Gαs, which transduces mitogenic signals, in 30% of the analyzed JGCTs.Ovarian granulosa cell tumors are the most common sex-cord stromal tumors and have juvenile (JGCTs) and adult forms. In a previous study we reported the occurrence of activating somatic mutations of We have searched for alterations in other proteins involved in ovarian mitogenic signaling. We focused on the PI3K–AKT axis. As we found mutations in AKT1, we analyzed the subcellular localization of the mutated proteins and performed functional explorations using Western-blot and luciferase assays.We detected in-frame duplications affecting the pleckstrin-homology domain of AKT1 in more than 60% of the tumors occurring in girls under 15 years of age. The somatic status of the mutations was confirmed when peritumoral DNA was available. The JGCTs without duplications carried point mutations affecting highly conserved residues. Several of these substitutions were somatic lesions. The mutated proteins carrying the duplications had a non-wild-type subcellular distribution, with a marked enrichment at the plasma membrane. This led to a striking degree of AKT1 activation demonstrated by a strong phosphorylation level and by reporter assays.AKT1 as a major event in the pathogenesis of JGCTs. The existence of AKT inhibitors currently tested in clinical trials opens new perspectives for targeted therapies for these tumors, which are currently treated with standard non-specific chemotherapy protocols.Our study incriminates somatic mutations of   •We detected duplications affecting the pleckstrin-homology domain of AKT1 in > 60% of the Juvenile Granulosa cell tumors studied.•The JGCTs without duplications carried point mutations affecting highly conserved residues.•Mutated proteins with duplications displayed an enrichment at the plasma membrane and a striking phosphorylation level. Our study points to AKT1 as a major driver in the pathogenesis of JGCTs.Sex cord stromal tumors involve granulosa, theca or stromal cells, alone or combined. The most common ones are ovarian granulosa cell tumors (GCTs), which represent up to 5–8% of all ovarian tumors . Two disof AGCTs . This muof AGCTs . With reof AGCTs . In anote latter . FSHR sie latter  that phoe latter . Becausee latter , we hypo22.1This study involves a cohort of 16 histologically proven JGCTs, occurring in girls under 15 years of age, collected between 1994 and 2014 . The available clinical data are displayed in 2.2Twelve samples were formalin-fixed paraffin-embedded (FFPE) (T1–T12). Four tumors were obtained as frozen samples (T13–T16). We isolated genomic DNA and RNA from FFPE tumors using the AllPrep DNA/RNA FFPE Kit (Qiagen) and the frozen ones were processed using standard procedures. To assess the somatic status of the mutations, we extracted DNA from manually isolated peritumoral tissue fragments (only available for 8 samples). We also analyzed the COV434 cell line, supposed to derive from a JGCT .Gαs gene, potentially harboring the activating mutations R201C and R201H, was performed as described , supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin .2.4AKT1RED–EcoR1-F: 5′AGCTTCGAATTCGCCACCATGAGCGACGTGGCTATTGTGAAGG3′AKT1-F2: GTGGACCACTGTCATCG3′AKT1RED–BamH1-R: 5′ACCGGTGGATCCCG GGCCGTGCCGCTGGCCGAGTAGGAGAAC3′InsertionT3R: 5′CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCAGCGGATGATGAAGG3′InsertionT8R: 5′CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCAGGCAGCGGATGATGAAGG3′InsertionT5R: 5′CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCTCGCAGCGGATGATGAAGGTGTTGG3′InsertT11R: 5′CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCTCCAGGCAGCGGATGATGAAGG3′InsertT12R: 5′CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCTCCGTCTTCATCAGCTGGCGGATGATGAAGGTGTTGGG3′InsertT14R: 5′CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCTGCAGGCAGCGGATGATGAAGG3′InsertT15R: 5′CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGATGATGAAGGTGTTGG3′InsertT16R: 5′CGATGACAGTGGTCCACTGCAGGCAGCGGATGATGAAGGTGTTGGGCCGGGGCCGCTGGCAGCGGATGATGAAGG3′E17K F: 5′GCCACCATGAGCGACGTGGCTATTGTGAAGGAGGGTTGGCTGCACAAACGAGGGAAGTACATCAAGACCTGG3′Q79K-F: 5′CATCATCCGCTGCCTGAAGTGGACCACTGTCATCG3′Q79K-R: 5′CGATGACAGTGGTCCACTTCAGGCAGCGGATGATG3′W80R-F: 5′CATCATCCGCTGCCTGCAGAGGACCACTGTCATCG3′W80R-R: 5′CGATGACAGTGGTCCTCTGCAGGCAGCGGATGATG3′Q79K–W80R-F: 5′CATCATCCGCTGCCTGAAGAGGACCACTGTCATCG3′Q79K–W80R-R: 5′CGATGACAGTGGTCCTCTTCAGGCAGCGGATGATG3′.The plasmids driving the expression of wild-type and mutated AKT1 fused to the mCherry protein were constructed by fusion PCR. Briefly, for the insertion mutations, two PCRs were performed to generate the 5′ and 3′ portions of the AKT1 coding sequence using, respectively, AKT1RED–EcoR1-F primer and the corresponding mutagenic R primer and AKT1-F2 primer along with AKT1RED–BamH1-R. After purification of the PCR products, they were quantified, mixed in similar amounts and allowed to undergo eight cycles of PCR in the absence of primers, to generate the full-length mutated coding regions. Then, a final PCR reaction was performed using the EcoR1–BamHI primers. For E17K, we used the primers E17K F and AKT1RED–BamH1-R to generate the amplicon in a single PCR. The amplified EcoR1–BamHIs were cloned (EcoR1–BamHI) into digested pDsRed vector to produce fusion proteins in frame with the mCherry. All constructs were sequenced to exclude the presence of PCR-induced mutations. The sequences of the primers used are the following:2.5HeLa cells were transfected with constructs driving the expression of AKT1 fused to the mCherry protein. Cells were seeded, in 24-well plates containing sterile coverslips, 16 h before transfection to be confluent at the time of transfection. Cells were transfected using the calcium-phosphate method with 250 ng of plasmid per well and rinsed 24 h after transfection. At this point, cells were serum-starved or not for 24 h. Forty-eight hours after transfection, cells were washed with phosphate-buffered saline solution (PBS) and fixed for 15 min with paraformaldehyde (PFA 4%). They were washed three times in PBS and nuclei were stained with Hoechst 33342 . Coverslips were mounted on microscope slides using the fluorescence mounting medium DakoCytomaton . Cells were visualized with an epifluorescence microscope provided with an ApoTome module.For Western-blot studies, HeLa cells were transfected with the constructs driving the expression of wild-type or mutated AKT1 forms. One day after transfection cells were rinsed and serum-starved or not for 24 h before lysis. Electrophoresis and Western-blot were performed as previously described . The priDual-Luciferase Reporter Assays  involved the reporter promoter 4XDBE-luc which contains 4 copies of the FOXO response element (DAF-16 family member-binding element or DBE) upstream of a minimal promoter driving the expression of the firefly luciferase gene . Each ex33.1Gαs altering protein position 201  samples, the allelic combinations (i.e. haplotypes) of the other co-occurring mutations could not be worked out, thus preventing their experimental exploration. In total, 14 out of 16 JGCTs harbor putative driver alterations of AKT1.Given the high degree of identity between and AKT2 . These m3.21H10, 3O96 and 4EJN3O964EJN). The tandem duplications described here involve the 6th beta strand, according to the ribbon model displayed in The tandem duplications described here alter the PHD of AKT1 . The PHDAKT1 in our experimental setting. The pervasive presence at the plasmalemma of the elongated AKT1 proteins suggested the existence of a high level of activation. To test this hypothesis, we analyzed the phosphorylation status of seven AKT1 variants carrying the in-frame duplications, which is supposed to reflect protein activation  or by the presence of a myristoylation signal, artificially introduced or provided by the viral Gag sequence fused to Akt in v-Akt . StructuAlthough JGCTs are of good prognosis, it would be interesting to study the properties of the filopodia-like processes induced by the mutated AKT1 carrying the duplications because of the known roles of filopodia/invadopodia in sensing, migration and intercellular interactions . These pAKT1 mutations and the involvement of the PI3K–AKT pathway in regulating cellular proliferation and survival suggests that the molecular lesions reported here are driver events. Irrespective of the precise role of AKT1 in the origin and/or progression of the JGCTs, which is yet to be studied, the uncovering of the AKT1 insertions will facilitate the molecular diagnostics of JGCTs. Our findings open also targeted therapeutic perspectives because inhibitors of the PI3K–AKT–mTOR pathway(s) are being tested in clinical trials  partners. Finally, we cannot exclude that granulosa cells may need a strong level of AKT1 activation to become transformed, which would only be achieved with the observed duplications or with several AKT1 mutations per cell, such as Q79K and W80R. The fact that most of the JGCTs harbor l trials .GEFLUC (Groupement des Entreprises Françaises dans la Lutte contre le Cancer), the Centre National de la Recherche Scientifique (CNRS), the Université Paris Diderot and the Agence Nationale pour la Recherche (Projet ICEBERG).This work was supported by LB, ALT, AA and RAV conceived and designed the study. LB, ALT, AA, LG, BL, DF and RAV developed the methodology. LB, ALT, AA, LG, BL and RAV acquired data. LB, ALT, AA, SS, DF, CS, NK, LG and RAV analyzed and/or interpreted data. All authors wrote and provided final approval of the manuscript.The authors declare no competing interests.Tumorothèque Necker-Enfants Malades and Tumorothèque Montpellier.This project was approved by the ethics committees of the tumor repositories The funding sources had no role in study design, data collection, data analysis, data interpretation, or writing of the manuscript. The corresponding author had full access to all data and had the final responsibility for the decision to submit for publication.


