Four RNA adenosine modifications, including m6A, m1A, alternative polyadenylation, and adenosine-to-inosine RNA editing, have been identified as potentially valuable in influencing colorectal carcinogenesis, immune infiltration, and response to drug therapy. However, the regulatory mechanisms and clinical significance of these four RNA modifications in ovarian cancer (OC) remain unknown.We comprehensively described the transcriptional and genetic modifications of 26 RNA modification “writers” in OC and assessed the expression patterns. We identified two RNA modification subtypes using an unsupervised clustering approach. Subsequently, using differentially expressed genes (DEGs) in both subtypes, we calculated RNA modification “writer” scores (RMW scores) to characterize the RNA modifications of single OC patients. RMW score-related gene expression was investigated by qRT-PCR. We explored the correlation between RMW score and clinical features, immune infiltration, and drug sensitivity. We drew a nomogram to more intuitively and accurately describe the application value of the RMW score.We found that molecular alterations in “writers” are strongly related to prognostic and immune-infiltrating features in OC patients. We identified two different clusters of RNA modifications. According to the immune infiltration characteristics in the two RNA modification isoforms, cluster A and cluster B can correspond to “hot” and “cold” tumors, respectively. With the median RMW score, we classified the patients into high- and low-score subgroups. A low RMW score was associated with good patient prognosis and lower immune infiltration. In addition, a low RMW score equated with a higher cancer stem cell index and a lower tumor mutation burden, which to some extent affected the sensitivity of patients to therapeutic drugs. Seven RMW score-related gene expressions were investigated by qRT-PCR in three OC cell lines. Compared to previously known models, our established RMW score has higher accuracy in predicting patient survival.A comprehensive analysis of four RNA modification patterns in OC reveals their potential value in OC prognosis, immune microenvironment, and drug sensitivity. These results could deepen our knowledge of RNA modification and yield fresh insights for new personalized therapeutic strategies. Ovarian cancer (OC) is a malignant tumor that grows on the ovaries, 90%–95% of which are primary . Becausevia their specific cellular compositions and perform critical functions in multiple biotic processes  and N1-methyladenosine (m1A) . These mrocesses . Among trocesses –11 and grocesses . Considerocesses , we set m6A is a methylation modification on the 6th nitrogen atom of adenine . m6A metm1A is a methylation modification that occurs on the first nitrogen atom of adenine. A high abundance of m1A modifications is present on tRNA and rRNA , 20. FurAPA is a widespread gene post-transcriptional regulation process in eukaryotes. Most APAs occur in the 3′UTR region . ThroughA-to-I type RNA editing is a fundamental biological phenomenon that is widespread in mammals and is considered a post-transcriptional modification mechanism capable of generating molecular diversity . It reguThe tumor immune microenvironment (TME) refers to the immediate ecological niche surrounding a tumor, consisting of various types of cells in the metabolic environment. TME contains a complex immune cellular environment that includes cells engaged in the innate immune response, such as natural killer (NK) cells and dendritic cells, and cells engaged in the adaptive immune response, such as T and B cells , 31. SomIn this study, we assessed expression levels and genomic alterations in 26 “writers” in OC specimens from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, and we compared expression levels with normal ovarian samples from the GTEx database. By comprehensively evaluating the two RNA modification patterns of OC samples, we revealed that RNA modification modes are related to not only tumor immune infiltration, which can correspond to different immune typing, but also cell proliferation and oncogenic mechanisms. Patients were then categorized into distinct gene clusters according to the expression profiles of differentially expressed genes (DEGs) in two RNA modification clusters. Considering individual differences in RNA modification, we calculated the RNA modification score to accurately quantitate RNA modification patterns in a single OC patient and proved that this score can correctly predict patient outcomes, immune characteristics, and treatment efficacy.The process of this research is illustrated in A total of 26 RNA modification “writers” were identified based on previous research . Table STo explore the biological functions between different RNA modification patterns, based on the “c2.cp.kegg.v6.2.symbols.gmts” gene set in the MsigDB database, with two RNA modification isoforms as phenotypic features, the Gene Set Variation Analysis (“GSVA”) package was used to determine biological process differences between