Clinical outcomes for mucosal melanomas are often poor due to a lack of effective systemic drug therapies. Identifying driver genes in mucosal melanoma may enhance the understanding of disease pathogenesis and provide novel opportunities to develop effective therapies.SF3B1 (6 of 27: 22%) as the most commonly mutated gene, followed by KIT (3 of 27: 11%). Other less frequently mutated genes (4% otherwise stated) included BRAF (7%), NRAS (7%), ARID2, CTNNB1, DICER1, MAP2K1, NF1, PTEN, SETD2 and TP53. Recurrent SF3B1 p.R625 hotspot mutations were exclusively detected in vulvovaginal (5 of 19: 26%) and anorectal melanomas (3 of 5:60%). The only other SF3B1 mutation was a p.C1123Y mutation that occurred in a conjunctival mucosal melanoma.Somatic variant analysis identified SF3B1-mutated patients were associated with shorter overall survival  and progression-free survival  compared to non-SF3B1-mutated patients .SF3B1 mutations occurred predominantly in the vulvovaginal region. SF3B1 mutations may have a negative prognostic impact.Molecular subgroups of mucosal melanoma with Formalin-fixed biopsies were collected from 27 pathologically-confirmed mucosal melanomas. Genomic DNA was isolated from the tumor tissue and sequenced using a novel dual-strand amplicon sequencing technique to determine the frequency and types of mutations across 45 target genes. Mucosal melanoma is a rare subtype of melanoma that originates from melanocytes in the epithelial lining of the conjunctiva, respiratory, alimentary, and genitourinary tracts. Although a majority of mucosal melanomas arise from the mucosa of the nasal cavity, oral cavity, anorectum, vulvovaginal, they can arise in any part of mucosal membranes. Mucosal melanomas comprise approximately 1% of all melanomas in European populations, but up to 25% in Asian populations . In contNRAS only occur in 10–20% of mucosal melanomas [GNAQ and GNA11 that are commonly detected in uveal melanoma, occur in approximately 9.5% of mucosal melanomas [KIT, are present in approximately 15% of mucosal melanomas [Several studies have shown that mucosal melanomas have distinct molecular profiles –11. Moleelanomas , 6, whilelanomas –14. Unfon =183) of cutaneous (n = 140), acral (n = 35) and mucosal melanomas (n = 8), and SF3B1 (splicing factor 3B subunit 1) was identified as significantly mutated gene in mucosal melanoma [SF3B1 mutations are the most common spliceosomal component gene mutation implicated in the pathogenesis of cancer and act by causing aberrant RNA splicing events [SF3B1 were identified in 20% of uveal melanomas [SF3B1 in subsets of solid tumors, as well as in myelodysplastic syndrome and chronic lymphocytic leukemia (CLL), in which they occurred in almost 15% of the reported cases [Recently, we performed whole-genome sequencing (WGS) on a large cohort , anorectal region , nasopharynx , conjunctiva  and palate . Fourteen (52%) patients had T4 disease, three (11%) had T3 disease, and the remaining patients had T0, T1 or T2 disease. Tumor thickness was >1 mm in 22 (82%) patients. Median mitotic rate was 15 mitoses/mm2 and ulceration was present in 16 of 22 patients (72%) with known ulceration status.There were 27 patients included in this study Table ; 22 femaet al or TCGA SKCM datasets that were detected using the MutSig, OncodriveFML or IntOGen driver detection tools for coding mutations and OncodriveFML for non-coding genes, as outlined in the prior studies [SF3B1 (6 of 27: 22%) as the most commonly mutated gene, followed by KIT (3 of 27: 11%) , NRAS (7%), ARID2, CTNNB1, DICER1, MAP2K1, NF1, PTEN, SETD2 and TP53  and KIT (67%) mutations were most frequently mutated in tumors of female genital origin and anorectal region (33.3% for SF3B1 and KIT) compared to a single SF3B1 mutant conjunctival melanoma in the upper body sites  that occurred in the known hotspot p.R625H/L originated in the anorectal or female genital region, while the conjunctival primary harbored a SF3B1 p.C1123Y mutation , nucleotide and amino acid changes are summarized in A 45 gene targeted NGS panel was designed to include all significantly mutated genes (SMG) identified in cutaneous, mucosal or acral melanomas in our previous publications , 27. The studies , 27, 28.) Figure . Other ls Figure . All fivn Figure . The KITSF3B1 mutations at codon 625 in anorectal and vulva/vaginal melanomasRecurrent hotspot SF3B1 as a significantly mutated gene in mucosal melanoma using OncodriveFML . Similar to The Cancer Genome Atlas cohort and our previous published work [SF3B1 mutations occured at codon 625, comprising four p.R625H and one p.R625L alterations  had T2 disease, one (16.7%) had T3 disease, and the remaining three (50%) had T4 disease. The depth of invasion of SF3B1 mutant tumors ranged from 1.9 to 12 mm. Melanomas with SF3B1 mutations had a similar mitotic rate when compared to non-SF3B1 mutated cases (15.6±4.4 vs 15.5±2.54 mitoses/mm2) but were more often ulcerated (SF3B1: 6 out of 6 cases vs non-SF3B1: 10 of 16 cases with ulceration data), and were comprised of heterogeneous cell types. We tested whether SF3B1 mutated and non-SF3B1 cases had different prognosis. Median overall survival (OS) was 34.9 months in patients harboring SF3B1 mutations compared to 79.7 months in patients with non-SF3B1 mutations . Five patients harbored a mutation at exon 14, including p.R625H (n = 4) and p.R625L (n = 1), while one patient carried a p.C1123Y mutation within exon 23 in SF3B1. Of particular interest is the confirmation that the SF3B1 hotspot mutant cases are apparently unique to mucosal melanomas of the lower body sites, hinting to divergent biology with those of upper body sites [KIT have been reported in 15–20% of mucosal melanomas, and are more commonly observed in anorectal and vulval/vaginal tumors (15–25%) [KIT mutations in 11% of mucosal melanomas, particularly in the vulval and vaginal sites. KIT functions as a receptor tyrosine kinase, which transmits signals from the cell membrane into the cell [Mutations within exons 12–15 of  cancers , 30. In dy sites . Further(15–25%) , 31–33. (15–25%) , 31–33, the cell , 35. Oncthe cell . The abethe cell ), howeveSF3B1 has functional impact in initiating aberrant 3’ splice site selection causing down-regulation of canonical protein expression to promote tumorigenesis [SF3B1 mutation in mucosal melanomas may provide more understanding of its role in tumorigenesis, and facilitate the development of new drugs (i.e. SF3B1 inhibitors) for mucosal melanomas with SF3B1 mutations.Our findings are in line with the growing evidence that the recurrent hotspot mutation at codon 625 of igenesis , 20, 24.igenesis , 24, 25.igenesis , 38. Phaigenesis , 38. FutSF3B1 mutations have different prognostic associations in different types of cancers [SF3B1 mutations are associated with a better prognosis, whereas in CLL, SF3B1 mutations are correlated with a worse prognosis [SF3B1-mutated patients had a worse PFS and OS outcomes, however, our study of these rare tumors was underpowered to detect statistically significant differences. Larger studies are required to address this issue. cancers , 39, 40.rognosis , 40, 41.SF3B1 C1123Y mutation in a conjunctival mucosal melanoma, and a recurrent SF3B1 R625 mutation, which predominantly occurred in female genital tract mucosal melanomas. We validated previous reports that the commonly known mutations in cutaneous melanoma, including BRAF, NRAS, NF1, GNAQ and GNA11, were rarely mutated in mucosal melanomas. This study provides additional insight into genetic alterations that occur in mucosal melanomas. Collectively, these findings provide a better understanding of the oncogenic drivers of tumor development and may the identification of effective systemic therapies for these rare melanomas.In summary, we discovered a This study was approved by the New South Wales Department of Health Human Research Ethics Committee (Protocol no. X15-0454). All human research procedures were performed according to the National Health and Medical Research Council of Australia guidelines. Samples were acquired with patients’ informed consent from the Melanoma Institute Australia Biospecimen bank and Royal Prince Alfred Hospital. Mucosal melanomas were defined as occurring in the mucosa membranes of the oral, conjunctiva, respiratory, gastrointestinal and urogenital tracts. The Melanoma Institute Australia’s research database was searched for primary melanomas originating in the aforementioned sites, and all available archival formalin-fixed biopsies were collected from these patients. The H&E slides of the primary melanoma of all cases were carefully reviewed independently by melanoma pathologists  to ensure they were from a primary mucosal site. Any melanoma that had arisen at the junction of mucosa and cutaneous skin was excluded.Formalin-fixed, paraffin-embedded tumor tissue was prepared in 10 μm sections and deparaffinized through xylene and ethanol according to standard procedures. After air-drying, the tumor tissue was manually macro-dissected, as previously reported . GenomicAmplicon dual-strand (DS) library preparation was performed using the TruSeq Custom Amplion Low Input Library Prep Kit according to the manufacturer’s protocol. Briefly, the amount of DNA was quantitated and diluted to a final concentration of 10−25 ng/μl. The DNA samples were hybridized to the custom amplicons across 45 target genes  using IlP = 1–CDF, where P is the probability that no variant is present given K or more observations, and CDF represents Poisson cumulative distribution function. The probability, P, is subsequently converted to a Q-score and only calls with above Q20 were included for further processing. Variants that were marked as passing quality control contained the following criteria: (i) each variant present in both pools, (ii) a cumulative depth of 1000, and (iii) at least 3% or greater variant frequency. Variants were annotated using human genome build UCSC hg19 with ANNOVAR [Sequence data were aligned to the UCSC hg19 assembly using BWA 0.7.13 , and SAM ANNOVAR . Variant ANNOVAR . The mut ANNOVAR .U test and Fischer exact tests for continuous and categorical variables as appropriate. The Kaplan–Meier survival curves were compared according to the type of mutation group using the log-rank test. All statistical analyses were performed using GraphPad Prism 7.0.The association of mutation status with available clinicopathologic characteristics was performed using Mann–Whitney


Subtelomeric transcription and chromatin can have a significant impact on telomere repeat maintenance and chromosome stability. We have previously found that tumor suppressor protein p53 (TP53) can bind to retrotransposon-like elements in a majority of human subtelomeres to regulate TERRA transcription and telomeric histone acetylation in response to DNA damage. TP53 also prevents the accumulation of γH2AX DNA-damage signaling at telomeres. We now show that the inherited TP53 polymorphism Pro47Ser (hereafter S47), which is enriched in populations of African descent, is associated with elevated marks of telomere dysfunction. We found that human and mouse cells carrying the S47 variant show increased γH2AX DNA-damage signals at telomeres, as well as reduced TERRA transcription and subtelomeric histone acetylation in response to DNA damage stress. Cell-lines containing inducible genes for P47 or S47 versions of p53, as well mouse embryo fibroblasts (MEFs) reconstituted with human p53, showed elevated telomere-induced DNA damage foci and metaphase telomere signal loss in cells with S47. Human lymphoblastoid cell lines (LCLs) derived from individuals homozygous for S47, show increased accumulation of subtelomeric γH2AX and unstable telomere repeats in response to DNA damage relative to age matched LCLs homozygous for P47. Furthermore, LCLs with S47 had reduced replicative lifespan. These studies indicate that the naturally occurring S47 variant of p53 can affect telomeric chromatin, telomere repeat stability, and replicative capacity. We discuss the potential evolutionary significance of the S47 variant to African populations with respect to telomere regulation and the implications for inherited health disparities. The tumor suppressor p53 (TP53) is among the most frequently mutated genes in human cancers . In addiA principal function of p53 is to protect the genome from excessive mutagenesis, degradation, and instability . We haveTo investigate the function of S47 in human cells, we utilized p53 null H1299 cancer cell lines that were reconstituted with doxycycline-inducible p53 genes carrying either WT p53 (P47) or the S47 variant. P53 was induced and activated by addition of both doxycycline and 1 μM etoposide for 24 hrs to induce DNA damage signaling . After 2H1299 cells treated as described above  were anaTo determine if DNA replication and/or DNA repair are impaired in S47 expressing H1299 cells, we assayed BrdU incorporation by DNA-IP (DIP) assay. Cells were treated with doxycycline and etoposide as above to induce p53 and DNA damage, followed by addition of BrdU and 24 hr recovery time. The BrdU-DIP assay was used to measure the rate of DNA synthesis at the 18q subtelomere. We focused on the 18q subtelomere since it was found to selectively accumulate γH2AX in S47, but not P47 expressing cells . The BrdTo corroborate the results above, we took advantage of mouse embryo fibroblasts (MEFs) isolated from P47 and S47 mice with knock-in humanized p53 . We usedEBV immortalized lymphoblastoid cell lines (LCLs) containing either homozygous P47 or S47, obtained from Coriell Institute, were described previously . Westernp53 is known to play a critical role in telomere regulation through a telomere-shelterin-ATM/ATR-p53 axis , 16. LosDirect binding of p53 to subtelomeres can alter telomere function through several mechanisms. p53 can induce TERRA transcription , 17. Dirp53 mutations and naturally occurring variants may alter site-specific functions of p53, including those at telomeres. Loss of function mutations found in cancers likely provide a permissive environment for telomere shortening and dysfunction in proliferating cells, a condition that facilitates genomic instability. However, it is not known how gain-of-function mutations and natural variants of p53 may alter telomere length and regulation. Here, we have found that cells harboring the S47 variant of p53 have dysfunctional telomere regulation in human and mouse cell culture models. In each model, we found that S47 impaired telomere transcription and DNA integrity. In human cells, we found that S47 increased the formation of telomeric histone H3K27Ac and decreased accumulation of γH2AX. Increased histone acetylation is likely due to the direct recruitment of histone acetylases by p53 at sequence-specific subtelomeric DNA binding sites. The accumulation of γH2AX is likely due to the reduced efficiency of S47 in DNA damage repair at telomeres. This effect on telomere DNA damage repair is likely due to the direct effect of p53 on TERRA transcription and histone acetylation. However, it is also possible that indirect, non-telomeric functions of p53 also contribute to telomere dysregulation observed with S47 variant.Other mutations in p53 have been shown to affect telomere structure and regulation. A dominant negative mutation of p53 (N340Q/L344R) was found to reduce TERRA expression and enhance hepatocellular carcinoma . p53 mutNatural genetic polymorphisms may contribute to population health disparities. The S47 variant of p53 has been shown to occur predominantly in individuals of African descent , 25. AltHCT116 +/+ and −/− cells were generously contributed by Bert Vogelstein’s laboratory (Johns Hopkins School of Medicine). HCT116 cells were cultured in Dulbecco’s Modification of Eagle’s Medium  with 10% fetal bovine serum (FBS), 1% Pen/Strep and 4.5 g/L glucose and L-glutamine. H1299 tet-inducible p53 cell lines were cultured in DMEM, 1% Pen/Strep and with Tet-system approved 10% FBS . A concentration of 0.75 ug/ml doxycycline was used to induce p53. Etoposide (Sigma) 50 mM in DMSO was added to cells, at 1, 2, or 50 μM final concentration for 24 hr or times indicated. MEFs was P47 and S47 mice, as well as LCLs from individuals homozygous for P47 and S47, were previously described [ChIP-qPCR was performed essentially as described previously . All qPCThe following antibodies were used: mouse Anti-p53 (Ab6) ; rabbit anti-phospho-p53 (Ser15) ; mouse anti-phospho-Histone H2A.X (Ser139) (Millipore 05-636); rabbit anti-Histone H2A.X (Millipore 07-627); rabbit anti-acetyl-Histone H3 (Lys9) (Millipore 07-352); rabbit anti-Histone H3 (K4me1) (abcam ab8895); rabbit anti-Histone H3 (K27Ac) (abcam ab4729); rabbit anti-Histone H3, CT, pan (Millipore 07-690); rabbit anti-GAPDH (14C10) .t method using Power SYBR Green (Applied Biosystems) and the primers have been described previously [RNA was isolated from cells by suspending well in 1 ml Trizol (Life Technnologies) at RT. 200 μL chloroform was added, and the sample shaken by hand for 15 sec. After 2–3 min at RT, samples were centrifuged at 12,000 × G for 15min at 4° C. 550 μL of the upper aqueous phase was transferred to a new eppendorf, mixed with 500 μL isopropanol, and incubated at RT for 10 min. RNA is precipitated at 12,000 × g for 10 min at 4° C, washed in 75% ethanol, dried briefly and dissolved in DNAse I reaction mix prepared in DEPC-treated water. DNAse I treatment proceeds at 37° C for 45 min, then inactivated at 65° C for 5 min. RNA was reverse transcribed with random primers using iScript reverse transcription supermix (Biorad) or for TERRA transcript detection a gene specific primer (GSP) designed to complement telomere repeats (ccctaaccctaaccctaaccctaaccctaa) and SuperScript III reverse transcriptase (Invitrogen). cDNA was quantified by qPCR using the ΔΔCeviously . Primerseviously , 33, 34.Telomere length, TIF and metaphase telomere signal intensity assays were performed as described previously .P-values were calculated by two-tailed student t-test with at least three replicates.Error bars indicate standard deviation.


Further studies showed that CAM and SCM could also inhibit infection by other influenza A viruses, including A/Shanghai/37T/2009(H1N1), A/Puerto Rico/8/1934(H1N1), A/Guizhou/54/1989(H3N2), and one influenza B virus, B/Shanghai/2017(BY). Mice were challenged intranasally with A/H7N9/4664T/2013 (H7N9) virus and intraperitoneally injected with CAM (10 mg/kg per day) or SCM (1 mg/kg per day) for 5 days. CAM or SCM (10 mg/kg per day) were fully protected against challenge with A/Shanghai/4664T/2013(H7N9). The results from mechanistic studies indicate that both could inhibit influenza virus infection by blocking viral entry into the target cell, the early stage of virus life cycle. However, CAM and SCM neither blocked virus attachment, characteristic of HA activity, nor virus release, characteristic of NA activity. Such data suggest that these two compounds may interfere with the endocytosis process. Thus, we have identified two FDA-approved antihistamine drugs, CAM and SCM, which can be repurposed for inhibiting infection by divergent influenza A strains and one influenza B strain with potential to be used for treatment and prevention of influenza virus infection.Influenza A viruses (IAV) comprise some of the most common infectious pathogens in humans, and they cause significant mortality and morbidity in immunocompromised people as well as children and the elderly. After screening an FDA-approved drug library containing 1280 compounds by cytopathic effect (CPE) reduction assay using the Cell Counting Kit-8, we found two antihistamines, carbinoxamine maleate (CAM) and S-(+)-chlorpheniramine maleate (SCM) with potent antiviral activity against A/Shanghai/4664T/2013(H7N9) infection with IC Although most influenza virus infections are self-limiting, they can sometimes be associated with severe, even fatal, disease outcomes, such as pneumonia and congestive heart failure  infection was reported in China in April of 2013. This has caused at least 1,564 deaths during the 5 major epidemic waves that followed  and influenza neuraminidase inhibitors  . M2 ion Drug discovery is extremely expensive and time-consuming. It generally takes 10–15 years and billions of dollars to reach clinical trials . Thus, f50  at low μM level.Using this strategy, some FDA-approved drugs for non-infectious diseases have shown efficacy against infection caused by such emerging viruses as Ebola virus and Zika viruses . FollowiCAM  and SCM  are H1-receptor blocking agent-based antihistamine drugs approved by the U.S. FDA in 1950s and 1982 for treatment of hay fever, rhinitis, urticaria, and asthma . At presThe FDA-approved drug library containing 1,280 compounds was obtained from MicroSource Discovery Systems, Inc. . Compounds were solubilized in dimethyl sulfoxide (DMSO) at 10 mM and stored in 96-well PCR Tubes at -80°C. Carbinoxamine maleate salt (CAM) and S-(+)-Chlorpheniramine maleate salt (SCM) were purchased from Sigma-Aldrich . Favipiravir (T-705) and Oseltamivir phosphate were purchased from TargetMol Company .50 endpoint dilution assay. Plasmid pNL4-3.Luc.R-E- was obtained from the NIH AIDS Reagent Program .Madin-Darby canine kidney (MDCK) cells, 293T cells, U87 cells and Huh-7 cells were obtained from ATCC . Cells were cultured in DMEM  containing 10% FBS , penicillin 100 U/ml, and streptomycin 10 μg/ml. Influenza virus strains A/Shanghai/37T/2009(H1N1), A/Puerto Rico/8/1934(H1N1), A/Guizhou/54/1989(H3N2), A/Shanghai/4664T/2013(H7N9), and B/Shanghai/2017(BY) were obtained from the Shanghai Public Health Clinical Center . The experiments on H7N9 IAV were conducted in the BSL-3 Laboratory of Fudan University. Live viruses were propagated in MDCK cells. Virus titers were determined by a TCID5 cells/ml) were dispensed into wells of a 96-well plate and incubated at 37°C/5% CO2 for 24 h. The test compound at the serial dilution in 100 μL of DMEM without serum was added to the cells. After culture for 72 h, cell viability was measured using the Cell Counting Kit-8 (CCK-8), which was purchased from Dojindo Laboratory , and using a spectrophotometer . The percent cytotoxicity (=100% – % cell viability) was calculated, and CC50  of the test compound was determined using the Calcusyn software program   .4 cells/well. After incubation at 37°C for 12 h, a mixture of a test compound (50 μL) at graded concentration in serum-free DMEM containing 2 μg/mL tosyl phenylalanyl chloromethyl ketone (TPCK)-Trypsin and 50 μL of a influenza virus (100 TCID50) was added to the monolayer cells, followed by an incubation at 37°C for 8 h. The supernatants were replaced with DMEM medium containing 2 μg/mL TPCK-Trypsin. After culture at 37°C for 72 h, the inhibitory activity of the compound on cytopathic effect induced by viral infection was measured using CCK-8 as described above. The IC50 values were calculated with the Calcusyn Program (Biosoft).The inhibitory activity of a test compound against influenza virus infection-induced death of MDCK cells was measured by CPE reduction assay using CCK-8 as previously described . PBS conA time-of-addition assay was performed as previously described . Briefly5/well) and cultured overnight. The cells were infected with approximately 100 pfu/ml of the influenza A/PR/8/34 (H1N1) virus for 1h in the absence or presence of CAM and SCM at the indicated concentration and then the medium was replaced with fresh DMEM containing 1.5 μg/ml TPCK-Trypsin. After incubation at 37°C for 12 h, total RNA was isolated from cells using Trizol Reagent . The First-stranded cDNA was reverse by TransScript First-Strand cDNA Synthesis SuperMix  according to the instructions. The qPCR assay was performed by using the TransStart Top Green qPCR SuperMix. The primer sequences used for the influenza A HA genes are 5′-TTCCCAAGATCCATCCGGCAA-3′ (forward) and 5′-CCTGCTCGAAGACAGCCACAACG-3′ (reverse); NP genes are 5′-GACCAGGAGTGGAGGAAACA-3′ (forward) and 5′-CGGCCATAATGGTCACTCTT-3′ (reverse); and the sequences for the GAPDH gene are 5′-AGGGCAATGCCAGCCCCAGCG-3′ (forward) and; 5′-AGGCGTCGGAGGGCCCCCTC-3′ (reverse).MDCK cells were seeded onto 12-well plates (2 × 104 cells/well) were seeded onto 96-well plates at 37°C under 5% CO2 for 8 h. The cells were washed twice with phosphate-buffered saline (PBS) and infected with 100 TCID50 of the influenza A/PR/8/34 (H1N1) virus in the presence or absence of CAM and SCM at the indicated concentration for 1 h. The supernatant was aspirated and covered with culture medium containing 1.5 μg/ml TPCK-Trypsin. After incubation at 37°C for 12 h, the cells were fixed with 4% paraformaldehyde for 10 min, then permeabilized with 0.1% TritonTM X-100 for 10 min, blocked with 1% BSA for 1 h and labeled with a primary antibody against influenza virus A nucleoprotein (NP)  for 3 h. Goat anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody Alexa Fluor® 488 conjugate (diluted 1:250) was added at 37°C for 1 h. After further washing, the nuclei were stained with DAPI for 10 min. The cells were observed and photographed by using a fluorescence microscope.MDCK cells (1 × 106 cells/well) and cultured overnight. MDCK cells were washed twice with PBS and incubated with approximately 100 pfu/ml of the influenza A/PR/8/34 (H1N1) virus in the presence or absence of CAM and SCM at the indicated concentration for 1 h. After incubation at 37°C for 1 h, the supernatant was aspirated. The cells were washed once with PBS and were covered with DMEM containing 1% low melting agarose and 1.5 μg/ml TPCK-trypsin. After 2–3 days post-infection, the cell monolayer was fixed with 4% formalin and stained with 1% crystal violet for 1 h and the plaque forming units were counted.Briefly, MDCK cells were plated onto 12-well culture plates (1 × 105 cells/well) were seeded to six-well plate the day before transfection, and co-transfected with 2.5 μg of pcDNA3.1-HA and NA of A/Shanghai/4664T/2013(H7N9), respectively, and 10 μg of lentivirus vector pNL4-3-Luc R-E- by using VigoFect reagents . For the pseudotyped Nipah virus or VSV, 293T cells were co-transfected with Nipah G protein or VSV G protein, respectively, and plasmid encoding Env-defective, lentivirus vector pNL4-3-Luc R-E- by using VigoFect reagents. After incubation at 37°C for 6 h, cells were washed with PBS 3 times and cultured in DMEM containing 10% FBS. After culture at 37°C/5% CO2 for 2 days, supernatants containing the pseudoviruses were collected and filtered to remove cells and cell debris. The pseudoviruses were concentrated by ultracentrifugation at 25,000 rpm at 4°C for 3 h, and then their titers were determined using the Luciferase Assay System , following the manufacturer’s instructions. Aliquots of the pseudoviruses were stored at -80°C.Pseudotyped H7N9 IAV, Nipah virus, and vesicular stomatitis virus (VSV) were constructed, and their infectivity was evaluated as previously described . For the4 cells each well, respectively. After incubation at 37°C for 12 h, a mixture of a test compound (50 μL) at a serial twofold dilution in serum-free DMEM and 50 μL of the corresponding pseudovirus was added to the corresponding cells. MDCK, Huh7 and U87 cells were infected by these control viruses, as described above, followed by incubation at 37°C for 12 h. The culture supernatants were replaced with free DMEM containing 2% FBS. After culture at 37°C for 48 h, the luciferase activity was measured using the Luciferase Assay System (Promega) with a microplate luminometer . The inhibitory activity of a test compound on entry of HIV-1 IIIB infection were determined as described previously .The inhibitory activity of a test compound on entry of H7N9, Nipah or VSV pseudovirus into their target cells was determined as described previously . Brieflyeviously ,b. SerieD-N-acetylneuraminic acid sodium salt hydrate , as the substrate dissolved in the MES (2-(N-Morpholino) ethanesulfonic acid buffer , were added to each well, followed by incubation at 37°C for 1 h in the dark. Subsequently, 50 μL of 14 mM NaOH were added to terminate the enzymatic reaction. The fluorescence intensity of the product 4-methylumbelliferone was recorded using a microplate reader with excitation and emission wavelengths of 340 and 440 nm, respectively. The IC50 values were calculated with the Calcusyn Program (Biosoft).Neuraminidase inhibition assay was performed to investigate the influence of a test compound on the release of newly produced viral particles, as described previously . BrieflyThe HI assay was performed to measure the inhibitory activity of a test compound on attachment of an influenza virus to red blood cells (RBC) through the interaction between HA on virus and receptor on RBC, as described previously . A/ShangThe animal experimental procedure was carried out according to ethical guidelines and approval by Shanghai Public Health Clinical Center Animal Welfare and Ethics Committee (2017-A046-01). The animal studies with infectious H7N9 IAV were conducted in a Biosafety Level 3 facility of Fudan University with Institutional Biosafety Committee approval.n = 12): mock-infection group, PBS group, CAM high dose group (10 mg/kg/day), CAM low dose group (1 mg/kg/day), SCM high dose group (10 mg/kg/day), SCM low dose group (1 mg/kg/day) and OSE group (1 mg/kg/day). Except for mice in the mock-infection control group that received 20 μL DMEM and 20 μL PBS, mice in the treatment groups were anesthetized by intraperitoneal injection of pelltobarbitalum natricum (7 TCID50 in 20 μL DMEM), following by i.p. application of 20 μL PBS (for PBS control group) or a compound in 20 μL PBS (for each compound-treatment group). On the 6th day post infection, 3 mice in each group were euthanized with CO2 inhalation and their left lungs were removed. The lungs were then immersed in TRIzolTM reagent and frozen at -80°C. Total RNA was isolated from lung homogenates. The levels of viral RNA were determined by two-step RT-qPCR kit . The right lungs were placed in 4% paraformaldehyde at 4°C for hematoxylin-eosin (H&E) staining and histological analysis. The body weight of the mice was monitored daily for 14 days after infection. Survival curves were generated according to the Kaplan–Meier method and the differences was calculated by Log-rank (Mantel-Cox) Test (GraphPad Prism 6.0).Female C57BL/6 mice  purchased from Shanghai Lingchang BioTech co., Ltd.  and randomly divided into 7 groups . Observation and description were made after scanning in the panoramic scanner . Blinded sections were examined by two independent pathologists and scored for the following indicative parameters: (A) alveolar congestion, (B) hemorrhage, (C) Neutrophilic infiltration, (D) hyaline membrane formation, (E) thickness of the alveolar wall . Ten non-overlapping microscopic images were obtained from each tissue sample with 40x objective for observation.Immunohistochemical staining of viral NP was performed. The right lung of each mouse was fixed in 10% buffered formalin solution for 7 days and embedded in paraffin. The embedded tissue was cut into 4 μm sections and stained with a primary mouse polyclonal antibody against NP of influenza A virus  at 1:1000 and a secondary HRP-labeled goat anti-mouse IgG antibody (Thermo Fisher Scientific) at 1:200. The integrated optical density (IOD) value was measured by Image-Pro Plus 6.0 software . The images were captured under an optical microscope (×200). The same brown and yellow color was selected as the unified standard to judge the positivity of all images. The IOD values in three randomly selected views were determined. The mean IOD value was considered as the relative expression levels of viral NP.50. We found two antihistamines, carbinoxamine maleate (CAM) and S-(+)-chlorpheniramine maleate (SCM)  against H7N9 influenza virus infection by CPE reduction assay using CCK-8 for identifying compounds with inhibitory activity against influenza virus infection-induced cell death at the concentrations of the compounds below their CCin vitro antiviral efficacy of CAM and SCM was also evaluated by using RT-qPCR assay for assessing the levels of viral gene expression. The results showed that CAM and SCM could downregulate viral RNA synthesis in a dose-dependent manner, compared with the PBS control  of CAM and SCM were in the range of 12 – 84  and intraperitoneally injected with CAM and SCM at both high dose (10 mg/kg/day) and low dose (1 mg/kg/day), respectively, daily for 5 days beginning 1 h after infection. Mice in the positive and negative control groups were treated with OSE, a neuraminidase inhibitor , or PBS, respectively. As shown in Figure To evaluate the We then examined the lung tissues of H7N9- or mock-infected mice treated or untreated with CAM or SCM for the virus gene (mRNA for HA) expression by using a two-step RT-qPCR kit. The results showed that both CAM and SCM could significantly downregulate the expression of viral gene in the mouse lung tissues  G protein was included as control. The result showed that CAM and SCM could effectively inhibit H7N9 pseudovirus infection in a dose-dependent manner with ICy Figure , while Cy Figure  or VSV-Gy Figure , suggesty Figure . CollectHA is a major membrane glycoprotein responsible for virus-cell receptor interaction and virus attachment to the target cell, the first step of viral entry , while N2. People infected with the H7N9 virus suffer from severe pneumonia and its complications, which can be fatal , 1934(H1N1), and 1989(H3N2) strains, as well as an influenza B virus, B/Shanghai/2017(BY), suggesting that both CAM and SCM have broad antiviral activity against divergent influenza A strains and one influenza B strain. The result from an animal study showed that i.p. injection of CAM or SCM at 10 mg/kg/day for 5 days resulted in full protection of mice against challenge with lethal dose of avian influenza virus H7N9, indicating that these two antihistamine drugs have good potential for clinical use in the treatment, or, possibly, prevention of influenza virus infection.By screening an FDA-approved drug library, we identified two antihistamine drugs, CAM and SCM, which have had clinical application in the treatment of hay fever, rhinitis, urticaria, and asthma, with potent antiviral activity against avian influenza virus H7N9 with ICThe life cycle of the influenza virus can be divided into seven stages, as follows: (1) Attachment - HA, which can be cleaved into HA1 and HA2 subunits , binds tIn summary, based on an FDA-approved drug library, we have identified two FDA-approved antihistamine drugs, CAM and SCM, that exhibit potent inhibitory activity against divergent influenza A strains and one influenza B strain and that protect mice from fatal challenge of avian H7N9 influenza virus. These antihistamine drugs inhibit influenza virus infection by targeting the early stage of virus life cycle, viral entry into the host cells. These results suggest that both CAM and SCM have potential for clinical use in the treatment and prevention of influenza virus infection.SJ and LL conceived and designed the experiments. WX, SX, JP, PL, and QW performed the experiments. WX, LL, and SJ analyzed the data and wrote the manuscript.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.


PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PIP2. At the cellular level, PIK3C2A is critical for the formation of cilia and for receptor mediated endocytosis, among other biological functions. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity. Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. In particular, the considerable phenotypic overlap, yet distinct features, between this syndrome and Lowe’s syndrome, which is caused by mutations in the PI-5-phosphatase OCRL, highlight the key role of PI metabolizing enzymes in specific developmental processes and demonstrate the unique non-redundant functions of each enzyme. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease. PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of the lipids phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PIP2 that are essential for a variety of cellular processes including cilia formation and vesicle trafficking. This syndrome is the first monogenic disorder caused by mutations in a class II PI3K family member and thus sheds new light on their role in human development.Identifying the genetic basis of rare disorders can provide insight into gene function, susceptibility to disease, guide the development of new therapeutics, improve opportunities for genetic counseling, and help clinicians evaluate and potentially treat complicated clinical presentations. However, it is estimated that the genetic basis of approximately one-half of all rare genetic disorders remains unknown. We describe one such rare disorder based on genetic and clinical evaluations of individuals from 3 unrelated consanguineous families with a similar constellation of features including short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations including stroke, among other findings. We discovered that these features were due to deficiency of the PIK3C2A enzyme.  PIK3C2A that cause a newly identified genetic syndrome consisting of dysmorphic features, short stature, cataracts and skeletal abnormalities.Identifying the genetic basis of diseases with Mendelian inheritance provides insight into gene function, susceptibility to disease, and can guide the development of new therapeutics. To date, ~50% of the genes underlying Mendelian phenotypes have yet to be discovered . The disPIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family of lipid kinases that catalyzes the phosphorylation of phosphatidylinositol (PI) [tol (PI) . PI3Ks atol (PI) . The comtol (PI) . PI(3)P,tol (PI) . Howevertol (PI) .FIG4) [MTM1) [PIK3CD) [FIG4 subsequent to its identification as a cause of Charcot-Marie-Tooth type 4J have revealed both genetic and physiological interactions with VAC14 and PIKFYVE, which together generate PIP2 and are required for melanosome homeostasis, oligodendrocyte differentiation, and remyelination [The functions of class II PI3Ks are poorly understood relative to many other kinases and phosphatases that regulate PI metabolism, in part because there was no causal link between any class II PI3K and a monogenic human disease. In contrast, a number of disorders of PI metabolism have previously been described that have provided invaluable insight into the physiological functions of specific PI metabolizing enzymes . These iFIG4) ,11, Cent) [MTM1) , and pri[PIK3CD) ,14, amonlination –18. CollPIK3C2A or any class II PI3K and a Mendelian disorder. Here, we describe the evidence that homozygous loss-of-function mutations in PIK3C2A cause a novel syndromic disorder involving neurological, visual, skeletal, growth, and occasionally hearing impairments.PIK3C2A has previously been attributed a wide-range of biological functions including glucose transport, angiogenesis, Akt activation, endosomal trafficking, phagosome maturation, mitotic spindle organization, exocytosis, and autophagy –28. In aFive individuals between the ages of 8 and 21 from three unrelated consanguineous families were found by diagnostic analyses to have a similar constellation of clinical features including dysmorphic facial features, short stature, skeletal and neurological abnormalities, and cataracts . The dysCYP17A1 gene (OMIM #202110) [CYP17A1 or have CAH, suggesting the presence of two independent and unrelated conditions in Family I. The co-occurrence of multiple monogenic disorders is not uncommon among this highly consanguineous population [In addition to the shared syndromic features described above in all three families, both affected daughters in Family I were diagnosed with congenital adrenal hyperplasia (CAH), due to 17-alpha-hydroxylase deficiency, and were found to have a homozygous familial mutation: NM_000102.3:c.286C>T; p.(Arg96Trp) in the #202110) ,30. The pulation .GALNS) in Family I. Additionally, since some of the features of patient II-II-3 were reminiscent of Noonan syndrome, Hennekam syndrome, and Aarskog-Scott syndrome, individual genes involved in these disorders were analyzed in Family II, but no pathogenic mutation was identified. In patient III-II-2, Williams-Beuren syndrome was excluded in childhood. Additionally, direct molecular testing at presentation in adulthood excluded Leri-Weill syndrome, Alstrom disease, and mutations in FGFR3.To identify the genetic basis of this disorder, enzymatic assays related to the mucopolysaccharidosis subtypes MPS I, MPS IVA, MPS IVB, and MPSVI were tested in Families I and II and found to be normal. Enzymatic assays for mucolipidosis II/III were also normal and no pathogenic mutations were found in galactosamine-6-sulfate sulfatase (CYP17A1 (p.Arg96Trp) mutation that is the cause of the CAH [ATF4, DNAH14, PLEKHA7, and PIK3C2A  truncates 1,492 amino acids from a protein that is 1,686 amino acids. This is predicted to eliminate nearly all functional domains including the catalytic kinase domain, and is expected to trigger nonsense-mediated mRNA decay [PIK3C2A mRNA are significantly decreased in both heterozygous and homozygous individuals carrying the p.Tyr195* variant  that leads to decreased mRNA levels  [PIK3C2A, which is consistent with total PIK3C2A deficiency causing severe early onset disease.In Family I, the nonsense mutation in NA decay . Accordi variant . The delpression . The varA levels . Deep seA levels . AlthougA levels . This isA levels . Thus, aAD v2.1) , none arPIK3C2A cause cellular phenotypes consistent with loss of PIK3C2A function, we examined PI metabolism, cilia formation and function, and cellular proliferation rates. PIK3C2A deficiency in the patient-derived fibroblasts decreased the levels of PIP2 throughout the cell P2 and PI(3)P, shortening of the cilia and impaired ciliary protein localization, and reduced proliferation capacity. Thus, based on the loss-of-function mutations in PIK3C2A, the phenotypic overlap between the three independent families, and the patient-derived cellular data consistent with previous studies of PIK3C2A function, we conclude that loss-of-function mutations in PIK3C2A cause this novel syndrome.Here we describe the identification of three independent families with homozygous loss-of-function mutations in PIK3C2A loss-of-function mutations in humans represents the first mutations identified in any class II PI-3-kinase in a disorder with a Mendelian inheritance, and thus sheds light into the biological role of this poorly understood class of PI3Ks [PIK3C2A in rare monogenic disorders, but also the potential contribution of common variants in PIK3C2A in more genetically complex disorders. There are now numerous examples where severe mutations in a gene cause a rare Mendelian disorder, whereas more common variants in the same gene, with a less deleterious effect on protein function, are associated with polygenic human traits and disorders [PPARG cause monogenic lipodystrophy, whereas less severe variants are associated with complex polygenic forms of lipodystrophy [PIK3C2A and schizophrenia [The identification of of PI3Ks ,37. Thisisorders –40. For ystrophy ,42. In tophrenia –45.OCRL and INPP5E, respectively [INPP5E is quite distinct, and includes cerebellar vermis hypo-dysplasia, coloboma, hypotonia, ataxia, and neonatal breathing dysregulation [Other monogenic disorders of phosphoinositide metabolism include Lowe’s syndrome and Joubert syndrome, which can be caused by mutations in the inositol polyphosphate 5-phosphatases ectively . All thrgulation . In contgulation ,48. The gulation . The simgulation ,51, demoPik3c2a knockout models that result in growth retardation by e8.5 and embryonic lethality between e10.5–11.5 due to vascular defects [PIK3C2A and the mouse ortholog. However, the involvement of both human and mouse PIK3C2A in cilia formation, PI metabolism, and cellular proliferation suggests a high degree of functional conservation at the cellular level [INPP5B that may uniquely contribute to PI metabolism in each species [PIK3C2A deletion, suggesting that this may partially compensate for the lack of PIK3C2A in humans, although it remains to be determined whether a similar compensatory pathway exists in mice.The viability of humans with PIK3C2A deficiency is in stark contrast to mouse  defects . One potar level ,28. An a species . AlternaIt is intriguing that both PIK3C2A and OCRL have important roles in primary cilia formation ,53,54. PThe study was approved by the Helsinki Ethics Committees of Rambam Health Care Campus (#0038-14-RMB), the University Hospital Institutional Review Board for Case Western Reserve University (#NHR-15-39), the Ethics Committee of the Friedrich-Alexander University Erlangen-Nürnberg (#164_15 B), and was in accordance with the regulations of the University Medical Center Groningen’s ethical committee. Written informed consent was obtained from all participants.Whole exome sequencing (WES) of two patients from Family I was performed using DNA (1μg) extracted from whole blood and fragmented and enriched using the Truseq DNA PCR Free kit (Illumina). Samples were sequenced on a HiSeq2500 (Illumina) with 2x100bp read length and analyzed as described . Raw fasWhole exome sequencing was performed on the two affected individuals of Family II and both their parents essentially as previously described . Target −3 or below (based on the ExAC database [The DNAs of Family III were enriched using the SureSelect Human All Exon Kit v6 (Agilent) and sequenced on an Illumina HiSeq 2500 (Illumina). Alignment, variant calling, and annotation were performed as described . The avedatabase  and an iAll candidate variants in Families I, II, and III were confirmed by Sanger sequencing . SNP array raw data was mapped to the reference human genome GRCh37 and analyzed using GenomeStudio (v. 2011/1). Signal intensity files with Log R ratio and B-allele frequency were further analyzed with PennCNV   supplemented with 10–20% Fetal Calf Serum, 1% Sodium Pyruvate and 1% Penicillin and streptomycin (P/S) in 5% CO2 at 37°C. Control fibroblasts were obtained from healthy age-matched volunteers. Fibroblasts from passages 4–8 were used for the experiments. To measure cell proliferation, cells were detached using trypsin and counted with an Automated Cell Counter (ThermoFisher). Cells (n = 2500) were plated in triplicate in 96-well plates. Viability was measured at day 2, 4, 6 and 8. Each measurement was normalized to day 0 (measured the day after plating) and expressed as a fold increase. Viability was assessed by using CellTiter-Glo Luminescent Cell Viability Assay (Promega). Three independent experiments were performed.HeLa cells were infected with lentiviral particles containing pLKO-TET-PI3KC2A-shRNA or pLKO-TET-scramble-shRNA in six-well plates . After two days, the medium containing lentiviral particles was replaced with DMEM 10% FBS, 1.5μg/ml puromycin. After 7 days of selection, cells were detached and 100,000 cells were plated in six-well plates in triplicate in the presence of doxycycline . Medium containing doxycycline was replaced every 48 hours. After 10 days of doxycycline treatment, cells were lysed and analysed by Western blot.GADPH.Total RNA was purified from primary fibroblasts using the PureLink RNA purification kit (ThermoFisher) or RNAPure peqGOLD (Peqlab). RNA was reverse transcribed into complementary DNA with random hexamer using a high-Capacity cDNA Reverse Transcription Kit (ThermoFisher). RT-PCR from lymphocytes to detect exon-skipping in family III was performed using primers flanking exon 6. The resulting product was sequenced on an Illumina HiSeq2500 (Illumina) to detect splicing variants with high sensitivity. Gene expression was quantified by SYBR Green real-time PCR using the CFX Connect Real-Time System (BioRad). Primers used are detailed in Protein was extracted from cultured primary fibroblast cells as described ,77. ExtrPrimary fibroblasts were grown on glass coverslips to approximately 80% - 90% confluency in DMEM + 10% FCS + 1% P/S, at which time the medium was replaced with DMEM without FCS for 48 hours to induce ciliogenesis. Cells were fixed in either methanol for 10 minutes at -20°C or 4% paraformaldehyde for 10 minutes at room temperature (RT). Fixed cells were washed in PBS, and incubated with 10% normal goat serum, 1% bovine serum albumin in PBS for 1 hour at RT. If cells were fixed with paraformaldehyde, blocking solutions contained 0.5% Triton X-100. Cells were incubated with primary antibody overnight at 4°C, washed in PBS, and incubated with secondary antibody including 4',6-diamidino-2-Phenylindole (DAPI) to stain nuclei for 1 hours at RT. Coverslips were mounted on glass slides with fluoromount (Science Services) and imaged on a confocal laser scanning system with a 63x objectives . Primary antibodies are detailed in To induce ciliogenesis, cells were grown in DMEM with 0–0.2% FCS for 48 hours. Cells were washed in PBS, then fixed and permeabilized in ice-cold methanol for 5 minutes, followed by extensive washing with PBS. After blocking in 5% Bovine Serum Albumin, cells were incubated with primary antibodies for 1.5 hours at RT and extensively washed in PBS-T. Primary antibodies used for Centrin and ARL13B are detailed in S1 FigPhotographic images of (A) teeth, (B) hands, and (C) feet are shown from the five individuals with PIK3C2A deficiency. (D) X-Ray images of the pelvis and (E) MRI images of the brain are shown when available. White arrows in the MRI images indicate regions of altered signal intensity.(TIF)Click here for additional data file.S2 FigPIK3C2A, indicated with the red line (B) Sanger sequencing confirmed homozygosity for the PIK3C2A c.585T variant in Family I. (C) Sanger sequencing confirmed homozygosity for the PIK3C2A c.1640+1 G>T variant in Family III.(A) CNV analysis confirmed a homozygous deletion encompassing exons 1–24 out of 32 total exons of (TIF)Click here for additional data file.S3 FigPIK3C2A (exons: yellow boxes) with transcripts of the patient of family III compared to controls. (B) Example of sequenced RT-PCR products from cDNA of fibroblasts from wild-type control and the patient of family III using primers located in exons 3 and 10. Skipping of exons 5 and 6 is the result of the mutation. Positions of primers are indicated by orange arrows and position of the splice site mutation is indicated by a black arrow.(A) Deep sequencing of RT-PCR products revealed 4 alternative transcripts in lymphocytes (red lines) compared to control samples (blue lines): *: r.1561_1704del; p.Ala521_Glu568del, **: r. 1640_1641ins1640+1_1640+27; p. Arg547SerinsTyrIleIle*, ***: r.1561_1640del; p.Ala521Thrfs*4, ****: r.1449_1640del; p.Asn483_Arg547delinsLys. Exon/intron structure of (TIF)Click here for additional data file.S4 FigQuantification of fluorescence intensity for (A) PI(3)P at the ciliary base, (B) RAB11 at the ciliary base, and (C) ciliary IFT88. Data is shown separately for each individual family as indicated.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Influenza activityU.S. World Health Organization (WHO) collaborating laboratories and National Respiratory and Enteric Virus Surveillance System laboratories, which include both clinical and public health laboratories throughout the United States, contribute to virologic surveillance for influenza. During September 30, 2018–May 18, 2019, clinical laboratories tested 1,145,555 specimens for influenza virus; among these, 177,039 (15.5%) tested positive, including 167,529 (95.0%) for influenza A and 9,510 (5.0%) for influenza B. The percentage of specimens testing positive for influenza each week ranged from 1.7% to 26.2%.Nationally, the percentage of clinical laboratory–tested specimens positive for influenza virus peaked during the weeks ending February 9–March 16 (surveillance weeks 6–11) (range = 25.1%–26.2%). Regionally,Public health laboratories tested 80,993 specimens during September 30, 2018–May 18, 2019; among these specimens, 42,303 (52.2%) were positive for influenza viruses, including 40,624 (96.0%) that were positive for influenza A and 1,679 (4.0%) for influenza B. Among the 38,995 seasonal influenza A viruses subtyped, 22,084 (56.6%) were influenza A(H1N1)pdm09, and 16,991 (43.6%) were influenza A(H3N2). Influenza B lineage information was available for 1,105 (65.8%) influenza B viruses; 406 (36.7%) of those were B/Yamagata lineage, and 699 (63.3%) were B/Victoria lineage. Whereas influenza A(H1N1)pdm09 viruses accounted for the majority of circulating viruses nationwide from October 2018 to mid-February 2019, influenza A(H3N2) viruses were detected more frequently than were A(H1N1)pdm09 viruses beginning in late February nationally  and in aAmong 38,564 (91.2%) patients whose test results were positive for seasonal influenza virus by public health laboratories and for whom age data were available, 4,844 (12.6%) were aged 0–4 years; 12,508 (32.4%) were aged 5–24 years; 13,382 (34.7%) were aged 25–64 years; and 7,830 (20.3%) were aged ≥65 years. Influenza A(H1N1)pdm09 virus was the most frequently reported virus among persons aged 0–4 years (57.1%) and 25–64 years (63.2%), whereas influenza A(H3N2) virus was the most commonly reported virus among persons aged 5–24 years (48.8%) and ≥65 years (51.3%). The age group with the largest proportion of reported influenza B viruses (6.3%) was persons aged 5–24 years.https://www.ncbi.nlm.nih.gov/genbank or EpiFlu: https://www.gisaid.org/). Antigenic characterizations were carried out by hemagglutination inhibition assays or virus neutralization–based focus reduction assays to evaluate whether genetic changes in circulating viruses affected antigenicity; substantial differences could affect vaccine effectiveness. CDC genetically characterized 2,750 influenza viruses collected and submittedGenetic characterization was carried out using next-generation sequencing, and the genomic data were analyzed and submitted to public databases  viruses indicated cocirculation of multiple clades/subclades. Circulating viruses possessed HA gene segments that belonged to clade 3C.2a , subclade 3C.2a1 , or clade 3C.3a . The frequency of 3C.3a viruses increased from 12.7% of the A(H3N2) viruses collected and sequenced by November 2018 to 81.9% of those collected and sequenced during December 2018–May 2019. Among the 505 A(H3N2) viruses antigenically characterized by focus reduction assays with ferret antisera, 191 (37.8%) were well inhibited by ferret antisera raised against A/Singapore/INFIMH-16-0019/2016 (3C.2a1), a cell culture–propagated reference virus representing the A(H3N2) component of 2018–19 Northern Hemisphere influenza vaccines. However, only 43 (11%) of the 388 viruses tested were well inhibited by antiserum raised against egg-propagated A/Singapore/INFIMH-16-0019/2016 reference virus, likely because of egg-adaptive amino acid changes in the HA protein of the egg-propagated virus. Three hundred fourteen (62.2%) viruses were poorly inhibited by ferret antiserum raised against cell culture–propagated A/Singapore/INFIMH-16-0019/2016 reference virus (at titers that were reduced eightfold or more when compared with the homologous virus); among those viruses, 312 (99.4%) belonged to clade 3C.3a, the prevalence of which increased throughout the season.Phylogenetic analysis of 203 influenza B/Yamagata lineage viruses determined that the HA gene segments belonged to clade Y3. All 178 B/Yamagata lineage viruses antigenically characterized were well inhibited by ferret antiserum raised against cell culture–propagated B/Phuket/3073/2013, the reference virus representing the B/Yamagata lineage component of quadrivalent vaccines for the 2018–19 Northern Hemisphere influenza season.Multiple genetically and antigenically distinct B/Victoria lineage viruses cocirculated during the 2018–19 season. Viruses with a two-amino acid deletion (162–163) in the HA protein belong to subclade V1A.1, and viruses with a three-amino acid deletion (162–164) in the HA protein belong to subclade V1A-3Del. Among the 272 influenza B/Victoria lineage viruses sequenced and phylogenetically analyzed, the HA gene segment belonged to genetic clade V1A , subclade V1A.1 , or subclade V1A-3Del . Among 191 B/Victoria lineage viruses antigenically characterized, 147 (79.1%) were well inhibited by ferret antiserum raised against cell culture–propagated B/Colorado/06/2017-like V1A.1 reference virus representing the B/Victoria lineage component of the vaccines for the 2018–19 Northern Hemisphere influenza season. Among the 44 (20.9%) viruses that reacted poorly, 17 were antigenically related to the previous vaccine virus B/Brisbane/60/2008 and belonged to clade V1A, and 27 belonged to subclade V1A-3Del.Testing of seasonal influenza A(H1N1)pdm09, influenza A(H3N2), and influenza B viruses for resistance to the neuraminidase inhibitors oseltamivir, zanamivir, and peramivir is performed at CDC using next-generation sequencing analysis, a functional assay pdm09, 1,016 influenza A(H3N2), 252 influenza B/Victoria, and 191 influenza B/Yamagata viruses collected in the United States since October 1, 2018, were tested for resistance to oseltamivir, zanamivir, and peramivir. Five (0.3%) influenza A(H1N1)pdm09 viruses had the amino acid substitution H275Y and displayed highly reduced inhibition by oseltamivir and peramivir. In addition, four (0.3%) influenza A(H1N1)pdm09 viruses displayed some reduction in inhibition by oseltamivir, and two influenza B viruses (0.4%) from different lineages had the amino acid substitution H273Y and displayed highly reduced inhibition by peramivir.During the 2018–19 influenza season, CDC began to test seasonal influenza viruses for resistance to the PA cap-dependent endonuclease inhibitor baloxavir using next-generation sequencing analysis, a phenotypic assay pdm09, 1,007 influenza A(H3N2), 255 influenza B/Victoria, and 198 influenza B/Yamagata viruses collected in the United States since October 1, 2018, and tested genetically for resistance to baloxavir, none contained amino acid substitutions in the PA protein previously associated with decreased susceptibility to baloxavir. All 191 influenza viruses tested by a phenotypic assay were susceptible to baloxavir.Vaccine recommendations were made based on factors including data from global influenza virologic and epidemiologic surveillance, genetic characterization, antigenic characterization, and the candidate vaccine viruses that are available for production. WHO recommended the Northern Hemisphere 2019–20 influenza vaccine composition  was at or above the national baselineILINet data are used to produce a weekly jurisdiction-level measure of ILI activity,State and territorial epidemiologists report the geographic distribution of influenza in their jurisdictions through a weekly influenza activity code.CDC monitors hospitalizations associated with laboratory-confirmed influenza infections through the Influenza Hospitalization Surveillance Network (FluSurv-NET),Complete medical chart abstraction data in FluSurv-NET will not be finalized until later in 2019; however, as of June 13, 2019, data were available for 7,531 (40.0%) hospitalized adults and children with laboratory-confirmed influenza. Among 6,399 hospitalized adults with information on underlying medical conditions, 92.6% had at least one reported underlying medical condition that placed them at high riskCDC tracks pneumonia and influenza (P&I)–attributed deaths through CDC’s National Center for Health Statistics (NCHS) Mortality Surveillance System data. The percentages of deaths attributed to P&I are released 2 weeks after the week of death to allow for collection of sufficient data to produce a stable P&I mortality percentage. During the 2018–19 season, according to NCHS data, the proportion of deaths attributed to P&I was at or above the epidemic thresholdDuring September 30, 2018–May 18, 2019, 116 laboratory-confirmed influenza-associated pediatric deaths were reported to CDC from Chicago, New York City, and 41 states. Two deaths occurred in non-U.S. residents. Twenty-five (22%) of the deaths were associated with influenza A(H3N2) infection, 43 (37%) with influenza A(H1N1)pdm09, 39 (34%) with an influenza A virus for which no subtyping was performed, eight (7%) with an influenza B virus, and one (1%) with an influenza virus for which the type was not determined. The mean age of the pediatric deaths reported this season was 6.1 years (range = 2 months–17 years); 75 (66%) children died after admission to the hospital. Among the 104 children with a known medical history, 53 (51%) had at least one underlying medical condition recognized by the Advisory Committee on Immunization Practices (ACIP) as placing them at high risk for influenza-related complications. Among the 89 children who were eligible for influenza vaccination (age ≥6 months at date of onset) and for whom vaccination status was known, 30 (34%) had received at least 1 dose of influenza vaccine before illness onset .https://www.cdc.gov/flu/professionals/classifies-flu-severity.htm). These intensity thresholds represent the historic chance that surveillance system data exceeded a certain threshold. CDC then classifies the severity of the current influenza season by determining which intensity threshold was exceeded by at least two of the peak values from these indicators. The severity of the 2018–19 season was thus classified as moderate overall, as well as by age group .In 2017, CDC implemented a new methodology to classify influenza season severity using three indicators: 1) the percentage of visits to outpatient clinics for ILI (from ILINet); 2) the rates of influenza-associated hospitalizations (from FluSurv-Net); and 3) the percentage of deaths resulting from pneumonia or influenza (from NCHS) pdm09 viruses predominated overall and represented the most frequently detected influenza A virus from October 2018 to mid-February 2019; influenza A(H3N2) viruses were reported more frequently than were A(H1N1)pdm09 viruses from late February through mid-May 2019. The predominant influenza A virus also differed by geographic region and age group. In contrast to the number of influenza A viruses reported, the number of influenza B viruses reported was low, compared with previous seasons, accounting for 4% of influenza viruses reported by public health laboratories.The 2018–19 influenza season was longer than recent influenza seasons, and ILI activity was at or above baseline for 21 consecutive weeks. Compared with hospitalization rates during the previous five influenza seasons, the 2018–19 cumulative influenza-associated hospitalization rate  was most similar to rates observed during 2014–15 (64.1) and 2016–17 (62.0) and well below those observed during 2017–18 (102.9). Hospitalization rates for children aged <17 years exceeded those during the 2013–14 through 2016–17 seasons and were similar to those during the 2017–18 season, whereas hospitalization rates for adults aged 18–64 years exceeded those in 2013–14 through 2016–17 but were less than those during the 2017–18 season. For persons aged ≥65 years, this season’s hospitalization rates were below those observed during the three most recent H3N2-predominant seasons  but higher than the two H1N1-predominant seasons (2013–14 and 2015–16). Compared with P&I-attributed mortality during the previous five seasons, 2018–19 P&I-attributed mortality was most similar to the 2015–16 season and was lower than that during the other four seasons.Most of the influenza A(H1N1)pdm09 viruses characterized (using hemagglutination inhibition tests with ferret antisera) were antigenically similar to the cell culture–propagated reference virus representing the 2018–19 Northern Hemisphere influenza vaccine virus, but considerable genetic diversity among currently circulating influenza A(H1N1)pdm09 viruses belonging to clade 6B.1A was observed. The increased circulation of clade 3C.3a viruses strongly contributed to the increasing proportion of A(H3N2) viruses that were antigenically distinct from the reference virus representing the A(H3N2) vaccine component of the 2018–19 Northern Hemisphere vaccines. Viruses from clade 3C.3a were well inhibited by ferret antisera raised against recent 3C.3a cell culture–propagated reference viruses, including A/Kansas/14/2017, the reference virus representing the A(H3N2) component for the 2019–20 Northern Hemisphere influenza vaccines . Additional information regarding influenza viruses, influenza surveillance, influenza vaccine, influenza antiviral medications, and novel influenza A infections in humans is available online (https://www.cdc.gov/flu).Influenza surveillance reports for the United States are posted online weekly pdm09 predominated from October 2018 to mid-February 2019, and A(H3N2) activity increased from mid-February through mid-May.Receiving a seasonal influenza vaccine each year remains the best way to protect against seasonal influenza and its potentially severe consequences. Testing for seasonal influenza viruses and monitoring for emergence of antigenic drift variant viruses should continue year-round.


Respiratory syncytial virus (RSV) subtypes, A and B, co-circulate in annual epidemics and alternate in dominance. We have shown that a subtype A RSV fusion (F) glycoprotein, stabilized in its prefusion conformation by DS-Cav1 mutations, is a promising RSV-vaccine immunogen, capable of boosting RSV-neutralizing titers in healthy adults. In both humans and vaccine-tested animals, neutralizing titers elicited by this subtype A DS-Cav1 immunogen were ~ 2- to 3-fold higher against the homologous subtype A virus than against the heterologous subtype B virus.To understand the molecular basis for this subtype difference, we introduced DS-Cav1 mutations into RSV strain B18537 F, determined the trimeric crystal structure, and carried out immunogenicity studies.The B18537 DS-Cav1 F structure at 2-Å resolution afforded a precise delineation of prefusion F characteristics, including those of antigenic site Ø, a key trimer-apex site. Structural comparison with the subtype A prefusion F indicated 11% of surface residues to be different, with an alpha-carbon root-mean-square deviation (RMSD) of 1.2 Å; antigenic site Ø, however, differed in 23% of its surface residues and had an alpha-carbon RMSD of 2.2 Å. Immunization of vaccine-tested animals with DS-Cav1-stabilized B18537 F induced neutralizing responses ~100-fold higher than with postfusion B18537 F. Notably, elicited responses neutralized RSV subtypes A and B at similar levels and were directed towards both conserved equatorial and diverse apical regions.We propose that structural differences in apical and equatorial sites–coupled to differently focused immune responses–provide a molecular explanation for observed differences in elicited subtype A and B neutralizing responses. Respiratory syncytial virus (RSV) was first identified by Chanock and coworkers in 1957  as the eAnalysis of human sera indicates that the majority of neutralizing antibodies elicited by natural RSV infection target the prefusion form of the F glycoprotein , 29, whiSeveral RSV-targeting monoclonal antibodies that are subtype specific, such as the highly potent monoclonal antibodies RSE20  and 5C4 oC. The reaction was stopped with 1mM Nα-p-tosyl-L-lysine chloromethyl ketone and 0.4mM leupeptin and the mixture passed over a protein A or protein G column to remove the Fc fragment. Flowthrough fractions containing Fab were concentrated and further purified to remove Lys-C by size-exclusion chromatography  in phosphate buffered saline (PBS) or a buffer containing 350mM NaCl, 2.5mM Tris pH7.1, 0.02% NaN3.We purchased 293F human embryonic kidney cell lines from Invitrogen  which were maintained in Freestyle 293 expression medium (Invitrogen). Monoclonal antibodies used in binding studies, AM22, D25, 5C4, motavizumab, and MPE8, were expressed by transient co-transfection of both the heavy and light chain gene-containing plasmids into 293F cells in suspension culture at 37°C. Supernatant was harvested after 6 days, and antibodies were purified from a protein A or protein G affinity column (Qiagen). Antigen-binding fragment (Fab) was produced by endoproteinase Lys-C digestion of purified antibody in 25mM Tris-Cl, 1mM EDTA (pH 8) for 6 hours at 37The RSV F gene constructs from subtype B strains B18537 and B1 were made by synthesis of DNA containing codons 1 to 513 from Uniprot sequence P13843 and O36634, respectively, followed by a T4 fibritin trimerization motif [g to remove cell debris, and then sterile filtered. The supernatant was buffer exchanged and concentrated using tangential flow filtration. RSV F glycoprotein was purified by nickel-(Roche) and Strep-Tactin-affinity (IBA lifesciences) chromatography. Purification tags were removed by thrombin digestion overnight at 4oC, and the protein was further purified by size-exclusion chromatography.Soluble RSV F proteins were expressed by transient transfection in Expi293F cells using True-Fect-Max . The culture supernatant was harvested 5 days post transfection, centrifuged at 10,000Robotic screening yielded crystals with the RSV strain B18537 F glycoprotein in 1.2M ammonium sulfate, 0.1M sodium acetate pH5.5, 0.1M lithium sulfate. Crystallization conditions were manually optimized in hanging drops by mixing 1 μL of protein complex with 1 μL of the reservoir solution. Crystals were harvested and flash-cooled in liquid nitrogen using 3.2M ammonium sulfate as a cryoprotectant. Data were collected at a wavelength of 1.00 Å at the Southeast Regional Collaborative Access Team (SER-CAT) beamline ID-22 .Diffraction data were processed using HKL2000, and crystal unit cell analysis  indicateThe final refined structures were analyzed with MolProbity . StructuMice were housed and cared for in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International at the Vaccine Research Center (VRC), National Institute of Allergy and Infectious Diseases, National Institutes of Health in accordance with local, state, federal, and institute policies. Animal experiments were performed in compliance with the Animal Welfare Act requirements for environment enhancement which were adequate to promote the psychological and physical well-being of small animals. The animal care protocol of this study was approved by the VRC Animal Care and Use Committee.To assess the immunogenicity of DS-Cav1-stabilized RSV F of B18537, we immunized CB6F1/J mice (10 in each group) by intramuscular injection with 10 µg RSV F combined with 50 µg poly I:C adjuvant at weeks 0 and 3. Serum samples were collected at week 5. RSV F B18537 postfusion was used as a control for a side-by-side comparison.4 HEp-2 cells in 30 μL minimal essential medium in each well of 384-well black optical bottom plates. The plates were incubated for 20-22 hours and read with 588 nm excitation and 635 nm emission . The EC50 for each sample was calculated by non-linear regression using GraphPad Prism . P values were determined by Student's t-test.For RSV neutralization assays, sera were serially diluted and mixed with an equal volume of recombinant mKate-RSV expressing F glycoprotein from strain A2 or B18537 and the Katushka fluorescent protein. The mixtures were incubated at 37°C for 1 hour, and 50 μL of each was added to 1.5x10A fortéBio Octet Red384 instrument was used to measure binding kinetics of RSV B18537, B1 and A2 DS-Cav1 F glycoproteins to antibodies AM22, 5C4, D25, MPE8, and motavizumab , 52–54. Serum antibody binding analysis was carried out in a similar manner to the kinetic analysis described above. RSV B18537 F DS-Cav1 and postfusion forms were immobilized to NTA bio-sensors through a 6x His-tag. The biosensor probes were then equilibrated for 300 seconds in PBS + 1% BSA buffer. Sera were diluted 50- or 100-fold in PBS + 1% BSA, and binding to the immobilized F proteins was assessed for 300 seconds. To analyze antibody competition for binding F trimers, RSV F-loaded probes were incubated in MPE8 or D25 solutions prior to assays in the diluted sera. Sera depletion was carried out by using 1 μg of DS-Cav1 or postfusion F proteins per 1 μL of animal sera. Parallel correction to subtract non-specific sera binding was carried out by subtracting binding levels of an unloaded probe incubated with the sera.The RSV F glycoprotein nucleotide sequences used in this study were downloaded from Gen-bank and the DNA databank of Japan. Accession numbers of utilized sequences are as follows for subtype B strains: JX576729 to JX576762, AF013254 (B1), AY353550 (9320), and for subtype A strains: JQ901447 to JQ901458, JX015479 to JX015499, M74568 (A2), AY911262 (Long), FJ614813 (Line19), NC_001803 (RSS-2), JX482018 to JX482038, JX682715 to JX682823, JX477455 to JX477594.The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model . The treSequence variability for RSV subtypes A and B F protein was analyzed using 2 approaches: Shannon entropy and mutual information . A totalMutual information scores take into account the association between mutations at a given RSV F residue position and the prevalence of a residue within a given subtype. Higher mutual information scores are given for a residue where particular mutations are preferentially found in 1 subtype. For example, residue 209 had a mutual information score of 0.56455, which indicates a substantially different amino acid distribution for that residue in RSV F subtype A versus subtype B. Specifically, in RSV F subtype A sequences, residue 209 is a completely conserved Lys, whereas in subtype B, that residue is 94% Gln and only 6% Lys. On the other hand, residue 276 had a mutual information score of 0.171243, indicating the existence of some, though not as pronounced, differences in amino acid distribution between A and B sequences; residue 276 is 46% Asn and 54% Ser in A sequences, whereas it is 100% Ser in B sequences.To stabilize the RSV glycoprotein of strain B18537 in its prefusion conformation, we introduced disulfide and cavity-filling mutations , and apWe determined antigenic recognition of RSV F using biolayer interferometry (Octet). The F glycoproteins were immobilized on the biosensor tip, and their affinity to a panel of RSV F reactive antibodies was measured . NotablyP4132 with 1 protomer per asymmetric unit  with neighboring protomers resulting in an interweaved structure, where the DIII domain was located directly above the DII domain of an adjacent protomer. The fusion peptide of each protomer was located in a central cavity, interacting with adjacent protomers. We determined the surface areas of antigenic sites on the prefusion trimer using the previously defined antigenic site residues [2, and it overlapped with sites II and IV. Antigenic site V [The F glycoprotein formed a homotrimer, each protomer comprising 2 different subunits, F2 and F1 . Residueresidues , 61–65  between the structures of B18537 (determined here) and A2 (PDB ID: 4MMU) was 1.18 Å over the Cα atoms of 454 residues present in both structures. Despite 28 residues being different in the mature sequence between the 2 proteins, their overall secondary structures were well conserved . Most ofComparison between the 2 structures of RSV F subtype B, B18537 and B9320 [Analysis of 352 RSV F sequences  showed that subtype A and subtype B sequences were separated into 2 distinct phylogenetic branches . From th2 of solvent accessible area, which is approximately 11% of the F trimer surface . The stThe RSV-neutralizing response elicited by B18537 prefusion-stabilized F in mice appeared not to be focused entirely on site Ø. Rather, equal responses against both equatorial and apical sites were observed, strongly competed by both MPE8 and D25. This targeting, together with the high conservation of sequence and structure in the equatorial regions, provide an explanation for the similar neutralizing responses elicited by B18537 DS-Cav1 F against both RSV subtypes. It remains to be seen if similar equatorial responses will be elicited by B18537 DS-Cav1 F in humans.The availability of prefusion-stabilized RSV F glycoproteins from subtypes A and B should enable the assessment of dual subtype immunization mixtures, the sequential immunizations of subtypes, as well as other immunization regimens aimed at generating improved neutralizing titers against both subtypes. In terms of optimizing the vaccine-generated protective response, the high-resolution RSV B18537 structure should allow vaccine designers to incorporate sub-type differences into structure-based efforts. The RSV F prefusion structures are well conserved between subtypes, with the observed structural differences resulting from sequence variation as well as the high flexibility at the apical regions . It may


The association between increased production of reactive oxygen species (ROS) and jaw dysfunction is still poorly known especially in youth population. The present study was aimed to assess different parameters of oxidative stress in malocclusion class II division 1 of adolescents .2−), superoxide anion radical (O2−), and hydrogen peroxide (H2O2)  with permanent dentition with or without malocclusion, examined at the Institute of Dentistry, Kragujevac, Serbia. According to degree of malocclusion, all participants were divided into the following groups: 1. control group - included healthy participants with overjet 2–4 mm; 2. Group A - included participants who had class II division 1 malocclusion with overjet 5–7 mm; 3. Group B - included participants who had class II division 1 malocclusion with overjet 8–10 mm; 4. Group C - included participants who had class II division 1 malocclusion with overjet > 10 mm. Malocclusion evaluation and classifications are based on the relationship of the mesiobuccal cusp of the maxillary first molar and the buccal groove of the mandibular first molara as previously described (e (H2O2) .All patients before inclusion in the study signed an informed consent. The study was approved by the Committee of Ethics of the local institution  and in compliance with ethical standards.P<0.05, P<0.01) , while the values of nitrites were significantly higher in groups A and C comparing to control (P<0.05)  . Values (P<0.05) . Present


To address this controversy, a new p58C266-464 construct containing all residues was crystallized under the conditions previously used for crystallizing p58C272-464, and the solution structures of both constructs were assessed using circular dichroism and NMR spectroscopy. In the new crystal structure, p58C266-464 exhibits the same elements of secondary structure near the DNA binding site as observed in the crystal structure of p58C272-464. Moreover, in solution, circular dichroism and 15N,1H-heteronuclear single quantum coherence (HSQC) NMR spectra show there are no significant differences in the distribution of secondary structures or in the tertiary structure or the two constructs. To validate that the two constructs have the same functional properties, binding of a primed DNA template was measured using a fluorescence-based DNA binding assay, and the affinities for this substrate were the same . The electrochemical properties of p58C266-464 were also measured and this p58C construct was able to engage in redox switching on DNA with the same efficiency as p58C272-464. Together, these results show that although p58C can be stabilized in different conformations in the crystalline state, in solution there is effectively no difference in the structure and functional properties of p58C constructs of different lengths.The regulatory subunit of human DNA primase has a C-terminal domain (p58C) that contains a [4Fe4S] cluster and binds DNA. Previous electrochemical analysis of a p58C construct revealed that its affinity for DNA is sensitive to the redox state of the [4Fe4S] cluster. Concerns about the validity of this conclusion have been raised, based in part on differences in X-ray crystal structures of the p58C DNA synthesis at the replication fork begins with the formation of 8–12 nucleotide (nt) RNA primers on the single-stranded DNA template –5. PrimaHuman DNA primase is composed of catalytic (p48) and regulatory (p58) subunits. The regulatory subunit has a C-terminal domain (p58C) that is unique to higher eukaryotes and contains a [4Fe4S] cluster –11. This272-464 and p58C266-456 constructs. The two structures are clearly very similar except for residues Leu318-His351, which are positioned near the DNA binding site. In the crystal structure of p58C272-464, these residues occupy a β-sheet-like structure that is stabilized by cross-strand interactions with another molecule in the unit cell  p58C. Samples were initially deposited onto multiplex chip quadrants in 20 μL volumes and the remaining sample deposited in a well of bulk solution above the chip surface, to a final volume of 200–300 μL.Multiplexed chips were fabricated as described previously . p58C co-1 cm-1) , 32. RecAll electrochemistry was performed using a CHI620D potentiostat and 16-channel multiplexer (CH Instruments) in an anaerobic glove chamber. Multiplex gold electrodes were part of a three electrode system with an external Ag/AgCl reference electrode  and platinum counter electrode. Cyclic voltammetry scans were performed at a scan rate of 100 mV/s over a potential range of +0.412 V to -0.288 V vs. NHE or +0.512 V to -0.188 V vs NHE. Bulk electrolysis on DNA was performed at an applied potential of +0.512 V vs. NHE for all electrochemical oxidation reactions. The oxidizing potential was applied for at least 8.33 minutes for single oxidation reactions on a surface, and 6.67 minutes for the iterative oxidation cycles of p58C variants. The reducing potential was applied for 8.33 minutes in all electrochemical reduction reactions. All bulk electrolysis and cyclic voltammetry was performed in previously deoxygenated p58C electrochemistry buffer (20 mM HEPES (pH 7.2), 75 mM NaCl). Charge transfer (nC) in the cathodic peak of oxidized samples CV scans was assessed using the area under the current wave of the reduction signal. Charge transfer was measured for oxidized samples using CHI software, assessing the area under the reductive peak in CV after electrochemical oxidation. NTP-dependence of electrochemical signals was measured by pipetting a small volume (1–3 μL) of a 0.1 M ATP stock solution into each quadrant of the multiplexed chip setup. Samples were added by quadrant, as physical barriers in the setup prevent diffusion of NTPs between electrode quadrants. After the volume of ATP stock was deposited onto the electrode quadrant, resulting in a 2.5 mM or 5 mM concentration of ATP in the quadrant, CV scans were measured (100 mV/s scan rate). Charge transfer was assessed using CHI software; charge values were determined by calculation of the area under the reductive and oxidative peak curves. Midpoint potentials of NTP-dependent redox signals were assessed using the peak selection function in CHI software.266-456 and p58C272-464 structures were a by-product of the differences in sequence, we produced, purified, and crystallized a p58C construct containing residues 266–464 in the conditions used to crystallize p58C272-464 2+ form was loosely associated with DNA and exhibited no DNA-mediated electrochemical signal in a cyclic voltammetry scan. To test whether the same redox switching function was present in p58C266-464, we characterized this construct using DNA electrochemistry. Using the multiplexed, DNA-modified Au electrode platform shown in 266-464 on a 20-nt duplex DNA substrate with a 3-nt, 5’-ssDNA overhang 2+ oxidation state, displays no redox signal on DNA, as observed with p58C272-464. However, upon electrochemical oxidation to the [4Fe4S]3+ state by bulk electrolysis at an applied potential of 512 mV vs. NHE, CV scans of p58C266-464 display a large reductive peak near -140 mV vs. NHE. As observed for p58C272-464, this peak disappears after one scan to negative reducing potentials  used originally for p58C272-464  cluster serves as a redox switch that modulates the DNA binding affinity of p58C, setting the stage to further asses the role this switch plays in the priming function of human DNA primase.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigSixteen Au electrodes  are divided into four quadrants, allowing for electrochemical measurements on different DNA substrates with replicates on a single surface.(TIF)Click here for additional data file.S4 Fig2 nC charge transport. All scans were performed under anaerobic conditions on 40 μM [4Fe4S] p58C in 20 mM HEPES (pH 7.2), 75mM NaCl, at 100mV/s scan rate (CV) or 15 Hz (SQWV).Upon addition of 2.5 mM ATP, a reversible redox signal generally appears in CV scans. The signal in the presence of 2.5 mM ATP was centered at an average midpoint potential measured near 142 ± 12 mV vs. NHE. The signal was observed at physiologically relevant redox potential, with a magnitude on the order of 10(TIF)Click here for additional data file.S5 Fig272-464 (blue) and p58C266-464 (red) plotted in the same order (right) and in reverse order (left) relative to 2H2O.Overlay of the spectra of p58C(TIF)Click here for additional data file.S6 FigTitrations of p58C272-464 (blue circles) and p58C266-464 (red squares) into 25 nM fluorescein in a buffer containing 20 mM MES (pH 6.5), 50 mM NaCl, 2 mM DTT, and 0.016% DMSO. Fluorescence anisotropy was measured at 25°C. Before plotting, the data were normalized by subtracting the fluorescence anisotropy value at [p58C] = 0 μM from each titration point. Error bars represent the standard deviation of three independent measurements.(TIF)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.S2 Dataset(ZIP)Click here for additional data file.S3 Dataset(XLSX)Click here for additional data file.S4 Dataset(XLSX)Click here for additional data file.S5 Dataset(ZIP)Click here for additional data file.S6 Dataset(ZIP)Click here for additional data file.S7 Dataset(ZIP)Click here for additional data file.