AbstractHeteranassa Smith , native to the southwestern United States and Mexico, includes two recognized species, namely Heteranassamima (Harvey) and Heteranassafraterna Smith. These are separated mainly by subtle differences in wing color and pattern, leading to speculation about the validity of the described species. This study examines variation in external and internal morphology across the geographic range of the genus, aiming to clarify species limits, describe morphology, and provide a comprehensive assessment of variation within the genus. Results indicate that Heteranassafraternasyn. n., is a junior synonym of Heteranassamima. Heteranassa Smith, 1899, is a genus of moths native to warmer desert regions of southwestern United States southward to southern Mexico, currently containing two valid species. Heteranassamima , described from Texas, and Heteranassafraterna Smith, 1899, described from Death Valley, California. PageBreakan additional species, Heteranassaminor Smith, 1899, with Heteranassafraterna. Heteranassa feed on mesquite (Prosopis sp.) and Acacia (Acacia sp.) (both Fabaceae) and are multivoltine , White Sands National Monument, Otero Co., New Mexico, , Cuatrocienagas Protected Area, Cuatrociénagas, Coahuila, Mexico, , Pima Co., Arizona, (July 2012), and Socorro Co., New Mexico (October 2012). Specimens were collected with a sheet trap using 15W UV fluorescent lamp, 175W Mercury Vapor lamp, or a 175W, 6500K metal halide lamp. Death Valley specimens were collected at incandescent or fluorescent outdoor lighting at the Furnace Creek Ranch Hotel.Specimen loans were generously provided by the following institutions: University of Alberta Strickland Entomology Museum, Edmonton, Alberta (F.A.H. Sperling)UASM Essig Entomology Museum, University of California, Berkeley (J. Powell)EMEC Natural History Museum of Los Angeles County, Los Angeles, California (B. Brown)LACM University of Arizona Insect Collection, Tucson (W. Moore)UAIC Arizona State University Entomology Collection, Tempe (T. Dowling)ASUT Cornell University Insect Collection, Ithaca, New York (J. Liebherr)CUIC Kansas State Entomological Museum, Manhattan (G. Zolnerowich)KSUCUnited States National Museum  and the Lepidoptera and Biodiversity McGuire Center for . A complete list of specimens examined is included in Suppl. material Heteranassa. A list of dissected specimens is included in Suppl. material Specimens were also examined during visits to the PageBreakwatch glass with distilled water, and scales were removed with a fine brush. Once clear of scales, the integument of the abdomen was cut along the left pleural membrane, and the genital capsule removed. On male specimens setae were carefully removed from the membranous costal region of valves with a fine camel’s hair brush. The aedeagus was separated from the valves by grasping the distal end with fine–tipped forceps, and gently pulling to separate from the juxta. The ductus seminalis was then cut where it enters the side of the proximal part of the aedeagus. The vesica was then carefully teased out of the aedeagus with a #20 minuten with the tip bent to a right angle, held in a standard pin vice, and with water pressure from a syringe. A syringe with a modified 30 gauge needle . Line drawings were made in Adobe Illustrator with a Wacom Intuos 4 drawing tablet from photographs or sketches made with a drawing tube attached to a Wild M5 stereomicroscope.Photographs of dissected specimens, genitalia, and adults were made using a Visionary Digital imaging system (Prosopisglandulosa Torr.) foliage and bark. Larvae were reared on Prosopisglandulosa foliage.Eggs were obtained from gravid females collected in Box Canyon, Pima Co., AZ (18 July 2012). The females were placed in brown paper bags with Honey Mesquite , Toxonprucha species and Coxina species are frequently misidentified as Heteranassa. Of these, Acritogrammametaleuca is the most similar to Heteranassa Homopteramima Harvey, 1876: 155–156.Eubolinamima ; Campometramima ; Elousamima ; Draudt and Gaede (in Seitz) 1923: 478.Heteranassamima ; Campometrafraternasyn. n.;  Smith, 1899: 104, Heteranassafraterna ; Elousafraterna ; Draudt and Gaede (in Seitz) 1923: 478.Campometraminor Smith, 1899: 104–105; Elousaminor : Draudt & Gaede (in Seitz) 1923: 478.Heteranassaminorsyn. n.;  , PageBreakThis is the only species in the genus and can be diagnosed with the generic combination (see above).Heteranassamima . Holotype,  labeled: “Homoptera mima, type, Harvey, Holotype, 15/9, 73.” The specimen and associated labels were examined through high-resolution photographs provided by the BMNH. Type locality: Texas [USA]pe, Fig.  ♀ in theHeteranassafraterna . Lectotype . Lectotype,  barcodes from California, Arizona, New Mexico, Texas, and northern Mexico showed a maximum divergence of less than 0.8%. One haplotype* dominated the sample, representing more than half of the specimens; the other barcodes included 36 haplotypes that had no more than two base-pair differences from each other. One haplotype, restricted to central and southern Texas, departed from this pattern in being 0.8% different from those from farther west. This is most probably the haplotype that should be associated with the name Heteranassamima, it being described from this part of Texas. However, this “eastern” haplotype is found with “western” haplotypes in central Texas and there is no indication in genital structural characters, or wing color or pattern, that Heteranassa includes more than a single species. The barcodes of Heteranassa are so divergent that they give no indication of a close relationship to any other erebid genus, other than belonging in the subfamily Erebinae, tribe Omopterini. Heteranassa specimens from Texas and Mexico are frequently confused with some species associated with the genus Coxina Guenée, which can have a similar superficial pattern, but the barcodes are more than 10% different and the two genera do not appear to be closely related. (D. Lafontaine pers. comm.).Barcode variation in *CNCNoctuoidea13382 PageBreakTATTATAATTTTCTTTATAGTTATACCAATTATAATTGGAGGATTTGGAAATTGATTAGTCCCCTTAATATTAGGAGCTCCTGATATAGCTTTCCCTCGAATAAATAATATAAGTTTCTGATTATTACCCCCATCTTTAACTCTTTTAATCTCAAGAAGAATCGTAGAAAATGGAGCAGGAACAGGATGAACAGTTTACCCCCCACTTTCATCTAACATTGCTCATAGAGGAAGATCAGTAGATTTAGCAATTTTCTCTCTTCATTTAGCTGGAATTTCATCAATTTTAGGAGCTATTAATTTTATTACTACTATTATCAATATACGATTAAATAGATTAATATTTGACCAAATACCTTTATTTGTTTGAGCTGTTGGTATTACTGCTTTTTTACTATTATTATCTTTACCTGTTTTAGCTGGAGCTATTACTATACTCTTAACAGATCGAAATTTAAATACTTCCTTTTTTGATCCTGCTGGAGGAGGAGATCCTATTCTTTACCAACATCTATTTAACTTTATATTTTATTTTTGGAATTTGAGCAGGAATAGTAGGAACCTCTTTAAGTTTATTAATTCGTGCTGAATTAGGAAACCCTGGTTCTTTAATTGGAGATGATCAAATTTATAATACTATTGTTACAGCTCATGCTTTLACM.Warm, arid habitats from California to Texas, northward to Oklahoma, and south as far as Oaxaca, Mexico Fig. . A singlHeteranassa wing pattern and coloration is continuous, with many specimens appearing intermediate to the phenotypes described by Heteranassa contains a single, highly variable species, Heteranassamima. Studies of another erebine genus, Catocala, have shown that pressure from avian predators may drive high levels of polymorphism in forewing pattern and coloration  than they are in Elousaschausi  and the other Coxina species we have dissected. We have examined 10 species in these genera from the Caribbean and South America. Specimens belonging to this group that we collected in the Nicaraguan highlands appear more similar to Elousaschausi specimens from Argentina and Coxina specimens from Mexico, south Texas, and Florida, than they do to Caribbean Elousa or North American Heteranassa. Future research could test the monophyly of Coxina and Elousa with respect to Heteranassa, and how these genera speciated in North and South America and the Caribbean.During the course of this research, we became aware of potential taxonomic affinities with the neotropical genera PageBreak


Staphylococcus aureus and vancomycin-resistant Enterococcus, there is not much information describing its activity against gram-negative bacteria. In this study, we report the effect of methylglyoxal against multidrug-resistant Pseudomonas aeruginosa (MDRP) using 53 clinically isolated strains. We also assessed the effect of deleting the five multidrug efflux systems in P. aeruginosa, as well as the efflux systems in Escherichia coli and Salmonella enterica serovar Typhimurium, on MICs of methylglyoxal. Our results indicate that methylglyoxal inhibits the growth of MDRP at concentrations of 128–512 μg/ml (1.7–7.1 mM) and is not recognized by drug efflux systems.Honey has a complex chemistry, and its broad-spectrum antimicrobial activity varies with floral source, climate, and harvesting conditions. Methylglyoxal was identified as the dominant antibacterial component of manuka honey. Although it has been known that methylglyoxal has antibacterial activity against gram-positive bacteria, including methicillin-resistant  Pseudomonas aeruginosa is endemic among critically ill patients, and multidrug-resistant strains are increasingly being isolated in intensive care units  has been defined as P. aeruginosa resistant to imipenem, amikacin, and ciprofloxacin  foraging on a shrub known as manuka (Leptospermum scoparium) that is indigenous to New Zealand. Manuka honey is broad in spectrum, able to inhibit a diverse range of bacterial and yeast pathogens, and equally effective against multidrug-resistant bacteria  from wounds  reported to be <3% (v/v)    and NKS233 (ΔtolC) were constructed as described previously  and NKE95 (ΔtolC), we performed gene disruption using procedures described previously ; acrB-P2 (GAACAGTCCAAGTCTTAACTTAAACAGGAGCCGTTAAGACCATATGAATATCCTCCTTAG); acrD-P1 (TGAAAAAGGCGACACATTGGCATGTCGCCTTTTTTATTGCGTGTAGGCTGGAGCTGCTTC); acrD-P2 (AAGCCTACAACGATACGCAGAAACACGAGGTCCTCTTTTACATATGAATATCCTCCTTAG); mdtA-P1 (ATCATTCCGCGAAACGTTTCAGGAAGAGAAACTCTTAACGGTGTAGGCTGGAGCTGCTTC); mdtC-P2 (GAGATACACCACCGGCGTGGTATACAGCGTAAGGAGCTGGCATATGAATATCCTCCTTAG); mdtE-P1 (TTAAAGAACCGTTATTTCTCAAGAATTTTCAGGGACTAAAGTGTAGGCTGGAGCTGCTTC); mdtF-P2 (AGGCTGAACCTTCATGTTCAACCTTACTCTCATTTACACGCATATGAATATCCTCCTTAG); acrE-P1 (TTGGGTAAATAACGCGCTTTTGGTTTTTTGAGGAATAGTAGTGTAGGCTGGAGCTGCTTC); acrF-P2 (AAATAATAAAGGCACCCGAAAGCGCCTTTATGTTTCTGATCATATGAATATCCTCCTTAG); tolC-P1 (ACTGGTGCCGGGCTATCAGGCGCATAACCATCAGCAATAGGTGTAGGCTGGAGCTGCTTC); and tolC-P2 (TTACAGTTTGATCGCGCTAAATACTGCTTCACCACAAGGACATATGAATATCCTCCTTAG). The chloramphenicol resistance gene cat or the kanamycin resistance gene aph, flanked by Flp recognition sites, was amplified by PCR using the primers listed above. The resulting PCR products were used to transform E. coli MG1655, which harbors plasmid pKD46, expressing Red recombinase. The chromosomal structures of the mutated loci were verified by PCR; cat and aph were further eliminated using the plasmid pCP20, as described previously . The inoculated agar plates were examined after incubation at 37°C for 16 h. MIC was the lowest concentration of a compound that inhibited cell growth.Antibacterial activities were determined on Muller Hinton II agar  plates containing methylglyoxal (32–2048 μg/ml), imipenem (0.0625–2048 μg/ml), amikacin (0.125–4096 μg/ml), or ciprofloxacin (0.0078–2048 μg/ml) . Agar plates were prepared using the two-fold agar dilution technique. Bacteria were grown at 37°C overnight and then tested at a final inoculum volume of 1 × 10E. coli  and S. enterica  strains were grown in