A; 2. inactivated vaccine with 5.75 μg HA/dose + AS03A; 3. inactivated vaccine with 11.5 μg HA/dose + MF59; and 4. recombinant virus like particle (VLP) vaccine with 15 μg HA/dose + ISCOMATRIX™. Vaccine group 1 had the highest antibody responses to the vaccine virus and the 3rd/5th wave drifted viruses. Notably, the relative levels of cross-reactivity to the drifted viruses as measured by the antibody GMT ratios to the 5th wave viruses were similar across all 4 vaccine groups. The 1st wave vaccines induced robust responses to the 3rd and Pearl River Delta lineage 5th wave viruses but lower cross-reactivity to the highly pathogenic 5th wave A(H7N9) virus. The population in the United States was largely immunologically naive to the A(H7N9) HA. Seasonal vaccination induced cross-reactive neuraminidase inhibition and binding antibodies to N9, but minimal cross-reactive antibody-dependent cell-mediated cytotoxicity (ADCC) antibodies to A(H7N9).Influenza A(H7N9) viruses remain as a high pandemic threat. The continued evolution of the A(H7N9) viruses poses major challenges in pandemic preparedness strategies through vaccination. We assessed the breadth of the heterologous neutralizing antibody responses against the 3rd and 5th wave A(H7N9) viruses using the 1st wave vaccine sera from 4 vaccine groups: 1. inactivated vaccine with 2.8 μg hemagglutinin (HA)/dose + AS03 Pandemics can occur when novel viruses emerge and spread efficiently among human population that has little or no pre-existing immunity. As we are combating the current unprecedented coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is imperative that we continue to monitor the pandemic risk of other respiratory viruses, notably influenza. Over the past 100 years, influenza viruses have caused four global pandemics. Despite increased surveillance efforts and poultry vaccination programs, novel zoonotic influenza virus infections in humans continue to occur and remain a public health threat. Influenza A(H7N9) viruses first emerged in March 2013 in China. Between 2013 and 2017, epidemics of A(H7N9) zoonotic human infections have occurred as distinct waves. The majority of human infections were zoonotic, as a result of direct contact with birds from live bird markets ,5. The CHost immunity is a critical parameter in assessing population susceptibility to influenza virus infections. Studies from an AH7N9)-affected area in China reported that poultry workers who were exposed to A(H7N9) had a low seroconversion rate (6.3%) and there was no sero-positivity in the general population in the region as measured by hemagglutination inhibition (HI) titers. Moreover, survivors of A(H7N9) infection had a higher percentage of HI antibody sero-positivity (65.8%) than did fatal cases (28.6%), suggesting HI antibodies contributed to protective immunity [N9-affectA and MF59, recombinant virus-like particle (VLP) vaccines, recombinant protein vaccines and mRNA vaccines [Vaccination is the most effective prophylactic measure available to lessen morbidity and mortality from influenza virus infections. The World Health Organization (WHO) has recommended several Candidate Vaccine Viruses (CVVs) to be developed against AH7N9) since its emergence in humans . Several since itvaccines ,11,12,13A or MF59, and recombinant VLP adjuvanted with a saponin nanoparticle, ISCOMATRIX™) and 4 antigen/adjuvant combinations. We also assessed the population immunity against A(H7N9) viruses in the US. Lastly, we evaluated cross-reactive antibody responses to A(H7N9) viruses following seasonal vaccination in various age groups using expanded immunological measurements including neutralizing antibodies, neuraminidase (NA) inhibition antibodies and antibody-dependent cell-mediated cytotoxicity (ADCC) antibody responses.In this study, we evaluated the breadth of the heterologous cross-reactive neutralizing antibody responses against the 3rd and 5th wave A(H7N9) viruses following vaccination with the 1st wave antigens using sera collected from 4 clinical trials representing 3 vaccine technology platforms  by SRID [AH7N9) vaccine sera were collected from healthy adults enrolled in 4 clinical trials: 1 trial sponsored by GlaxoSmithKline (GSK) Vaccines , 2 trials sponsored by the National Institute of Allergy and Infectious Diseases (NIAID) , and 1 trial sponsored by Novavax Inc . Stored sera from the trials were used in the current study. From each clinical trial, a subset of sera was selected by the sponsors from strong responders (achieved seroconversion by HI to CVV) in the target vaccine arms based on the original immunogenicity evaluations of the trials . In the A , an oil-A . In the  vaccine  by SRID . The vac by SRID , sera we by SRID .A, we also assessed the addition time point at 180 days post-the first vaccination from the same participants. The study was approved by Human Subject Research Determination Review Board of CDC.In all 4 vaccine groups, vaccines were administered with 2 doses 21 days apart. Paired sera were collected before the first dose and 21–28 days after the second dose. From the GSK trial with inactivated vaccine formulated at 2.8 μg HA/dose with AS03Anonymous influenza seasonal vaccination sera were collected pre-vaccination and 28 days post-vaccination from 3 age groups: children (6–35 months), adults (18–49 years) and elderly (≥65 years) who received the 2012–2013 and 2015–2016 Northern Hemisphere egg-based seasonal trivalent inactivated influenza vaccines (IIV3).To assess the population immunity to A(H7N9) viruses in the US, 900 sera from healthy individuals aged 6–102 years collected in 2010 by the National Health and Nutrition Examination Survey (NHANES) were used in the study. NHANES sera collection is a population-based survey conducted nationwide using a complex, multistage, probability sample design to select participants representative of the civilian, non-institutionalized US population. Sera were stratified by nine age groups: 6–11, 12–19, 20–29, 30–39, 40–49, 50–59, 60–69, 70–79 and ≥80 (in years). The study was approved by Human Subject Research Determination Review Board of CDC.Six wild-type A(H7N9) viruses were used in the study, two from the 1st epidemic wave, A/Anhui/1/2013 (AH1) and A/Shanghai/2/2013 (SH2); one from the 3rd wave: A/British Columbia/1/2015 (BC1), three from the 5th wave: A/Hong Kong/61/2016 , A/Hong Kong/125/2017 , and a HPAI A(H7N9) virus A/Guangdong/17SF003/16 (GD17SF003) also from the YRD lineage. Seasonal A(H3N2) viruses A/Victoria/361/2011, A/Perth16/2009 and A/Switzerland/9715293/2013 were also used. Viruses were propagated in 10–11 day old embryonated eggs. All studies using A(H7N9) viruses were conducted in Biosafety level 3 Enhanced (BSL3E) laboratories, and studies using seasonal viruses were conducted in Biosafety Level 2 (BSL2) laboratories.https://bioedit.software.informer.com, accessed on 26 October 2022).Virus stocks used in the study were sequenced and HA sequences were analyzed using the BioEdit software (HI assays for A(H3N2) viruses were performed using 0.5% turkey erythrocytes . HI assa50) of influenza viruses. The virus-sera mixture was used to infect 1.5 × 104/well Madin-Darby canine kidney cells and incubated overnight. The plates were fixed with cold 80% acetone and the presence of viral nucleoprotein was quantified by enzyme-linked immunosorbent assay (ELISA). Microneutralization titers were defined as the reciprocal of the highest serum dilution that showed 50% neutralization. Antibody titers <10  were reported as 5.MN assays were performed as previously described . Heat inNeuraminidase inhibition antibodies (NAI) were detected using ELLA assay . ReassorNickel plates were coated overnight at 4 °C with his-tag recombinant NA at 200 ng/well, plates were then washed and blocked with 5% milk and 0.1% Tween20 in Phosphate buffer saline for one hour and washed again. Serial diluted sera were transferred to each well (100 uL/well) and incubated at room temperature for one hour. After 3 washes, HRP conjugated goat anti-human IgG was added and incubated for one hour. The reactivity was revealed by TMB substrate and read at an absorbance of 450 nm with a spectrophotometer.v/v) FcγRIIIa receptor was mixed with PE-conjugated mouse anti-human CD107a. Following incubation and washing, the percentages of CD107a expression on NK cells were determined on a flow cytometer using a high throughput sampler. The ADCC antibody titers were defined as the highest serum dilution that achieved 3% threshold of CD107a + NK cells.Influenza HA-specific ADCC-mediating antibodies were quantified using an ADCC-NK cell activation assay as described previously ,20. Two Geometric mean antibody titers (GMTs) and 95% confidence intervals (CIs) were calculated. Seroconversion was defined as ≥4-fold rise in titers between pre- and post-vaccination with a post-vaccination titer ≥40. Seroconversion rate (SCR) is the proportion of the participants that seroconverted in the current study. Seroprotection rate (SPR) is the proportion (%) of the current study participants that achieved titers ≥40 by HI or MN ,21,22. PAntigenic characterization using ferret antisera showed that the two 5th wave viruses in the YRD lineage are antigenically drifted from the 1st wave virus A/Anhui/1/2013 (≥8 fold reduction in MN titer); the HPAI YRD lineage virus, A/Guangdong/17SF003/2016, is antigenically the most distant ; group 2. 5.75 μg HA/dose + AS03A (NIAID); group 3. 11.5 μg HA/dose + MF59 (NIAID), and group 4. 15 μg HA VLP/dose + ISCOMATRIX adjuvant (Novavax). Prior to vaccination, all participants were immunologically naive to A(H7N9) viruses (baseline titers <10). Following two doses of the 1st wave vaccines, participants from the vaccine group 1 who received 2.8 μg HA/dose + AS03A had the highest antibody responses to the homologous 1st wave vaccine virus  (A}), a 3rd wave virus A/British Columbia/1/2015 was also tested and had the highest post-vaccination titers (MN GMT 95%CI: 329 {216–502})  . In all {18–34}) , in one 16–502}) , suggestA had the highest heterologous antibody responses, achieved 36% seroconversion and 44% SPR in both HI and neutralizing antibodies against the HPAI 5th wave virus at 42 days post-vaccination (21 days post-second dose)  .A  viruses waned to almost baseline levels (GMT < 20) for most viruses, except that MN titers to the 3rd wave A(H7N9) virus (which had the highest post-vaccination titers) remained detectable at GMT > 40.We then assessed antibody waning at 180 days post-1st dose in participants from group 1 who received 2.8 μg HA/dose adjuvanted with AS03A . At 6 moLastly, post-vaccination HI and MN titers to A(H7N9) viruses correlated well; spearman correlation R values ranged from 0.5949 to 0.9248 for all 5 A(H7N9) viruses .n = 900), indicating that the US population is largely immunologically naive to the HA of A(H7N9) viruses. Because H7 and H3 are both group 2 influenza viruses, we also included a seasonal A(H3N2) virus representing the circulating A(H3N2) virus at the time of the sera collection in the analysis as a control. In contrast to A(H7N9) viruses, all age groups had pre-exiting antibodies to this seasonal A(H3N2) virus, demonstrating an age-related pattern  viruses in 9 age groups  as measured by HI and neutralization antibodies to A(H7N9). No cross-reactive HI and neutralization antibodies were detected to A(H7N9) HA with 0% SPR in almost all 9 age groups  viruses, we expanded our analysis to measure both HA-targeting neutralizing antibodies (by HI and MN) and other functional antibodies including NAI and ADCC antibodies. Seasonal vaccination with 2012–2013 IIV3 did not induce any cross-reactive HI or MN antibodies to both 1st wave A(H7N9) viruses tested  in children (6–35 months), adults (18–49 years) or elderly (≥65 years) .SH2 and H6N9AH1), whereas 17% elderly (≥65 years) and 20% children had ≥4 fold rise to H6N9SH2. To verify that the NA antibody rise detected was not H6NA reassortant virus dependent, we tested two reassortant N9 viruses bearing different strains of H6 and N9  with adult sera. Similar NA antibody rises were detected by both H6NA reassortant viruses . Although vaccination with 2015–16 IIV3 in adults induced robust ADCC antibodies to homologous A(H3N2) vaccine virus (61% ≥ 4 fold rise), it induced low cross-reactive ADCC antibodies to A(H7N9); only 4% (N = 1) of participants achieved ≥ 4 fold rise in H7-mediated ADCC antibodies to A/Shanghai/2/2013 .Influenza viruses continue to pose a serious pandemic threat. As we are learning from the current unprecedented COVID-19 pandemic, vaccination has been proven to be the essential public health measure to combat the global pandemic . The COVA, MF59, VLP and even mRNA technologies have already been developed and evaluated in the clinical trials for immunogenicity [A and MF59), and one newer technology platform based on VLP antigens with ISCOMATRIX™ adjuvant. Our results showed that vaccines containing the 1st wave A(H7N9) viruses can induce robust heterologous cross-reactivity against the 3rd wave and the PRD lineage 5th wave A(H7N9) viruses but low cross-reactivity against the YRD lineage 5th wave viruses especially the HPAI virus. Importantly, we found that the relative ratios of the heterologous neutralizing antibody responses induced to the drifted 5th wave viruses to the homologous wild type vaccine virus, as measured by post-vaccination GMT ratios, were similar across all 3 vaccine platforms . It is also encouraging that the newer vaccine platform based on recombinant VLP and a saponin-based adjuvant also elicited heterologous antibody responses to the drifted 5th wave viruses.Inactivated AH7N9) vaccines alone induce poor immune responses in immunologically naïve populations; currently, potent adjuvants and 2 dose vaccine regimens are needed to provide sufficient immunogenicity against A(H7N9) . At the  vaccinesgenicity ,12,13,14genicity . Here, wlatforms A. TherefInfluenza viruses continue to undergo antigenic drift. In the event of an influenza pandemic caused by the A(H7N9) viruses, the pandemic strain is likely to be antigenically drifted from the current A(H7N9) vaccines. One scenario of A(H7N9) pandemic vaccination strategy is to rapidly deploy the existing vaccines as the priming dose as soon as possible at the onset of the pandemic, followed by a booster dose that is antigenically matched with the pandemic strains once it becomes available. In such a scenario, instead of requiring two doses, one booster dose with an antigenically matched pandemic strain could potentially elicit sufficient immunogenicity against the new pandemic viruses. Our previous study reported that heterologous prime-boost with different antigens could induce broader antibody responses than homologous prime-boost for HPAI A(H5N1) influenza viruses ,22. FurtA (group 1), HI and neutralizing antibodies to both homologous vaccine virus and heterologous 5th wave viruses waned almost to the baseline level, suggesting one or more booster doses may be needed in the event of a prolonged pandemic.A pandemic can be prolonged; it is therefore also critical to understand the duration of immunity. Few A(H7N9) clinical trials have reported immune responses beyond 42 days post-1st dose of vaccination (21 days after the second dose). Here, we were able to include sera collected at 6 months post-1st dose of vaccination to assess the waning of cross-reactive antibodies. At 6 months following vaccination with 2.8 μg HA/dose + AS03Neutralizing antibodies targeting HAs on the surface of the influenza viruses, often measured as HI or MN titers, are considered correlated with protection against influenza infection . An HI tA or MF59 elicited robust cross-reactive NAI antibodies and HA-specific ADCC antibody responses to the 5th wave A(H7N9) viruses [In addition to neutralizing antibodies, multiple immune mechanisms may contribute to immune protections from influenza infection. We previously reported that vaccination with inactivated 1st wave A(H7N9) virus adjuvanted with either AS03 viruses . The VLP viruses . Here, w viruses . NAI ant viruses . HoweverOur study has limitations, first, for the A(H7N9) vaccine responses we only focused on the strong responders selected from the immunogenicity evaluation of the original study, because other participants that did not respond well to the vaccine virus likely had low cross-reactive responses to drifted viruses, thus, the level of heterologous cross-reactive responses from the strong responders represent a best case scenario. It also should be noted that in the original immunogenicity studies reverse genetics CVVs were used, in the current study we used wild type A(H7N9) vaccine viruses, therefore the titers may differ. Second, the 4 A(H7N9) vaccine groups were selected from 4 different clinical trials, a vaccine trial with a randomized comparison of optimized doses of the different vaccine technology platforms are not available for direct comparison of the responses.Taken together, the continued antigenic evolution of influenza viruses with pandemic potential poses challenges for effective influenza pandemic mitigation through vaccination. Effective pandemic vaccines need to provide timely, broad, and long-lasting protection against antigenically drifted emerging variants. Immunological markers with demonstrated correlates of protection against influenza illness can be used to assess the vaccine immunogenicity and predictive efficacy against emerging drifted variants, and protective immunity. Such correlates of protection assessments can be expanded beyond the traditional HI and neutralization assays to include NAI and ADCC antibodies, and other relevant immunological measurements. Vaccines that can induce multiple arms of immune responses can not only prevent illness but may also interrupt transmission or alleviate the diseases severity to further mitigate the pandemic impact. Although the data presented in the current study do not represent a randomized comparison of the vaccines studied; nonetheless, they illustrate that both classical inactivated virus and recombinant technologies can address pandemic threats. Concerted efforts are needed to be better prepared for future influenza pandemics.


Figure 3. Change Illustrative example of a patient in the poor group with a calcified disc herniation. T2-weighted sagittal (A) and axial (B) magnetic resonance images show an L5/S1 disc herniation. Sagittal (C) and axial (D) computed tomography myelography helped to confirm the calcified disc herniation”. The word change was added by mistake. The correct legend is shown below. The authors state that the scientific conclusions are unaffected. This correction was approved by the Academic Editor. The original publication has also been updated.In the original publication , there wFigure 3. Illustrative example of a patient in the poor group with a calcified disc herniation. T2-weighted sagittal (A) and axial (B) magnetic resonance images show an L5/S1 disc herniation. Sagittal (C) and axial (D) computed tomography myelography helped to confirm the calcified disc herniation.”“


Single Cell Implementation to Find Expressed Retrotransposons (SCIFER). SCIFER aligns scRNA-Seq reads uniquely to the genome and extracts alignments from single cells by cell-specific barcodes. In contrast to the alignment performed using default parameters, this alignment strategy increases accuracy of L1 locus identification by retaining only reads that are uniquely mapped to individual L1 loci. L1 loci expressed in single cells are unambiguously identified using a list of L1 loci manually validated to be expressed in bulk RNA-Seq datasets generated from the same cell line or organ.Endogenous expression of L1 mRNA is the first step in an L1-initiated mutagenesis event. However, the contribution of individual cell types to patterns of organ-specific L1 mRNA expression remains poorly understood, especially at single-locus resolution. We introduce a method to quantify expression of mobile elements at the single-locus resolution in scRNA-Seq datasets called Validation of SCIFER using MCF7 cells determined technical parameters needed for optimal detection of L1 expression in single cells. We show that unsupervised analysis of L1 expression in single cells exponentially inflates both the levels of L1 expression and the number of expressed L1 loci. Application of SCIFER to analysis of scRNA-Seq datasets generated from mouse and human testes identified that mouse Round Spermatids and human Spermatogonia, Spermatocytes, and Round Spermatids express the highest levels of L1 mRNA. Our analysis also determined that similar to mice, human testes from unrelated individuals share as much as 80% of expressed L1 loci. Additionally, SCIFER determined that individual mouse cells co-express different L1 sub-families and different families of transposable elements, experimentally validating their co-existence in the same cell.SCIFER detects mRNA expression of individual L1 loci in single cells. It is compatible with scRNA-Seq datasets prepared using traditional sequencing methods. Validated using a human cancer cell line, SCIFER analysis of mouse and human testes identified key cell types supporting L1 expression in these species. This will further our understanding of differences and similarities in endogenous L1 mRNA expression patterns in mice and humans.The online version contains supplementary material available at 10.1186/s13100-022-00276-0. Long INterspersed Element-1 (LINE-1 or L1) mRNA has multiple negative consequences for genome stability. L1 mRNA expression can lead to retrotransposition of L1 transcripts or trans-retrotransposition of transcripts from other mobile elements, such as Alu or SVA [Expression of u or SVA . Additiou or SVA –7. Experu or SVA , 9. Prevu or SVA –14, but L1 sequences are abundant in the human genome, which contains 500,000 truncated and full-length copies, as well as in the mouse genome, which harbors 600,000 truncated and full-length copies –17. A crWe developed a method to rigorously analyze L1 mRNA expression at a single-locus resolution by filtering out truncated and passively transcribed L1 sequences , 20, 30.Methods to quantify transposable element expression in single cells, such as the study by Shao, et al,.  implemenSingle Cell Implementation for Finding Expressed Retrotransposons (SCIFER). SCIFER extracts scRNA-Seq reads generated from traditional 10X Chromium Single Cell 3′ Genomics sequencing along with their barcodes and realigns them to the reference genome using Bowtie [Here, we report a new bioinformatics tool designed for analysis of L1 mRNA expression quantification from individual L1 loci in single cells called g Bowtie  with unig Bowtie , 20, 30.g Bowtie , 20, 30.Single Cell Implementation to Find Expressed Retrotransposons) is a method to quantify L1 mRNA expression at the locus-specific level in scRNA-Seq datasets. SCIFER aligns data from a standard 10X Chromium Single Cell 3′ RNA-Seq dataset to the reference genome using procedures optimized to deal with the very high genomic copy number of L1 elements  by cross referencing the list of L1 loci that were assigned same-sense RNA-Seq alignments in the scRNA-Seq dataset with a list of full-length L1 loci validated to be expressed in MCF7 cells from a previous study  were down sampled in 10% intervals and the average number of expressed L1 loci detected in each resulting interval sample was compared Fig. A. This aP < 0.0001, comparing Fig. To determine limitations imposed by lower sequencing coverage, we performed SCIFER analysis on a low coverage MCF7 scRNA-Seq dataset with 10-fold less sequencing coverage and 5-fold more sequenced cells compared to the High coverage MCF7 scRNA-Seq dataset Fig.  