarr1-ΔFL and M2V2R–βarr1-DDD complexes showed similar ability to enhance C-Raf activity compared with the M2V2R–βarr1-WT complex . These results suggest that the interactions neither between the GPCR transmembrane core and βarr1 nor between the C-edge of βarr1 and the lipid bilayer are necessary for the allosteric activation of C-Raf by βarr1. These findings suggest that the binding of the phosphorylated GPCR C terminus to βarr1 plays a critical role in the allosteric activation of C-Raf. To test this, V2Rpp was used to activate βarr1 in vitro. We found that the V2Rpp-bound active βarr1 could allosterically activate C-Raf, whereas inactive βarr1 failed to do so. These results also show that V2Rpp alone was sufficient to activate C-Raf, whereas Fab30 alone did not have a significant effect to this end . These results demonstrate that the interaction between the phosphorylated GPCR C terminus and βarr1 is necessary and sufficient for the allosteric activation of C-Raf.Previous studies revealed that GPCR–βarr complexes adopt two different conformations: in the “core” conformation, βarrs bind to the phosphorylated C terminus and the transmembrane bundle of GPCR; in the “tail” conformation, βarrs bind only to the phosphorylated GPCR C terminus . Recent Fig. 3A) , 27. By ormation . By intr2V2R–βarr1 complex or GTPγS-bound H-Ras. The kinetics and calculated initial rate of C-Raf activity show that both M2V2R–βarr1 and GTPγS-bound H-Ras enhance the catalytic activity of C-Raf toward MBP (D). The activation effect of GTPγS-bound H-Ras is slightly but significantly higher than that of the M2V2R–βarr1 complex. C-Raf activity is enhanced but not significantly when both the M2V2R–βarr1 and GTPγS-bound H-Ras are added together (D). In our study, the nonhydrolyzable ATP analog ATPγS was used during the purification process to stabilize C-Raf. These results show that the M2V2R–βarr1 complex or GTPγS-bound H-Ras allosterically activate ATPγS-bound C-Raf in a comparable extent. Taken together, our findings demonstrate that in addition to the canonical scaffolding function, the GPCR–βarr complexes serve as direct allosteric activators of C-Raf.Active GTP-bound H-Ras plays a critical role in the activation process of C-Raf , 20. To ward MBP D. The actogether D. In ourβarrs have been demonstrated to interact with the C-Raf/MEK/ERK cascade after coupling to multiple GPCRs and thereby mediate activation of this pathway either independent of or codependent on G proteins , 15. Pre2V2R–βarr1 complex enhance the enzymatic activity of C-Raf. Our results indicate that the interaction between βarr1 and the phosphorylated GPCR C terminus is sufficient to activate C-Raf. The precise mechanism by which a GPCR–βarr complex activates C-Raf must be the subject of future studies.Previously, βarrs have been considered adaptors and scaffold proteins that link the receptors to different signaling partners and bring various signaling components into close contact to facilitate the signal transduction process . HoweverScaffold proteins facilitate signal transduction through tethering signaling components into specific complexes. Recent studies reveal that several scaffold proteins could allosterically regulate their partner molecules in addition to tethering , 23, 33.The construct expressing FLAG-M2R with C-terminal sortase ligation consensus sequence (LPETGGH) followed by a 6× His-tag has been previously described . WT HumaEscherichia coli BL21 (DE3) cells and purified on glutathione sepharose beads (GoldBio) followed by further purification using SEC. Expression and purification of Fab30  was ligated on the C terminus of M2R by enzyme sortase as previously described (2V2R was performed as previously described (2V2R (5 mM) was incubated with twofold molar excess atropine at 4 °C for 30 min. Subsequently, M2V2R (5 mM) was mixed with a 3:2 M ratio of 8 mM 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine with 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) and 80 μM MSP1D1E3 on ice for 1 h. Bio-Beads (Bio-Rad) were added to remove the detergent, and the mixture was incubated overnight at 4 °C. High-density lipoprotein-M2V2R was further purified by M1-FLAG and SEC.The synthetic phosphopeptide GGG-Vescribed . The higescribed . In shor2V2R–βarr1 complexes or activated H-Ras was measured with a Kinase/ADP kit . In short, 100 nM C-Raf was mixed with 5 μM MBP (MilliporeSigma) as a substrate in the reaction buffer phosphine hydrochloride). ATP with the final concentration of 20 μM was added to initiate the reaction. Same volume of commercially supplied “Detection solution” was added to the reaction, then the fluorescence intensity (excitation of 540/emission of 590) was measured continuously. The concentration of βarr1 or M2V2–βarr1 complexes in all experiments was 500 nM.C-Raf activity regulated by M2Rpp and 30 μM Fab30 at room temperature for 30 min. About 7 μM GST-C-Raf-RBD or other GST-tagged C-Raf variants were then added to the reaction mixture and incubated for 1 h at room temperature. Subsequently, 10 μl of Glutathione Sepharose beads (GoldBio) were added and incubated for another hour at 4 °C with end-to-end rotation. The beads were collected and washed three times using wash buffer . Finally, the proteins were eluted in elution buffer  and visualized by Western blotting using anti-GST antibody  and anti-βarr1 antibody .About 10 μM βarr1-8× His was incubated with 30 μM Vin vitro, purified C-Raf protein (100 nM) was incubated with [γ-32P] ATP  and MBP (5 μM) at 30 °C for 30 min. Incorporation of 32P into MBP was analyzed by SDS-PAGE and autoradiography.The kinase activity of C-Raf was tested with MBP (MiliporeSigma). To measure C-Raf kinase activity lefko001@receptor-biol.duke.edu).All data presented are available upon request from Robert J. Lefkowitz (The authors declare that they have no conflicts of interest with the contents of this article.