Luria–Bertani broth , and P. aeruginosa (PAO1 and PMX52) strains were grown in Muller Hinton II (MHII) broth . Bacterial cells were cultured overnight at 37°C, and then 100 μl of cell cultures were diluted in 5 ml of the same medium. The diluted bacterial cells were incubated at 37°C until OD600 reached 0.5. Then, the bacterial cells were diluted in the same medium to an OD600 of 0.05. This diluted bacterial cells were incubated in NUNC Edge 96-well plates  with shaking at 37°C for 7 h. Bacterial growth was monitored using an Infinite M200 Pro plate reader .P. aeruginosa resistant to imipenem , amikacin (≥32 μg/ml), and ciprofloxacin (≥4 μg/ml) . Although PMX52 was more susceptible to amikacin and ciprofloxacin than PAO1, MIC of methylglyoxal for these strains was the same. This suggests that methylglyoxal is not recognized by drug efflux systems in P. aeruginosa. To confirm whether same phenomenon could be observed in other gram-negative bacteria, we determined MICs of methylglyoxal for the efflux-deficient mutants of E. coli and S. enterica serovar Typhimurium. There are five RND-type drug efflux systems  in E. coli, and all of them require the TolC outer membrane channel for their function , NKE1329 (ΔacrB acrD mdtABC mdtEF acrEF), and NKE95 (ΔtolC) strains. The susceptibility of NKE1329 and NKE95 to methylglyoxal was same as that of the wild-type strain, although they were more susceptible to ciprofloxacin than the wild-type strain. S. enterica serovar Typhimurium harbors at least nine drug efflux systems belonging to RND, multidrug and toxic compound extrusion, and ATP-binding cassette (ABC) superfamilies , NKS196 (ΔacrAB acrEF acrD mdtABC mdsABC emrAB mdfA mdtK macAB), and NKS233 (ΔtolC) strains. Although NKS196 and NKS233 were more sensitive to ciprofloxacin than the wild-type strain ATCC14028s, MICs of methylglyoxal for ATCC14028s, NKS196, and NKS233 were the same. In addition to MIC determination using agar plates, we tested the effect of methylglyoxal on bacterial growth in liquid medium. The growth of E. coli  and Salmonella  strains was inhibited by methylglyoxal at a concentration of 256 μg/ml, and the growth of P. aeruginosa (PAO1 and PMX52) strains was inhibited at 512 μg/ml, which is consistent with MICs determined . Methylglyoxal is a key antimicrobial component of manuka honey, and manuka honey has previously been suggested as a topical treatment option for burn patients infected with P. aeruginosa  (Mavric et al., P. aeruginosa, E. coli, and S. enterica.In this study, we showed that methylglyoxal equally inhibits drug-susceptible The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The correct sentence is: Then an exchange of ADP with ATP in ATP domains of MutS takes place  shown in the “Synthetic DNA Fragments” subsection of the Materials and Methods. Please see the correct structures here.GGGTGCC I (G/C)CAAGCCTATGCCCTCAGCACCCACCCACGGGTTCGGATACGGGAGTCGTGGGTGGGTGCC II (G/T)CAAGCCTATGCCCTCAGCACCCATCCACGGGTTCGGATACGGGAGTCGTGGGTG-GGTGCC III (G/Tg)CAAGCCTATGCCCTCAGCACCCATgCCACGGGTTCGGATACGGGAGTCGTGGGTA-GGTGCC IV (A/Tg)CAAGCCTATGCCCTCAGCACCCATgCCACGGGTTCGGATACGGGAGTCGTGGGT


Correction: Genome Biol 19, 205 (2018)https://doi.org/10.1186/s13059-018-1581-3Following the publication of the original paper , the autATCTTGTGGAAAGGACGAAACAThe sequence for Primer 1: SAMlibrary-HiSeq_50bp-F1 has two extra bases inserted (in bold) ACACTCTTTCCCTACACGACGCTCTTCCGATCTThe correct sequence should be:ACACTCTTTCCCTACACGACGCTCTTCCGATCTCTTGTGGAAAGGACGAAACA


Retinopathies are multifactorial diseases with complex pathologies that eventually lead to vision loss. Animal models facilitate the understanding of the pathophysiology and identification of novel treatment options. However, each animal model reflects only specific disease aspects and understanding of the specific molecular changes in most disease models is limited. Here, we conducted transcriptome analysis of murine ocular tissue transduced with recombinant Adeno-associated viruses (AAVs) expressing either human VEGF-A, TNF-α, or IL-6. VEGF expression led to a distinct regulation of extracellular matrix (ECM)-associated genes. In contrast, both TNF-α and IL-6 led to more comparable gene expression changes in interleukin signaling, and the complement cascade, with TNF-α-induced changes being more pronounced. Furthermore, integration of single cell RNA-Sequencing data suggested an increase of endothelial cell-specific marker genes by VEGF, while TNF-α expression increased the expression T-cell markers. Both TNF-α and IL-6 expression led to an increase in macrophage markers. Finally, transcriptomic changes in AAV-VEGF treated mice largely overlapped with gene expression changes observed in the oxygen-induced retinopathy model, especially regarding ECM components and endothelial cell-specific gene expression. Altogether, our study represents a valuable investigation of gene expression changes induced by VEGF, TNF-α, and IL-6 and will aid researchers in selecting appropriate animal models for retinopathies based on their agreement with the human pathophysiology. Accordingly, anti-VEGF treatment has emerged as standard-of-care for wet AMD5 and DME6. Another common hallmark of many retinopathies is inflammation: proinflammatory cytokines such as TNF-α as well as IL-6 proteins are upregulated in the vitreous of DR8 and uveitis patients10.Retinopathies such as diabetic retinopathy (DR), age-related macular degeneration (AMD), uveitis or retinopathy of prematurity (ROP) display complex pathologies in patients including vasculopathies, inflammation, neurodegeneration, and fibrosis, ultimately leading to blindness. A number of molecular changes have been associated with these retinal pathologies, for example changes in expression of Vascular Endothelial Growth Factor (VEGF), Tumor Necrosis Factor alpha (TNF-α), or Interleukin-6 (IL-6). VEGF causes vascular leakage and pathological neovascularization, and VEGF protein has been shown to be upregulated in wet AMD12. Transgenic mice expressing VEGF15, intraocular injection of recombinant VEGF protein18 or recombinant adeno-associated viruses (AAVs) expressing VEGF23 further demonstrated that VEGF is not only necessary but also sufficient to cause vasculopathies. Accordingly, anti-VEGF treatment prevents neovascularization in the OIR model24 and these preclinical studies paved the way for modern anti-VEGF therapies. Furthermore, inflammatory processes observed in patients of retinopathies can also be modeled in rodents, for example by uveitis mouse models such as endotoxin- or antigen-induced uveitis27 or transgenic mice lacking the Aire gene29. Similarly, recombinant proteins or AAV-mediated expression of TNF-α and IL-6 induces inflammation in the rodent eye, although the direct function of IL-6 is more controverse32. Again, anti-TNF-α or anti-IL-6 treatment improves the induced pathologies in diverse uveitis models35.Various pre-clinical rodent models have directly or indirectly shed light on the function of VEGF, TNF-α, or IL-6 in retinopathies. One frequently used animal model displaying vascular pathologies, similar to those observed in proliferative retinopathies such as wet AMD or ROP, is the oxygen induced retinopathy (OIR) model19. We further demonstrated that AAV-mediated expression of human VEGF, TNF-α, and IL-6 in the murine eye leads to pathway-specific, human-relevant retinal pathologies. In brief, AAV-driven expression of VEGF induced vascular leakage and neovascularization. On the other hand, pro-inflammatory cytokines TNF-α and IL-6 both activated immune cells. TNF-α additionally led to vasculitis, fibrosis, and development of fibrotic epiretinal membranes.In the context of pre-clinical research, AAV mediated gene transfer has recently emerged as a powerful method to create novel animal models. As a main benefit, AAVs allow for long-term expression of a transgene in a tissue- and cell-type-specific manner. Previously, we have shown that AAV-mediated expression leads to constant and long-term expression of human transgenes at 1, 3 and 6 weeks after IVT-injection38. Similarly, the transcriptome of multiple rodent models of retinopathies has been sequenced and analyzed42. For the OIR model, several studies have identified time point-dependent gene expression changes related to hypoxia, angiogenesis, and inflammation47. However, comparisons with other mouse models of retinopathies are still missing. Additionally, modern single cell RNA-sequencing (scRNA-Seq) approaches gave unprecedented insights into the cellular organization of healthy and diseased mammalian ocular tissues54. In more detail, Heng et al. demonstrated by single cell sequencing of Aire-/- mice, a spontaneous uveoretinitis model, that a very diverse population of immune cells invades the retina of Aire-/- mice and that Th1 cells represent the main effector T cells in this model, highlighting the great potential of such single cell sequencing approaches. As the number of available bulk and single-cell datasets in the context of retinopathies increases, so does the potential to integrate data from various retinopathy models and compare them on a molecular level.In the recent years, next generation sequencing techniques allowed for a detailed investigation of the molecular changes in retinopathies and brought about a better understanding of disease progression in AMD and DR patientsIn this study, we provide transcriptomic analysis of intravitreally injected recombinant AAVs expressing human VEGF, TNF-α, or IL-6 in mice. We observed that AAV-mediated expression of human transgenes introduced distinct transcriptomic responses: While TNF-α and IL6 displayed overlapping gene expression changes, VEGF overexpression led to a more distinct response compared to TNF-α and IL-6. In more detail, investigating pathways affected by each experimental condition, VEGF led to a specific regulation of extracellular matrix (ECM)-related genes, while TNF-α and IL-6-induced changes in interleukin signaling. We further identified specific gene expression changes associated with TNF-α that included cellular adhesion molecules, such as Madcam1 and we further demonstrated a conserved regulation of MAdCAM-1 by TNF-α in human retinal endothelial cells. By integration of single cell RNA-Sequencing data, we could show an increase of T-cell-specific genes following TNF-α expression and, indeed, TNF-α mediated T-cell invasion was validated by immunofluorescence. In addition to assessing transcriptome changes in the various transgenic mice models, we generated a detailed time-course of transcriptome changes in the established OIR model. Comparing transcriptome changes in AAV overexpression models and OIR mice, we observed the largest overlap between AAV-VEGF treated eyes and the late response (P16) in the OIR model, most prominently including changes in the ECM pathway and endothelial cell specific marker genes. Prominently, OIR-specific changes in gene regulation included neuronal signaling pathways which were not observed after AAV-VEGF transgene expression. In conclusion, our study suggests that each animal model produces distinct gene expression profiles and a careful selection of models according to the requirements of different research questions is necessary.8 VG/eye), while AAV-TNF-α and AAV-IL-6 were injected at 1 × 109 VG/eye. Eyes of all animals were imaged in vivo directly before tissue dissection and RNA sequencing to validate expected pathologies and grade phenotype severity. In line with our previously published study19, AAV-stuffer injections did not induce obvious retinal pathologies at both concentrations based on Blue Autofluorescence (BAF) imaging . Since no mRNA staining was observed in AAV-stuffer control treated eyes , choroid, sclera and the ciliary body. Apart from two samples that were excluded due to a low RNA integrity number (RIN), six biological replicates per group were sequenced. Sequencing libraries were sequenced at an average depth of 31.3 million reads, with 80.1% mapping to mRNA transcripts and low ribosomal content (2.1%), suggesting overall good quality of the RNA sequencing data. As expected, human transgenes showed strong expression in their respective treatment groups  indicated the largest variance in gene expression between retina and eye cup samples Fig. d. The reVcam1 and Madcam1 and chemokines such as Ccl2 were among the strongest deregulated genes, supporting the well-known function of TNF-α in regulating immune cell adhesion to endothelial cells56. To confirm the RNA sequencing results, Ccl2 expression was validated by qRT-PCR, which demonstrated similar expression patterns with the highest expression of Ccl2 found in AAV-TNF-α treated eyes  genes in the three treatment groups AAV-VEGF, TNF-α, and AAV-IL-6 in comparison to their respective AAV-stuffer control, as well as AAV-stuffer compared to non-injected controls  by AAV-VEGF, AAV-TNF-α or AAV-IL-6 treatment. Only 1 pathway was enriched in the retina of all three treatment groups Fig. a, namelySince only very few genes were significantly differentially regulated in AAV-VEGF transduced eye cup tissue, only AAV-TNF-α and AAV-IL-6 treatment was compared in the eye cup tissue  of endothelial and perivascular cell-specific marker genes after AAV-VEGF treatment was observed  were mostly positive , P13, P14, P15 and P16. PCA demonstrated a clear separation between the OIR model and controls  model, presenting both neovascularization and neurodegeneration. Neonatal mice are exposed to 75% oxygen from postnatal day 7 to 12, and thereafter return to room air (21% oxygen) until day 17. At day 7, the retinal vasculature of mouse pups is still immature and vulnerable to high oxygen conditions. Loss of capillaries in the center of the retina leads to the formation of an avascular area. Upon return to room air at day 12 the area becomes hypoxic and Hif1α dependent VEGF expression has been shown to trigger angiogenesis, peaking at P17ols Fig. . At late54 demonstrated that, among others, also vascular cell-specific marker genes were enriched in the OIR data set  in OIR retina compared to those identified in the AAV-VEGF model Fig. c,d. The Finally, we aimed to understand which pathways were affected in the AAV-VEGF driven retinopathy models compared to the OIR model  into mouse eyes with a biologically inactive sequence, produced very few gene expression changes. AAV-mediated expression of hTNF-α, hIL-6, or hVEGF, led to high expression of these human transgenes, which in turn produced strong and distinct transcriptome changes. Although measurement errors in RNA-Seq caused by the simultaneous presence of human and murine versions of the AAV-expressed transgene are theoretically possible, we expect no large mis-quantification, since human transgenes are codon optimized and possess a V5 tag, hence they are sufficiently dissimilar to their endogenous transcript sequence. In addition, we did not observe any reduction in the percentage of uniquely mapped reads in the presence of AAV expressed transgenes (data not shown).AAVs have emerged as a useful tool to quickly generate novel animal models that allow a high degree of flexibility regarding timing and tissue-specific expression. In our study, we chose the ShH10 capsid for expression of the three human transgenes. Even on a global scale, measured via a high-throughput method such as RNA-Seq, injection of the control capsid  secondary to AMD72. In the past, transgenic animals modifying the expression of complement genes, or mice expressing human variants of complement-related genes, have proven extremely useful to understand their contribution to disease progression78. To further develop novel and effective therapies, animal models with robust activation of the complement pathway are needed. In LPS-induced uveitis model, complement-related genes including C3 are upregulated39, suggesting complement activation. Similarly, experimental autoimmune uveitis (EAU) leads to activation of the complement system and blockage of the complement system ameliorates disease pathology79. Activation of the complement system is also visible in the laser-induced choroidal neovascularization (CNV) model, and inhibition of various complement factors had beneficial effects82. However, both the experimental uveitis models and laser CNV model are transient and only allow for short-term investigations. In contrast, in the presented AAV-TNF-α model, C3 showed strong and sustained upregulation at 3 and 6 weeks after IVT injection of AAV-TNF-α, thus, allowing long-term studies of the complement system. In addition, as a proof-of-mechanism we showed that treatment with the neutralizing TNF-α antibody golimumab reduces C3 expression, demonstrating that complement activation is reversible in our model. In summary, the AAV-TNF-α induced mouse model provides a valuable tool to study the role of the complement system in disease progression, featuring long-term TNF-α and C3 expression, which is comparable to the human pathology that develop over decades.In our analysis, significantly changing genes present in the complement cascade were strongly and specifically upregulated in eyes injected with AAV-TNF-α. Among them, the complement pathway factor C3 was one of the strongest upregulated genes in AAV-TNF-α injected eyes. C3 plays a central role in the activation of the classical and alternative complement pathway and mutations in C3 and other members of the complement cascade correlate with higher risk of AMDIcam1, Vcam1 and Madcam1 were highly upregulated in AAV-TNF-α injected eyes, strengthening the previous observation of immune cell infiltration and vasculitis in AAV-TNF-α injected eyes by histology19. MAdCAM-1 is induced by TNF-α in diverse endothelial cells and recruits T-cells to inflamed tissues85 and, therefore, has recently been identified as an interesting target to treat inflammatory bowel diseases86. In addition to Madcam1 upregulation, we observed T-cell infiltration in our AAV-TNF-α driven uveitis-like model, in line with well-described role for T-cells in uveitis patients and uveitis rodent models88. Although there is general interest in the role of TNF-α and CAMs in the context of retinopathies, to our knowledge there exists only one recent study by Peng et al. demonstrating a direct function for murine Madcam1 in retinal degeneration83. In line with Peng et al., we demonstrated upregulation of Madcam1 in AAV-TNF-α injected eyes correlating with an invasion of T-cells. Expanding on this observation, we demonstrated that MAdCAM-1 is also upregulated in TNF-α stimulated HRMECs, suggesting that the TNF-α induced regulation of MAdCAM-1 may also be relevant in human retinal cells.In addition to C3, cellular adhesion molecules (CAMs), such as 12. To capture transient expression dynamics present in the OIR model, we collected mouse eyes at 5 subsequent time points after hyperoxic treatment. Indeed, other authors have performed analysis of OIR gene expression, however, many studies relied on microarray data only assessing part of the transcriptome in contrast to the whole transcriptome analysis presented here. On the other hand, OIR RNA-Seq studies included less dense sampling of timepoints compared to our study, in which we provide more detailed insights into time-dependent gene expression changes. The study of Yang et al.46 focused on gene expression changes during and shortly after hyperoxic treatment, while our study was mostly interested in gene expression changes after the return to normoxic conditions and a detailed comparison to AAV induced gene expression changes. Nevertheless, we observed shared gene expression signatures of key genes between the two studies, for example VEGF and Edn2 gene were both upregulated at P13.In order to better understand the differences between various retinopathy disease models, we compared gene expression profiles of the generated AAV-driven retinopathy models to RNASeq data of the frequently used OIR model24. Thus, as expected, DE genes in the AAV-VEGF model showed the largest overlap with those identified in the OIR model. By integration of single-cell RNA-Seq expression data, we showed that within the time-course measured for the OIR model, endothelial cell-specific genes were first down-regulated and then up-regulated starting from P15. Also other studies have found downregulation of angiogenesis-related genes such as CD34 in the OIR model at P12, while endothelial cell-specific genes such as Esm1 or Edn2 were upregulated at P1789. Esm1 is a well-known gene induced by VEGF in tip cells with a crucial role in angiogenesis and its blockage inhibits neovascularization of diverse rodent models of neovascularization including the OIR, laser CNV and the rho/VEGF transgenic mouse91. Accordingly, we also observed upregulation of the Esm1 gene in both the OIR dataset but also the AAV-VEGF driven model, resembling very well the observed neovascularization phenotype. Since we provide a high time-resolution of gene expression changes in the OIR model, we were able to pinpoint the switch between down- and up-regulation of endothelial cell-specific genes to P14. These gene expression changes fit very well with the phenotypic changes present in the OIR model, where at P12 the development of the vasculature is impaired and avascular areas are present, and later on, neovascularization peaks at around P1793.It is well known that VEGF is a major driver of the OIR model and anti-VEGF treatment prevents neovascularization in the OIR model95. Furthermore, neuronal death and decreased retinal function has been observed in the OIR model97, probably due to ischemia and malnutrition induced by hypoxia. Accordingly, the OIR model has been discussed as the more appropriate model for retinopathy of prematurity (ROP)93, a disease affecting human preterm infants. While very young mouse pups are needed for the OIR model, old or diabetic mice may be combined with AAV overexpression of human transgenes, to mirror more closely age-related or diabetes-associated human retinopathies such as AMD or DR in the future.Although many pathways including ECM-related pathways and endothelial cell-specific responses were similar between the OIR model and AAV-VEGF treated mice, we also observed differences between these disease models: For example, in the OIR model, but not AAV-VEGF, neuronal signaling pathways and synapse-related genes were significantly enriched for DE genes. In line with this observation, we observed a significant overlap of neuronal cell-specific genes  and DE genes identified in the OIR model. For the OIR model pups at age P12 to P17 are used, a timepoint which corresponds to a time-point where vascular and neuronal networks are still developing in the murine retinaDespite our efforts to ensure optimal experimental design, certain limitations apply, most prominently that AAV and OIR sequencing data sets were generated in independent experiments. Furthermore, also from a biological perspective, the models differ, since the OIR model uses very young mouse pups during development, in contrast to our AAV-induced models that were developed using adult mice. While in the OIR study 3 or 4 samples where included at each time-point, the AAV study was performed with 5 or 6 replicates per group. In addition, library preparation and sequencing was dependent on different protocols. To alleviate these limitations, our analysis is based on relative fold changes between treatment and respective controls, rather than on absolute expression values, thereby accounting for differences in library preparation.Altogether, here we characterized the gene expression profiles of AAV-VEGF, TNF-α, or IL-6 driven retinopathy mouse models, and combined these measurements with publicly available singe-cell RNA expression data. Our study gives unique insights into the molecular and cell-specific changes leading to retinal pathologies, hereby uncovering novel potential treatment options for retinal diseases, and at the same time offering the means to test them in a stable disease model comparable to the human pathology. By further measuring gene expression changes in the well-established OIR model, we compared new and established animal models for retinopathies and provide researchers with important information guiding the decision of which animal model best suits the need of a given pre-clinical research project.C57BL/6 J mice were purchased from Charles River . 6–8-week-old male mice were used for the AAV study. Pregnant females were purchased for the OIR study and male and female pups at P12 were used for the OIR experiments. Mice were housed in individually ventilated cages in groups of 2–5, constant temperature and a 12-h light/dark cycle. Mice had ad libitum access to standard rodent chow and water. Animal experiments were performed in accordance with the German animal welfare act, the guidelines of the Federation of the European Laboratory Animal Science Association (FELASA), the ARRIVE guidelines and were reviewed and approved by the governmental body responsible for animal welfare in the state of Baden Württemberg .19.The AAV-stuffer control construct includes a non-coding sequence from the 3’ UTR of the UBE3A gene and has been described elsewhere (Strobel 2015). AAVs used in this study were packaged into the ShH10 capsid (Klimczak 2009). AAV production and quantification by quantitative PCR was done according to previously published protocols (Strobel 2019). The following human codon-optimized sequences were included in each of the recombinant AAVs:Plasmids with the ubiquitous CAG promoter were generated encoding for three human, codon optimized transgenes  that have a V5-tag sequence at the 3’ end of the gene were generated previouslyAAV-hIL6—ATGAACAGCTTCAGCACCAGCGCCTTCGGACCTGTGGCTTTTTCTCTGGGACTGCTGCTGGTCCTGCCTGCCGCTTTTCCAGCTCCTGTTCCTCCTGGCGAGGACAGCAAAGATGTGGCCGCTCCTCATAGACAGCCTCTGACCAGCTCCGAGCGGATCGATAAGCAGATCCGGTACATCCTGGATGGCATCAGCGCCCTGCGGAAAGAGACATGCAACAAGAGCAACATGTGCGAGAGCAGCAAAGAGGCCCTGGCCGAGAACAACCTGAACCTGCCTAAGATGGCCGAAAAGGACGGCTGCTTCCAGAGCGGCTTCAACGAGGAAACCTGCCTGGTCAAGATCATCACCGGCCTGCTGGAATTCGAGGTGTACCTGGAATACCTGCAGAACAGATTCGAGTCCAGCGAAGAACAGGCCAGAGCCGTGCAGATGAGCACCAAGGTGCTGATCCAGTTCCTGCAGAAGAAGGCCAAGAACCTGGACGCCATCACCACACCTGATCCTACCACAAATGCCAGCCTGCTGACAAAGCTGCAGGCCCAGAATCAGTGGCTGCAGGACATGACAACCCACCTGATTCTGCGGAGCTTCAAAGAGTTTCTGCAGAGCAGCCTGCGGGCCCTGAGACAAATGGGAGGCGGAGGATCTGGCGGAGGCGGATCTGGAAAGCCCATTCCTAATCCTCTGCTGGGCCTCGACAGCACCTGATGATAA.AAV-hTNFa—ATGAGCACCGAGAGCATGATCAGAGATGTGGAACTGGCCGAGGAAGCCCTGCCTAAGAAAACAGGCGGACCTCAGGGCAGCAGAAGATGCCTGTTTCTGAGCCTGTTCAGCTTCCTGATCGTGGCAGGCGCCACCACACTGTTCTGTCTGCTGCACTTTGGAGTGATCGGCCCTCAGAGAGAGGAATTCCCCAGAGATCTGTCCCTGATCTCTCCACTGGCTCAGGCTGTGCGGAGCAGCTCTAGAACACCTAGCGATAAGCCTGTGGCTCACGTGGTGGCCAATCCTCAGGCTGAAGGACAGCTGCAGTGGCTGAATAGAAGGGCCAACGCTCTGCTGGCCAACGGCGTGGAACTGAGAGATAATCAGCTGGTGGTGCCCAGCGAGGGCCTGTACCTGATCTATAGCCAGGTGCTGTTCAAAGGCCAGGGCTGCCCTTCTACACACGTGCTGCTGACCCACACCATCAGCAGAATCGCCGTGTCCTACCAGACCAAAGTGAACCTGCTGAGCGCCATCAAGAGCCCCTGTCAGAGAGAAACACCTGAGGGCGCCGAAGCCAAGCCTTGGTACGAACCTATCTATCTCGGCGGCGTGTTCCAGCTCGAGAAGGGCGATAGACTGAGCGCCGAGATCAACAGACCCGACTACCTGGATTTTGCCGAGAGCGGCCAGGTGTACTTCGGCATTATTGCTCTCGGAGGCGGAGGAAGTGGTGGCGGAGGATCTGGCAAGCCCATTCCTAATCCTCTGCTGGGCCTCGACTCCACCTGATGATAA.AAV-hVEGF—ATGAACTTCCTGCTGAGCTGGGTGCACTGGTCACTGGCTCTGCTGCTGTATCTGCACCACGCCAAATGGTCACAGGCCGCTCCTATGGCTGAAGGCGGAGGACAGAATCACCACGAGGTGGTCAAGTTCATGGACGTGTACCAGCGGAGCTACTGTCACCCCATCGAGACACTGGTGGACATCTTCCAAGAGTACCCCGACGAGATCGAGTACATCTTCAAGCCTAGCTGCGTGCCCCTGATGAGATGCGGCGGCTGTTGTAACGATGAAGGCCTGGAATGCGTGCCCACCGAGGAATCCAACATCACCATGCAGATCATGCGGATCAAGCCCCACCAGGGCCAGCATATCGGCGAGATGTCTTTCCTGCAGCACAACAAGTGCGAGTGCAGACCCAAGAAGGACCGGGCCAGACAAGAGAATCCTTGCGGCCCTTGCAGCGAGCGGAGAAAGCACCTGTTTGTGCAGGACCCTCAGACCTGCAAGTGCTCCTGCAAGAACACCGACAGCAGATGCAAGGCCCGGCAGCTGGAACTGAACGAGAGAACCTGCAGATGCGACAAGCCTAGAAGAGGTGGCGGAGGATCTGGCGGAGGCGGATCTGGAAAGCCCATTCCTAATCCTCTGCTGGGCCTCGACAGCACCTGATGATAA.8 viral genomes (VG)/eye (low dose) or 1 × 109 VG/eye (high dose) in 1 µL AAV buffer. Note that AAV-VEGF was injected at a concentration of 1 × 108 VG/eye and AAV-TNF-α and AAV-IL-6 at 1 × 109 VG/eye with matching AAV-stuffer controls. In an independent mouse cohort, 2 weeks after AAV-TNF-α treatment, eyes were IVT injected with 1 µL of vehicle or neutralizing anti-TNF-α Golimumab . If IVT injection failed (e.g. due to damage of a major blood vessel or the lens), the eye was excluded from analysis.Mice were intravitreally injected with the different AAVs under isoflurane anesthesia and after local anesthetic eye drops were applied . Both eyes of 6 mice per group were injected with either 1 × 1019. In brief, mice were anesthetized by intraperitoneal injection with 60–90 mg/kg ketamine (Medistar) and 6–8 mg/kg xylazine . Pupils were dilated with 5 mg/mL tropicamide  and phenylephrine . A Spectralis HRA/OCT device (Heidelberg Engineering) equipped with a 55° widefield lens was used for recording of Optical Coherence Tomography (OCT) pictures and Autofluorescence (AF) images. For Fundus Fluorescein Angiography (FFA), 200 µL of a 0.2% fluorescein solution (Alcon) were injected subcutaneously and recorded 90 s after injection with the Spectralis HRA/OCT (Heidelberg Engineering). Mice were euthanized by cervical dislocation. The retina and the eye cup  were dissected, and flash frozen in liquid nitrogen.In vivo imaging was done as described previously11). In brief, 7-days-old male and female pups with their mothers were transferred to a hyperoxic chamber (75% oxygen). At P12, oxygen concentration was slowly reduced back to normoxic conditions (21% oxygen) within approximately 3 h. Control mice remained at normoxic conditions for the whole experiment. Mice were euthanized at P12, P13, P14, P15 and P16 by cervical dislocation. Dissected retinae were stored in RNAlater (Invitrogen).The OIR experiments were performed according to previously published protocols  were homogenized in 700 µL Qiazol (Qiagen) using a Precellys Evolution homogenizer (Bertin Technologies). Retina samples were homogenized by ceramic beads MPbio, 6913-500) and eye cup samples by metal beads  and processed in 4 separate batches. Total RNA was extracted using the miRNeasy Micro Kit  according to the manufacturer’s tissue protocol including on-column DNase treatment . Total RNA samples  were quantitatively and qualitatively assessed using the fluorescence-based Broad Range Quant-iT RNA Assay Kit (Thermo Fisher Scientific) and the Standard Sensitivity RNA Analysis DNF-471 Kit on a 96-channel Fragment Analyzer (Agilent), respectively. Concentrations averaged at 48.7 ng/µL while RIN ranged from 5.2 to 9.6, with a median at 9.2. Two samples with RIN below 6 were excluded from downstream processing: AAV-stuffer (high) eye cup replicate 4 and AAV-VEGF eye cup replicate 5. See Table 3-500 andRetinae were dissected from 12 to 16-day postnatal C57BL/6 J mice (n = 32). RNAlater-preserved tissues were disrupted and homogenized in CK14 tubes  with RLT buffer using a Precellys Evolution homogenizer (Bertin Technologies). Total RNA was then extracted using the MagMAX-96 total RNA isolation kit  including a DNase digestion prior to final elution . Total RNA samples were quantitatively and qualitatively assessed on a Synergy microplate reader with a Gen5 Take3 module (BioTek), and the Eukaryote total RNA 6000 Nano microfluidic chip  on a 2100 Bioanalyzer system (Agilent), respectively. Concentrations averaged at 136.3 ng/µL while RIN of selected samples were above 8.4. All samples were further processed for library preparation. See Table 36.70 retina- and eye cup-derived RNA samples were normalized on the MicroLab STAR automated liquid platform (Hamilton). Total RNA input of 250 ng was used for library construction with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina #E7760, together with the NEBNext Poly(A) mRNA Magnetic Isolation Module #E7490 upstream and the NEBNext Multiplex Oligos for Illumina #E7600 downstream . The only deviation from the manufacturer’s protocol was the use of Ampure XP beads (Beckman Coulter) for double-stranded cDNA purification, instead of the recommended SPRIselect Beads. The index PCR was performed with 12 cycles, while the final library was eluted in 35 µL. mRNA libraries were then quantified by the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). Library molarity averaged at 149 nM. mRNA libraries were also assessed for size distribution  and adapter dimer presence (< 0.5%) by the High Sensitivity Small Fragment DNF-477 Kit on a 96-channel Fragment Analyzer (Agilent). All 70 sequencing libraries were then normalized on the MicroLab STAR (Hamilton), pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered on an S4 Flow Cell and sequenced on a NovaSeq 6000 Sequencing System (Illumina) with dual index, paired-end reads at 2 × 100 bp length , reaching an average depth of 31.3 million Pass-Filter reads per sample (7.0% CV). The description of RNA library preparation and sequencing closely aligns with the one given in Becker et al.Total RNA input of 100 ng was used for library construction with the TruSeq Stranded mRNA LT kit—Set A , together with the SuperScript II Reverse Transcriptase , according to the manufacturer’s protocol. The quality of mRNA libraries was assessed for adapter and heterodimer presence using the DNA 1000 microfluidic chip , while library molarity was measured using the Quant-iT PicoGreen dsDNA Assay Kit . All 32 sequencing libraries were then normalized, pooled and spiked in with PhiX Control v3 (Illumina). The library pool was subsequently clustered using the TruSeq SR Cluster Kit v3 , and sequenced with TruSeq SBS Kit v3 reagents  on a HiSeq 2000 Sequencing System (Illumina) with single index, single-read reads at 1 × 51 bp length , reaching an average depth of 24.9 million Pass-Filter reads per sample (13.1% CV).2. The LUNA-FL cell counter (LogosBio) with Acridine Orange dye  was used for cell quantification before seeding. HRMECs were stimulated with 10 ng/mL recombinant human TNF-α  for 24 h at 37 °C. Cells were washed with PBS and lysed in RLT buffer provided by Qiagen and RNA extracted using the RNeasy Mini Kit  according to the manufacturer’s manual.Primary human retinal microvascular endothelial cells  were cultivated in Endothelial Cell Growth Medium  on plates pre-coated with 0.1% gelatin . Cells were cultivated in a humidified chamber at 37 °C and 5% COcDNA synthesis was done using the high-capacity cDNA Kit  and qRT-PCR was performed with the Taqman Universal PCR Master Mix  on a QuantStudio 6 Real-Time PCR system (Applied Biosystems). Relative expression (fold induction) was calculated using the ΔΔCT method and the 18S (for mouse tissue) and POLR2A (for HRMECs) genes were used as a normalization controls. The following Taqman probes were used in this study: 18S (Hs99999901_s1), POLR2A (Hs00172187_m1), Madcam1 (Hs00369968_m1), CCL2 (Mm00441242_m1) and a custom-made Taqman probe detecting human codon-optimized VEGF with a V5 tag .57. In brief, 2 weeks after IVT injection of AAV-TNF-α, 100 µg golimumab (Simponi) were injected intravitreally. 6 weeks after AAV injection, whole eyes were enucleated, flash frozen in liquid nitrogen, and homogenized in lysis buffer  supplemented with proteinase inhibitor Pefabloc SC  with metal beads  in a Precellys Evolution tissue homogenizer (Bertin Technologies). C3 expression was measured with the Mouse C3 SimpleStep ELISA Kit  according to the manufacturer’s protocol and measured with a SpectraMax Plus 384 plate reader (Molecular Devices).Eyes for validation of C3 expression were generated from an independent mouse cohort that is published elsewhere19. Eyes were enucleated 3–6 weeks after IVT injection and directly fixed in 4% paraformaldehyde  for 48 h at 4 °C. Eyes were dehydrated, incubated in xylol, and infiltrated with paraffin using a tissue processing machine . Immunohistochemical stainings were performed on 3 µm sections with the Opal Multiplex IHC Kit (Akoya Biosciences) and carried out on the automated Leica Bond platform . Antigen retrieval was done with the BOND Epitope Retrieval Solution 1  at 95 °C and pH 6.0 for 20 min. Polyclonal rabbit anti-CD3 antibody  was diluted 1:200 and used as a pan-T-cell marker. OPAL polymer anti-rabbit-HRP secondary antibody  and OPAL 570 reagent  were used to develop the fluorescent signal. Nuclei were stained with spectral DAPI  and slides were mounted with ProLong Antifade Mounting Medium . Immunofluorescence stainings were imaged using a laser-scanning microscope LSM700 . RNAscope in situ hybridizations for human VEGF, TNF-α and IL-6 were performed externally at ACDBio/Biotechne using the RNAscope 2.5 LSx Red assay. Custom-made probes were designed based on the codon-optimized, V5-tagged human transgenes expressed by each AAV. As technical controls, ACD Positive Control  and ACD Negative Control  probes were used. Epitope retrieval was done for 15 min at 88 °C and proteolysis was performed with Protease III for 15 min at 40 °C at ACDBio. Slides were imaged using an AxioScan.Z1 slide scanner .Eyes for histological analysis were generated in an independent experiment that was published elsewhereIn case of the OIR dataset GRCm38.96 was used as the reference genome. For the AAV study, a custom genome was created by merging codon optimized sequences for hIL6, hTNFa, and hVEGF into the mouse genome. Generation of genomic indices and read alignment was carried out via STAR (version 2.5.2b). Quantification on gene level was performed using FeatureCounts (version 1.5.1) and RSEM (version 1.3.0), while discarding multimapping reads. Quality control metrics were obtained using MultiQC (version 1.0), with information assembled from STAR, picardmetrics (version 0.2.4), and fastQC (version 0.11.5), among other sources.Genes with a read count value of below 10 across all samples in either the AAV or OIR study were removed from our analysis. We normalized counts expression values using the voom function provided by limma (version 3.44.3). In the AAV study we corrected for an identified batch effect associated with the RNA extraction batch (variable: rna_extraction_batch) using the ComBat function (sva package version 3.40.0). Principal component analysis was performed using the pcaMethods R package (version 1.80.0) selecting for the top 1000 most variable genes.Differential gene expression was calculated using the lmFit function provided by the limma R package. Significantly changing genes were selected based on a BH-adjusted p-value < 0.05. Pathway information was retrieved using the misgdbr R package (version 7.1.1). Gene set enrichment analysis was performed using the over-representation analysis (ORA) function provided by Clusterprofiler version 4.0.2. Direct comparisons between gene sets derived from the AAV experiment and the OIR study were carried out using a hypergeometric test provided by the stats R package (version 4.1.0). Venn diagrams were plotted using ggvenn (version 0.1.9).http://feb2021.archive.ensembl.org/. Raw counts were further processed using the Seurat R package (version 4.0.3). Cell specific genes were identified for each individual dataset and cell type via the FindMarkers function. Genes were defined as cell specific if the BH-adjusted p-value < 0.01 and the gene was not identified as specific to any other cell type (unique genes only). As above, the overlap between cell specific marker genes and genesets derived from the AAV or OIR studies were quantified by the ORA function part of clusterProfiler.Single cell RNA-Seq datasets were obtained from GSE1352229, GSE130636, and GSE135922. If required, human ensembl IDs were mapped to mouse homolog using the biomaRt R package (2.48.2), with host parameter set to Supplementary Figure 1.Supplementary Table 2.Supplementary Table 3.


E. coli genome codes for four different POTs, known as Di- and tripeptide permeases A-D (DtpA-D). DtpC was shown previously to favor positively charged peptides as substrates. In this study, we describe, how we determined the structure of the 53 kDa DtpC by cryogenic electron microscopy (cryo-EM), and provide structural insights into the ligand specificity of this atypical POT. We collected and analyzed data on the transporter fused to split superfolder GFP (split sfGFP), in complex with a 52 kDa Pro-macrobody and with a 13 kDa nanobody. The latter sample was more stable, rigid and a significant fraction dimeric, allowing us to reconstruct a 3D volume of DtpC at a resolution of 2.7 Å. This work provides a molecular explanation for the selectivity of DtpC, and highlights the value of small and rigid fiducial markers such as nanobodies for structure determination of low molecular weight integral membrane proteins lacking soluble domains.Proton-coupled Oligopeptide Transporters (POTs) of the Major Facilitator Superfamily (MFS) mediate the uptake of short di- and tripeptides in all phyla of life. POTs are thought to constitute the most promiscuous class of MFS transporters, with the potential to transport more than 8400 unique substrates. Over the past two decades, transport assays and biophysical studies have shown that various orthologues and paralogues display differences in substrate selectivity. The  Membranes of cells compartmentalize metabolic processes and present a selective barrier for permeation. To preserve the characteristic intracellular milieu, membrane transporters with specialized functions have evolved to maintain the nutrient homeostasis of cells . Many ofE. coli genome codes for four different POTs named Di- and tripeptide permease A-D (DtpA-D), also known as YdgR (=DtpA), YhiP (=DtpB), YjdL (=DtpC) and YbgH (=DtpD). They cluster in pairs, DtpA and B (sequence identity 51%), and DtpC and D (sequence identity 56%) with around 25% identity between them. DtpA and B exhibit a prototypical substrate preference similar to the human PepT1 transporter  fused the transporter to split-sfGFP , ii) rai. This resulted in a 52 kDa Pro-macrobody (short Mb26), and we expected it to bind to the periplasmic side of the transporter as seen in other MFS transporter-Nb complexes , or ten residues (split sfGFP-DtpC1-470) of the transporter were deleted. We then assessed proper folding and complementation by monitoring the fluorescence of the chromophore on an HPLC system (FL) and lowest in the most truncated version (split sfGFP-DtpC1-470). In order to extend this observation to other MFS transporters, we repeated this experiment with the human POT homologue PepT1, and noticed a similar trend upon shortening of the termini. Yet, since the decrease of fluorescence was only minor in split sfGFP-DtpC1-475 in comparison to split sfGFP-DtpCFL, we proceeded to imaging with the shorter construct in the presence of Nb26.Since MFS transporters typically lack additional domains outside their transport unit, which is a major impediment for accurate particle alignment in single particle cryo-EM approaches, we assessed three fiducial marker strategies introducing additional density outside of detergent micelles containing DtpC, by analyzing the quality of 2D class averages . To obtaomplexes . In a thC system . All con(1–475)-Nb26 sample allowed clear visualization of the transmembrane helices after clustering a small subset of particles, but the majority of particles clustered in classes with blurry density for the split sfGFP fiducial, or with the two complementary parts β1-6 and β7-11 not assembled   . HoweverAs we obtained the best 2D class averages for DtpC with the fiducial marker Nb26, we proceeded to a large data collection  and coulα-atoms). The peptide binding site of DtpC is exposed to the cytoplasmic side  organized in two helical bundles and additional two TMs specific for the POT family (known as HA and HB domains). It is highly similar compared to the previously determined DtpD structure  with an mic side , 4. Almomic side .Shewanella oneidensis (PepTSo), where the inter-bundle periplasmic salt bridge is formed between TM5 and TM7, the IF state is in all other analyzed structures stabilized by a salt bridge on the tip of TM2 and TM7  and R294  and hydrogen bonds between H37  and D293 (TM7) as well as R28 (TM1) and N421  . We also and TM7 . Additio1XXE2R motif on TM1 involved in proton coupling and ligand binding, and ii) the ability to accommodate dipeptides, tripeptides, and peptidomimetics, which relies on a set of conserved residues located in the central binding cavity. In DtpC, the E1XXE2R motif, has evolved to Q1XXE2Y . In all high resolution X-ray structures of canonical POTs, R is in salt-bridge distance to E2 and the C-terminus of substrate peptides. Mutation of either E1 or E2 in the conventional E1XXE2R motif to glutamine residues abolishes uptake  the presence of the Es uptake . A revereases it . In addiproposed . This bi1XXE2Y motif instead of E1XXE2R.In DtpC, we now observe that the side chain pocket has a different architecture and characteristic in comparison with the one of canonical POTs. It displays an overall more acidic groove caused by the presence of the aspartate residue 392, conserved among atypical POTs. This residue has been predicted to be involved in substrate coordination and mutation of this residues in DtpC and homologues DtpD (corresponding residue is D395) abolished transport activity . CanonicE. coli POTs structures. Lastly, it displays some of the challenges related to high resolution cryo-EM structure determination of MFS transporters devoid of soluble domains, and manifests once again, the benefit of fiducial markers in overcoming those.In summary, our work provides new insights into promiscuous versus selective substrate recognition in POTs and constitutes a step forward towards completing the family of Escherichia coli genome, and cloned into a pNIC-CTHF vector by ligation-independent cloning (LIC). This vector contains a C-terminal His-Tag and a Tobacco Etch virus (TEV) cleavage site and a kanamycin resistance gene as selectable marker. The first 6 N-terminal beta strands of sfGFP were fused to the N-terminus of DtpC, and the beta strands 7 to 11 fused to the C-terminus. We named this construct split sfGFP-DtpCFL. Two additional constructs were cloned with truncations of 5 (split sfGFP-DtpC1-475), and 10 residues (split sfGFP-DtpC1-470), on the C-terminal side of DtpC.The full-length cDNA of DtpC wild type (WT) was amplified from the HsPepT1 was previously cloned into a pXLG vector containing an expression cassette composed of an N-terminal Twin-Streptavidin tag followed by the HRV-3C protease recognition sequence  a C-terminal truncation of 36 residues (split sfGFP-HsPepT11-672), and ii) a C-terminal truncation of 36 residues and a N-terminal truncation of 10 residues (split sfGFP-HsPepT110-672) were cloned.sequence . SimilarE. coli C41(DE3) cells grown in terrific broth (TB) media supplemented with 30 μg/ml kanamycin according to established procedures , and the pellet was stored at -20°C until further use. Cell pellets were resuspended in lysis buffer  glycerol, 15 mM imidazole, with 3 ml of lysis buffer per Gram of wet weight pellet), supplemented with lysozyme, DNase and 0.5 mM tris(2-carboxyethyl)phosphine (TCEP). The cells were lysed by three cycles using an Avestin Emulsiflex homogenizer at 10,000–15,000 psi. Recovered material was centrifuged to remove non-lysed cells  and the supernatant was subjected to ultracentrifugation to separate the membrane fraction . Membranes were resuspended in lysis buffer supplemented with cOmplete EDTA-free protease inhibitors (Roche), and solubilized by adding 1% n-Dodecyl-β-D-Maltoside (DDM) detergent (Anatrace). The sample was centrifuged for 50 min at 90,000 × g, and the supernatant was applied to Ni-NTA beads for immobilized-metal affinity chromatography (IMAC) on a gravity column. The beads were pre-equilibrated in lysis buffer and incubated with the solubilized membrane proteins for one hour at 4°C on a rotating wheel. Loaded beads were washed with buffer with increasing imidazole concentrations . The proteins were eluted from the column with a buffer containing high imidazole concentration  and combined with 1 mg of TEV protease to perform the His-tag cleavage during dialysis overnight at 4°C. The dialysis buffer contained 20 mM HEPES at pH 7.5, 150 mM NaCl, 5% glycerol, 0.5 mM TCEP, 0.03% DDM. The cleaved protein was recovered by negative IMAC, concentrated to 4 ml using a 50 kDa concentrator (Corning® Spin-X® UF concentrators) and run on an ÄKTA Pure system , using a HiLoad 16/ 600 Superdex 200 column for DtpC, and a Superdex 200 Increase 10/300 column for the split sfGFP-DtpC constructs. Fractions containing the protein were pooled, concentrated, flash frozen and stored at -80°C until further use.Recombinant DtpC, and the three split sfGFP-DtpC constructs were expressed in ocedures . CultureHsPepT1 constructs, expression was done in mammalian cells as described previously . After 20 min of incubation on a rotating wheel, the suspension was transferred to a gravity column. Following two wash steps with 300 mM NaCl, 20 mM HEPES (pH 7.5), 0.03% DDM, and 0.003% CHS, split sfGFP-HsPepT1comnstructs were eluted with 0.03% DDM, 0.003% CHS, 150 mM NaCl, 20 mM HEPES (pH 7.5), and 10 mM desthiobiotin (Sigma-Aldrich).For the split sfGFP-eviously . BrieflyTo generate DtpC specific nanobodies, two non-inbred llamas were injected six times at weekly intervals with a mixture of 94 different proteins including DtpC purified in the detergent DDM (50 µg of each antigen weekly). After 6 weeks of immunization, two separate phage display libraries were constructed, one from each animal, in the pMESy2 vector, which is a derivative of pMESy4 that contain a C-terminal EPEA-tag for affinity purification. After pooling both libraries, nanobodies were selected against individual antigens in two rounds of parallel panning in 96-well plates containing one immobilized antigen in each well. After two selection rounds on DtpC, 60 clones were picked for sequence analysis, 13 clones encoded antigen-specific nanobodies as tested in ELISA, grouping them in 5 different sequence families. A nanobody family is defined as a group of nanobodies with a high similarity in their CDR3 sequence . Nanobodies from the same family derive from the same B-cell lineage and likely bind to the same epitope on the target. Immunizations, library construction, selection by panning and nanobody characterization were performed according to standard procedures . Five naE. coli WK6 cells and purified following standard procedures. Specifically, the cell pellet was resuspended in TES buffer  supplemented with one protease inhibitor tablet (Roche). Osmotic shock was performed by the addition of diluted TES buffer to release the periplasmic proteins. The solution was first centrifuged for 20 min at 10,000 × g and additionally for 30 min at 100,000 × g. The supernatant was applied to CaptureSelect beads (Thermo Fisher Scientific), which were equilibrated with wash buffer . After three column volumes of washing, the nanobody was eluted with 20 mM HEPES, pH 7.5, 1.5 M MgCl2. The nanobodies were further purified on a HiLoad 16/600 Superdex 75 pg column in 20 mM HEPES, pH 7.5, 150 mM NaCl, 5% glycerol, concentrated with a 5 kDa cut-off concentrator, flash-frozen and stored at -80°C until further use.The nanobodies were expressed in E. coli WK6 cells as above. The cell pellet was resuspended in TES buffer  supplemented with one protease inhibitor tablet (Roche). Osmotic shock was performed by the addition of diluted TES buffer to release the periplasmic proteins. The solution was first centrifuged for 20 min at 10,000 × g and additionally for 30 min at 142,000 × g. The supernatant was further purified by immobilized-metal affinity chromatography (IMAC) on a gravity column. The beads were pre-equilibrated in 20 mM NaPi at pH 7.5, 300 mM NaCl, 5% glycerol, 15-30 mM imidazole, 0.5 mM TCEP and incubated. Loaded beads were washed with increasing imidazole concentrations . The proteins were eluted from the column with a buffer containing high imidazole concentration  and combined with 1 mg of 3C protease to perform the His-tag cleavage. The cleaved protein was recovered by negative IMAC, concentrated to 0.5 ml using a 30 kDa concentrator (Corning® Spin-X® UF concentrators) and run on an ÄKTA Pure system , using a Superdex 75 Increase 10/300 column. Fractions containing the protein were pooled, concentrated, flash frozen and stored at -80°C until further use.The nanobody 26 (Nb26) was first inserted into a pBXNPH3 vector containing a C-terminal penta-histidine tag preceded of a HRV-3C protease recognition sequence. The maltose binding protein (MBP) was then inserted in frame with the 3’ end of the nanobody, with two proline residues as a linker between the two genes similar as described in . The resulting Pro-macrobody 26 (Mb26) was expressed in 1-475-Nb26 with a Prometheus NT.48 device . The purified proteins were diluted to 16 µM, and the complexes were formed using a 1:1.5 M ratio of membrane protein: fiducial. The fluorescence at 330 and 350 nm was recorded over a temperature gradient scan from 15° to 95°C and processed in GraphPad Prism 9.0 (GraphPad Software).The differential scanning fluorimetry method was used to follow the thermal unfolding event  of Nb17,Structures with the following sequences were used as input for AlphaFold2 structure prediction , and AMBMSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNKTPSQPRAIYYIVAIQIWEYFSFYGMRALLILYLTHQLGFDDNHAISLFSAYASLVYVTPILGGWLADRLLGNRTAVIAGALLMTLGHVVLGIDTNSTFSLYLALAIIICGYGLFKSNISCLLGELYDENDHRRDGGFSLLYAAGNIGSIAAPIACGLAAQWYGWHVGFALAGGGMFIGLLIFLSGHRHFQSTRSMDKKALTSVKFALPVWSWLVVMLCLAPVFFTLLLENDWSGYLLAIVCLIAAQIIARMMIKFPEHRRALWQIVLLMFVGTLFWVLAQQGGSTISLFIDRFVNRQAFNIEVPTALFQSVNAIAVMLAGVVLAWLASPESRGNSTLRVWLKFAFGLLLMACGFMLLAFDARHAAADGQASMGVMISGLALMGFAELFIDPVAIAQITRLKMSGVLTGIYMLATGAVANWLAGVVAQQTTESQISGMAIAAYQRFFSQMGEWTLACVAIIVVLAFATRFLFSTPNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNKTPSQPRAIYYIVAIQIWEYFSFYGMRALLILYLTHQLGFDDNHAISLFSAYASLVYVTPILGGWLADRLLGNRTAVIAGALLMTLGHVVLGIDTNSTFSLYLALAIIICGYGLFKSNISCLLGELYDENDHRRDGGFSLLYAAGNIGSIAAPIACGLAAQWYGWHVGFALAGGGMFIGLLIFLSGHRHFQSTRSMDKKALTSVKFALPVWSWLVVMLCLAPVFFTLLLENDWSGYLLAIVCLIAAQIIARMMIKFPEHRRALWQIVLLMFVGTLFWVLAQQGGSTISLFIDRFVNRQAFNIEVPTALFQSVNAIAVMLAGVVLAWLASPESRGNSTLRVWLKFAFGLLLMACGFMLLAFDARHAAADGQASMGVMISGLALMGFAELFIDPVAIAQITRLKMSGVLTGIYMLATGAVANWLAGVVAQQTTESQISGMAIAAYQRFFSQMGEWTLACVAIIVVLAFATRFLFSTPTNMIQESNDNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNKTPSQPRAIYYIVAIQIWEYFSFYGMRALLILYLTHQLGFDDNHAISLFSAYASLVYVTPILGGWLADRLLGNRTAVIAGALLMTLGHVVLGIDTNSTFSLYLALAIIICGYGLFKSNISCLLGELYDENDHRRDGGFSLLYAAGNIGSIAAPIACGLAAQWYGWHVGFALAGGGMFIGLLIFLSGHRHFQSTRSMDKKALTSVKFALPVWSWLVVMLCLAPVFFTLLLENDWSGYLLAIVCLIAAQIIARMMIKFPEHRRALWQIVLLMFVGTLFWVLAQQGGSTISLFIDRFVNRQAFNIEVPTALFQSVNAIAVMLAGVVLAWLASPESRGNSTLRVWLKFAFGLLLMACGFMLLAFDARHAAADGQASMGVMISGLALMGFAELFIDPVAIAQITRLKMSGVLTGIYMLATGAVANWLAGVVAQQTTESQISGMAIAAYQRFFSQMGEWTLACVAIIVVLAFATRFLFSTPTNMIQESNDGGGGGNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK.One hour before vitrification, the purified protein complexes were thawed on ice and run on a Superdex Increase 200 5/150 column in 0.015% DDM, 100 mM NaCl, 10 mM HEPES (pH 7.5), 0.5 mM TCEP in order to remove the excess of empty detergent micelles earlier generated upon sample concentration. The top fraction reached a concentration ranging between 3 and 6 mg/ml, and for each sample, 3.6 μl were applied to glow-discharged gold holey carbon 2/1 300-mesh grids (Quantifoil). Grids were blotted for 4 s at 0 force and 1-s wait time before being vitrified in liquid propane using a Mark IV Vitrobot (Thermo Fisher Scientific). The blotting chamber was maintained at 4°C and 100% humidity during freezing.All movies were collected using a Titan Krios (Thermo Fisher Scientific) outfitted with a K3 camera and BioQuantum energy filter (Gatan) set to 10 eV. Automated data acquisitions were set using EPU (Thermo Fisher Scientific). The applied defocus ranged between -0.9 µm and -1.8 µm in all datasets.2 was used with 2.8 s exposure time, fractionated in 50 frames. For split sfGFP-DtpC1-475-Nb26, movies were collected at a nominal magnification of ×,130,000 and a physical pixel size of 0.67 Å, with a 50-μm C2 aperture and 100-μm objective aperture at a dose rate of 19.0 e−/pixel per second. A total dose of 57 e−/Å2 was used with 3 s exposure time fractionated in 40 frames.For DtpC-Nb26 and DtpC-Mb26, movies were collected at a nominal magnification of ×105,000 and a physical pixel size of 0.85 Å, with a 70-μm C2 aperture and 100-μm objective aperture at a dose rate of 19.5 e−/pixel per second. A total dose of 75 e−/ÅAll movies were motion-corrected using Relion-3.1  own impl1-475-Nb26, 7602 movies were collected, 1,049,399 coordinates were picked and used for 2D averaging and clustering. For DtpC-Nb26, 24,333 movies were collected, 6,464,070 coordinates were picked and used for 2D averaging and clustering, and 878,428 particles were used in the final 3D reconstruction. Briefly, DtpC-Nb26 dimeric population was clustered using 3D class averaging in Relion3.1  were collected on the EMBL P12  beamlineGraphs were generated using GraphPad Prism 9.0 (GraphPad Software). Molecular graphics and analyses performed with UCSF ChimeraX-1.2.5 . Figures


Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals. Neural stem cells in modern humans have a longer metaphase and fewer chromosome errors compared with Neanderthals and chimpanzees. FOXP2  Kinesin 8], KNL1 (a.k.a. CASC5), and SPAG5 (a.k.a. astrin), which are highly expressed in the germinal zones of the developing neocortex and are associated with mitotic spindle, kinetochore, and chromosome segregation functions. The kinetochore is a complex, three-dimensional (3D), multiprotein structure mediating the attachment of chromosome centromeres with the ends of kinetochore microtubules  that carry the ancestral variants of KIF18a and KNL1 are used to generate cerebral organoids, shorter metaphases, less SAC-positive kinetochores, and more chromosome segregation defects are observed in APs. Together, our data suggest that the three amino acid substitutions in KIF18a and KNL1 cause fewer chromosome inheritance errors to occur in APs of modern humans than in archaic humans and apes.P > 0.05; P > 0.05; fig. S1, A, B, and D). Organoids can therefore be used to study the number of SAC-positive kinetochores in cerebral tissue APs.An active SAC, triggered, e.g., by one or more kinetochores not being properly attached to kinetochore microtubules, delays anaphase onset and therefore prolongs metaphase  in the fetal human neocortex and human organoid APs, respectively, than in chimpanzee organoid APs . Thus, human metaphase APs have more SAC-positive kinetochores than chimpanzee APs.To investigate whether the previously described metaphase prolongation in human relative to chimpanzee APs  mice; P < 0.001; P < 0.001) that did not differ (P > 0.05) from the hKIF18a-hKNL1-hSPAG5 mice with the six amino acid substitutions , two in KNL1 (H159R and G1086S), and three in SPAG5 , albeit not significantly so  versus 4.6 min (hKNL1 substitution 2) versus 4.6 min (wt), respectively; fig. S4, see also In contrast, in hSPAG5 mice, the length of the AP metaphase was 4.4 min and hence similar to wt mice , indicatNotably, the durations of prometaphase, which precedes metaphase, and of anaphase, which follows metaphase, did not show any significant changes in any of the mouse lines analyzed .We also explored whether the modern human-specific changes in KIF18a and KNL1 may influence general aspects of cortical development. However, we did not observe any effects on the overall morphology, thickness, or perimeter of the neocortex in adult mice humanized for KIF18a and KNL1 . Consistent with this, the adult mice did not show any change in the distribution or numbers of Ctip2KIF18a and the two codons in KNL1 back to the ancestral, Neanderthal-like state in the H9 ESC line. Individual cells subjected to the relevant RNA guides and donor DNAs were expanded to cell lines, among which we selected two independent ancestralized lines [aKif18a-aKNL1 lines 1 (L1) and 2 (L2)] and two lines where none of the three codons had been changed  at the midpoint of the dark period . After positive plug detection in the morning, the cumulus complexes were isolated and zygotes were removed with a treatment of hyaluronidase [final concentration of 0.1% (801 U/ml)]. CRISPR-Cas9 solutions were injected into the male pronucleus of fertilized zygotes by using a motor-driven manipulator-based microinjection stage. About 2 hours after injections, the surviving embryos were transferred into Crl:CD1(ICR) pseudopregnant recipient female mice (ca. 20 embryos per recipient).The recipient mice were mated with vasectomized males Crl:CD1 (ICR). After detection of a copulation plug in the morning of the transfer day, pseudopregnant mice were used for unilateral surgical embryo transfer into the oviduct as described  with 2 μl of genomic tail DNA, 0.25% dimethyl sulfoxide, and 0.5 μM forward and reverse primer. Primer sequences (target-seq-primer) and PCR conditions are in table S1. Products were Sanger sequenced either with the forward and reverse PCR primers or if required with internal sequencing primers. Primary candidates have been identified by sequence alignments of potentially modified PCR fragments against the wt and modified reference sequence. The products from putatively edited mice were cloned (TOPO) and verified by Sanger sequencing of 10 to 20 randomly selected clones. Founders carrying the respective genome modification either homo- or heterozygously were propagated and genotyped by Sanger sequencing of PCR fragments. From the second generation onward, allele-specific PCRs were performed on 2 μl of crude buccal DNA in a total of 10 μl using the REDExtract-N-Amp PCR ready Mix (Sigma-Aldrich) according to the manufacturer’s protocol using 0.5 μM forward, reverse, and mutant primer. The readout is done on standard agarose gels. Primers and PCR conditions are in table S1.Kif18a, Knl1, and Spag5 in the wt and humanized embryonic mouse telencephalon was confirmed by PCR, using generated cDNA as templates, followed by Sanger sequencing of the PCR products. Wt and gene-edited sequences were mapped to the Kif18a, Knl1, and Spag5 genes, respectively, using Geneious Prime (version 2020.2.4). Data showed the expression of only the wt Kif18a, Knl1, and Spag5 in E11.5 wt embryonic mouse telencephalon, and the expression of only the gene-edited hKif18a, hKnl1, and hSpag5 in E11.5 humanized hKIF18a-hKNL1-hSPAG5  and LightCycler 96 Instrument (Roche). Gene expression analyses by qPCR showed that the relative mRNA levels of Kif18a, Knl1, and Spag5 were not significantly different between E11.5 wt and humanized embryonic mouse telencephalon .For Sanger sequencing and real-time qPCR, total RNA was isolated from E11.5 wt and humanized mouse dorsolateral telencephalon using the RNAeasy Micro Plus Kit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using the Maxima First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The expression of PAG5 see  embryoniThe following primers were used for PCR:Forward primer for Kif18a (5′-AGAAAAGGCGGTGCAGTTCT-3′),Reverse primer for Kif18a (5′- GGAATCTTCCCGGACAGCAA-3′);Forward primer for Knl1 H159R (5′-CCCCAGACAAGTCAAGCAGAA-3′),Reverse primer for Knl1 H159R (5′-TCAACTCCATACACTCATTGCC-3′);Forward primer for Knl1 G1086S (5′-TGGATATCACCAAGAGTTGCAC-3′),Reverse primer for Knl1 G1086S (5′-CAAAACTGAAGCCCTTTCTGTC-3′);Forward primer for Spag5 D410H and E162G (5′-TGAATCTCGGTTTGTCGCCT-3′),Reverse primer for Spag5 D410H and E162G (5′-TTCCTCCCCTGGATCGACAT −3′);Forward primer for Spag5 P43S (5′-GTTCAAATAGAGGCGGCGGG-3′),Reverse primer for Spag5 P43S (5′-GTTCTTTCCCACCAGCTACAAG-3′).The following primers were used for qPCR:Forward primer for Kif18a (5′-CAAACTCAGGACCACTTGCTGT-3′),Reverse primer for Kif18a (5′-ATGGGAACGAGAAGACACTGC-3′);Forward primer for Knl1 (5′-CCTCTGGGGGAGATGGCTACAT-3′),Reverse primer for Knl1 (5′-GATGGACTTTGTTGGGCTGAGA-3′);Forward primer for Spag5 (5′-GTCTCACCCTCTTCTTACAGGC-3′),Reverse primer for Spat5 (5′-GCTGGTTCTGGCACTTCATCTA-3′);Forward primer for Actb (5′-CGGGACCTGACAGACTACCTC-3′),Reverse primer for Actb (5′-GGTGGTGAAGCTGTAGCCACG-3′).AAVS1 locus from the H9 human ESC line  that we generated as previously described :KIF18A_sg1: 5′-TGTTATAAAGAAACAAAATAKIF18A_sg2: 5′-GATTTGTAGTTTTCTTTCCAKNL1_H109R_sg1: 5′-ATTTGCATGTTTCCTTTCACKNL1_G1086S_sg1: 5′-TGAATGAACCTCTATCAAGCAssDNA KIF18A_K67R: 5′-AGTTGACGTTTCATCAAAAACAGCATCAAATACAAATTTAAGATCCTTGTTTTGTCTCTTTATAACATTTTGATTTGTAGTTTTCTTTCCATGGAAAAAACTGACTssDNA KNL1_H159R: 5′-TGTTGTAAAATGCCTTTTTAAAAGTTTGCTTTTGTTCTGATCTCTTATATTTTGCTTTCATTATAGTTTTCAATTATAGAACATACCCATGAAAGGAAACATGCAAATGACCAGACAGTCATTTTTTssDNA KNL1_G1086S: 5′-TCTTGTCATTTTTTAGCTTAAGGCTTTTTCTTCTCTGACTTTTGCCTGATAGAGGTTCGTTCAGAAATCCAGGACTTTGTACATCTTTGATo derive colonies from single cells, cells were incubated with StemFlex with supplement  and CloneR  for 1 day and then sorted with a Cytena cell printer.Genomic DNA of each colony was isolated, a region of ~200 base pairs (bp) around the cut site was amplified and sequenced, and from sequences, the editing state was evaluated as described  amplification included 1× ddPCR Supermix for probes , 0.2 μM of each primer and 0.2 μM probe (both IDT) for target and reference, together with 1 μl genomic DNA in QuickExtract DNA Extraction Solution (Lucigen). After droplet generation with the QX200 Droplet generator (Bio-Rad), the PCR was for 5 min at 95°C, followed by 42 cycles of 35 s at 95°C (at a ramp rate of 1.5°C/s) and 65 s at 54°C for KIF18A and KNL1_G1086S or 65 s at 56°C for KNL1_H109R (at a ramp rate of 1.5°C/s), and 5 min at 98°C. Readout was in a QX200 Droplet reader (Bio-Rad), and allele copy numbers were determined relative to the FOXP2 reference and unedited controls.Deletions bigger than ~200 bp and therefore not detected by the amplicon sequencing KNL1_G10862_upSNP_F: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGCAATCCCACACCTGACTKNL1_G10862_upSNP_R: 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTTCGTCCTCTAACACATCCAKNL1_G10862_downSNP_F: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCCCTGGAGGATAAAGAGGAKNL1_G10862_downSNP_R: 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAATCGGTGACTTCCAGATCAKNL1_H159R KNL1_H159R_upSNP_F: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTAAGACCCTTCAGAATCCTACCCKNL1_H159R_upSNP_R: 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGTATCCTCTGTGTGGCTGAAKNL1_H159R_downSNP_F: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAGAGCCTGTCAAATCCTTKNL1_H159R_downSNP_R: 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCAGGTGTCGGTTAAGCTGTKIF18A KIF18A_upSNP_F: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGTGGGTAAGGGAGTGGGAATKIF18A_upSNP_R: 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCTTCAGAGCCTGCACTCTTKIF18A_downSNP_F: 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATCTGCAACCGTTTTAGCCAAGATKIF18A_downSNP_R: 5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCCCCGAATGTCTTTCTCATo find large-scale chromosomal deletions, duplications, or karyotype abnormalities, we performed “shallow” whole genome sequencing to an average genome coverage 0.10 to 0.15 of edited and nonedited colonies as well as the cell line from which the colonies were derived, as described (KIF18a and KNL1 in edited and nonedited cell lines as well as their “mother” cell line was measured with CellsDirect One-Step qRT-PCR Kit (Invitrogen) using TaqMan probes for KIF18a (FAM Hs0105428_m1), KNL1 (FAM Hs00538241_m1), and a “housekeeping gene” PPIB (VIC Hs00168719_m1). The producer’s protocol was followed to set up reactions. For a subset of reactions, reverse transcriptase was inactivated by 15-min incubation at 70°C. The cycler program for RT-qPCR was 50°C for 10 min, 95°C for 2 min, then 50 cycles of 95°C for 3 s, 53°C for 15 s, and 60°C for 30 s.Expression of Organoids were generated from the H9 ESCs and cultured as described  and immunohistofluorescence (fixed). Mouse neocortex was fixed, embedded, cryosectioned, and prepared for immunohistofluorescence as described  for multiphoton excitation, and using 63× Plan-Apochromat 1.4 numerical aperture (NA) oil or 40× C-Apochromat 1.2 NA W objectives .http://imagej.nih.gov/ij/). Brightness and contrast were recorded and adjusted linearly. Measurements of mitotic phases were performed as described . Because at least some datasets in most experiments did not pass normality tests (Shapiro-Wilk and Kolmogorov-Smirnov tests), nonparametric Mann-Whitney