B. The cr = 0.55, P < 0.0001 Additional file P < 0.0001). The positive L1 FPKM-RPM correlation and high percent of shared expressed loci detected between Low coverage scRNA-Seq and bulk RNA-Seq establishes that L1 expression detection, averaged across a population of cells, is not dramatically impacted by reduced sequencing depth. However, the number of expressed L1 loci per cell and their levels of expression are underestimated when a low coverage dataset is used. Additionally, similar to the High coverage scRNA-Seq dataset, L1 loci expressed in bulk RNA-Seq but missed by SCIFER in lower sequencing coverage scRNA-Seq datasets are those L1 loci that have low expression levels.We next compared the total L1 expression levels detected by SCIFER in the Low coverage MCF7 dataset with L1 expression levels detected in the bulk RNA-seq dataset. Despite reduced sensitivity of detection of L1 expression per cell in the Low coverage dataset, RPM per expressed L1 locus in the Low coverage dataset was positively correlated with bulk RNA-Seq L1 FPKM  to determine the extent of variation in L1 expression between individual cells Fig. C. This aWe also detect expression of housekeeping genes (HKGs) in cells with read numbers downsampled in 10% increments, the same three cells used in Fig. P < 0.0001, Additional file P < 0.0001, Additional file P < 0.0001, Additional file Previously, our comprehensive analysis of locus-specific L1 mRNA expression in mouse organs determined that testes express the highest levels of L1 mRNA compared to other mouse organs, including male and female brains, male and female lungs, ovaries, and uteri . To deteP < 0.0001, P < 0.0001, P < 0.0001, respectively, Fig. P = 4.39E-99, P = 1.20E-28, P = 3.89E-94, respectively, Additional file P < 0.0001, P < 0.0001, P < 0.0001, respectively, Fig. SCIFER analysis was performed on the scRNA-Seq mouse testis samples to discover the levels and patterns of L1 mRNA expression in different cell types using a list of L1 loci validated to be expressed in mouse testes from a previous publication . SCIFER To understand potential changes in L1 expression during spermatogenesis, we compared the identity of L1 loci expressed in different sperm cell types identified in our single cell pool. Comparison of L1 loci expressed in different sperm cell types determined that in the Mouse 1 dataset, Spermatocytes, Round Spermatids, Elongating Spermatids, and Condensing Spermatids share only 2% 5 of 212) of L1 loci expressed in testes, with 157 L1 loci being detected to be expressed only in Round Spermatids alone Fig. D. In the of 212 or = 0.51, P < 0.0001, Fig. r = 0.72, P < 0.0001, Fig. P < 0.0001, Fig. Previously we found that in MCF7 cells SCIFER detects 88–93% of expressed L1 loci detected in bulk RNA-Seq and the L1 FPKM-RPM levels of expressed L1s are positively correlated between SCIFER-detected L1 loci expression in single cells and bulk RNA-Seq  that were manually validated to be expressed in bulk RNA-Seq were analyzed in scRNA-Seq datasets  and Spermatocytes 0.029) compared to Spermatogonia (0.025), Elongated Spermatids 0.28), and Condensing Spermatids (0.007)  (P < 0.0001) , which allows unique mapping of scRNA-Seq reads to the genome while retaining cell-specific barcodes. SCIFER analysis also includes validating L1 expression detected in scRNA-Seq with the analysis of L1 expression in bulk RNA-Seq from a matching tissue sample using our existing approach for identifying expressed L1 mRNAs from individual sites, while eliminating those that are passively expressed [Using RNA-Seq to measure the expression of L1 elements derived from their own promoter is technically challenging, both because of difficulties properly aligning short-read sequences to a single, specific locus and the high levels of passive inclusion of L1 sequences in other transcripts that create high levels of background , 20–22. xpressed , 20, 30.We validated SCIFER as a method for measuring L1 expression in single cells by analyzing a scRNA-Seq dataset generated from a pooled sample of MCF7 and HEK293 cells to determine whether our method was sensitive enough to detect the same loci as seen in the bulk RNA-Seq studies .These findings have important implications for experimental design and comparative analysis of L1 expression in single cells. For example, comparisons between scRNA-Seq with significantly different average sequencing depth per cell are likely to produce artifacts regarding the number of expressed L1 loci per cell and the number of cells expressing specific L1 loci. However, both High and Low coverage datasets can be used for organ-derived studies to identify relative contribution of different cell types to L1 expression and the diversity and cell type specificity of expressed L1 loci in a specific organ Fig. .Our single-cell analysis also provides evidence that SCIFER can be adapted for detection of other repetitive sequences and assist in answering outstanding questions regarding mobile element biology. It has been proposed that L1 promoter evolution is driven in part by co-expression of different L1 subfamilies, which leads to their competition for cellular transcription factors and other transcriptional machinery –65. UsinWe also performed SCIFER analysis on human testes to observe whether L1 expression patterns in testes are conserved between mice and humans. Bulk RNA-Seq of human testes from two unrelated 20 yo donors showed a high level of similarity in the L1 loci expressed  of origin of L1 expression in different organs and species. Additionally, SCIFER has the ability to uncover evolutionary niches occupied by different classes of TEs or TE subfamilies, as well as to understand L1 impact on function and genome stability of single cells.MCF7 (ATCC HTB-22) and HEK293-FRT-LacZeo cells were maintained in DMEM with high glucose (Gibco) supplemented with 10% fetal bovine serum (Gibco), sodium pyruvate, essential and nonessential amino acids, and L-glutamine. Cells at 100% confluency were detached using accutase (Innovative Cell Technologies) and resuspended in DPBS. MCF7 and HEK293-FRT-LacZeo cells were combined at a 9:1 ratio with 180 MCF7 and 20 HEK293 cells per uL. The single cell suspension was confirmed to be 84.4–83.1% viable prior to RNA preparation and sequencing.Human testis bulk RNA samples from two 20-year-old (yo) donors were obtained from AMSBIO (product codes: CR562159 and CR562389). RNA was then poly-A selected prior to stranded, paired-end RNA sequencing using an Illumina NextSeq 2000 in the Tulane NextGen sequencing core.Single-cell RNA sequencing was carried out on individual cells using the 10X Genomics Chromium™ Single Cell 3′ Library & Gel Bead Kit v3 and 150 cycle kit by the Tulane NextGen sequencing core. Eight hundred fifty-three cells were sequenced and barcoded. Sequencing was carried out on an Illumina NextSeq 2000. Pooled MCF7 and HEK293-FRT-LacZeo cells were separated by aligning reads to an FRT-LacZeo genome. Cell barcodes with reads aligning to the FRT-LacZeo locus were classified as HEK293-FRT-LacZeo cells.The shallow sequenced MCF7 scRNA-Seq dataset and mouse and human testes scRNA-Seq datasets were obtained from NCBI SRA. The shallow sequenced MCF7 scRNA-Seq dataset is listed under SRR10018060. The mouse testes datasets are listed under SRR6129050 (Mouse 1) and SRR6129051 (Mouse 2). The human testes datasets are listed under SRR6860521 (24 yo) and SRR6860523 (25 yo).The strategy for detection of L1 mRNA used here has been described previously . BrieflyBioinformatic Analysis for bulk RNA-Seq , resulting in unambiguous assignment of reads to their best genomic location , as previously reported , 30. ForThe described formula for RPM is shown below:10X Chromium Single Cell 3′ Genomics data was demultiplexed and converted into fastq format using cellranger v3.1.0 mkfastq tool. The cellranger v3.1.0 count program was then used to analyze scRNA-Seq reads and alignment of reads to the appropriate reference genome to generate a gene expression matrix. The total number of L1 UMIs from SCIFER analysis were loaded into the gene expression matrix and analyzed using Seurat v4.0.5. The datasets were normalized  and L1 expression was compared between cell clusters and cell types (FindAllMarkers).n = 2) were made using the normalized gene expression levels downloaded from the Seurat object and Welch’s t-tests.Seurat v4.0.5 was used to compare gene expression levels between clusters and cell types . Cells wt-test with Welch’s correction when making comparisons between two groups and by one-way ANOVA with multiple comparisons and “two-stage” Benjamini, Krieger, & Yekutiel for controlling the false discovery rate when making comparison within a n > 2 group. To measure linear correlation between two variables and determine the r value, a two-tailed Pearson correlation test was performed. Data presented from Seurat was analyzed using a non-parametric Wilcoxon rank-sum test with Bonferroni correction and the adjusted P-values from Bonferroni correction are plotted. Statistical analysis was performed with GraphPad Prism and Seurat v4.0.5.Bar graphs are presented with the mean and standard deviation bars. Data were analyzed by two-tailed Student’s Additional file 1. Single cell RNA-Seq reads align to the 3′ end of genes and L1 loci. A. Alignment of scRNA-Seq reads (top), bulk RNA-Seq reads (middle), and DNA-Seq reads (bottom) to the L1 consensus sequence using Bowtie v0.12.8. The sequencing read scale is indicated in the bottom left corners. B. Alignment of MCF7 bulk RNA-Seq and MCF7 scRNA-Seq reads to the actin (ACTB) gene locus (top) and an L1 locus (bottom). Images were taken from IGV and the visible tracks include, from top to bottom, chromosome location indicated by the red line, scale in base pairs, mappability from a DNA-Seq samples aligned with the same bowtie settings used for RNA-Seq , RNA molecules per cell (middle), and percent of mitochondrial reads per cell (right) for each MCF7 cell cluster. C. The number of expressed L1 loci per cell for all High coverage MCF7 cell clusters is shown in the individual value plot . D. The L1 mRNA expression level per cell for all MCF7 clusters is shown in the individual value plot . E. The normalized expression levels for MCF7 Clusters 1, 3, and 4 from Seurat analysis are shown . F. The number of reads per HKG averaged between 3 MCF7 cells downsampled in 10% intervals is shown. G. The average number of expressed HKGs detected in 3 MCF7 cells is shown with decreasing read depth . The black bars have significantly higher numbers of detected HKGs compared to the gray bars . The lightest gray bar (10%) indicates detection of less than half of expressed HKGs detected with 100% of reads. H. A correlation matrix of HKGs expressed in 10 MCF7 cells. The HKGs are listed at the top of the matrix and the 10 cells are listed along the left side. Filled boxes indicate that the HKG was expressed in the corresponding cell.Additional file 3. Low depth sequencing reduces sensitivity of L1 mRNA expression detection in MCF7 single cells. A. A t-SNE plot of the Low coverage MCF7 scRNA-Seq dataset. Cell clusters are indicated in the figure legend. B. The number of million mapped reads per cell in each cluster is shown in the individual value plot. C. L1 mRNA expression quantified by RPM for each cell is shown in the individual value plot. D. The number of expressed L1 loci per cell in each cluster is shown in the individual value plot E. The L1 FPKM values for bulk RNA-Seq (left y-axis) and L1 RPM values for Low coverage MCF7 scRNA-Seq (right y-axis) are shown in the dot-plot. Orange circles indicate L1 loci with detected expression in the bulk dataset that were not detected to be expressed in scRNA-Seq. The nested Venn diagram shows the number of shared expressed L1 loci from the bulk and scRNA-Seq datasets. The violin plot shows the FPKM values for expressed L1 loci unique to the bulk dataset and those shared between bulk and scRNA-Seq .Additional file 4. Expression patterns of genes involved in spermatogenesis in mouse testes. Cell types are abbreviated as follows: Spermatocytes (Spcyt), Round Spermatids (RS), Elongating Spermatids (ES), Condensing Spermatids (CS), Sertoli (Ser), Leydig (Ley). A. Mouse 1 gene expression patterns for TATA-Box Binding Protein Like-1 (TBPL1) (A1), Protamine (PRM1) (A2), and Transition Protein 1 (TNP1) (A3). B. Mouse 2 gene expression patterns for TBPL1 (B1), PRM1 (B2), and TNP1 (B3). C. The heat map shows the normalized expression patterns for each cell type and gene in Mouse 1 (top) and Mouse 2 (bottom). The asterisks indicate a significant change in gene expression in the cell type compared to the preceding cell type in the row (P < 0.0001). D. The normalized expression levels for Mouse 1 testis cell types from Seurat analysis are shown .Additional file 5. SCIFER analysis of L1 mRNA expression in mouse testis biological replicates. A. The number of million mapped reads per cell for each testis cell type is shown for Mouse 1 . B. The number of million mapped reads per cell for each testis cell type is shown for Mouse 2 ANOVA,, ***, < 0.0005, ****,< 0.0001). C. L1 mRNA expression measured by RPM per cell for all testis cell types is shown in the scatter plot for Mouse 1 . D. L1 mRNA expression measured by RPM per cell for all testis cells types is shown in the scatter plot for Mouse 2 . E. The number of expressed L1 loci per cell for each testis cell type is shown in the scatter plot for Mouse 1 . F. The number of expressed L1 loci per cell for each