N-substituents lie on opposite sides of the pyrazole ring. Intermolecular hydrogen bonds from the amino group to an S=O group and to the oxo substituent lead to a layer structure.The  14H17N3O5S, the five-membered ring is essentially planar. The substituents at the nitrogen atoms subtend a C—N—N—S torsion angle of −95.52 (6)°. The amino group forms an intramolecular hydrogen bond to a sulfonyl oxygen atom; two intermolecular hydrogen bonds from the amino group, to the other S=O group and to the oxo substituent, form a layer structure parallel to the ab plane. The structure determination confirms that the title compound is N- rather than O-alkylated.In the title compound, C N-sulfonylamino- and N-sulfonyl moieties. The products may be biologically active, displaying for instance anti-viral activity . The substituents S1 and C6 of the five-membered ring, which is effectively planar (r.m.s. deviation 0.026 Å) lie significantly outside the ring plane  in opposite directions; the corresponding torsion angle C6—N1—N2—S1 is −95.52 (6)°. The side chain at N1 exhibits an extended conformation. An intramolecular hydrogen bond is formed from the amino group to the sulfonyl oxygen atom O4  are not shown in the Figure. The shortest distance between ring centroids is 3.97 Å for the ring C10–C15 .The molecules of ne Fig. 2. The hyd4, and bearing the same substituents at C5 (oxo) and C3 (amino), gave eight hits involving uncharged species. However, none of these was substituted at both ring nitrogen atoms. Two  and C(=O)NH-nBu  torsion angles are very variable; in four cases the absolute value lies between 0 and 11°, whereas for the bulky substituents in DOJKIW and VOQGOZ the values are 63.7 and 32.1°, respectively.A database search (CSD Version 5.41) for the same ring system as in H-pyrazol-3-one 1 (0.01 mol), ethyl bromoacetate 2 (0.01 mol) and anhydrous potassium carbonate (0.01 mol) in N,N-dimethylformamide (5 mL) was stirred at room temperature for 3 h. The mixture was poured onto ice–water; the solid thus formed was filtered off and recrystallized from ethanol to give pale yellow crystals in 64% yield, m.p. = 415–416 K. IR : ν 3460, 3297 (NH2), 1752 (ester C=O), 1700 (ring C=O); 1H NMR (DMSO-d6): δ = 1.17 , 2.41 , 4.09 , 4.31 , 4.40 , 7.15 , 7.45 , 7.73 . Analysis: calculated C14H17N3O5S (339.36); C, 49.55; H, 5.05; N, 12.38; S, 9.45. Found: C, 49.38; H, 5.23; N, 12.59; S, 9.27%.A mixture of 5-amino-1-tosyl-1,2-dihydro-32 group were refined freely. The methyl groups were refined as idealized rigid groups allowed to rotate but not tip . Other hydrogens were included using a riding model starting from calculated positions . The Uiso(H) values were fixed at 1.5 (for the methyl H) or 1.2 times the equivalent Ueq value of the parent carbon atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989021004795/zl5011sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989021004795/zl5011Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021004795/zl5011Isup3.cmlSupporting information file. DOI: 2082046CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


New spiros have been synthesized with high regio- and stereospecificity from simple starting materials in a one-step process. Different combinations of hydrogen bonds link the molecules to form either sheets or chains of rings. S)-pipecolic acid and trans-3-benzoylacrylic acid, and subsequently characterized using a combination of elemental analysis, IR and 1H and 13C NMR spectroscopy, mass spectrometry and crystal structure analysis. -2′-Benzoyl-5-fluoro-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro-1′-carboxylic acid, C23H21FN2O4, (I), and -2′-benzoyl-5-methyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro-1′-carboxylic acid, C24H24N2O4, (II), are isomorphous, as are -2′-benzoyl-1-methyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro-1′-carboxylic acid, C24H24N2O4, (III), and -2′-benzoyl-5-chloro-1-methyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro-1′-carboxylic acid, C24H23ClN2O4, (IV). Within each isomorphous pair, the spiro ring systems show some conformational differences. In each of (I) and (II), the molecules are linked into complex sheets by a combination of four types of hydrogen bond, and in each of (III) and (IV), a combination of O—H⋯O and C—H⋯π(arene) hydrogen bonds links the molecules to form a chain of centrosymmetric rings. In -2′-benzoyl-1-hexyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro-1′-carboxylic acid, C29H34N2O4, (V), a combination of five hydrogen bonds links the molecules into sheets of alternating R22(16) and R66(46) rings. A mechanism is proposed for the formation of compounds (I)–(V) and some comparisons with related structures are made.Five new spiros have been synthesized with high regio- and stereospecificity in one-pot three-component reactions between a substituted indole-2,3-dione, ( Here we report the synthesis and spectroscopic charaterization, and the molecular and supramolecular structures of five representative examples, namely, -2′-benzoyl-5-fluoro-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro-1′-carboxylic acid, (I)SR,2′SR,3RS,8a′RS)-2′-benzoyl-5-methyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro-1′-carboxylic acid, (II)SR,2′SR,3RS,8a′RS)-2′-benzoyl-1-methyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro-1′-carboxylic acid, (III)SR,2′SR,3RS,8a′RS)-2′-benzoyl-5-chloro-1-methyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro-1′-carboxylic acid, (IV)SR,2′SR,3RS,8a′RS)-2′-benzoyl-1-hexyl-2-oxo-1′,5′,6′,7′,8′,8a′-hexahydro-2′H-spiro-1′-carboxylic acid, (V)Multicomponent reactions can provide versatile and efficient routes to new heterocyclic systems, permitting the incorporation of a wide variety of functionalities by the combination of three or more simple building blocks –(V) were formed in yields between 48 and 69% in one-pot reactions between an appropriately substituted isatin (see Scheme 2trans-3-benzoylacrylic acid [(E)-4-oxo-4-phenylbut-2-enoic acid]  in acetonitrile (10 ml) were heated under reflux until the reactions were complete, as judged by thin-layer chromatography (TLC) monitoring . The reaction mixtures were allowed to cool to ambient temperature, giving the crystalline products (I)–(V), which were collected by filtration and then dried in air. No further purification was required, as judged by TLC and spectroscopic examination, and crystals suitable for single-crystal X-ray diffraction were, in each case, selected directly from the synthesized samples.All reagents and solvents were obtained commercially and all were used as recieved. For the synthesis of compounds (I)–(V), mixtures of pipecolic acid , the appropriately substituted isatin (0.5 mmol) , which crystallizes as a partial dichloromethane solvate, the heterocyclic molecules are linked by N—H⋯O hydrogen bonds to form RS,2′RS,3SR,7a′SR)-2′-benzoyl-1-hexyl-2-oxo-1′,2′,5′,6′,7′,7a′-hexahydrospiro-1′-carboxylic acid, the molecules are linked by O—H⋯O hydrogen bonds to form cyclic S6) symmetry, which are further linked by C—H⋯O hydrogen bonds to form a three-dimensional framework structure.Overall, therefore, the supramolecular assembly is one-dimensional in each of compounds (III)In summary, therefore, we have developed a new application of the ylide/alkene procedure, which we have now used for the formation of spiros in a single step, using simple and readily available starting materials. This approach permits the incorporation of a wide variety of substituents and other functional groups for further elaboration. Five representative examples have been fully characterized spectroscopically and structurally, and their patterns of supramolecular assembly have been analysed, described and illustrated.10.1107/S2053229621007142/ky3207sup1.cifCrystal structure: contains datablock(s) global, I, II, III, IV, V. DOI: 10.1107/S2053229621007142/ky3207Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2053229621007142/ky3207IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2053229621007142/ky3207IIIsup4.hklStructure factors: contains datablock(s) III. DOI: 10.1107/S2053229621007142/ky3207IVsup5.hklStructure factors: contains datablock(s) IV. DOI: 10.1107/S2053229621007142/ky3207Vsup6.hklStructure factors: contains datablock(s) V. DOI: Click here for additional data file.10.1107/S2053229621007142/ky3207Isup7.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229621007142/ky3207IIsup8.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229621007142/ky3207IIIsup9.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229621007142/ky3207IVsup10.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2053229621007142/ky3207Vsup11.cmlSupporting information file. DOI: 2095737, 2095736, 2095735, 2095734, 2095733CCDC references: