Lactobacillus-depleted CST IV had ~ 90% dominance in participants with high-grade squamous intraepithelial lesions and high-risk HPV. The increasing concentration of pro-inflammatory cytokines was associated with a decrease in L. crispatus. In contrast, dysbiosis-associated bacteria such as Gardnerella, Prevotella, Atopobium concomitantly increased with pro-inflammatory cytokines. Our study highlights that the cervical microbiota of Hispanics living in Puerto Rico is composed mostly of diverse CST profiles with decreased Lactobacillus and is associated with a higher pro-inflammatory environment. The joint host-microbe interaction analyses via cytokine and microbiota profiling have very good translational potential.Cervical cancer (CC) is women’s fourth most common cancer worldwide. A worrying increase in CC rates in Hispanics suggests that besides Human papillomavirus infections, there may be other cofactors included in the epithelial microenvironment that could play a role in promoting the disease. We hypothesized that the cervical microbiome and the epithelial microenvironment favoring inflammation is conducive to disease progression in a group of Hispanics attending gynecology clinics in Puerto Rico. Few studies have focused on the joint microbiota and cytokine profile response in Hispanics outside the US, especially regarding the development of precancerous lesions. We aimed to investigate the relationship between the cervicovaginal microbiome and inflammation in Hispanic women living in PR while considering cervical dysplasia and HPV genotype risk. Cervical samples collected from 91 participants coming to gynecology clinics in San Juan, underwent 16S rRNA genes (V4 region) profiling, and cytokines were measured using Luminex MAGPIX technology. Cytokines were grouped as inflammatory , anti-inflammatory , and traffic-associated . They were related to microbes via an inflammation scoring index based on the quartile and tercile distribution of the cytokine’s concentration. We found significant differences in the diversity and composition of the microbiota according to HPV type according to carcinogenic risk, cervical disease, and cytokine abundance. Community State Types (CSTs) represents a profile of microbial communities observed within the vaginal microbiome ecological niche, and  Cervical cancer (CC) is the 4th most common cancer among women and the 4th leading cause of cancer death in females worldwide . AlthougPersistent infection with oncogenic Human Papillomavirus (HPV) is the most important cause of CC. HPV infections range from 90–100% prevalence in cervical abnormalities, from high-grade squamous intraepithelial lesion (HGSIL) to cancer , where tLactobacillus species. This concept was introduced in 2011 by Ravel et al. to categorize vaginal microbial communities among reproductive-age women [L. crispatus (CST-I), CST-II by L. gasseri, CST-III by L. iners, CST-V by L. jensenii (CST-V), or non-Lactobacillus dominated and highly diverse profiles such as CST-IV. Furthermore, CST-IV is often characterized by three sub-CSTs: 1) CST IV-A has a high relative abundance of Candidatus Lachnocurva vaginae and a moderate relative abundance of G. vaginalis and Atopobium vaginae, 2) CST IV-B also has a moderate relative abundance of G. vaginalis and Atopobium vaginae, while 3) CST IV-C has low relative abundance of Lactobacillus spp., as well as low levels of G. vaginalis, A. vaginae, and Ca. L. vaginae with high facultative and strictly anaerobic bacteria [Lactobacillus dominates the cervicovaginal microbiome of Caucasians, Hispanic and black women have higher diversity and reduced Lactobacillus populations [Atopobium vaginae and Gardnerella vaginalis [Women of different ethnic groups differ in their vaginal microbiome signatures . The cerge women . CST-I rbacteria . While Lulations . A previaginalis ,20. Howeaginalis –24.Lactobacillus. To test this hypothesis, we investigated the relationship between the cervicovaginal microbiome with cervical neoplasia and HPV infections and characterized the host immune profiles through profiling cytokine expression analyses from cervical lavages.We hypothesized that there would be changes in the cervical cytokine responses and the microbiota associated both with cervical disease and HPV carcinogenic risk types, and that markers of inflammation would be associated with a decrease in This study provides insight into specific bacterial taxonomic changes associated to pro-inflammatory states and cervical dysplasia and represents the first effort to combine microbiome and cytokine profiling in Hispanics living in Puerto Rico.Females seeking gynecologic care at the University of Puerto Rico and San Juan City Hospital clinics were enrolled in our study. Only women who did not meet the exclusion criteria were recruited and evaluated. All women were HIV-negative. Exclusion criteria included: 1) Active urinary infections, 2) history of common urinary incontinence, 3) sexually transmitted diseases (STDs), 4) antibiotics taken in the prior month, 5) candidiasis, 6) being less than 21 and more than sixty years old, and 7) treatment for or suspicion of prior toxic shock syndrome. All subjects were informed of the study’s sampling procedure, risks, and benefits with written informed consent and a verbal explanation of the document. A detailed questionnaire was administered. Women recruitment and samples used in this study were collected between November 2017 and February 2020. DNA extractions and data analyses was done between March 2020 and October 2022.This recruitment was conducted in compliance with the regulations of the Human Subjects Protection Office at the University of Puerto Rico, under the Institutional Review Board Medical Sciences Campus IRB protocol #1050114. This protocol was initially approved on June 24, 2014, and it has been continually renewed and remains in effect until 2024. The biosafety committee of the Medical Sciences Campus also approved the Protocol (IBC) # 94620. All procedures were done according to the Declaration of Helsinki.A total of 91 women, 21–60 years old and able to provide informed consent, were enrolled in our study. Cervical samples were collected through speculum access during the gynecological examination by using cotton tip collection swabs. Sterile Catch-All Specimen Collection Swabs  were placed in the cervix, rotated along the lumen with a circular motion, and then placed in sterile 2 milliliters (mL) microtubes with cap. We collected approximately 10mL of cervical lavages (CVL) by injecting pure certified nuclease-free water  into the vaginal canal. Collected lavages were stored in a clean 15mL collection tube and pH was measured with hydrion wide range pH paper strips short after sample collection at room temperature . All samples were kept from 1–4 hrs in a cooler with ice packs before transportation to the lab, where aliquots were prepared and further long-term storage was done at −80°C. Information from the medical records and questionnaires and the results from cervical cytology and pathology reports were gathered. These reports were used to categorize participants according to cervical disease status. Disease status was defined using cytology and HPV HR status . If patient cytology was abnormal (LGSIL or HGSIL) and or HPV HR-positive colposcopy was received and biopsy results were used in this case. In this study, the groupings included Negative for intraepithelial lesion or malignancy (NILM); low-grade squamous intraepithelial lesion (LGSIL), high-grade intraepithelial lesion (HGSIL).® system. Cytokines/chemokines analyzed were IL-1β, IL-4, IL-6, IL-8, IL-10, TNF-α, IFNγ, IP10, MIP1, and MCP1. Also, 79 samples were tested in duplicate using Bio-Plex Pro TGF-β 3-plex immunoassay to quantify TGF-β 1 . We performed all analyses following the manufacturer’s recommendations.A total of 79 cervical lavages were tested in duplicate with a multiplex solid-based immunoassay  using the same volume of CVL across samples (80 uL) for the Luminex MAGPIXData were analyzed with the Bio-Plex Manager 6.1 software  using a 5-parameter logistic curve that was verified for normality before statistical testing . Inflammt-test. All statistical analyses used GraphPad Prism 6.0 .Cytokine profiles were evaluated according to cervical disease, CSTs, and HPV infection. These analyses were done using cytokine concentration (pg/mg protein) instead of the fluorescence intensity (FI) used for the inflammation scores. For comparisons between multiple groups, we used ANOVA, and for comparisons between two groups, we used the student’s ® dsDNA HS (High Sensitivity) Assay  (ranging from 5-100ng/μL). An average of 10-30ng/μL genomic DNA was shipped to an outsourced sequencing facility.Genomic DNA extractions from swabs were performed with the Qiagen Power Soil Kit  following an optimized protocol for these types of samples as described in a previous study . DNA wasGTGCCAGCMGCCGCGGTAA3’) and 806R (5’GGACTACHVGGGTWTCTAAT3’) as in the Earth Microbiome Project (http://www.earthmicrobiome.org/emp-standard-protocols/16s/) [DNA from cervical samples was normalized to 4nM during 16S library prep. The V4 hypervariable region of the 16S ribosomal RNA marker gene ~291bp) was amplified using universal bacterial primers: 515F (5’ls/16s/) . The 16S1bp was aWe used a kit based on a highly sensitive short-polymerase chain reaction-fragment assay . It uses SPF10 primers to amplify a 65-bp fragment of the L1 open reading frame of HPV genotypes, followed by a Reverse-Hybridization step to determine which HPV type is present by comparing to kit-provided controls. This assay allows for the identification of the following common mucosal HPV genotypes: 14 High-risk types including  and 11 Low-risk types . Previous studies explain the method in detail .In this paper we refer to HPV types as HPV type according to carcinogenic risk, hence we categorize it as HPV negative (HPV-), HPV+ Coinfection (having both low-risk and high-risk types) and HPV+ High risk exclusively.Raw read pre-processing of demultiplexed files was done with a Phred offset of 33 and default parameters using split libraries FASTQ (QIIMEq2 1.9.1) in QIITA . SequencThe taxonomy of cervical microbial communities was sorted into categories termed community state types (CSTs). This categorization collapses a taxonomic profile into a single categorical variable, allowing data exploration and statistical modeling . In addiFor community-level analyses, we computed the pairwise Bray-Curtis distances (Beta diversity) which quantify the compositional dissimilarity between samples . We visu® system) and community state types (CSTs).The most important metadata variables included in our study were cervical disease, HPV carcinogenic types and cytokine levels. Cervical disease was grouped as: a) negative for intraepithelial lesion or malignancy (NILM) who are HPV negative, b) NILM participants who are HPV positive were considered in the same category as participants with low-grade squamous intraepithelial lesion (LGSIL) and c) high-grade squamous intraepithelial lesion (HGSIL); HPV risk, was defined as HPV negative, HPV positive with co-infection (low and high-risk HPV simultaneously), and HPV positive high-risk (exclusively high-risk types). Microbiota was also associated with cytokine ranks  in R (ggplot and reshape2 libraries), correcting for age and body mass index (BMI). Differential abundance analysis was performed using amplicon sequence variants corresponding to L. crispatus, L. iners, Sneathia, Atopobium, Gardnerella and Prevotella. They were evaluated for their differential presence across the cytokine groups. Initially, a summary of each one of the ASVs of interest was used to perform groupings due to abundance. The groupings were separated as follows ASV 0–50 counts = very low abundance; ASV count 51–500 = low abundance; and >500 = high abundance. They were evaluated for their differential presence across the cytokine groups.For beta diversity analyses, we tested if distances between sample groups were statistically significant using PERMANOVA, a non-parametric multivariate ANOVA statistical test . Using aTo note despite 91 women were recruited and samples collected the number of samples analyzed by the different methods differ. Specifically, for prevalence analysis of the metadata variables, we used the entire study cohort n = 91. In the case of cytokine profile analysis, we ended up with less samples due to cytokine concentration availability n = 79. When using fluorescence intensity in analyses where Transforming growth factors are included, we used n = 88. We chose fluorescence as a method to avoid altering the data before conducting correlation analyses. These same 88 samples were utilized for analyzing physiological status, cervical disease, and cytokines .A total of 91 women were included in the study. The mean age was 39 years old. Women had an average pH value of 5.49. Overall, 11% of our participants smoke, while 44% occasionally drink alcohol. The average age of first sexual encounter was 19 years old and the average number of sexual partners was 5. Most subjects were self-declared heterosexuals (98.0%). This study corresponds to an analyses of a subgroup of the nearly 300 patients which we recently characterized for only cervicovaginal microbiota and physiological status . The disSignificant variations in cytokine concentrations (measured in pg/mg of protein) were observed in women with high-grade disease compared to healthy participants. Notably, there were significantly higher concentration levels in HGSIL compared to , and the values were nearly significant in participants with low-grade disease (p = 0.059) see . AdditioWhen examining cytokines based on CSTs, notable distinctions in IL-1β and IL-4 concentrations were identified, see A total of 2,067,074 high-quality reads of 16S rRNA sequences and 8,167 OTUs were subjected to analysis, with a rarefaction threshold set at 7,400 reads per sample. For most analyses the sample pool primarily consisted of women of reproductive age (n = 70) and menopausal women (n = 18). We examined the cytokine profiles related to menopausal status . We obseFurthermore, we assessed the differences in microbiota based on cervical disease, considering HPV infections. Beta diversity analyses related to cervical disease showed that the structure of the bacterial community significantly differed in cases of high-grade cervical disease (HGSIL) compared to healthy controls (NILM/HPV-) (p = 0.003) and when compared to low-grade cervical disease (LGSIL) (p = 0.021) . Both riLactobacillus crispatus and Lactobacillus iners. However, at the species level, we observed an increase in L. iners communities and a decrease in L. crispatus in LGSIL  and IFNγ (p = 0.022) (L. iners were significantly associated with lower levels of IL-6 (p = 0.021) and IFNγ (0.015), also pro-inflammatory cytokines (L. iners correlate with lower expression of IFNγ (p = 0.039)  (of Prevotella correlated with higher concentrations of IL-1β (p = 0.020), IFNγ (p = 0.011) and TNFα (p = 0.003) in our samples (Prevotella correlated to IL-1β (p = 3.00e-05). Low (p = 8.34e-06) and high (p = 0.002) levels of Atopobium, a mostly pathogenic bacteria, were significantly associated with high levels of IL-1β. As for Sneathia, another dysbiosis associated taxa, for which low (p = 0.0009) and high (p = 0.006) abundances had a correlation with higher levels of IL-1β  . Low levytokines . On the = 0.039) . High le.62e-05) . Low abu samples . Also, hof IL-1β .L. crispatus decreases with higher cytokine levels. Participants with high levels of cytokines were dominated by L. iners, as L. crispatus nearly disappears with increased levels of cytokines. Additionally, this group of participants were dominated by Prevotella sp., A. vaginae and G. vaginalis. Simultaneously, Lachnospiraceae sp. was detected mainly on participants with High cytokine levels, confirming the dysbiosis typical of Lactobacillus depleted communities (In community composition (beta diversity), X axis of the NMDS1 plot separated samples according to their cytokine levels . We founmunities .Cervical cancer (CC) is a complex disease where the cervical environment is modified, including cytokine expression that leads to a local immunosuppression state . More thLactobacillus bacteria and associated physicochemical modifications that typically coincide with microbial imbalances, leads to changes in the vaginal and cervical milieu. This situation creates a specific microbial environment that exerts selective pressures on the microbiota [Modifying the host innate immune system is a well-known feature of numerous malignancies, including HPV-driven cancers . HPV intcrobiota .Several studies have already established background data on how the microbiome is modified by HPV infections, stating that HPV infections alone are not enough to drive significant changes that translate into cervical disease progression ,57. NoneLactobacillus species have several mechanisms that function as a defensive barrier for invasive bacteria, such as, the production of significant amounts of bacteriocins that contributes to colonization the use of available glycogen to produce lactic acid, lowering vaginal pH, responsible of lysing bacteria other than Lactobacillus [Lactobacillus population reduction, may explain the increase of Atopobium vaginae, Prevotella spp., and Gardnerella spp.. Also, our findings coincide with most studies suggesting an association between L. iners and HGSIL [L. iners exhibits a reduced capacity to consistently maintain the acidic vaginal environment. Conversely, the prevalence of L. crispatus is linked to higher acid levels, a lower pH, and reduced microbial diversity within the vaginal microbiome. Moreover, research indicates that L. crispatus displays superior adherence to vaginal epithelial cells, enhancing its ability to outcompete potential pathogens for space and resources within the vaginal environment. In alignment with our findings, previous studies have associated L. crispatus with the modulation of the local immune response, promoting a well-balanced and healthy microbiome. Conversely, our data demonstrates a correlation between L. iners and a pro-inflammatory state.Concerning cervical lesions, we found a strong microbiome association with the cervical phenotype (lesion severity), with a significantly higher diversity value (Shannon) in HGSIL compared to LGSIL participants, but not substantially different from NILM/HPV-. In contrast, richness (Chao1) was also significantly higher in HGSIL when compared to LGSIL and NILM/HPV-. Overall, bacillus –67. The nd HGSIL ,69. L. iLactobacillus can prevent the colonization of exogenous pathogens by producing lactic acid, bacteriocins and reactive oxygen species (ROS) [L. crispatus that revealed a significant correlation between high bacterial concentrations and low abundances of pro inflammatory cytokines IL-1β and IFNγ. Our study confirms cervicovaginal microbial dysbiosis in high-risk HPV and cervical disease severity associated with pro-inflammatory cytokines, validating other studies in other cohorts [Lactobacillus validating previous studies where high concentrations IL-1β were detected in women with cervical neoplasia [L. iners. IL-6 is essential for the growth, metastasis, and initiation of cancer; affecting tumor growth, differentiation, and the host immune defense mechanism [L. iners) may be stimulating the production of these mediators, this could be due in part to their heterogeneous genome, that, unlike the protective counterparts, do not produce as much lactic acid or hydrogen peroxide [Previous studies have described that es (ROS) ,70,71, p cohorts ,72. IL-1eoplasia –75. The eoplasia  or metaseoplasia ; as welleoplasia  associateoplasia , reactiveoplasia . We founechanism . Many imechanism ,83. IL-6echanism . In addiechanism . The lesperoxide .Anti-inflammatory cytokine IL-4 was highly elevated on participants with HGSIL. IL-4 within the cervicovaginal microbiome regulates the immune response , upholdsLactobacillus profile) and CST IV-A , which confirms previous work of our lab [Lactobacillus spp., G. vaginalis, and A. vaginae, while instead is characterized by the abundance of a diverse array of facultative and strictly anaerobic bacteria, thus revealing that Hispanic women living in PR are in constant dysbiosis with a microbiome dominated by anaerobic non-lactobacillus communities. Overall, the high prevalence of CST-IV across all samples reflects a cervical microbial environment with low stability and less likelihood to return to its equilibrium state after a perturbation, which increases the risk of developing adverse health outcomes [Taxonomic profiles of cervical microbial communities were sorted into community state types (CSTs), allowing a valuable categorization for statistical modeling and comparisons between datasets 18,19). The vaginal microbiota in this Hispanic women cohort of women living in Puerto Rico was dominated by CST IV-C  were related to significant bacterial community composition changes and high diversity levels (Shannon). High and low levels of aginosis , a classaginosis . This reaginosis . Increasammation . A studytococcus , assumintococcus . Prevoteaginosis , and thiLactobacilli and the presence of diverse anaerobic taxa. We acknowledge that the main limitation of our study is the relatively low sample number, especially in the categorical groups (n = 91), besides lack of information regarding other viral infections and sexual behavior and menstrual health. The cervical microbiota in Hispanic women living in Puerto Rico is highly diverse and dominated by CSTs III and IV. Further changes of the microbiota with HR-HPV and inflammation reveal that the joint host-microbe interaction analyses via cytokine signaling and microbiota in precancerous lesions have great translational potential.Cervical dysplasia is a complex multifactorial process, and further research, including time-series analyses of longitudinal sampling, is needed to improve our understanding of its etiology. Our study evidence that high concentrations of all three cytokine groups  associates with cervical dysbiosis, which occurs through dramatic losses of protective S1 FigCSTs according to category groups were used to compute Fisher’s test using pairwise analysis to compare between groups. Results were depicted in boxplots for cervical disease. Significant differences are highlighted by brackets and corresponding p-values.(PDF)Click here for additional data file.S2 FigCytokine concentrations (pg/mg protein) were used to compute multiple comparison analysis using ordinary one-way ANOVA with Tukey’s multiple comparisons test. Significant differences are highlighted by brackets and corresponding p-values.(PDF)Click here for additional data file.S3 FigCytokine concentrations (pg/mg protein) were used to compute multiple comparison analysis using ordinary one-way ANOVA with Tukey’s multiple comparisons test (A-C). Beta and alpha diversity analyses are represented by non-metric multidimensional scaling (NMDS) (D) and Shannon index boxplots (E). Relative abundance of bacteria at the species level is shown in a relative abundance bar plot (F).(PDF)Click here for additional data file.S4 FigBray-Curtis analysis represented by Non-metric multidimensional scaling (NMDS), alpha diversity (Shannon) and bar plots showing relative abundance of bacteria at the species level were grouped using pro inflammatory (A), anti-inflammatory (B) and trafficking cytokines (C) as metadata categories.(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableP values were adjusted using Bonferroni corrections.(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Specifically, the 90-70-90 targets set out to see that 90% of girls are fully vaccinated with HPV vaccine by age 15 years, 70% of women are screened with a high-performance test at 35 years and again at 45 years and that 90% of women identified with cervical disease receive treatment.1In Nigeria, an estimated incidence of 12,100 patients and 8,000 deaths in 2020 puts cervical cancer as the second leading cause of female cancer morbidity and mortality behind breast cancer.7 Evidence from population studies including post-vaccination follow-up has shown HPV vaccines to be highly immunogenic, safe, and effective in preventing HPV infection, cervical lesions, anogenital warts, and other HPV-related diseases.8 Currently, six HPV vaccines are licensed for use by the WHO—bivalent HPV vaccines , quadrivalent HPV vaccines (gardasil and cervarax), and nonavalent HPV vaccine (gardasil9).7 Providing protection against high-risk (oncogenic) HPV types 16 and 18 that cause the majority of precancers, cervical cancers, and other HPV-related cancers is a unique characteristic across the licensed vaccines. WHO's guidelines recommend that adolescent girls between 9 and 14 years receive two doses of vaccine to be fully immunized with at least a 6-month interval between doses, with exceptions for special populations including immunocompromized individuals and those age 15 years or older, for whom three doses are recommended.7 In 2022, following evidence that a single dose of HPV vaccine provided similar protection against HPV infection as seen in multidose regimen,9 the WHO updated its HPV vaccination recommendations to include a single-dose schedule  for individuals age 9-20 years.7 The single-dose schedule update is timely and can help countries such as Nigeria to introduce the HPV vaccine, improve coverage, and cut associated costs.Vaccination of adolescent girls with HPV vaccines before the onset of sexual activity is the most effective long-term intervention for reducing the risk of developing cervical cancer. Knowledge of the causal relationship between HPV infection and cervical cancer has stimulated advances in the development, licensing, and usage of HPV vaccines.10 Although more than 80% of high-income countries (HICs) have introduced HPV vaccines, LMICs still lag behind and have introduced HPV vaccines at a slower pace as only 41% achieved introduction as of 2019.10 Regrettably, 70% of girls worldwide reside in countries that are yet to nationally introduce HPV vaccines including Nigeria.10 From 2006 when the first HPV vaccine was licensed to 2017, more than 100 million girls globally have been reached with at least one dose of HPV vaccine, and it is worrisome that 95% of vaccinated girls were resident in HICs, and this further reflects inequities in HPV vaccination access.1 Financial and programmatic barriers including high vaccine cost, lack of political will, vaccine logistics challenges, health workforce gaps, and global HPV vaccine supply constraints have been adduced as major reasons for inequitable national introduction of HPV vaccination especially in LMICs where vaccines are needed the most.12As of June 2020, the number of WHO member states considered to have partially or fully introduced the HPV vaccine nationally stood at 107 (55%) of 194.13 At the heart of Gavi partnerships are its core members—WHO, UNICEF, World Bank, and Bill and Melinda Gates Foundation and other key development partners including governments, civil society organizations, vaccine manufacturers, research agencies, and other private sector players. Gavi achieved a historic agreement with vaccine manufacturers—GlaxoSmithKline and Merck to reduce the price of the bivalent (cervarix) and quadrivalent (gardasil) HPV vaccines which can cost more than $100 (USD) in HICs to less than $5 for Gavi-eligible countries where cervical cancer burden is the highest.13 Despite such historic agreements, it has been argued that the price is still high and not sustainable for LMICs including Nigeria within the context of pervasive poverty, overstretched health budgets, and competing needs for scarce resources.14The interventions of Gavi, The Vaccine Alliance—a global public-private partnership targeted at addressing major precursors of vaccine inequity such as high vaccine prices and vaccine delivery logistics have been hailed as a masterstroke toward improving global access to HPV vaccines especially in LMICs.13 The national introduction pathway involves eligible countries to meet a Gross National Income per capita (GNIpc) of ≤$1,580 (USD), at least a 70% diphtheria-tetanus-pertussis third dose coverage, and have shown the capacity to deliver a multidose vaccine to at least 50% of 9- to 14-year-old girls.14 However, Gavi's funding model and reliance on national income to determine eligible countries for support have been criticized as exclusionary on the basis that the GNIpc metric may not reflect the true economic realities of LMICs.14 Such exclusion potentially translates to widening inequities when countries that bear the greatest burden cannot afford the negotiated vaccine price and are not eligible to access vital support to roll out life-saving HPV vaccines.Beyond the agreement to make HPV vaccine affordable for target LMICs, Gavi provides significant funding and technical support for eligible countries that indicate interest to introduce HPV vaccines via two pathways—one for the national introduction of HPV vaccines and the other as a demonstration program.6 Gavi covers the vaccine supply cost until the port of entry and provides further cash grant that supports about 80% of operational costs under the demonstration program. New Gavi guidelines released in 2016 now allow for countries yet to introduce HPV vaccines like Nigeria to apply directly for Gavi support to fund national or phased introductions rather than starting with a demonstration program from 2017 onwards.13 The phased introduction approach and availability of empirical evidence of what works for successful HPV vaccination implementation represent critical opportunities for LMICs including Nigeria yet to introduce HPV vaccines to leverage.Gavi's demonstration program pathway is a 2-year project which allows countries the opportunity to gain experience in delivering HPV vaccines to a target population (9- to 14-year-old girls) not usually involved in routine immunization schedules and make decisions to either introduce HPV vaccine nationally or not. Precisely, country decisions on national introduction are expected to be informed by the key lessons provided by the demonstration program on issues such as vaccine costs and logistics, coverage, acceptability, and feasibility.15 and National Cancer Control Plan (2018-2022),16 had set the roadmap for Nigeria's vision for cervical cancer prevention including specific plans and targets for HPV vaccination. For instance, the National Strategic Plan on Prevention and Control of Cervical Cancer in Nigeria (2017-2021) highlighted Nigeria's intention to leverage high-volume catchment areas toward delivering two doses of quadrivalent HPV vaccine to 4.5 million girls age 9-13 years over a 5-year period at a financial cost of $18 million (USD) ($3.98 per fully immunized girl). Similarly, the National Cancer Control Plan (2018-2022) boldly set an objective of attaining 90% HPV vaccine coverage for girls age 9-13 years in Nigeria by 2022. These ambitious goals outlined by both strategic documents were never achieved.17 Nevertheless, such documents could serve as the foundation for future national cervical cancer prevention plans especially HPV vaccine introduction.Two strategic documents, National Strategic Plan on Prevention and Control of Cervical Cancer in Nigeria (2017-2021)17 This announcement was met with renewed optimism and expectations. However, more than 2 years later, HPV vaccination has not been rolled out in Nigeria. When the 2021 deadline was missed, Faisal Shuaib, executive director of the National Primary Health Care Development Agency (NPHCDA), made a similar announcement for a new date on July 14, 2022.17 Unsurprisingly, much skepticism exists about the likelihood of introducing HPV vaccination by the new date. Specifically, Shuaib announced that HPV vaccines for Nigerian girls (9-14 years) will be introduced into Nigeria's expanded immunization program by the third quarter of 2023.18 Concerns around the feasibility of implementation considering high vaccine cost which is the largest contributor to vaccine delivery costs and if Nigeria will get needed support including partnerships to roll out HPV vaccines in 2023 persist. However, to address these concerns, the NPHCDA executive director recently stated that the country had met the conditions for support from Gavi and obtained provisional approval for a phased HPV vaccine introduction and subsequently, provide the same as part of routine immunization after 2024.18During the national stakeholder's forum on the elimination of cervical cancer held in November 2020, Nigeria's health minister Osagie Ehanire in his keynote speech announced the country's plan to introduce HPV vaccines by 2021.19 Although low awareness of HPV and HPV vaccines has been reported among parents of Nigerian girls as barriers to vaccinating their kids, their willingness to vaccinate their daughters is high.20 Similarly, evidence from a pilot school-based HPV vaccination program revealed the high acceptability of HPV vaccines among parents and adolescent girls in Nigeria.21 Such a high level of willingness and acceptability of HPV vaccines further strengthens the rationale for the national introduction of HPV vaccines in Nigeria.In some areas in Nigeria, HPV vaccines are sparsely provided in private and public health facilities at an average cost of $13 (USD) which is expensive considering the high poverty levels in the country and further impedes access for individuals who need them the most but cannot afford it.14 The success of the HPV vaccine rollout in Rwanda since 2011 which put the country on a path toward the elimination of cervical cancer after high HPV vaccine coverage (92.23%) of the target population serves as a key example of what strong political commitment, government ownership, and leadership can accomplish.22Moving forward, if Nigeria is to make considerable progress in fully immunizing the estimated 15 million Nigerian girls age 9-14 years, the country must be deliberate in leveraging every opportunity that puts them in a good position to sustainably roll out HPV vaccines nationally. Strong political commitment to prioritize and drive needed actions for the introduction of HPV vaccines and sustaining the gains of such introduction in Nigeria is critical.22 Hence, Nigeria must leverage collaborative partnerships and ensure coordination between government and relevant development partners including bilateral and multilateral organizations, vaccine manufacturers, private sector stakeholders, and community-based and civil society organizations in driving advocacy, policy, and program implementation efforts that creates access to HPV vaccination especially for the most disadvantaged girls. Again, Rwanda's partnership with Merck  that led to the latter agreeing to supply HPV vaccines for the first 3 years of national introduction at no cost to the country highlights the power of partnerships and provides lessons for Nigeria.22 Rwanda's agreement with Merck which made the country the first low-income country to introduce HPV vaccines also demonstrated how partnership can facilitate the achievement of shared goals. For instance, although Merck was able to show the feasibility of introducing HPV vaccines in LMICs, Rwanda made huge progress in protecting girls from cervical cancer through lifesaving HPV vaccines. Furthermore, Nigeria must also prioritize interministerial collaboration particularly involving the finance, health, and education ministries as part of the foundations for HPV vaccine rollout and improved HPV vaccination outcomes.12To meet global and national HPV vaccination targets, the significance of global partnerships that stimulate national action, address funding concerns including vaccine costs, and strengthen equitable vaccine supply access for countries with the highest cervical cancer burden has been emphasized.23Nigeria's intention to introduce HPV vaccines must rely on evidence-based and comprehensive introduction plans. These plans should recognize the peculiarities of the target population (girls age 9-14 years) through microplanning, clearly outline financing strategies and cost implications for introduction and sustainability including operational and logistics costs, and prioritize diverse vaccine delivery mechanisms including integrated approaches, community outreaches, and school-based vaccination programs. In addition, the plan must provide for health system strengthening including health workforce and infrastructure initiatives and include a robust communication and social mobilization approach underlined by meaningful community engagement that improves vaccine acceptability, increases population awareness, and translates willingness to vaccinate into action.As Nigeria prepares to roll out HPV vaccination, it is equally important to establish strong accountability mechanisms including robust monitoring and evaluation frameworks that highlight progress, identify multilevel challenges across the HPV vaccine introduction process, and proffer solutions to improve program operations and sustainability.


We previously demonstrated the convergent validity of a fully automated voice recognition analogue of the Symbol Digit Modalities Test (VR-SDMT) for evaluating processing speed in people with multiple sclerosis (pwMS).We aimed to replicate these results in 54 pwMS and 18 healthy controls (HCs), demonstrating the VR-SDMT’s reliability.r = −0.771, p < 0.001) and HC  groups.Significant correlations were found between the VR-SDMT and the traditional oral SDMT in the multiple sclerosis (MS) (Taken collectively, our two studies demonstrate the reliability and validity of the VR-SDMT for assessing processing speed in pwMS. Although the test is quick to complete, the need for a trained tester can hinder its use in clinical settings. We previously developed a fully automated voice recognition SDMT analog (VR-SDMT) and demonstrated the VR-SDMT’s convergent validity with the traditional oral SDMT in English speakers and French–Canadian speakers. We now present new data in English aiming to replicate the results from the earlier study, thereby enhancing the measure’s reliability.Cognitive dysfunction can have serious consequences in the lives of people with multiple sclerosis (pwMS). The Symbol Digit Modalities Test (SDMT) evaluates information processing speed and is considered a key neuropsychological test predictive of future cognitive decline, employability, and activities of daily living in pwMS.A sample of 54 pwMS and 18 healthy controls (HCs) were recruited at two tertiary care neuropsychiatry and multiple sclerosis (MS) clinics in Ontario. MS diagnosis according to the revised McDonald criteria was confirmed by a MS neurologist. All participants were 18–60 years old and provided informed consent. Exclusion criteria included prior completion of the SDMT within a year, history of traumatic brain injury, severe psychiatric illness, learning disability, or concurrent neurological disorder. The program begins by administering an eye test to confirm adequate visual acuity, which the participant must pass to advance to the test. A symbol digit code is displayed in a box with two rows: the top row has nine unique symbols and the bottom row has numbers 1–9. Below the code, there is another row with the same nine symbols in a different order, and the participant must orally report what number goes with each symbol in the row, according to the key. The program takes them through a tutorial and a practice trial to ensure they understand what needs to be done before beginning the test. The participants are instructed to proceed as quickly as possible. The test goes on for eight sequential trials, and each trial features the nine symbols arranged in a different order. The program uses Google’s online speech recognition software to time the responses. At the end, the program records the total time and mean time per trial. To control for practice effects, half of the participants completed the traditional SDMT first, and the other half completed the VR-SDMT first.The details of the VR-SDMT analog have been described previously.t-tests or chi-square tests as appropriate. The convergent validity of the VR-SDMT and traditional SDMT was assessed with Pearson’s correlation coefficients. The relationships between test scores, Expanded Disability Status Scale (EDSS), and employment status were assessed with Spearman’s rank correlations.Demographic comparisons between the MS and HC groups were performed with independent samples 2 = 5.908; p = 0.015)  .r = −0.771, p < 0.001) and HC  groups. Scatterplots of these relationships are shown in p = 0.021), but the difference between groups did not reach significance on the traditional SDMT (p = 0.069).There were significant correlations between the traditional SDMT and the VR-SDMT, for both the MS (p = 0.022) and traditional SDMT . Furthermore, unemployed participants were more likely to have slower VR-SDMT scores . Employment status did not correlate with traditional SDMT scores.In addition, EDSS scores of the MS group correlated significantly with scores on both the VR-SDMT  scores are better.57 and SDMT scores and employability. Here, we have replicated the correlation between the EDSS, on one hand, and SDMT and VR-SDMT scores, on the other hand. We also found a correlation between VR-SDMT scores and employment status, which enhances the test’s ecological validity. The failure to find a similar result with the traditional SDMT may reflect a small sample size.Previous studies have demonstrated correlations between SDMT scores and EDSS scoresz-scores, from which impairment can be determined. Furthermore, although we have shown here the consistency of the test across different samples, indicative of good reliability, we do not yet have test–retest reliability in the same sample. As with the traditional oral SDMT version, significant dysarthria is a major barrier to the test’s use. This limitation is offset by the absence of an upper limb motor component, something which needs to be taken into account with an analogous assessment, the iPad-based Processing Speed Test.This study is limited by its modest sample size and the relatively mild level of disability. Establishing a larger normative database would allow us to update the program to calculate participants’ In summary, the utility of the VR-SDMT analog is that it does not need a trained tester to administer and score the test. This eliminates any inter-rater variability and makes it easy to use in a busy clinical setting. We have now shown that the test has good reliability to add to its previously demonstrated validity, making it a promising tool for clinicians to incorporate into their evaluation of processing speed in pwMS.


Human papillomavirus (HPV) is a viral infection that, if does not go away, can cause health problems like genital warts and cancer. The national immunization schedules for individuals before sexual debut, significantly decreased HPV-associated mortality and it will be affordable. However, immunization programs remain vulnerable to macroeconomic factors such as inflation, fiscal policy, employment levels, and national income. This review aims to investigate the association between national income in lower-middle-income countries to explore recent advances and potential issues, as well as how to deal with challenges. Human papillomavirus (HPV), with a global prevalence of 11.7%, is the cause of one of the most common sexually transferred diseases worldwide . It is kCervical cancer is the fourth most common cancer among women in the world and especially in low and middle-income countries (LMICs) such as Brazil, India, China, South Africa (SA), and Iran –9 Figu. OverallHPV immunization could control nearly 70% of all cervical cancers  TablesTables2Due to girls aged 9 to 14 not yet sexually involved, World Health Organization (WHO) suggested these ages as the most suitable time for vaccination against HPV . The natHowever, according to the most current research, which was published in December 2022, WHO recommends: a one or two-dose schedule for girls aged 9 to 14 years; a one or two-dose schedule for girls and women aged 15 to 20 years; and two doses with a 6-month interval for women over 21 years.WHO recommended that HPV vaccination be incorporated into national immunization schedules for the preliminary target individuals of girls 9 to 14 and the secondary target individuals of women over 15 years of age . In mid-Collectively, the timely, accurate introduction and implementation of HPV vaccination in national programs is an essential step in eliminating cancer and other diseases caused by HPV. Recent developments in this field are promising, although still associated with challenges. Hence, this study reviews different aspects of HPV vaccination status in low- and middle-income countries.2via School Health Services (SHS) with enhanced vaccine databases  have included the HPV vaccine in their routine national immunization schedules. Otherwise, despite accounting for most of the disease burden in LMICs, the adoption rate remains lower than in HICs . The staThe most critical problem in HPV vaccination is identifying eligible individuals; otherwise, opportunities for immunization may be missed, and it is beneficial to define narrow criteria for determining the target population. For example, grade-based eligibility can be helpful in school-based strategies in countries with high school attendance and a narrow age spectrum of girls in each class. However, for community or facility-based approaches, or where out of school (OOS) girls constitute a significant proportion of the population, age-based eligibility is usually preferable . Usually2.2® with proprietary AS04 adjuvant, indicated for girls and women, boys and men between 9-45 years of age. 1.2) Innovax’s Cecolin® with aluminum-containing adjuvant, indicated for girls and women aged 9-45. 1.3) Walvax Biotechnology’s product with aluminum-containing adjuvant, indicated for girls and women aged 9-30 years (developed by its subsidiary Shanghai Zerun Biotech), 2) One quadrivalent (HPV4) vaccine: Merck’s Gardasil® with aluminum-containing adjuvant, indicated from 9-45 years of age for girls and women, boys and men, 3) one nonavalent (HPV9) vaccine: Merck’s Gardasil 9® with aluminum-containing adjuvant, indicated from 9-45 years of age for girls and women, boys and men . However, many countries still struggle with enumerating eligible girls, obtaining consent when necessary, identifying eligible girls if age is a criterion, and keeping records. Private schools can be more challenging to deal with and less willing to engage than public schools, and they may have stricter consent requirements. To develop effective remedies, more data and case studies are required , 44.3.4The effect of the on-time conduct of HPV vaccine in LMIC will be significant because every five-year deferment in supply leads to the death of 2 million people with cervical cancer . TherefoPrior research from HICs and theoretical acceptance research in LMICs both predicted that sociocultural cases would be the greatest barrier to vaccine schedules; however, purposeful sensitization schedules have been victorious in overwhelming this challenge and reaching high vaccine acceptability covering levels in several LMICs .Health system financial obstacles may disable the capability of some LMICs to effective implementation of high-quality HPV vaccine schedules. Securing endurable financing for the vaccine such as health system charges and holding donation schedules, represents a substantial barrier. A record low cost for as small as US $4.50 per dose for LICs, in comparison with over $100 in HICs, was reported in May 2013, by the GAVI Alliance . In otheThe vaccine also introduces new political points, especially challenging the harmony of numerous stakeholder groups to maintain the motivation for supporting vaccination campaigns. The influence of political engagement in maintaining the integrity of vaccination campaigns is reflected in Rwanda’s successful HPV vaccination program, which reached over 93% of vaccine coverage after its first three-dose rollout in 2011. The success was attributed to the government agreement with Merck Company for providing the first three-dose for free, a population sensitization program powered by local government officials and healthcare professionals, and the development of technical working groups with other ministries to design a school-based delivery program . Thus itvia schools, particular campaigns, healthiness centers, or several techniques can achieve a considerable proportion of qualified girls and that support and sensitivity are essential. GAP nations such as Uganda, Lesotho, and Cameroon have lately communicated lessons from experimental measures such as the significance of gaining political commitment, mustering resources, and suggesting merging the vaccine within existing immunization networks for stability , a completely owned subsidiary of Beijing Wantai Biological Pharmaceutical Co., LTD. (Wantai), and is ready to defend against HPV types 16 and 18, the most usual virus types which lead to cervical cancer. Governments facing obstacles to national introduction or developing their HPV vaccine schedule to the whole cohort owing to cost or supply constraints will now have another choice for cost-effective, stable access. A summary of progress, ongoing challenges, and prospects of national HPV vaccination programs is presented in The main burden (86%) of cervical cancer is in LMICs, while < 30% of these nations have presented the vaccine. The condition is aggravated by limitations in the vaccine, supply possible to continue until 2022/25 . Also, L4There are potential scientific and manufacturing improvements that could have a significant effect on future HPV vaccination schedules. Despite targeting a single cohort in specific periods that may sound more cost-saving for LMICs facing financial and technical obstacles, inbitiating multi-age cohorts may lead to more rapid and comprehensive immunization coverage among target groups . In addiA summary of progress, ongoing challenges, and future prospects of national HPV vaccination programs is presented in 5Various approaches can efficiently deliver the HPV vaccine to young adolescent girls. Gavi funding has been critical for covering the costs of vaccinations and early distribution in low and lower-middle-income countries. The possibility of new, more effective vaccine entry into the market may enable overcoming this barrier, while other issues or factors may be preventing some governments from doing so. The lessons learned from successful vaccination programs in HIC could guide health policymakers to take more proactive steps. Regarding the speed of vaccine introduction, LMIC should accelerate the process since they outnumber HIC, has more population, and have initiated the program later. Although current school-based programs have exhibited promising results in LMIC, the vaccine delivery route may also benefit from more engaging facility-based services compared to its dominant school-based form in LMIC . This isIn conclusion, several approaches should be considered. Educating and teaching people about compliance with public health, testing, and routine checkups in overcrowded places like pools, dormitories, schools, and even armies, teaching self protections in sex, making protected sex products available and reducing the cost of them, increasing screening tests, especially before marriage, mass vaccination, developing new effective vaccines in theire own country, and predicting appropriate treatment budgets for cervical cancer. On the other hand, WHO and high-income countries should regard supporting and increasing HPV vaccine availability for low-and middle-income countries. We must remember that if we neglect it, the next pandemic may be caused by HPV infection.NEb, ZY, GK, FE, MG, MA, MS, BG, and NEs conceptualized the study and wrote the manuscript. NEb, ZY, GK, FE, MG, and MA contributed to drafting of the manuscript. All authors contributed to the article and approved the submitted version.


OGG1 expression in CRC participants (p < 0.005) and a downregulation of OGG1 in normal-weight healthy patients (p < 0.05). Interestingly, the methylation analysis showed the hypermethylation of OGG1 in CRC patients (p < 0.05). Moreover, expression patterns of OGG1 were found to be regulated by vitamin D and inflammatory genes. In general, our results showed evidence that OGG1 can regulate CRC risk through obesity and may act as a biomarker for CRC.DNA damage has been extensively studied as a potentially helpful tool in assessing and preventing cancer, having been widely associated with the deregulation of DNA damage repair (DDR) genes and with an increased risk of cancer. Adipose tissue and tumoral cells engage in a reciprocal interaction to establish an inflammatory microenvironment that enhances cancer growth by modifying epigenetic and gene expression patterns. Here, we hypothesize that 8-oxoguanine DNA glycosylase 1 (OGG1)—a DNA repair enzyme—may represent an attractive target that connects colorectal cancer (CRC) and obesity. In order to understand the mechanisms underlying the development of CRC and obesity, the expression and methylation of DDR genes were analyzed in visceral adipose tissue from CRC and healthy participants. Gene expression analysis revealed an upregulation of  According to the last Global Cancer Statistics 2020, colorectal cancer (CRC) is the third most frequent cancer  and the OGG1 gene on chromosome 3p25) is a key enzyme in correcting this mismatch. This multifunctional protein repairs the guanine lesions in mammalian cells through stepwise base excision repair (BER) [OGG1 gene in cancer tissue [OGG1 polymorphisms and the risk of CRC [ROS can damage DNA in different pathways. However, 8-oxoguanine (8-oxoG) stands out as one of the most common DNA lesions observed as a result of exposure to ROS products . 8-OxoG ir (BER) ,10,11. Ar tissue . Furtherk of CRC ,14,15,16k of CRC . Hence, k of CRC ,19. Howek of CRC ,21.OGG1 gene in VAT in the context of cancer, ultimately contributing to DNA damage and increasing the risk of CRC. Then, our study aims to examine the gene expression and methylation of OGG1 in the adipose tissue of patients with CRC. In addition, we intend to evaluate the Ser326Cys OGG1 polymorphism to understand its association with obesity and cancer. Finally, we investigate an in vitro model in adipocytes to investigate the role of vitamin D in DNA repair.Therefore, we hypothesize that obesity could alter the expression and methylation profile of the p = 0.001). Furthermore, there were significant differences in biochemical variables related to glucose and lipid metabolism. Accordingly, patients with CRC had increased glucose levels compared to healthy participants (p < 0.001). Conversely, total cholesterol and LDL were higher in healthy participants in comparison with patients with CRC (p < 0.001). Furthermore, CRC patients also had lower triglycerides and HDL than healthy participants (p < 0.001). Finally, serum levels of 25-hydroxyvitamin D (25(OH)D) were lower in patients with CRC when compared to healthy participants (p < 0.001). OGG1 Ser302Cys polymorphism in all subjects are summarized in p = 0.023*) and in patients with CRC (p = 0.006 *), but not in healthy participants (p = 0.45). The minor allele frequencies of the OGG1 Ser302Cys polymorphism were 0.07 for all participants, 0.05 for the healthy participants, and 0.11 for patients with CRC.The frequencies of the OGG1 expression was upregulated in VAT patients with CRC compared to healthy participants (p < 0.01). We also found that OGG1 was upregulated in whole blood in patients with CRC compared to healthy participants (p < 0.05)  had a BMI ≥ 25 Kg/m2. Our analysis showed that healthy participants with Ow/Ob had higher OGG1 expression than lean healthy participants (p < 0.01). Nevertheless, no significant differences were observed between lean and Ow/Ob patients with CRC  . Furtherwith CRC B. The coOGG1 gene. Our analysis revealed that the promoter methylation of the OGG1 gene was higher in patients with CRC in comparison with healthy participants (p < 0.05)   C. Howeve < 0.05) D. The co= 0.039) . SurprisOGG1 and biochemical variables. We observed that OGG1 expression in VAT was negatively associated with 25(OH)D  and insulin levels  . OGG1 expression in whole blood was negatively associated with IL10  and positively associated with IL6 . Furthermore, OGG1 body methylation was negatively associated with IL10 expression  , LIG1 , PARP1 , WRN , MBD4 , CPT1ɑ , and TFAM expression . OGG1 body methylation was associated with CPT1ɑ expression   A. As for= 0.008) B. Finall= 0.031) C.OGG1 in the adipose tissue and the risk of CRC (OGG1 expression in VAT was positively associated with an increased risk of CRC (β = 7.21 (3.09), p < 0.05), explaining up to 30% of the variability. The area under the curve of this model was 0.686 (0.586–0.787)  was not associated with overall survival (p = 0.33). However, overall survival was associated with OGG1 expression in whole blood . High OGG1 expression was associated with worse survival compared to low OGG1 expression (p = 0.016) (OGG1 expression in adipose tissue was lower in late-stage cancer when compared with patients with early-stage cancer (p = 0.028)  . Howeversurvival  (p = 0.3= 0.016) D. We als= 0.028) .OGG1 Ser302Cys polymorphism and serum biomarkers for oxidative stress, such as Hb1Ac, transferrin, ferritin, and alkaline phosphatase (ALP). We did not observe significant differences between the CC (dominant group) and CG/GG (recessive group) groups (p = 0.066) (p = 0.029)  groups . However= 0.066) . In addi= 0.029) .OGG1 compared to explants from lean (p < 0.001) and obese healthy participants (p < 0.001)  .OGG1 expression of the treated explants compared to the controls (OGG1 expression when compared to the controls (p < 0.01)  (CYP24A1 compared to the controls (p < 0.001)  B. Furthe4A1 gene . In the ctively) . As for < 0.001) D.MBD4, PARP1, WRN, and LIG1 genes (p < 0.05) (MBD4 and LIG1 had higher expression in obese patients compared to lean participants and cancer patients (p < 0.05). Furthermore, PARP1 expression was increased in explants from cancer patients when compared to lean participants and those with obesity (p < 0.05)   . As for  < 0.05) . Finallyctively) .OGG1 in participants with obesity, as well as in patients with CRC. We found that OGG1 was upregulated and hypermethylated in adipose tissue from patients with CRC when compared to healthy participants, which was confirmed in adipose tissue explants. In addition, OGG1 expression was associated with the majority of genes in the BER pathways, suggesting a cooperative mechanism. Furthermore, gene expression in adipose tissue was linked to an increased risk of CRC, while gene expression in whole blood was a promising prognostic biomarker, with higher expression linked to worse survival. These findings revealed dysregulated mechanisms of BER genes in obesity-associated CRC, in which these genes could be potentiated in the obesity state associated with CRC.In the present study, we conducted a complete analysis of the transcriptional and epigenetic profiles of OGG1 gene in CRC. For instance, a study reported that OGG1 was upregulated in adenomas with severe dysplasia and adenocarcinomas compared to normal adjacent tissue [OGG1 expression was higher in 70 tumor tissue samples compared to matched adjacent tissue, which was consistent with our findings. Accordingly, we found that OGG1 was upregulated in adipose tissue from patients with CRC in comparison with normal participants, indicating increased OGG1 activity to mitigate the high levels of 8-oxo-2′-deoxyguanosine observed in the cancer context [OGG1 in tumor tissue from newly diagnosed patients with sporadic CRC when compared with controls [OGG1 expression was significantly downregulated in tumor samples (stage III and IV) compared to normal tissue and early-stage tumor samples [OGG1 expression could vary depending on confounding variables, as well as other clinical factors. It is important to consider these factors when interpreting the relationship between OGG1 expression levels and CRC.Several studies have reported the transcriptional profile of the t tissue . Furthert tissue  discover context . In contcontrols . In addi samples . TherefoOGG1 promoter methylation, we discovered that the OGG1 promoter was hypermethylated in adipose tissue from CRC patients compared to healthy participants, which was consistent with the findings of a study conducted by Slyskova et al. (2012) [OGG1 in 56% of tumors using microsatellite instability methods, although other methods did not further confirm these findings. It is important to note that the relationship between promoter methylation and gene expression is complex. In some cases, promoter methylation may actually be associated with increased gene expression, particularly if the DNA methylation affects the binding of specific repressor proteins [OGG1 gene was increased, along with an increase in gene expression. This finding is interesting because increased promoter methylation is typically associated with decreased gene expression. However, it is possible that the increased methylation in this case may affect the binding of key transcription factors that affect both chromatin accessibility and the initiation of transcription. However, future studies are needed to clarify this observation. However, only a few studies have reported the status of OGG1 methylation, on which it is relatively premature to make a statement.In terms of . (2012) . The latproteins . In thisOgg1 in mice seems to play a protective role against obesity. Accordingly, a study by Sampath et al. (2012) found that −/−Ogg1 mice had increased adiposity and insulin resistance compared to wild-type animals when exposed to a high-fat diet [OGG1 in the mitochondria had significant protection against obesity and adipose tissue inflammation when exposed to a high-fat diet, demonstrating a critical role in energy homeostasis [−/−Ogg1 mice preadipocytes differentiated more efficiently and accumulated more lipids than wild-type preadipocytes. In contrast, OGG1 overexpression significantly decreased adipogenic differentiation and lipid deposition in both preadipocytes from transgenic mice overexpressing human OGG1 and 3T3-L1 cells through a significant alteration in cellular PARylation, indicating a role in adipogenesis [OGG1 was upregulated in adipose tissue from participants with obesity in comparison with healthy participants and associated with insulin levels, indicating a role in obesity and insulin sensitivity in the human context. This difference might be due to the fact that the adipose tissue of people with obesity has an increased inflammatory profile. Accordingly, Ogg1-knockout mice had fewer inflammatory cells and lower expression of cytokines (including IL6 and NF-κB), implying that OGG1 may play a role in inflammation [OGG1 in protecting and/or resistance against endogenous obesity- and cancer-related oxidative stress, suggesting that this DNA glycosylase may be a promising target for developing DNA glycosylase inhibitors [OGG1 expression response [As for obesity, fat diet . Specifieostasis . Furtherogenesis . In our ammation . Previouhibitors . Howeverresponse .OGG1 in adipose tissue was associated with an increased risk of having CRC, according to our logistic regression, whereas high expression of OGG1 in whole blood was associated with an increased risk of worse survival when compared to low OGG1 expression. Upregulation of OGG1 in patients with CRC could indicate increased DNA damage. However, even with increased OGG1 activity, the repair of all of the DNA damage may not be completely achieved, leading to persistent genomic instability, which could increase the risk of cancer progression and metastasis. Most studies found an association between OGG1 polymorphisms and the risk of CRC. Accordingly, several polymorphisms have been associated with an increased risk of CRC, such as R154H [We also found that increased expression of as R154H , Arg46Glas R154H , or Ser3as R154H ,15. Howeas R154H ,36,37, was R154H  or no asas R154H ,40. ThesOGG1 expression in visceral adipose tissue was associated with 25-hydroxyvitamin D, we decided to test this relationship in vitro due to the role of vitamin D in oxidative stress and inflammation [OGG1 protein expression compared to those with vitamin D sufficiency [OGG1 in PBMC [OGG1 but increased OGG1 expression in adipocytes. This could be because the activation of CYP24A1 in explants was moderate compared to that in adipocytes, which was hugely increased. Therefore, our experiment with the explants might need more exposure to vitamin D to activate the expression of vitamin-D-targeted genes. Then, these findings can shed light on vitamin D’s role in DNA repair. However, more studies are needed to clarify the mechanisms behind this observation.Finally, given that ammation . A studyficiency . In cont in PBMC . In our Three hundred and ten participants from the “Virgen de la Victoria” University Hospital  were enrolled in this study. Two hundred and thirty were healthy participants, whereas eighty were patients with CRC. Healthy participants were included who underwent hiatus hernia surgery or a cholecystectomy. Patients with CRC underwent surgery with curative intent. The pathological specialist used colonoscopy and biopsy to diagnose patients with CRC. The weight of all participants was stable in the last three months prior to their participation in the study.The exclusion criteria were patients with diabetes mellitus, acute or chronic inflammatory diseases, renal and infectious diseases, and patients who had received treatment that altered their glucose and lipid metabolism, such as metformin or statins. Furthermore, patients who had changes in other metabolic parameters and who consumed more than 20 g of ethanol per day were excluded. All participants were anonymized prior to typing and gave their written informed consent. The study was performed in accordance with the guidelines of the “Declaration of Helsinki” and approved by the Ethics Committee of “Virgen de la Victoria” University Hospital .Blood samples were obtained from all of the participants, and serum samples were extracted by centrifugation at 4000 r.p.m. for 15 min at 4 °C. Fasting glucose, triglycerides, total cholesterol, and high-density lipoprotein (HDL) cholesterol levels were measured using the Dimension Autoanalyzer . We calculated low-density lipoprotein (LDL) cholesterol using the Friedewald equation . FastingEpiploic adipose tissue was obtained from healthy participants when they underwent hiatus hernia surgery or cholecystectomy. Epiploic adipose tissue from patients with CRC was obtained during surgery. Adipose tissue samples were frozen in liquid nitrogen until they reached the laboratory. Then, the samples were cleaned in sterile PBS, cut, and stored at −80 °C until analysis.OGG1, Hs.PT.58.38797078), nuclear factor kappa B subunit 1 , interleukin 1 beta , interleukin 6 , interleukin 10 , sirtuin 3 , DNA ligase 1 , poly-ADP-ribose polymerase 1 , Werner syndrome helicase , methyl-CpG binding domain 4 , carnitine palmitoyltransferase 1A , cytochrome P450 family 24 subfamily A member 1 , transcription factor A mitochondrial , and peptidylprolyl isomerase A  as an endogenous control. Gene expression was performed by real-time PCR with QuantStudio 6 Pro  using Premix Ex Taq™ (Probe qPCR) , according to the instructions of the manufacturer. Changes in gene expression were calculated using the 2−ΔCt method [Total RNA was isolated from VAT using an RNeasy Lipid Tissue Mini Kit according to the manufacturer’s instructions . cDNA was generated from 1000 ng of total RNA using a PrimeScript RT-PCR Kit  according to the manufacturer’s instructions. The gene expression quantification was carried out using a commercially available TaqMan technology primer/probe mix  for the quantification of 8-oxoguanine DNA glycosylase (OGG1 rs1052133 SNP was provided by the SNPedia Database System http://www.snpedia.com (accessed on 12 January 2023), dbSNP, and Ensembl [Genomic DNA was extracted from 200 µL of peripheral blood using a QIAamp DNA Blood mini kit  according to the manufacturer’s instructions, and from VAT using a QIAamp DNA Tissue Kit according to the manufacturer’s instructions . DNA purity was measured by the A260/A280 and A260/A230 ratios. The information on the  Ensembl . The SNPOGG1 gene, a genome-wide methylation analysis was conducted. To do this, high-quality genomic DNA samples (500 ng) in the adipose tissue from healthy participants (N = 25) and patients with CRC (N = 29) were treated with bisulfite reagent using the EZ-96 DNA Methylation kit . Subsequently, DNA methylation was analyzed by microarray assays using Infinium Human Methylation 450K bead chip technology . After obtaining data from the OGG1 gene, DNA quality was checked, normalized, and filtered using the minfi package [OGG1 gene were extracted from both the promoter region and the whole gene.To extract the whole methylation of the  package . After t3 cells/well in 6-well plates for three days under standard culture conditions. After this period, the expansion medium was replaced by means of induction (72 h) and adipogenic differentiation to adipocytes (18 days) for 21 days. After that, we treated adipocytes for each condition with vehicle (DMSO) and 0.5 µM of calcitriol for 24 h. The experiments were conducted in three independent replicates using N = 1.Human mesenchymal stem cells from adipose tissue  were seeded at a density of 15 × 10As for adipose tissue explants, epiploic adipose tissue from 3 healthy lean participants, 3 healthy participants with obesity, and 3 patients with CRC were used in this study. The adipose tissue was cut into 5–10 mg pieces to do this. Then, these pieces were incubated in PBS supplemented with 5% BSA for 30 min. After that, a serum-free medium supplemented with 1% P/S and 200 mmol/L of L-glutamine was added to the pieces and kept at 37 °C. For the vehicle, DMSO was added to the medium. For treatment, 1 µM calcitriol was added to the medium. After 24 h of treatment, RNA was extracted using the RNeasy Lipid Tissue Mini Kit  according to the manufacturer’s instructions . cDNA wa2 test. The Kruskal–Wallis test was used to evaluate the differences between groups for non-parametric variables. Pearson’s correlation coefficients were calculated for parametric variables, or Spearman’s correlation for non-parametric variables. Univariate and multivariate logistic regression models estimated hazard ratios to assess the association of the OGG1 methylation and expression with increased CRC risk. Analyses and graphical representations were created using R 4.2.1 software  and GraphPad Prism, and the significance threshold was set at p < 0.05.Continuous variables are presented as means ± standard deviations. The Welch (parametric) or Wilcoxon (non-parametric) two-sample tests were used to determine differences between healthy participants and patients with CRC. Differences in qualitative variables and the Hardy–Weinberg equilibrium (HWE) were tested using the χOGG1 expression and methylation were upregulated in adipose patients with CRC compared to healthy participants. OGG1 seems to be a risk factor for CRC, since increased OGG1 expression in VAT was associated with an increased risk of CRC, while high OGG1 expression in whole blood was associated with poor overall survival. Furthermore, calcitriol treatment increased OGG1 levels in adipocytes, suggesting a protective link between vitamin D and DNA repair in adipose tissue. Therefore, OGG1 is a promising biomarker in obesity-related CRC and can act as a protective factor against CRC. However, further studies are needed to understand this relationship better.


The four-subunit negative elongation factor (NELF) complex mediates RNA polymerase II (Pol II) pausing at promoter-proximal regions. Ablation of individual NELF subunits destabilizes the NELF complex and causes cell lethality, leading to the prevailing concept that NELF-mediated Pol II pausing is essential for cell proliferation. Using separation-of-function mutations, we show here that NELFB function in cell proliferation can be uncoupled from that in Pol II pausing. NELFB mutants sequestered in the cytoplasm and deprived of NELF nuclear function still support cell proliferation and part of the NELFB-dependent transcriptome. Mechanistically, cytoplasmic NELFB physically and functionally interacts with prosurvival signaling kinases, most notably phosphatidylinositol-3-kinase/AKT. Ectopic expression of membrane-tethered phosphatidylinositol-3-kinase/AKT partially bypasses the role of NELFB in cell proliferation, but not Pol II occupancy. Together, these data expand the current understanding of the physiological impact of Pol II pausing and underscore the multiplicity of the biological functions of individual NELF subunits. Followio Pol II . Consisto Pol II , 15, 16.o Pol II .via genetic ablation of various NELF subunits, especially NELFB. NELFB is essential for cultured cell proliferation and embryonic stem cell differentiation  and subjected them to functional assays including cell proliferation, transcription, and Pol II pausing in immortalized mouse embryonic fibroblasts (MEFs) . UnexpecA and B) (A). First, a previous crosslinking study identified several NELFB lysine residues in close proximity with the neighboring NELF subunits . Conversely, MT486 failed to bind to NELFE but retained at least its partial affinity for NELFA and NELFC (B and C–E). In addition to these alanine-substitution MTs, we also generated amino (N)- and carboxyl (C)-terminal truncated MTs of NELFB. The N-terminal truncation Δ-N78 was able to bind to NELFE, but not NELFA or NELFC (C). On the other hand, the C-terminal truncation Δ-C60 was co-IPed with NELFA and NELFC, but not NELFE (C). In the last mutational approach, we targeted the LXXLL sequence, a well-known motif that is critical for protein interactions (C). Thus, as summarized in Figure 1A, we generated a panel of NELFB MTs with the crippled ability of binding to specific NELF subunits.NELFB is defined as a common essential gene in the Cancer Dependency Map Project , A and BA and B) . HoweverA and B) A. First,subunits . We thersubunits . We assesubunits B and C–EFC B and , C–E. Inor NELFC C. On theot NELFE C. In theractions . Of the ractions C. Thus, Nelfbf/- MEFs, which contain a null and a floxed Nelfb allele. We then examined the ability of ectopic WT and MTs to support cell proliferation upon Cre-mediated deletion of the endogenous floxed Nelfb allele (D). As expected, these NELFB MTs were not able to stabilize the other NELF subunits in MEFs depleted of endogenous NELFB (F and G). Surprisingly, most of these MTs, except for Δ-N78, largely retained the ability of WT NELFB to support cell proliferation (E and F). In particular, MT486, which is devoid of its NELFE-binding ability, is indistinguishable from the WT protein in rescuing the proliferative defect of Nelfb−/− cells (E). Following infection of MEFs with the Cre-expressing virus, we were able to isolate single Nelfb−/− clones expressing ectopic NELFB MTs, including MT183, MT194, MT486, and Δ-C60 (H and 1G). Stable passaging of these clones as homogeneous cell populations further confirms the ability of the NELFB MTs to support long-term cell growth.Next, we ectopically expressed WT and various NELF complex–defective NELFB MTs  in b allele D. As expus NELFB , F and Gferation , E and F/− cells E. FollowNelfb−/− cells exhibited significantly lower levels of NELF subunits versus the WT clones . Nevertheless, these MT cell clones proliferated equally well compared to their WT counterparts . To determine whether the same was true for human NELFB, we used CRISPR-Cas9 in human breast cancer cells MDA-MB-231 to disrupt the corresponding C-terminal portion of human NELFB (hNELFB), which is over 90% identical to its mouse counterpart (A and B). Although the gene editing significantly destabilized the truncated hNELFB (Δ-C60) and the other hNELF subunits (C and D), the resulting MDA-MB-231 cell clones proliferated as robustly as the WT control (E). Collectively, our mutational study strongly suggests that an intact NELF complex is dispensable for NELFB-dependent cell proliferation.Stable clones of Δ-C60–expressing nterpart , A and Bsubunits , C and D control E. CollecA and A and B) (A and C), thus further validating the ChIP-seq specificity. Second, compared to WT cells, there was a substantial decrease in global Pol II density in all three MT MEFs examined (B and C and C–E). However, Pol II binding in Δ-C60 cells was reduced proportionally at both the transcription start sites (TSS) and GB (D and E), resulting in negligible changes in the Pol II traveling ratio (TR) (F). In contrast, MT194 and MT486 MT cell clones displayed a preferential reduction of Pol II binding at TSS over GB (D and E), giving rise to significantly diminished TRs (F). This suggests a multi-step nature of the NELF-dependent effects on Pol II movement. It is also worth noting that MT486, which lacks NELFE binding, was less defective in Pol II occupancy than MT194 (D and E). Furthermore, unlike MT194, MT486 also retained significant chromatin binding at TSS (G), supporting the notion that disruption of the NELFB-E interaction is less consequential than that of NELFA-C-B. This is in line with the previous finding that the NELFE tentacle is not required to stabilize NELF complex at the TSS  to assess the impact of various NELFB MTs on Pol II pausing. First, we confirmed that chromatin binding of ectopic WT NELFB as detected by flag-based ChIP-seq was concordant with our previously published finding from ChIP-seq of endogenous NELFB Figs. 2A and B) . As expeA and B) , A and CA and B .) and GB , D and Etio (TR) F. In con over GB , D and Eshed TRs F. This san MT194 , D and Eg at TSS G, suppor the TSS .Figure 2H). Upon rapid depletion of endogenous WT NELFB, ectopic Δ-C60 failed to stabilize the other NELF subunits (F) or support their chromatin binding at TSS (I and J). Likewise, unlike ectopic WT NELFB, Δ-C60 did not restore overall Pol II occupancy at either TSS or GB in NELFB-depleted cells (G–I). Despite the total loss of its Pol II–pausing capability, Δ-C60 still supported cell proliferation as effectively as ectopic WT NELFB upon rapid depletion of endogenous NELFB (J). Taken together, these data clearly show that NELFB-dependent Pol II pausing and cell proliferation are functionally separable.To rule out caveats associated with possible compensatory changes in stable MT cell clones, we established an auxin-induced protein degradation (AID) system in MEFs H. Upon rsubunits F or suppg at TSS , I and Jed cells , G–I. Deus NELFB J. Taken versus Δ-C60–expressing MEFs (K), despite the lack of NELF chromatin binding and the compromised Pol II occupancy in Δ-C60–expressing KO cells (Nelfb (D). Consistent with previous studies . As expected, ectopic NELF WT in KO cells rescued expression of most KO-affected genes . Of note, neither MT194 nor MT486 MTs displayed any obvious preference in transcriptional rescue toward NELFB-bound genes in Nelfb KO MEFs (H), which is consistent with the lack of chromatin binding for MT194 (F). These data strongly suggest that NELFB-dependent transcriptome in MEFs does not entirely depend on the integrity of NELF complex, NELF chromatin binding, or Pol II pausing at TSS.Given the well-documented role of NELFB in transcriptional regulation, we asked to what extent the separation-of-function MTs could support NELFB-dependent transcriptome. Surprisingly, our initial transcriptomic study showed little difference between stable clones of WT NELFB ing MEFs K, despitKO cells . To valis . In contrast, MT486, which retained binding to NELFC and chromatin, was predominantly localized in the nucleus . Despite their subcellular mis-localization, the cytoplasmic MTs (MT183 and MT194) were competent in supporting cell proliferation (E and S5A). This is consistent with the notion that the Pol II pausing function of nuclear NELFB is not absolutely required for NELFB-dependent cell proliferation.Our data so far suggest a nongenomic function of NELFB. In support, super resolution 3D–structured illumination microscopy imaging showed that while WT NELFB was mainly present in the nucleus, a fraction of it was also detectable in the cytoplasm . Because MT194 lacks NELFC binding but still binds to NELFE, we hypothesized that NELFC could be responsible for nuclear retention of the NELFB-E subcomplex. To test this, we established a NELFC-AID cell line that can be induced for rapid NELFC degradation (B). Acute NELFC depletion led to accumulation of cytoplasmic NELFB and NELFE and concurrent reduction of their nuclear pools (D). The effect of NELFC depletion on stabilizing NELFB persisted 3 days after incubation with auxin (C). These data strongly suggest that NELFC is required for nuclear recruitment and/or retention of NELFB-E. However, cytoplasmic NELFB is likely sufficient to support cell proliferation.Compared to their WT counterparts, MT194-expressing MEFs displayed enriched cytoplasmic NELFE, but not NELFA or NELFC C. Becausradation B. Acute ar pools D. The efth auxin C. These A). These NELFB-specific co-expressing genes are enriched in endocytosis, neurotrophin, and ErbB signaling pathways . Based on a functional prediction method from GeneMANIA (D). Using apurinic/apyrimidinic endodeoxyribonuclease 2 (APEX2)-mediated proximity labeling . Consistent with our co-IP result, NELF MTs defective in NELFC binding and mis-localized in the cytoplasm (MT144 and MT194) did not exhibit proximity with NELFA, NELFC, or Pol II . However, the same MTs retained the physical proximity with PI3Kα and AKT . Thus, our results strongly suggest that NELFB’s association with signaling molecules can be uncoupled from its interactions with its known nuclear partners involved in Pol II pausing.Recent work demonstrates that co-expression of genes is informative for functional interactions . The topFig. 5eneMANIA , these meneMANIA D. Using labeling , we deteNelfb WT and Cre/loxP-induced KO cells. There was a significant reduction in p-AKT, p-MEK1/2, and p-ERK1/2 in KO cells versus WT (A–D). Notably, NELFB depletion did not affect either mRNA or total protein levels of these signaling molecules (A and S7B), thus ruling out a direct role of NELFB in transcriptional regulation of these genes. Using the AID-based rapid degradation system, we found that reduction in phosphorylation of these signaling proteins occurred as early as 2 h after NELFB depletion . In contrast, rapid NELFC depletion resulted in increases in the phosphorylation intensity , likely due to increased cytoplasmic retention of NELFB (D). Despite their differential effects on the signaling molecules, both NELFB and NELFC depletion showed similar reduction of Pol II occupancy at TSS and GB (F and G and S7C). A similar effect of rapid NELFB degradation on p-AKT was observed in human cells (H and S1D). Lastly, we found that both WT NELFB and MT194, a MT predominantly retained in the cytoplasm, were capable of rescuing phosphorylation of these signaling molecules in Nelfb KO MEFs . Thus, our data strongly suggest physical and functional interactions between cytoplasmic NELFB and several progrowth signaling kinases, which can be genetically separable from the NELF nuclear function.To determine whether NELFB has any influence on the activity of these signaling molecules, we compared the phosphorylation status of several key signaling molecules in ersus WT , A–D. Noolecules A and S7Bof NELFB D. Despitan cells H and S1DA) .Myristoylation-mediated membrane recruitment is known to enforce activation of various cytoplasmic kinases including PI3Kα and AKT , 46. We Fig. 7A) . In addiA) , 30, 48.A). Therefore, the rescue of cell proliferation by Myr-AKT1 was unlikely due to augmentation of NELF expression. It is also noteworthy that although Myr-AKT1 fully restored phosphorylation of GSK3β, a direct downstream target of AKT, phosphorylation of MEK1/2 and ERK1/2 was still compromised in NELFB-depleted cells with Myr-AKT1 overexpression (A). Furthermore, Pol II ChIP-seq showed that Myr-AKT1 overexpression in NELFB-depleted cells did not restore the loss of Pol II occupancy at either TSS or GB (B and C). Thus, the rescuing effect of Myr-AKT1 on cell proliferation was not due to restoration of NELF-dependent Pol II pausing.Ectopic expression of Myr-AKT1 in NELFB-depleted MEFs did not increase the abundance of NELFB or the other three NELF subunits A. Therefpression A. FurtheSS or GB , B and CC and D). Ectopic expression of either WT NELFB or Myr-AKT1 substantially reduced this population of cells (C and D), suggesting that AKT1 acts downstream of NELFB to promote cell cycle progression. Taken together, our results support the notion that the function of NELFB in supporting cell proliferation is at least partially mediated by key signaling molecules in the PI3K/AKT pathway.We previously reported that NELFB-depleted cells exhibited defects in cell cycle progression in multiple phases including S-phase DNA replication . Consistof cells , C and DNelfb KO cells. Therefore, by genetically uncoupling NELFB’s function in cell proliferation from its role as part of an intact NELF complex in Pol II pausing, our findings highlight the multifaceted nature of NELFB and thus call for the need to reevaluate the preponderant Pol II pausing–centric paradigm in NELF biology.Biochemical and genetic approaches are complementary to each other in elucidating the functional relationships in biological processes . A wealtD) . F. FD) . This nongenomic activity of NELFB, which could be evolutionarily more conserved than its Pol II pausing activity, may also contribute to various context-dependent functions of NELFB in multicellular organisms in vivo.Our data strongly indicate that cytoplasmic NELFB supports cell proliferation by physically associating with and activating progrowth signaling molecules such as PI3K/AKT. This is distinct from the previously reported role of NELF in transcription of the genes involved in the MAPK pathway  for the KO cells A. This nPaused Pol II is considered as an equilibrium between Pol II recruitment, early termination, and release . NELF, tE). NELF subunits are known to undergo dynamic shuttling among different subcellular compartments (E) and a yeast exportin1–like domain in NELFB, following its dissociation from NELFC . C-terminal 3× flag-tagged mouse NELFB was introduced into the pLenti-CMV-GFP-neo (Addgene: 17447) backbone. NELFB truncations and site-specific point mutagenesis were generated using PrimeSTAR GXL DNA Polymerase  and different sets of primers  and S10.Nelfbflox/- mouse embryonic fibroblasts (iMEFs) were described previously  was introduced into Nelfbflox/- iMEFs by lentiviral infection and selected with 800ug/ml G418 (Gibco: 10131027). The established cell lines were used in the Cre-induced NELFB MT-swapping experiments. After removing the endogenous floxed Nelfb allele by Adeno-Cre, single colonies were isolated and propagated from WT and MT stable cell lines.Nelfbflox/- iMEFs via lentiviral infection and selected in media containing 5ug/ml Blasticidin (Gibco: A1113903) and 2ug/ml puromycin (Gibco: A1113803). Single colonies were isolated upon deletion of the endogenous floxed Nelfb allele by Adeno-Cre. To establish NELFB MT stable cell lines on top of NELFB-AID parental cells, cells were further infected with lentiviruses expressing NELFB, APEX2-NELFB, or the following MTs: Δ-N78, Δ-C60, MT144, MT183, MT194, APEX2-MT144, or APEX2-MT194. To establish Myr Kinases stable cell lines on top of the NELFB-AID parental cells, cells were infected with retroviruses expressing the following kinases: AKT1, PKM2, RPS6KB1, AKT3, PIK3CB, PIK3CG, CDK9, MAP2K7, PAK4, PDK1, and NTRK3 .To establish the NELFB-AID system in MEFs, NELFB with C-terminal mini-AID-tag (pLenti-mNELFB-AID-BSD) and OsTIR1-mcherry (pCDH-OsTIR1-mcherry-T2A-puro) were cointroduced into For NELFC-AID cells, human NELFC with the mini-AID tags (pLenti-HNELFC-AID-BSD) was introduced into NELFB-WT iMEFs or NELFB-Δ-C60 iMEFs, together with OsTIR1-mcherry (pCDH-OsTIR1-mcherry-2A-puro). Single colonies were isolated upon deletion of endogenous NELFC gene by Cas9 and sgRNA transfection .NELFB gene by Cas9 and sgRNA transfection  was first introduced into OsTIR1 stable MDA-MB-231 cells. Single colonies were then isolated upon deletion of the endogenous sfection .g for 4 h with 8 ug/ml polybrene. For adenoviral transduction, 1.5 × 105/well cells were seeded in a 6-well plate and adenovirus  was added at 100 pfu/cell in the media for overnight at 37 °C.The lentiviral infection procedure was described previously . BrieflyA450 reading was taken using BioTek Synergy H1 every 24 h for 4 days. A450 readings were processed using GraphPad Prism https://www.graphpad.com/. Doubling time was generated using nonlinear regression of Exponential  growth.MEFs or MDA-MB-231 cells were seeded in 96-well plates at a concentration of 1500 cells/well. Auxin, if indicated, was added at a final concentration of 0.5 mM after cells were attached to the plate and incubated for a period of time as indicated. Cell Counting Kit-8 (Dojindo: CK04) was diluted 1:10 with 10% fetal bovine serum Dulbecco's modified Eagle's medium media and incubated with cells for 1 h. The following antibodies were used in our study. NELFB , NELFB , NELFC , NELFE , NELFA , flag , Tubulin , PI3 Kinase p110α (C73F8) , PI3 Kinase p110γ (D55D5) , Akt (pan) (C67E7) , p-Akt (Ser473) (D9E) XP , p-GSK-3β (Ser9) (D85E12) , GSK-3α/β (D75D3) , p-MEK1/2 (Ser217/221) , ERK1/2 , p-ERK1/2(Thr202/Tyr204) , ATP5B, LaminB1, GAPDH , ACTIN , Vinculin , and Streptavidin-IRDye 800CW . Secondary antibodies include IRDye 680RD Goat anti-rabbit immunoglobulin G , IRDye 800CW Goat anti-mouse immunoglobulin G , and Goat anti-rabbit immunoglobulin G (H + L)-horseradish peroxidase .Protein samples were prepared using Laemmli buffer with protease/phosphatase inhibitors . After lysis, protein concentration was quantified using a bicinchoninic acid protein assay kit . SDS-PAGE and Western blotting procedure were described previously  following the manufacturer’s instructions. GAPDH, ATP5B, and LaminB1 were used as cytoplasm, membrane, and nuclear markers, respectively.g for 10 min. Lysates were then incubated with flag M2 magnetic agarose beads  at 4 °C overnight. Immune complexes were collected using a magnetic separator and washed three times with lysis buffer. To elute immunoprecipitated proteins, 3× flag peptide  was diluted at a final concentration of 200 ng/μl in Tris-buffered saline  buffer and incubated with beads for 30 min at 4 °C. The eluted lysates were analyzed by SDS-PAGE and Western blots as described.Whole-cell lysate was obtained by lysing cells in 500 ul NP-40 lysis buffer  with a protease/phosphatase inhibitor cocktail . Lysate was kept on ice for 15 min. Insoluble material was removed by centrifugation at 10,000Total RNA isolation was performed using a RNeasy Plus Mini kit . For mRNA-Seq (with PolyA selection), libraries were constructed and sequenced at the University of Texas Health San Antonio Genome Sequencing Facility using 50 bp single-read sequencing. For RT-quantitative polymerase chain reaction (qPCR), RNA was reverse-transcribed into complementary DNA using a Maxima H Minus First Strand cDNA Synthesis kit . Each cDNA sample was amplified using Thermo Fisher Scientific Luminaris Color HiGreen qPCR Master Mix, high ROX  in CFX384 Touch Real-Time PCR Detection System  following the manufacturer’s instructions and using specific primers . RelativNelf-b null background were generated as described above. 1.5 × 105 cells were seeded in a glass-bottom dish 35 mm  for overnight at 37 °C. Cells were then fixed using 4% paraformaldehyde/PBS at room temperature (RT) for 15 min and permeabilized with 0.5% Triton X-100/PBS for 10 min. Permeabilized cells were then blocked in blocking buffer (PBS with 10% goat serum) for 1 h at RT and incubated with 1:50 NELFB antibody  at 4 °C overnight in blocking buffer. Cells were washed three times, 10 min for each with PBS, and incubated with an Alexa 568–conjugated anti-rabbit secondary antibody  in the blocking buffer for 1 h. Cells were washed three times for 10 min in PBS before stained with 4′,6-diamidino-2-phenylindole (DAPI) 0.1 μg/ml in PBS for 10 min. DAPI was then removed and cells were imaged using Nikon Eclipse Ni fluorescent microscope.WT, MT183, MT194, and MT486 MEFs in 2, 37  °C. Image rendering was performed using ImageJ (https://imagej.net/ij/) (NIH). The 3D-structured illumination microscopy microscope was custom-built by the Chung Lab on a Zeiss Axio Observer inverted microscope platform with an ASI motorized stage and a 100×/1.46 NA oil immersion Zeiss objective (Alpha Plan-APO). NELFB excitation was performed at 568 nm and 405 nm excitation for DAPI, respectively. Sequential excitation at the two wavelengths was enabled by a software-controlled filter wheel (Finger Lakes Instrumentation). Excitation grating patterns at each wavelength were generated by a spatial light modulator (Forth Dimension Display) as previously described  with an iXon DU-897-BV monochrome CCD (Andor Technology) was used. The imaging environment was maintained at 5% COescribed . Fluoresescribed , 60.g for 10 min then diluted with ChIP-lysis buffer  with protease inhibitors. Antibodies used for ChIP include anti-Pol II  and anti-flag , NELFC , NELFE . Diluted chromatin was incubated with antibodies at 4 °C overnight. Dynabeads Protein A and Protein G  was added the following day and incubated for 2 h at 4 °C. After incubation, Dynabeads was washed as previously described  with protease inhibitors  was added to the cell suspension and incubated on ice for 10 min. Chromosomal DNA was fragmented with micrococcal nuclease  at 50 unit/ul and incubated at 37 °C for 15 min. The reaction was stop with EDTA and EGTA at 5 mM each and sonicated using Qsonica Sonicator Q500 for 40 s with 0.5 S on, 0.5 S off at 40%. Supernatant was collected by centrifuging at 14,000escribed . Samples2, 0.5 mM MgCl2) three times and then incubated with or without 1 mM H2O2 at room temperature for 1 min. The reactions were then stopped and washed with quenchers  in PBS . Cells were then harvested and lysed by 1 ml lysis buffer  containing protease inhibitors and quenchers. 50 ul of streptavidin agarose beads  slurry was washed and incubated with cell lysates on rotation at room temperature for 1 h. The beads were subsequently washed with radioimmunoprecipitation assay buffer, 1 M KCl, 0.1 M Na2CO2, 2M urea in 10 mM Tris–HCl pH 8. After a final wash with radioimmunoprecipitation assay buffer, beads were eluted with 200 mM 1,4-DTT and 10 mM biotin with 4× lithium dodecyl sulfate buffer (Thermo Fisher Scientific: NP0007) for 10 min at 95 °C. Cells without H2O2 exposure were served as negative control. The streptavidin pull-down lysates and input were analyzed by Western blotting.Proximity labeling by APEX2 was carried out following a published protocol . Five miCells were first incubated with 10 μM EdU in cultured medium at 37 °C. After 30 min, cells were washed, trypsinized, harvested, and processed following the manufacturer's instructions . Samples were then analyzed using BD Celesta Cell Analyzer.p-value (p-adj) less than 0.05. These differentially expressed genes were used for further Gene Ontology term enrichment analysis by R package clusterProfiler  and restored by WT NELFB (WT-Cre versus EV-Cre), we defined them as NELFB-dependent gene. The relative restoration for this gene was set as 100% in WT-Cre and 0 in EV-Cre. To calculate the GRC, the relative restoration of this gene in either MT194-Cre or MT486-Cre was calculated and normalized with the relative restoration in WT-Cre, using the following formula:We used the method below to calculate the gene rescue capability (GRC) for MT194 and MT486 in Figure 3The GRC is calculated for all NELFB-dependent genes in each MT. Next, we draw a diagram to present the entire distribution of the GRC from 0% to 100%.Raw ChIP-seq data in FASTQ format was aligned to mm10 reference genome using bowtie2 . The SAMThe locations for TSS regions  and GB regions (3000 bp) were located from the GRCm38 annotation file. The TSSR, GB region, and associated gene information were stored in bed files separately. Next, the read counts were calculated from BAM files and only the reads with MAPQ score greater than 30 were chosen. The read counts were further normalized if the input file was available. Lastly, minimum ChIP signal at TSSR was set as 0.001 to filter the weak peaks and the TR was calculated based on following formula :TR=ReadChttps://depmap.org/portal/). The RNA expression data 21Q4 or 22Q1 were used for gene coexpression analysis. We obtained the top 1000 coexpressing genes from 1378 cell lines for NELFA, NELFB, NELFC, and NELFE, respectively. A Venn diagram was generated using UpSet Plot package to analyze the overlapping of coexpressing genes for different NELF subunits. Gene Ontology analysis was performed using web-based tool g:Profiler for top 1000 coexpressing genes of NELFB or coexpressing genes that are specific for each NELF subunit.The public available database is from Cancer Dependency Project  and GEO Submission (GSE205504).This article contains The authors declare that they have no conflicts of interest with the contents of this article.