testis cell type is shown in the scatter plot for Mouse 2 . G. The L1 RPM values for L1 loci indicated on the X-axis are shown for each sperm cell type in Mouse 2. The Venn Diagram shows the number of expressed L1 loci shared by the sperm cell types with the percentage of shared expressed L1 loci indicated above the diagram. H. L1 mRNA expression compared between Mouse 1 and Mouse 2 for Spermatocytes (SC) (H1), Round Spermatids (RS) (H2), Elongating Spermatids (ES) (H3), and Condensing Spermatids (CS) (H4).Additional file 6. Expression patterns of genes involved in limiting L1 expression and integration in mice. Cell types are abbreviated as follows: Spermatocytes (Spcyt), Round Spermatids (RS), Elongating Spermatids (ES), Condensing Spermatids (CS), Sertoli (Ser), Leydig (Ley). A. Mouse 1 gene expression patterns for Phospholipase D Family Member 6 (PLD6) (A1), Heat Shock Protein 90 Alpha Family Class A Member 1 (HSP90AA1) (A2), and Ubiquitin Like with PHD And Ring Finger Domains 1 (UHRF1) (A3). B. Mouse 2 gene expression patterns for PLD6 (B1), HSP90AA1 (B2), and UHRF1 (B3). C. The heat map shows the normalized expression patterns for each cell type and gene in Mouse 1 (top) and Mouse 2 (bottom). The asterisks indicate a significant change in gene expression in the cell type compared to the preceding cell type in the row (P < 0.0001).Additional file 7. Expression of LTRs and L1 subfamilies is non uniform across single cells in mouse testis. A. The number of expressed L1 loci per L1 subfamily is shown for Mouse 1. The L1 subfamilies are indicated in the legend. B. The number of Round Spermatids expressing L1 loci from each mouse L1 subfamily is shown in the Venn diagram. C. The number of reads per LTR locus is shown for bulk RNA-Seq, Mouse 1 Single Cells, and Mouse 2 Single Cells. The Venn diagram shows the number of expressed LTR loci shared between the three datasets. D. Cells expressing at least one LTR loci are shown for each cell type in Mouse 1 (D1) and Mouse 2 (D2). LTR expression is indicated with purple and L1 co-expression is indicated with teal.Additional file 8 Expression patterns of genes involved in spermatogenesis in human testes. Cell types are abbreviated as follows: Spermatogonial Stem Cells (SSCs), Differentiating Spermatogonia (Diff Spg), Spermatocytes (Spcyt), Round Spermatids (RS), Elongating Spermatids (ES), Condensing Spermatids (CS), Macrophages (Macro), Endothelial (Endo), Myoid (Myo), and Sertoli (Ser). A. 24 yo Human Testis gene expression patterns for PRM1 (A1), SPAG6 (A2), TNP1 (A3), and TNP2 (A4). B. 25 yo Human Testis gene expression patterns for PRM1 (B1), SPAG6 (B2), TNP1 (B3), and TNP2 (B4). C. The heat map shows the normalized expression patterns for each cell type and gene in 24 yo Testis (top) and 25 yo Testis (bottom). The asterisks indicate a significant change in gene expression in the cell type compared to the preceding cell type in the row (P < 0.0001). D. The normalized expression levels for 24 yo testis cell types from Seurat analysis are shown .Additional file 9. Analysis of L1 mRNA expression in human testes with bulk RNA-Seq and scRNA-Seq. A. The L1 FPKM values for L1 loci listed on the X-axis are shown for two 20 yo human testis bulk RNA-Seq samples. The number of expressed L1 loci shared between the two replicates is shown in the Venn diagram with the percentage of shared expressed loci indicated above the diagram. B. The L1 RPM values for L1 loci listed on the X-axis are shown for a 24 yo and a 25 yo human testis scRNA-Seq sample. The number of expressed L1 loci shared between the two replicates is shown in the Venn diagram with the percentage of shared expressed loci indicated above the diagram. C. The million mapped reads per cell for all cell types in the 24 yo scRNA-Seq dataset is shown in the scatter plot. Statistics are shown for sperm cells and sperm progenitor cell types only. D. The L1 mRNA expression level per cell for all 24 yo testis cell types is shown in the scatter plot. E. The number of expressed L1 loci per cell for all 24 yo testis cell types is shown in the scatter plot. F. The million mapped reads per cell for all cell types in the 24 yo scRNA-Seq dataset is shown in the scatter plot. Statistics are shown for sperm cells and sperm progenitor cell types only. G. The L1 mRNA expression level per cell for all 25 yo testis cell types is shown in the scatter plot. H. The number of expressed L1 loci per cell for all 25 yo testis cell types is shown in the scatter plot. I. The L1 RPM values for L1 loci indicated on the X-axis are shown for each sperm cell type in the 25 yo human testis sample. The Venn Diagram shows the number of expressed L1 loci shared by the sperm cell types with the percentage of shared expressed L1 loci indicated above the diagram. J. The L1 FPKM values for bulk RNA-Seq (left y-axis) and L1 RPM values for scRNA-Seq (right y-axis) of human testis are shown in the dot-plot. Orange circles indicate L1 loci with detected expression in the bulk dataset that were not detected to be expressed in scRNA-Seq. The nested Venn diagram shows the number of shared expressed L1 loci from the bulk and scRNA-Seq datasets. K. The violin plot shows the FPKM values for expressed L1 loci unique to the bulk dataset and those shared between bulk and scRNA-Seq .Additional file 10. Expression patterns of genes involved in limiting L1 expression and integration in humans. Cell types are abbreviated as follows: Spermatogonial Stem Cells (SSCs), Differentiating Spermatogonia (Diff Spg), Spermatocytes (Spcyt), Round Spermatids (RS), Elongating Spermatids (ES), Condensing Spermatids (CS), Macrophages (Macro), Endothelial (Endo), Myoid (Myo), and Sertoli (Ser). A. 24 yo Human Testis gene expression patterns for DNMT1 (A1), MECP2 (A2), KDM1A (A3), TP53 (A4), TRIM28 (A5), ERCC4 (A6), MOV10 (A7), and RNASEH2B (A8). B. 25 yo Human Testis gene expression patterns for DNMT1 (B1), MECP2 (B2), KDM1A (B3), TP53 (B4), TRIM28 (B5), ERCC4 (B6), MOV10 (B7), and RNASEH2B (B8). C. The heat map shows the normalized expression patterns for each cell type and gene in 24 yo Testis (top) and 25 yo Testis (bottom). The asterisks indicate a significant change in gene expression in the cell type compared to the preceding cell type in the row (P < 0.05).Additional file 11. Directions for running Single Cell Implementation to Find Expressed Retrotransposons (SCIFER).Additional file 12. Coordinates of L1 loci identified to be expressed in bulk RNA-Seq of MCF7 cells and included in SCIFER analysis.Additional file 13. Coordinates of LTR loci identified to be expressed in bulk RNA-Seq of 2 mo mouse testes and included in SCIFER analysis.Additional file 14. Coordinates of L1 loci identified to be expressed in bulk RNA-Seq of 2 mo mouse testes and included in SCIFER analysis.Additional file 15. Coordinates and FPKM of L1 loci identified to be expressed in bulk RNA-Seq of 20 yo human testes and included in SCIFER analysis.Additional file 16. Results of comparing gene expression values in mouse testes using unpaired t-test with Welch’s correction.Additional file 17. Results of comparing gene expression values in human testes using unpaired t-test with Welch’s correction.


In the subcutaneous model of human CC, KMT2B overexpression significantly promoted tumor growth and increased tumor vascular density. Meanwhile, KMT2B enhanced the migration and invasion of CC cells and promoted their metastasis to bone in a tail-vein-metastasis model. Mechanistically, the genes upregulated by KMT2B were significantly enriched in PI3K-AKT pathway. Using H3K4me3 ChIP-seq analysis, we found increased H3K4me3 level at EGF promoter region in KMT2B-overexpressing HeLa cells. ChIP-qPCR experiments not only confirmed the increased H3K4me3 level of EGF promoter but also determined that in KMT2B-overexpressing HeLa cells, KMT2B increased binding with the EGF promoter. Blocking EGFR diminished the KMT2B-induced PI3K-AKT signaling activation and CC cell migration and invasion. Moreover, EGFR inhibitors abolished the KMT2B-drived tube formation capacity of HUVECs. In conclusion, KMT2B facilitates CC metastasis and angiogenesis by upregulating EGF expression, and may serve as a new therapeutic target for CC.Evidence has indicated that lysine methyltransferase 2B (KMT2B), a major H3K4 tri-methyltransferase (H3K4me3), contributes to the development of various cancers; however, its role in cervical cancer (CC) is unclear. In this study, increased KMT2B expression was observed in human CC specimens and significantly associated with poor prognosis. The condition medium of KMT2B-overexpressing cells facilitated angiogenesis Cervical cancer (CC) ranked as the fourth most common cancer and the fourth cause of cancer mortality in women, with an estimated 604,000 new cases and 342,000 deaths worldwide in 2020 Multiple epigenetic alterations, including global DNA hypomethylation, chromatin remodeling, as well as histone modifications, occur during whole stages of cervical carcinogenesis H3K4 methylation is mainly catalyzed by the lysine methyltransferase 2 (KMT2) family  family), which contains six members  In the field of cancer research, KMT2A, KMT2C and KMT2D have been intensively studied in different types of tumors It is well established that the epidermal growth factor (EGF) contributes to a number of processes which are important to cancer development and progression Table S1.To analyze the KMT2B expression in CC tissues, a commercially available tissue microarray  containing 30 pairs of CC tissues and the adjacent non-tumorous specimens was used for the IHC staining of KMT2B. The tissue samples were collected from Taizhou Hospital from 2006 to 2009 which were approved by Research Ethics Committee of Taizhou Hospital. The clinical characteristics of the tissue samples included in the tissue microarray are listed in The tumor tissues from nude mice with subcutaneous and myelogenous transplants tumors were resected, fixed in 10% neutral-buffered formalin, embedded in paraffin and sectioned at 5 μm. IHC was performed according to standard protocols. Briefly, the procedures for deparaffinization, rehydration, antigen retrieval and blocking were carried out as described previously 2 at 37 °C. For functional inhibition experiments, cells were treated with the PI3K inhibitor LY294002  or the EGFR inhibitor gefitinib  at final concentrations of 20 μΜ or 200 nΜ, respectively.The human CC cell line C-33A was obtained from the Cell Bank of the Chinese Academy of Sciences . The normal human cervical epithelial cell line HcerEpic cell was obtained from Mingzhou Biotechnology Co. . The Human Umbilical Vein Endothelial Cell line HUVEC and human CC cell line HeLa were purchased from the American Type Culture Collection . C-33A, HUVEC, and HeLa cells were maintained in DMEM medium supplemented with 10% FBS. HcerEpic cells were maintained in MEM medium supplemented with 10% FBS. All cells were cultured in a humidified atmosphere of 5% COhttps://www.ncbi.nlm.nih.gov/geo/). We searched the GEO profiles of the gene KMT2B within the GDS3323 dataset and obtained expression values  from GEO profile data pages. This dataset was generated on the Affymetrix Human Genome U133A Array (HG-U133A) platform, which has 3 probe sets designed to detect KMT2B. According to the JetSet scoring method http://kmplot.com/analysis/) The mRNA expressions of KMT2B in CC tissues (n = 24), noncancerous cervical tissues (n = 28), and CC cell lines (n=9) were analyzed using the Gene Expression Omnibus (GEO) dataset GDS3233, which contains the largest sample size from the GEO (The KMT2B-overexpressing CC cells (HeLa-KMT2B and C33A-KMT2B cells) were conducted by GeneChem Corporation  using a modified version of the CRISPR/dCas9 synergistic activation mediator (SAM) system http://n2t.net/addgene:48138; RRID:Addgene_48138) Table S2. Then, 1μg of plasmids were transfected into C-33A and HeLa cells via electroporation using NucleofectorTM 2b Device . The control HeLa (HeLa-SC) and C-33A (C-33A-SC) cells were transfected with scrambled sgRNA plasmids. At day 3 post electroporation, GFP positive cells were sorted using flow cytometry. The cutting efficiency was determined by T7EI assay in sorted cells. After 2 weeks of cell culture, the knockdown of KMT2B in sorted cells was confirmed by western blot analysis.KMT2B was knocked out by the CRISPR/Cas9 gene editing system to conduct the HeLa-KMT2BKO and C-33A-KMT2BKO cells. The pSpCas9(BB)-2A-GFP (PX458) was a gift from Feng Zhang  were injected into the right flanks of five-week-old female BALB/c nude mice (n = 5). These nude mice have a genetic mutation that cause a deteriorated or absent thymus, resulting in an immunecomprised phenotype due to a greatly reduced number of T lymphocytes. Tumor volumes were measured at the indicated time intervals. Tumor volume was calculated using the equation volume = 0.5 × length × width2. The mice were sacrificed when the tumor size reached approximately 1 cm3 and the tumors were excised and weighed.The animal studies were approved by the Animal Care and Use Committees of Shanghai Cancer Institute. To construct a subcutaneous CC model, HeLa-VC and HeLa-KMT2B cells (3 × 106 (in 150 μl PBS) of the HeLa-VC-luc and HeLa-KMT2B-luc cells were injected through tail vein of female BALB/c nude mice . In vivo imaging system (IVIS)  was used to monitor the development of tumors every week from day 21 after tumor cell injection. Micro-computed tomograpgy scanner  was used to confirm the bone metastases in the limbs. A mice model of CC tumor intraosseous growth was established by intra-tibia injection of 1×106 (in 20 μl PBS) HeLa-VC-luc and HeLa-KMT2B-luc cells (n = 8). After two weeks of tumor growth, IVIS was used to quantitated photon flux to monitor the tumor growth in bone marrow.HeLa cells were transduced with a lentiviral vector containing the GFP-P2A-luciferase cassette which was purchased from GeneChem Corporation  to generate HeLa cells expressing luciferase (HeLa-luc cells). Then KMT2B-overexpressing and control HeLa-luc cells (HeLa-KMT2B-luc and HeLa-VC-luc cells) was conducted by SAM system as previous described in 2.4. For the metastasis model, 3×10Western blot analysis was performed according to the procedure described previously P ≤ 0.05 and fold-change ≥ 2 or ≤ 0.5 were used to determine the differential expression of genes https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE209928).Total RNA from HeLa-VC and HeLa-KMT2B cells was isolated using TRIzol reagent  according to the manufacturer's instructions, and subjected to NovelBio lab  for RNA-seq. Briefly, cDNA library sequencing was performed using an Illumina, HiseqXTen platform. High-quality reads were aligned to the human reference genome GRCh38 NCBI) using hisat2. Gene expression was calculated from fragments per kilobase of transcript per million (FPKM) by expectation maximization (RSEM). The filter criteria of  NCBI usiTable S3. Relative mRNA expression levels of genes were analyzed by the 2(-ΔΔCt) method RNA isolation from CC cells and quantitative real-time PCR (qPCR) analysis of KMT2B and EGF genes were performed according to our previous description 7 cells in four 10 cm dishes were crosslinked in freshly prepared formaldehyde solution  and then quenched with 125 mM glycine. For sonication, pellets were resuspended into microcentrifuge tubes and then fragmented with a Bioruptor Pico  sonicator at 4 °C. Protein G Dynabeads  were added to the chromatin lysate with the antibody of H3K4me3  or KMT2B  and incubated overnight at 4 °C. ChIP-DNA was purified by a standard protocol using the QIAquick Gel Extraction Kit .ChIP assay was performed as previously described Table S3.For ChIP-qPCR experiments, Immunoprecipitated DNA was analyzed by q-PCR. The amount of DNA fragment co-precipitated with antibody was calculated and compared with the amount of the same genomic fragment in total input DNA, resulting in percentage of input. Primers for ChIP-qPCR are listed in For ChIP-seq, the ChIP assay was performed using anti-H3K4me3 antibody as described above. ChIP-seq library preparation and sequencing were performed by RiboBio . Briefly, the eluted ChIP DNA was used to construct the libraries and then sequenced on Illumina HiSeq 2500. Reads were mapped to hg19 human genome using Bowtie2 software 38. The mapped reads which needed compared were then selected for further analysis.4 cells/insert) were suspended in serum-free medium and plated in the top chamber of the non-coated culture inserts  5 cells/insert) were plated in the top chamber of the Matrigel -coated inserts for the cell invasion assay For the cell migration studies, CC cells  at a concentration of 2 × 106) were detached and transferred to microfuge tubes. The tubes were centrifuged to pellet the cells. Lysis buffer  were added to lyse cells. The cell lysate was collected and detected immediately according to the EGF ELISA kit instruction manual.The EGF levels in the cell lysate prepared from HeLa-VC or HeLa-KMT2B cells were quantitated using a human EGF ELISA kit . Briefly, cells  to generate EGF promoter luciferase reporter (pGL3-EGF). HeLa-VC cells or HeLa-KMT2B cells (1× 106) were co-transfected with pGL3-EGF (2 µg) and pRL-TK Renilla luciferase expression plasmid (2 µg) via electroporation using NucleofectorTM 2b Device (Lonza). At 48 h post-electroporation, firefly and Renilla luciferase signals were determined with a Dual Luciferase Reporter Assay Kit (Promega).The human EGF promoter regions spanning nucleotides -1022 to 214 (relative to the transcription initiation site) were amplified by PCR from HeLa cells and cloned into thet test  or the Wilcoxon rank sum test . The level of significance was set at 0.05 for all analyses.The statistical analysis was performed using GraphPad Prism  and presented as mean ± SD. The significance of the differences between groups was assessed using the student's p < 0.05, Fig. ). In parallelly, we examined the protein expressions of KMT2B by immunohistochemical staining on tissue microarray (n = 30). Consistently, the protein levels of KMT2B were remarkably increased in CC tissues compared with adjacent noncancerous tissues . As expected, two CC cell lines, namely HeLa and C-33A, exhibited significantly higher mRNA level of KMT2B than the normal cervical epithelial HcerEpic cells . Furthermore, the survival analysis based on gene expression profiling and clinical data from TCGA database indicated that the patients with high KMT2B expression have significantly shorter overall survival  and relapse-free survival  compared with those carrying low KMT2B expression . The collective results indicated a potential role of KMT2B in CC progression.Based on the microarray-based gene expression profiles (GDS3233) with the largest sample size from GEO database, we found that the KMT2B mRNA expression was remarkably increased in CC tissues (n = 28) compared with noncancerous cervical tissues (n = 24). Meanwhile, CC cell lines also had a significantly higher level of KMT2B than noncancerous cervical tissues  and Western blot , slightly promoted the proliferation in C-33A cells  but not in HeLa cells . However, KMT2B overexpression significantly increased the migration abilities of both HeLa cells  and C-33A cells  by about 35% and 63.5%, respectively. KMT2B overexpression remarkably enhanced the invasion abilities of both HeLa cells  and C-33A cells  by about 91% and 93%, respectively. We also investigated the effect of KMT2B on CC-associated angiogenesis using an in vitro tube formation assay. HUVECs were incubated with the conditioned medium collected from CC cells and the results showed that the conditioned medium from KMT2B-overexpressing HeLa and C-33A cells significantly resulted in increased capillary tube formation by about 52% and 42%, respectively , suggesting KMT2B promoted the migration, invasion, and pro-angiogenetic abilities of CC cells.To investigate the functional role of KMT2B, we overexpressed KMT2B in human CC cell line HeLa and C-33A using a CRISPR-dCas9-SAM system. The overexpression of KMT2B, which was confirmed by qRT-PCR  and C-33A cells . Between the two sgRNA which specially targeted to the KMT2B gene, sgRNA1 reduced about 72% KMT2B expression in HeLa cells and 74.5% in C-33A cells and sgRNA2 reduced about 69% and 67.5% KMT2B expression in HeLa and C-33A cells. Therefore, the knockdown of KMT2B CC cells in subsequent experiments was constructed using sgRNA1. In contrast to the effect of KMT2B overexpression, the knockdown of KMT2B significantly suppressed the migration, invasion and pro-angiogenic abilities of HeLa and C-33A cells .To further verify the role of KMT2B in regulating the malignant behaviors of CC cells, we knock down KMT2B using CRISPR/Cas9 system in HeLa . The tumors were harvested and weighed at the time of sacrifice (18 days post-inoculation) . Consistent with the results of tumor growth, a significant increase in tumor weight was observed in KMT2B overexpression group compared to control group , which was associated with markedly elevated Ki67 protein expression in KMT2B-overexpressing tumors . A significant increase in the number of blood vessels (p < 0.001) was detected in KMT2B-overexpressing tumors by immunohistochemical staining of the endothelial cells by using the marker CD31 . In contrast, the knockdown of KMT2B significantly decreased the growth rate after 15 days of tumor growth, resulting in a 43% reduction in tumor weight  at the time of sacrifice (21 days post-inoculation) . We also observed a significant decrease in the percentage of proliferating tumor cells (p < 0.01) and the number of blood vessels (p < 0.001) in KMT2B-knockdown tumors as determined by immunohistochemical staining for Ki67 and CD31 . These results indicated that KMT2B could accelerate tumor growth and promote angiogenesis in vivo.To investigate thein vivo. HeLa-VC-luc and HeLa-KMT2B-luc cells (expressing the comparable levels of luciferase) were injected into nude mice by the tail vein. The bone metastatic nodules were visualized by IVIS and further confirmed by micro-computed tomography . Interestingly, it showed that KMT2B overexpression significantly increased the incidence of bone metastasis  rather than lung metastasis (0% vs. 0%) of Hela cells (Table S4). Meanwhile, the average number of bone metastatic nodules in KMT2B-overexpressing group is remarkable greater than that of control mice (1.5 vs. 0.17) . These results suggested that KMT2B facilitated the CC cells colonization in the bone marrow. To confirm the influence of KMT2B on intraosseous growth of CC cells, we constructed a xenograft model using intra-tibia tumor cell implantation. Fourteen days after intramedullary injection of tumor cells, the animals were checked for quantitatively evaluating luciferase bioluminescence intensity by IVIS every week to monitor tumor growth. The results showed that the mice injected with HeLa-KMT2B-luc cells had a significantly higher bioluminescence intensity . The myelogenous CC xenografts in both groups were confirmed by histopathological examination . Moreover, there were significantly more Ki67 positive cells in HeLa-KMT2B group than the control group , indicating that KMT2B promoted HeLa cell growth in the bone marrow microenvironment.We next tested whether KMT2B promote CC metastasis in vivo.Collectively, these findings demonstrated that KMT2B promoted CC bone metastasis and angiogenesis Fig. ) with 176 genes significantly upregulated and 347 genes downregulated by KMT2B overexpression . The KEGG pathway enrichment analysis revealed that the most significantly enriched pathways of upregulated genes was PI3K/AKT signaling pathway . Furthermore, the levels of phosphorylated PI3K (p-PI3K) and phosphorylated AKT (p-AKT) was significantly increased in HeLa-KMT2B and C-33A-KMT2B cells, and significantly decreased in HeLa-KMT2BKO and C33A-KMT2BKO cells , indicating that KMT2B activated the PI3K/AKT signaling pathway in CC cells. Specific inhibition of PI3K activity by LY294002 markedly impeded the KMT2B-induced migration  and invasion  of HeLa cells, suggesting that the PI3K/AKT signaling pathway was required for KMT2B-mediated CC cell migration and invasion.To determine how KMT2B promotes the malignant behaviors of CC cells, we compared the whole genome mRNA expression profile of HeLa-VC and HeLa-KMT2B cells using RNA-seq. A distinct transcriptome difference was observed . Since KMT2B upregulates genes expression by catalyzing tri-methylation of lysine 4 on histone 3 at the promoter regions of target genes p < 0.001). (Table S5). There were 1063 gene promoter regions with increased H3K4me3 level , among which 26 genes showed transcriptional up-regulation by KMT2B as revealed by our RNA-seq. Then we investigated the overlapping gene set between these 24 upregulated genes and PI3K/AKT signaling pathway associated genes. We found that only one target gene EGF in the set, which not only showed increased H3K4me3 peak at promoter region but also be related to PI3K/AKT signaling pathway . To confirm the ChIP-seq results, we measured the levels of H3K4me3 at EGF promoter region using anti-H3K4me3 antibody in ChIP-qPCR experiment. In HeLa-KMT2B cells, increased H3K4me3 levels were confirmed in the promoter region of EGF . Then, ChIP-qPCR analysis was performed using anti-KMT2B antibody to determine whether KMT2B bound to the EGF promoter region. The results showed that KMT2B-overexpression resulted in a significant increase of KMT2B binding at EGF promoter region . Also, the results from luciferase report assay showed that KMT2B overexpression significantly enhanced the luciferase activity driven by EGF promoter fragment . These findings indicated that KMT2B physically bound to EGF promoter region and enhanced the transcriptional activity of EGF in CC cells. Further, to evaluated whether KMT2B upregulated the expression of EGF, qPCR assay was performed to detect the mRNA level of EGF in HeLa-VC and HeLa-KMT2B cells. Consistent with RNA-seq results, the qPCR result showed that KMT2B overexpression markedly promoted the transcription of EGF . Moreover, ELISA assay showed that KMT2B significantly promoted EGF protein level in HeLa-KMT2B cells . These data demonstrated that EGF was a key target gene of KMT2B and was upregulated in KMT2B overexpression HeLa cells.KMT2B is responsible for tri-methylation of H3K4 Fig. ), suggesting that KMT2B activated PI3K/AKT signal pathway through upregulated the expression of EGF. To evaluate the effect of EGF on KMT2B-mediated of CC cells migration and invasion, we treated HeLa-VC and HeLa-KMT2B cells with gefitinib. Transwell migration and invasion assays showed that gefitinib significantly hindered the ability of migration  and invasion  in HeLa-KMT2B cells. The result showed that KMT2B promoted CC cells migration and invasion through EGF/PI3K/AKT axis. In addition, we also treated HUVECs with gefitinib to investigate whether EGF also mediated the KMT2B-ibduced enhancement of tube formation. It showed that gefitinib remarkably abolished the KMT2B-induced HUVECs tube formation capacity .EGF is an upstream regulator of PI3K/AKT signaling pathway and plays an important role in tumor metastasis Collectively, KMT2B promoted the CC cells migration, invasion and pro-angiogenic effect by upregulated the expression of EGF.In recent years, KMT2B has attracted increasing attention because of its important role in depositing H3K4me3 at promoters that active gene expression, rationalizing its widespread involvement in various pathological processes Most previous studies have described KMT2B as a positive regulator of cell proliferation in colon cancer and breast cancer The present study also provided evidence demonstrating KMT2B as a novel pro-angiogenic factor in CC. Solid tumor growth can be separated into two stages, avascular and vascular. Avascular is the early stage of tumor growth, in which most solid tumors seem capable of living by simple diffusion until they reach a diameter of a few millimeter We identified EGF for the first time as a target gene which is regulated by KMT2B. KMT2B is responsible for the deposition of H3K4me3 at gene promoters to upregulate the target genes transcription. For instance, in non-small cell lung cancers, KMT2B increased the c-Myc transcription in vitro and in vivo by upregulated EGF . These results indicated a dual role of KMT2B in CC progression and suggested that KMT2B and EGF has potential therapeutic value in the treatment of CC.In conclusion, the present study demonstrated that KMT2B is upregulated in CC tissues or cells and associated with poor prognosis. It also showed that KMT2B promoted CC cells metastasis and angiogenesis both Supplementary figure and tables.Click here for additional data file.


Scientific Reportshttps://doi.org/10.1038/srep40464, published online 13 January 2017Correction to: This Article contains an error in Figure 3(C) and (E) and 5(E) where original biological duplicate data results were incorrect.The correct Figure This change does not affect the conclusions of the Article.


L target M2 protein, which was displayed on the yeast surface. The scFv/VL proteins were obtained with high yield and high purity through soluble expression in E. coli BL21 (DE3) pLysE strains. A single-domain VL-M2-specific antibody, NVLM10, exhibited the highest binding affinity to influenza virions and was engineered into a bivalent format (NVL2M10) to improve antigen binding. Both antibodies inhibited virus replication in a dose-dependent manner, determined using plaque reduction- and immunocytochemistry assays. Furthermore, bivalent anti-M2 single-domain VL antibodies significantly reduced the plaque number and viral HA protein intensity as well as viral genome (HA and NP) compared to the monovalent single-domain VL antibodies. This suggests that mono- or bivalent single-domain VL antibodies can exhibit neutralizing activity against influenza virus A, as determined through binding to virus particle activity.Flu disease, with high mortality and morbidity, is caused by the influenza virus. Influenza infections are most effectively prevented through vaccination, but it requires annual reformulation due to the antigenic shift or drift of hemagglutinin and neuraminidase proteins. Increasing resistance to available anti-influenza drugs was also recently reported. The M2 surface protein of the influenza virus is an attractive target for universal vaccine development as it is highly conserved and multifunctional throughout the viral life cycle. This study aimed to discover a single-chain variable fragment (scFv) targeting the M2 protein of influenza A H1N1/PR8, showing neutralizing activity through plaque inhibition in virus replication. Several candidates were isolated using bio-panning, including scFv and single-domain V Orthomyxoviridae family are segmented negative-stranded RNA viruses. There are eight viral RNA gene segments encoded all viral proteins [Human seasonal influenza viruses cause approximately 3 to 5 million severe flu cases and 290,000 to 650,000 annual deaths worldwide . The CDCproteins , 5. Vaccproteins , 8, promH or VL Abs. These smaller entities can be produced in bacteria with high solubility and retain the antigen-binding properties of parental mAbs, resulting in numerous medical and diagnostic applications [Biswas et al. reviewed valuable, broadly neutralizing monoclonal antibodies (bnAbs) against the influenza virus, including modifications using variable domains of heavy chain only antibodies (VHH) and multidomain antibodies [Antibody-based therapy is a promising alternative for the development of a universal influenza vaccine and novel antiviral drugs. Monoclonal antibodies (mAbs) can be engineered into smaller sizes in many formats, such as antigen binding fragments (Fabs), single-chain variable fragments consisting of variable regions of heavy and light chains connected by a flexible peptide linker, and single-domain Vications –11. Biswtibodies .in vitro and reduced lung virus titers in mice [The M2 protein is also a potential target for monoclonal antibody-based treatment of influenza . M2, whi in mice , 26. Var in mice –32. The  in mice , 33–35.  in mice .L specific to the M2 protein of influenza H1N1/PR8 strains, which were expressed on a yeast surface display (YSD) system. The candidates exhibited a binding affinity for influenza virions. Single-domain M2-VL (NVLM10), which showed the highest affinity, was engineered into a bivalent format (NVL2M10) that enhanced the binding affinity to the influenza particles. The binding strength of bivalent M2-VL (NVL2M10) to influenza virus particles resulted in a dose-dependent reduction in plaque number, indicating neutralizing activity.In this study, we used bio-panning method to isolate several candidates from human scFv-Tomlinson I+J libraries, including the single-chain variable fragment (scFv) and a single-domain VNheI and BamHI  sites of a pCTCON plasmid (ampicillin-resistant) for YSD (M2::pCTCON). The M2::pCTCON plasmid was transformed into Saccharomyces cerevisiae EBY100 competent cells, prepared according to the Clontech manual  as previously described [600 and made competent by chemical solution . The antigen M2::pCTCON plasmids were transformed to the competent yeast in ice-cold 1 M Sorbitol/1 mM CaCl2 using electroporation . The electroporated yeast cells were added to yeast-peptone-glucose media containing 1 M sorbitol before plating on selective SD media  without tryptophan supplementation and incubated at 28°C for 3 days. The yeast was cultured in the selective media and induced to express in SGCAA media containing 2% galactose.To optimize antigen expression in yeast, one N-glycosylation site of the M2 protein sequence of influenza virus A H1N1/PR8 (288 bp) (Accession number AY768951.1) was changed from asparagine (Asn-N) to glutamine (Gln-Q) (N20Q) before synthesis. The DNA fragment was amplified using PCR and cloned between the escribed . BrieflyThe M2 antigen expressed on the yeast surface (M2::YSD) was analyzed using western blotting. Briefly, yeast cells were treated with a protein sample loading buffer and denatured by heating. The supernatants were loaded into 12% polyacrylamide gel electrophoresis (PAGE) wells and transferred to a nitrocellulose membrane. The membranes were blocked in 5% skim milk and Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBS-T). Influenza A M2 polyclonal antibodies  were used at a 1:5000 dilution as primary antibodies and detected using goat anti-rabbit IgG-HRP conjugated secondary antibodies .M2::YSD cells were resuspended in PBS to confirm expression using enzyme-linked immunosorbent assay (ELISA). A total of 100 μL of the yeast suspension at an OD of range 0.4–0.6 was coated onto a maxibinding immunoplate  at 4°C and incubated in a humid box overnight. EBY100 yeast (without pCTCON plasmid) was used as a negative control under the same conditions. Each sample was analyzed in triplicate. The plate was washed with TBS-T and blocked with 3% BSA in TBS-T buffer at room temperature (RT) for 1 h. The plate was then incubated with primary influenza A M2 polyclonal antibodies  at a 1:3000 dilution for 2 h at RT (25°C). The wells were rinsed with TBS-T five times before adding goat anti-rabbit IgG-HRP conjugated antibodies  for 2 h at RT. After washing five more times, TMB substrate solutions  were added for 15 min and the reactions were stopped with 1 M sulfuric acid. The absorbance of the plate was measured at 450 nm using a microplate spectrophotometer. Each sample was analyzed in triplicate and the expressions were confirmed in every batch of experiment with the same method. GraphPad Prism 8.0 software  was used to analyze the results.E. coli cells (tetracycline-resistant). The phage libraries were infected with XL-1 blue E. coli cells and amplified in 2TY media with an M13K07 helper phage. The phages were precipitated using 20% PEG6000 and 2.5 M NaCl solution. Phage titers were determined using kanamycin-resistant colony forming units (CFUs) in XL-1 blue. Bio-panning methods were applied to screen M2-specific phage scFvs (M2::scFv), as previously described [600 of 0.6 were coated onto a 96-well Maxibinding immunoplate  and incubated overnight in a humid box at 4°C. The plates were washed three times and blocked with a blocking buffer (3% BSA in TBS-T) for 2 h at RT. Approximately 1 × 109 CFU/mL of scFv phage suspended in the blocking buffer was added to blank samples (PBS) and incubated for 2 h at RT. The non-binding scFv phages in the supernatant were transferred to a negative sample plate and incubated overnight at 4°C. The non-binding supernatant phages from negative plates were transferred to M2::YSD-coated plates (positive) for 2 h at RT. The plate was washed five times with TBS-T and dried thoroughly. Elution buffer (100 μL of 100 mM triethylamine solution) was added to each well to elute the M2::scFv phages. The eluted phages were neutralized with 1 M Tris-HCl buffer (pH 7.4) and used for the next round of bio-panning. The M2 -specific scFv selection was completed after three rounds of bio-panning.The human single-fold scFv libraries, obtained from Tomlison I+J, were provided by Prof. Myung-Hee Kwon . The libraries contain over 100 million different scFv fragments cloned in a phagemid vector pIT2, fused with His and Myc tag with ampicillin resistance, and were grown in XL1 escribed , 37. Bri600 of 0.6 for 30 min at 37°C, without shaking, and spread on LB agar plates supplemented with tetracycline (25 μg/mL), ampicillin (100 μg/mL), and 1% glucose. The phages were incubated overnight at 37°C. Single XL-blue colonies were randomly selected from each bio-panning round and grown in 96-well plates . The colonies were cultured in 2TY growth media containing 25 μg/mL tetracycline, 50 μg/mL ampicillin, and 1% glucose until an OD600 of 0.6 was attained. The M13K07 helper phage was then added to express scFvs. The scFvs were displayed on the phage by reducing glucose concentration to 0.1% in 2TY growth media, after culturing at 30°C overnight. The collected supernatant was used as a primary antibody for the phage ELISA. Each random phage clone bound to the M2::YSD was detected using anti-M13 antibodies HRP-conjugated in a 1:1000 blocking buffer dilution  for 1 h at RT. The phage and yeast ELISA were performed according to the same protocol in following steps.The M2::scFv selected phages were infected with XL-1 blue at an OD5’-CAGGAAACAGCTATGAC-3’, and pHEN, 5’-CTATGCGGCCCCATTCA-3’, under the following conditions: 95°C for 5 min, 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 2 min, and then 72°C for 10 min using 2X premix . The PCR products were run using 1% agarose gel electrophoresis. The plasmids with bands of approximately 900 bp or 400 bp were selected for sequencing by Macrogen (Republic of Korea). Each candidate sequence was analyzed using the IgBlast tool from NCBI [H and VL.Plasmids from each clone were extracted to detect and identify the sequences of M2-specific candidates. The individual clones of selected candidates were verified using PCR and the recommended primer pair, LMB3, rom NCBI , 39 to iH fragment at FR2. Therefore, to be soluble expression in BL21, site-directed mutagenesis was performed to introduce a stop codon to the glutamate residue using the Q5 site-directed mutagenesis kit , according to the manufacturer’s instructions. Two different single-domain VL only candidates, NVLM9 (324 bp) and NVLM10 (324 bp), were cloned in a pIg20 vector fused with protein A and PhoA leader signal peptides. Bivalent fragments (NVL2M10) were composed of two identical single domains VL of NVLM10, attached by a flexible glycine-serine linker, (G4S)3. The bivalent fragment sequences (693 bp) with the XmaI and NcoI enzyme  sites were synthesized and introduced into the pIg20 vector. The expressed plasmids were transformed into BL21 (DE3 pLysE) to produce soluble scFv. Protein expression was induced by adding 1 mM IPTG at an OD600 of 0.8. The soluble proteins in cell supernatants were collected and loaded into open columns containing Capto L resins  for NscFvM8 construct, and IgG Sepharose 6 resins . The eluted protein purity was confirmed by loading on SDS-PAGE with coomassie blue staining, or western blotting with primary antibodies, anti-6X His tag at a 1:3000 dilution . The ImageJ software was used to determine the purity of the purified protein.One full-length scFv candidate in the phagemid pIT2 vector, NscFvM8 (720 bp), contained a stop codon in the V5 plaque-forming unit PFU/well) in PBS buffer were coated onto a Maxibinding Immunoplate  and incubated at 4°C overnight. The plate was washed five times with TBS-T and blocked with blocking buffer (5% skim milk in TBS-T buffer) for 2 h at RT. Then, 100 μL of the indicated proteins in a 2X serial dilution (starting from the 100 μg/well) containing blocking buffer was added and incubated at 37°C for 1 h. Polyclonal rabbit anti-HA antibodies  were used as a control to ensure that the virions were present and intact for the assay. The proteins were detected using anti-6X His tag antibodies at a 1:3000 dilution  for 2 h at RT. After washing, 100 μL of a 1:5000 goat anti-mouse IgG H&L (HRP)  dilution for anti-6X His tag, and goat anti-rabbit IgG-HRP conjugated antibodies  for anti-HA antibodies were added and incubated for 2 h at RT. The TMB substrate and 1 M sulfuric acid were added last.Virus particles  to full confluence. H1N1/PR8 viruses with multiplicity of infection (MOI) of 0.1 were incubated with serial dilutions of NVLM10 and NVL2M10  for 24 h at 37°C. The mixture was then diluted to achieve countable plaque dilutions (approximated 0–100 PFU/well). One milliliter of the virus-protein mixture was inoculated into PBS-washed MDCK cells in duplicate. Following 1 h incubation at 37°C, the mixture was withdrawn from the cell and overlaid with 1% Seaplaque agarose containing 1 μg/mL TPCK-trypsin in DMEM. Plates were incubated at 37°C for 3 days to allow plaque formation. The cells were then fixed with 4% paraformaldehyde for 1 h and stained with 0.5% crystal violet for 30 min. Plaque assay were conducted three times with different batches of the protein expression. Plaques were counted and expressed as the percentage of PFUs.MDCK cells grown in complete Eagle’s minimal essential medium , supplemented with 10% fetal bovine serum  and 0.1% antibiotic-antimycotic , were seeded at 1 × 10in vitro. H1N1/PR8 (MOI 0.1) was treated with 10 μg NVLM10 and NVL2M10 for 24 h, followed by infection with MDCK cells grown in 8-well chamber slides . Following incubation for 1 h at 37°C, the inoculum was removed and the cells were cultured in MEM containing TPCK-Trypsin (1 μg/mL) and BSA (0.2%) for 24 h. The cells were washed with PBS and fixed for 15 min using ice-cold methanol. Intracellular Staining Perm Wash Buffer  was added for 15 min to permeabilize the cells, followed by blocking with 1% BSA and glycine in PBST buffer for 1 h. The cells were treated with polyclonal rabbit anti-HA antibodies at a 1:1000 dilution  for 24 h at 4°C. After washing rigorously, the cells were incubated with goat anti-rabbit IgG TRITC secondary antibodies  for 1 h at 25°C. Vectashield Antifade mounting medium with DAPI  was added to the cells and visualized using a Zeiss LSM 700 confocal microscope. The viral protein signal was converted to relative intensity percentages using the histogram function in the Zen 3.1 (blue edition) program, by normalizing the sum of HA protein intensity (red color) to the DAPI signal (blue color) and using untreated samples as a calibrator (100%). The relative intensity percentage was measured based on the whole cells presented in the image.ICC was conducted to assess the influenza HA protein in the presence of NVLM10 and NVL2M10 4 PFU/mL was neutralized by 10 μg for 24 h at 37°C. The mixture was then incubated to MDCK cells (MOI 0.1) for 1 h at 37°C. After removal of protein/virus complexes, the cells were cultured with MEM-free media supplemented with 0.2% BSA and TPCK-treated trypsin (1 μg/ml). The cells were harvested after 24 hpi virus challenge and stored at -20°C for further RNA extraction.Influenza virus A H1N1/PR8 at 2x10HA and NP) were amplified using the indicated primers  according to the manufacturer’s instructions. The RNA concentration was determined using a spectrophotometer and diluted in distilled water (DW) at final concentration 10 ng/ μL. One-step quantitative real time PCR was performed using Accupower GreeenStar RT-qPCR Premix and Master mix  and Rotor-Gene Q system (Qiagen) with 50 ng of total RNA template. Data were analyzed using Rotor-Gene Q series software version 2.3.1 . The influenza viral genes . This stop codon likely does not interfere with the phage binding affinity to M2 antigen. Two clones (M9 and M10) yielded a band at 400 bp and belonged to the VL sequences. The two single-domain VLs exhibited highly conserved sequences in the framework region but differed in the CDR2 and CDR3 regions by only a few amino acids (YSD systems were used to express antigens for bio-panning. M2 of the influenza virus A H1N1/PR8 gene sequence was synthesized (288 bp) (Accession number AY768951.1) with a change at one N-glycosylation site (N20Q) (asparagine to glutamine). The DNA fragment was amplified using PCR and introduced into a pCTCON plasmid vector for YSD . M2 antit 400 bp . The clono acids .L clones, NVLM9 and NVLM10, were constructed with a pIg20 vector containing PhoA signal peptide and protein A to ease expression and purification. For the NscFvM8 candidate, site-directed mutagenesis changed the stop codon to a glutamate residue, similar to the phagemid vector that contained a stop codon. The NscFvM8 sequences fused with His and Myc tag at the C-terminal in the pIT2 vector were expressed in the E. coli BL21 (DE3) pLysE strain  of 2X lower than that of bivalent NVL2M10 (D) value of NVL2M10 (500 μg/mL) was not significantly different from that of NVLM10 (580 μg/mL).As expected, the bivalent protein (250 μg/mL) demonstrated a high affinity for different densities of the virus H1N1/PR8 coated through the virion ELISA method. The virion ELISA sensitivity was approximately 1.25 × 10 protein . Virion  protein . Single- NVL2M10 . Howeverin vitro. A plaque inhibition assay was performed to identify whether two different M2-specific VLs (NVLM10 and NVL2M10) each neutralized virus infection. Purified NVLM10 and NVL2M10 at a 10X serial dilution were preincubated with H1N1/PR8 viruses. MDCK cells were infected with the protein/virus mixtures to perform the plaque reduction assay. The plaque numbers were visibly reduced in the presence of NVLM10 and NVL2M10 in a dose-dependent manner . NVL2M10 inhibited virus growth, which was confirmed by the absence of a red signal. However, HA proteins were detected at a lower intensity with 10 μg NVLM10 treatment . The relar ratio . These rH or VL) have the simplest forms that can retain antigen binding among Fab and scFv Abs. Approximately 61 sdAbs target the respective antigens with potential therapeutic applications [in vitro and in animal models [HH) reportedly neutralized the influenza A virus by specifically binding to the M2 protein [L Abs, NVLM10, and its engineered bivalent molecule, NVL2M10, exhibited neutralization activity against H1N1/PR8 with plaque and viral protein, viral genome expression reductions in vitro by binding to influenza virions.Single-domain antibodies (sdAbs) , consisting of VH and VL domains, was expected to show a higher affinity to virus particles than single-domain VL (NVLM9 and NVLM10). However, the virion ELISA indicated that single-domain NVLM10 had the highest affinity. Moreover, the NVLM9 single-domain did not exhibit binding affinity to the influenza virus particles  were reported with rheumatoid arthritis treatment in vivo, compared to monovalent molecules [L could enhance the antigen-binding affinity, at least 2-fold, compared to the monomer form. The bivalent protein, NVL2M10, was produced by connecting two identical single-domain VLs (M10) through a flexible linker that is commonly used in scFv structures. The protein was obtained in a soluble form with high yield and exhibited binding affinities to different virus particle concentrations  is the maximum binding affinity of the antibody to the constant antigen. The equilibrium dissociation constant (KD) is defined by the antibody concentration that exhibits 50% antigen binding affinity [max values and lower KD values. The Bmax value of NVL2M10 at OD450  was 2X greater than that of NVLM10, while the KD value of NVL2M10 was slightly lower than that of NVLM10 , 39.5 nM for VhHs [D values, implying that longer timer or higher temperatures were required to enable tight binding to the influenza virus particles. Furthermore, the presence of a small proportion of M2 proteins, 16–67 molecules per 500 HA molecules per virion [in vitro . The bivalent single-domain NVL2M10 was designed using NVLM10 molecules to improve binding efficiency, resulting in a 2X higher Bmax value  , 50. Furarticles . The dif binding . Becauseolecules . Therefotrations . Compareaffinity . Therefof NVLM10 . While t is 1 nM , the KD for VhHs , or lowefor VhHs . Unfortur virion  and the tro Figs  and 7. Tax value . NotablyL targeting M2 protein. However, the viral infection mechanisms of these candidates require further investigation. Further studies on the improvement of virion binding efficiency to enhance neutralizing activity are also necessary.Taken together, our findings potentially provide an additional approach for the development of neutralizing antibodies against influenza viruses using the single-domain VS1 Raw images(PDF)Click here for additional data file.


Human post-mortem anterior cingulate (AC) and corpus callosum (CC) from control subjects (CN), n = 6; age 81–90 LB = 0 and PD, n = 6, age 77–89, LB = III–IV were sectioned (10 μm slices). Brain slices were immunostained with anti-ubiquitin for LB (UIHC) and analyzed using QuPath for percent anti-ubiquitin per unit area (μm2). Adjacent brain slices were incubated with [18F]FAZIN3 and cortical layers I–III, IV–VI and CC (white matter) regions were quantified for the binding of [18F]FAZIN3. UIHC was correlated with [18F]FAZIN3 binding. All PD brains were positively UIHC stained and confirmed presence of LB. Outer cortical layers (I–III) of PD AC had 21% UIHC while inner layers (IV–VI) had >75% UIHC. In the CN brains LB were absent (<1% UIHC). Increased [18F]FAZIN3 binding to MAO-A in AC was observed in all PD subjects. [18F]FAZIN3 ratio in PD was AC/CC = 3.57 while in CN subjects it was AC/CC = 2.24. Increases in UIHC μm2 correlated with [18F]FAZIN3 binding to MAO-A in DLU/mm2. Increased [18F]FAZIN3 binding to MAO-A in PD is a potential novel “hot spot” PET imaging approach.Lewy bodies (LB) play a neuropathological role in Parkinson’s disease (PD). Our goal was to evaluate LB using anti-ubiquitin immunohistochemistry (UIHC) and find correlations with monoamine oxidase-A (MAO-A) using imaging agent, [ To the The sporadic nature of PD development can make studying specific pathologies challenging ,22. Aggr18F]FAZIN3.In anterior cingulate (AC), LB accumulation may be used to predict cognitive deficits in PD . These LR)-deprenyl were purchased from Research Biochemicals . All other chemicals were obtained commercially from Sigma Aldrich, St. Louis, MO, USA. All solvents used were provided by Fisher Scientific. Fluorine-18 autoradiographic studies were carried out by exposing tissue samples on storage phosphor screens . The apposed phosphor screens were read and analyzed by OptiQuant acquisition and analysis program of the Cyclone Storage Phosphor System .Clorgyline and  were used. Semi-quantitative scores of none, sparse, moderate and frequent were converted to numerical values 0–3 for each brain region. Numerical values 0–3 for each region were summed to provide total LB. All PD brains had a primary diagnosis of PD with some clinical history of dementia. Adjacent slices were used for immunostaining with anti-ubiquitin and anti-α-synuclein. All postmortem human brain studies were approved by the Institutional Biosafety Committee of University of California, Irvine.Human postmortem brain tissue samples were obtained from Banner Sun Health Research Institute (BHRI), Sun City, AZ brain tissue repository for in vitro experiments. Age and gender matched PD brain and cognitively normal (CN) brain tissue samples were selected for end-stage pathology ,28. HumaImmunostaining of all brain sections were carried out by University of California-Irvine, Pathology services using Ventana BenchMark Ultra protocols. Neighboring slices were immunostained for Ubiquitin  and α-synuclein . All IHC stained slides were scanned using the Ventana Roche instrumentation and analyzed using QuPath. Adjacent slices were also immunostained for Aβ plaques and total Tau. All IHC stained slides were scanned using the Ventana Roche instrumentation and analyzed using QuPath [18F]FAZIN3 was carried out in house. Fluorine-18 in H218O from PETNET was used to react with the precursor, AZIN3 Tosylate (1–2 mg in 0.5 mL of anhydrous acetonitrile) for 15 to 30 min at 96 °C. Product was purified in a reverse-phase HPLC C18 Econosil column 250 mm × 10 mm  with 60% acetonitrile: 40% water containing 0.1% triethylamine with a flow rate of 2.5 mL/min. Retention time of [18F]FAZIN3 was found to be 10.5 min. Batches of ~370 MBq [18F]FAZIN3 were produced in specific activity of >74 TBq/mmol, and was stable for in vitro studies. Solvents from the HPLC purified fraction were removed and [18F]FAZIN3 was taken in 5 mL of 0.9% sterile saline and passed through a 0.22 micron Millipore filter into a sterile dose vial.The radiosynthesis of [18F]fluoroethyl harmol ([18F]FEH) was produced. N-BOC-harmol-O-ethyl tosylate precursor (prepared in-house) was radiolabeled using [18F]fluoride in acetonitrile containing Kryptofix/K2CO3. The N-BOC-[18F]FEH was purified by HPLC  and purity was confirmed by radio TLC. Deprotection of N-BOC-[18F]FEH was carried out using trifluoroacetic acid in CH2Cl2 to provide [18F]FEH in modest radiochemical yields. [18F]FEH was taken in 5 mL of 0.9% sterile saline and passed through a 0.22 micron Millipore filter into a sterile dose vial.Using a two-step procedure, [18F]FAZIN3 (111 kBq/cc) was added to all the chambers and incubated for 60 min. After incubation, slides were washed twice (each wash lasting 3 min) and were rinsed in cold deionized water, air dried and exposed to a phosphor screen for 24 h. Using the Optiquant program , regions of interest were drawn in cortical layers I–III, IV–VI and white matter regions and digital light units/mm2 (DLU/mm2) were used to quantify the percentage binding of [18F]FAZIN3. Drug challenge studies in the presence of clorgyline or deprenyl were also analyzed in the same manner.Brain slices were placed in six separate incubation chambers  and were allowed to thaw from −80 °C to ambient temperature for 10 min. Subsequently they were pre-incubated in PBS (pH 7.4) buffer at 25 °C for 10 min. Fresh PBS buffer (pH 7.4) containing [18F]FEH at 25 °C for 1 h. Nonspecific binding was measured in the presence of 10 µM clorgyline. After incubation, slides were washed twice (each wash lasting one minute) with ice-cold buffer. Slides were then quickly dipped in cold deionized water, air dried and exposed to a phosphor screen for 24 h. The amount of binding was evaluated in digital light units (DLU/mm2).Similarly slides were incubated in the buffer at room temperature for 10 min and then in buffer with 111 kBq/cc FAZIN3 autoradiographs using Optiquant. Measurements of image intensity in DLU/mm2 of AC cortical layers I–III, IV–VI and CC regions were obtained. These correspond to [18F]FAZIN3 binding to MAO-A. Significant differences between groups were confirmed by Student’s t-test (p < 0.05). Correlation of UIHC area (μm2) with [18F]FAZIN3 for MAO-A area (in mm2) was carried out.Using QuPath, LB annotations were drawn on UIHC images. QuPath was then used to train a machine learning classifier for LB for each of the brain slices. Measurements of the area of UIHC were obtained for AC (gray matter) and CC (white matter) regions of interest (ROIs). These measurements corresponded to the presence of LB. The UIHC images of each brain slice provided percent anti-ubiquitin area  were determined using Microsoft Excel 16. Statistical power was determined with Student’s t test and 18F]FAZIN3 and [18F]FEH (All brain samples (6 PD and 6 CN) were immunostained with anti-ubiquitin (UIHC) for Lewy bodies. All PD brains were positively stained with UIHC A, while [18F]FEH F.18F]FAZIN3 in CN and PD subjects are shown in 18F]FAZIN3 in all subjects. [18F]FAZIN3 exhibited binding across the cortical layers in all subjects. 18F]FAZIN3 binding in 18F]FAZIN3 binding in 18F]FAZIN3 binding between the CN subjects (18F]FAZIN3 binding in the GM compared to the PD subjects (2 (DLU/mm2) were used to quantify [18F]FAZIN3 binding. 18F]FAZIN3 binding of all 6 PD and CN subjects in GM and WM regions. Ratio of PD GM/CN GM = 2.78, indicating >200% increase in PD, while average GM/WM = 2.18 for all CN subjects and average GM/WM = 3.15 for all PD subjects, suggesting a 45% increase in PD using ratios, which is more relevant to in vivo imaging where typically a reference region for quantification. All PD subjects showed lack of [18F]flotaza binding [125I]IPPI binding FEH, a known fluorine-18 analog of [11C]Harmine MAO-A radiotracer [18F]FEH in PD brains was observed -deprenyl, a known irreversible MAO-B inhibitor. Shown in 18F]FAZIN3 in these PD subjects is shown in 18F]FAZIN3 in the cortical regions is evident. Clorgyline at 1 μM concentration displaced over 80–90% of [18F]FAZIN3 binding (18F]FAZIN3 to MAO-A. In the presence of 1 μM (R)-deprenyl, there was little effect on the binding of [18F]FAZIN3, as seen in R)-deprenyl were observed in the CN brains, confirming that [18F]FAZIN3 binds to MAO-A in PD  in CN and PD brain regions of WM and GM . These mitochondria are labeled by [18F]FAZIN3 and because of their localized higher concentration in PD (compared to CN subjects), higher MAO-A binding by [18F]FAZIN3 is observed.Using QuPath, the UIHC stained brain sections were analyzed to provide UIHC μmM and GM A. CorrelLewy bodies (LB) are present in the cell bodies in various brain regions in PD patients ,32. A gr11C]harmine was used to study depression [11C]harmine was measured in several brain regions, including anterior cingulate. This was considered indicative of an increase in MAO-A (either more MAO-A per mitochondrion or more mitochondria) causing a decrease in monoaminergic neurotransmitters, such as dopamine, serotonin and norepinephrine. A molecular mechanism causing an increase in MAO-A in affective disorders is not clearly understood. PET imaging studies of MAO-A in PD have not been carried out.Reversible MAO-A PET radiotracer [pression . In a PE18F]FEH has only been used in small animal imaging. Rapid metabolism and poor brain penetration may have limited further development of [18F]FEH analogs. The lack of reversible fluorine-18 PET imaging radiotracers for MAO-A has been an impediment and there is now ongoing effort to develop such MAO-A PET imaging agents [18F]FAZIN3 as a new PET radiotracer that binds reversibly and selectively to MAO-A.The harmine-related fluorine-18 analog [18F]FAZIN3 LB in the anterior cingulate of well characterized PD subjects and normal controls -deprenyl, a MAO-B irreversible inhibitor [18F]FAZIN3 for MAO-A (by virtue of being displaced >90% by clorgyline). Because of a lack of effect of (R)-deprenyl on [18F]FAZIN3, it may be safely inferred that the increased binding of [18F]FAZIN3 in the PD subjects are elevations in MAO-A binding. The findings of [18F]FAZIN3 in the PD patient shown in 18F]FEH which demonstrated binding in the anterior cingulate of PD brain of the same patient .Our results suggest that MAO-A imaging is a potential surrogate biomarker for PD and LB. Several PET imaging probes have been used for PD that measure loss of monoaminergic targets and are thus “cold spot” imaging agents . Increas