different RNA modification .https://gdc.cancer.gov/about-data/publications/panimmune)  algorithm was applied to estimate the immune infiltration of each OC sample, and an enrichment score was used to indicate the degree of enrichment of each immune cell . The CIBnimmune) . ESTIMATnimmune) .Rosenberg et al. built a set of gene sets associated with biological processes, including epithelial–mesenchymal transition (EMT) markers, DNA damage repair, nucleotide excision repair, and CD8 T-effector signature –47. A coEmpirical Bayesian methods in the “limma” package were used for identifying DEGs of different RNA-modifying isoforms . A totalAll OC patients were equally randomized into training and test groups, and then RNA modification-related RMW scores were constructed using the training group. First, in the training set, a univariate Cox regression analysis of 1,641 DEGs identified 10 RNA modification “writers”-related genes significantly linked to prognosis (p < 0.001). The model fit was then minimized using the least absolute shrinkage and selection operator (LASSO) regression analysis . Finally−ΔΔCT method. The primer sequences are listed in TRIZOL reagent  was used to isolate total RNA from cell lines, and Revert Aid First Strand cDNA Synthesis kit  was used to synthesize cDNA. GAPDH was chosen as the internal reference. The relative expression of the target gene was estimated using the 22.The OC cell lines  were purchased from China Center for Type Culture Collection (CCTCC) and CRC/PUMC . The normal ovarian cell line (IOSE) was obtained from Shanghai Yaji Biotechnology Co., Ltd. All cells were maintained in Dulbecco’s modified Eagle medium (DMEM)  containing 10% fetal bovine serum (FBS)  in a humidified incubator at 37°C and 5% COThe “rms” package was used to integrate clinical characteristics and risk scores and draw a nomogram to visualize the relationship between variables in the prediction model . The calhttps://xenabrowser.net/datapages/).Cancer stem cell scores, including mRNA expression-based RNAs and DNA methylation-based DNAs, were designed to gauge cancer stem cell association . Scores The CellMiner database is based on the 60 types of cancer cells (NCI-60) listed by the National Cancer Institute’s Center for Cancer Research (NCI) . The NCISpearman’s and distance correlation analyses were performed to estimate correlation coefficients between the expression of RNA modification “writers” and immune infiltrating cells. Wilcoxon’s test was performed to analyze the variation between the two groups. Survival curves were drawn using the Kaplan–Meier (K-M) method, and a log-rank test was performed to determine the significance of the differences. ROC curve was performed to verify the validity of the model. p < 0.05 was considered statistically significant. All data were processed using R 4.0.1 software.Altogether, 26 RNA modification “writers” were included in this study , 28, 36.To reveal whether genetic variation in these “writers” in OC interferes with gene expression, we compared these “writers” expressions in normal and OC tissues. Compared with normal tissues, the number of “writers” with increased, decreased expression was approximately equal in OC samples, and the difference in expression of these genes in both tissues was essentially statistically significant . These gThe above analysis shows that the expression and genetic changes of RNA modification “writers” in normal and OC are highly heterogeneous, implying that RNA modification “writers” play a certain role in the occurrence and pathogenesis of OC.To better characterize RNA modifications in the development of OC, we integrated samples from TCGA-OC and GSE9891 cohorts and used them for further analysis. The Kaplan–Meier analysis revealed that 14 “writers” expression levels correlate with OC survival  and cluster B (n = 340)  and the external validation group (GSE26193) according to the formula and categorized the patients into two score groups , the greater the number of neoantigens in the tumor, and the better the patient’s susceptibility to immunotherapy . TherefoTo explore whether the RMW score can predict the immunotherapy response to ICIs, we used the TIDE score to model the two main mechanisms of tumor immune evasion and provide predictive outcomes for immunotherapy. We found that TIDE scores in the high-score group were much higher, suggesting that the low RMW score patients were more likely to respond to immunotherapy . From FiWe next selected chemotherapeutic agents available for OC to assess the sensitivity of the two groups to these agents. Interestingly, we found that with the exception of paclitaxel, the IC50 values of the rest of the drugs, including rucaparib, veliparib, vinblastine, cisplatin, docetaxel, and gemcitabine, were significantly elevated in the low RMW score group  can be found in the article/supplementary material.JL and XN conceived the study and participated in the study design, performance, and manuscript writing. CC, GC, and WD conducted the bioinformatics analysis. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Pleural mesothelioma (PM) is a cancer where epithelioid, biphasic and sarcomatoid histotypes are observed. Sarcomatoid PM is characterized by mesenchymal features. Multi-omics have been used to characterize the epithelial-to-mesenchymal (EMT) phenotype at the molecular level. We contribute to this effort by including the analysis of RNA editing. We extracted samples with the highest vs. lowest Epithelial score from two PM cohorts and observed increased RNA editing in introns and decreased RNA editing in 3′UTR upon EMT. The same was observed in primary PM primary cultures stratified by transcriptomics analysis into two groups, one of them enriched with mesenchymal features. Our data demonstrate that, as has been observed in other cancer types, RNA editing associates to EMT phenotype in PM. Pleural mesothelioma (PM) is the most common cancer arising from the mesothelial cell layer . TraditiBRCA-associated protein 1 (BAP1) status. In this study, our aim was to provide additional information on the molecular profile associated with different RNA editing patterns.Transcriptome heterogeneity can be further enhanced by RNA editing. A-to-I RNA editing is the most prevalent form of RNA editing in higher eukaryotes. It is catalyzed by enzymes known as adenosine deaminases acting on double-stranded RNA (dsRNA) (ADAR). More than 85% of human primary transcripts undergo RNA editing. A large proportion of these editing sites are within repetitive elements in untranslated regions (UTR) and introns of transcripts, such as Alu elements, which have the ability to form secondary dsRNA structures . The molWe have recently described  how unsuAltogether, these data indicate that the genomic distribution of RNA editing events varies with epithelial to mesenchymal transition (EMT).Of note, consistent with a low E-score being associated with a disrupted NF2/Hippo pathway , and theBAP1 alterations and the C2 group presents a mesenchymal phenotype [To further support the effect of different RNA editing patterns upon EMT, we next analyzed the rate of the different RNA editing clusters in the primary mesothelioma cultures that we have robustly classified into two groups (C1 and C2) with different molecular profiles, gene alterations and histology subtypes. The epithelioid histotype is found in both groups, and tumors classified in the C2 group are associated with a worse survival prognosis. The C1 group exhibits more frequent henotype . As for Consistent with the observation on low E-score, higher expression levels of ADAR2 were observed in C2 primary mesothelioma cultures .In this study, we provide evidence that higher levels of RNA editing in introns and lower levels of RNA editing in 3′UTR are associated with the mesenchymal pleural mesothelioma phenotype. The differential editing in these regions had been already associated with EMT in seven other cancer types , but hadlncRNA GAS5, CALB2 and RBM8A [NF2 [BAP1 [BAP1Δ splice variant are more sensitive to poly(ADP-ribose) polymerase inhibition by olaparib, indicating functional consequences of altered splicing [Increased editing in introns is inversely correlated with pre-mRNA splicing , includind RBM8A ,18,19, oM8A [NF2  and BAP1F2 [BAP1 . Splice splicing .Filamin B (FLNB), which was shown to be associated with the epithelial to mesenchymal phenotype in breast cancer [The contribution of splice variants to EMT has been described in lung cancer (reviewed in ) but hast cancer .In breast cancer patients, different cancer subtypes can be distinguished based on exon skipping splicing features within the four main subtypes, with the luminal subtypes closely connected and the basal subtype clearly separated . The subBAP1 [BAP1 is more common. The editing of specific features in 3′UTR can influence 3′UTR folding, potentially altering their interaction with RNA binding proteins. This has downstream biological consequences, as we have recently shown in PM [We had previously described how RNA editing in 3′UTR is higher in tumors with mutated BAP1 , and in wn in PM , where twn in PM .The implications of RNA-related processes in PM can be inferred from the observation that the DDX3X RNA helicase is mutated in some mesothelioma tumors . HoweverFuture studies will likely implement long-read sequencing of full-length cDNAs, including in single cells, enabling the detection of cell type specific isoforms and of aberrant splicing isoforms in cancer cells. These isoforms are occasionally translated, presented by HLA molecules, and recognized as neoantigens, as has been recently described in non-small-cell lung cancers . This imCOL5A2, ITGAV, and SPARC genes, which is correlated with mesenchymal phenotype in mesothelioma [https://ccsm.uth.edu/CAeditome/index.html, accessed on 10 January 2023), leading to, e.g., a loss of miRNA targeting, thereby potentially contributing to increased expression levels.Additional validation studies may include the analysis RNA editing and alternative splicing of the epithelioid vs. sarcomatoid regions within a biphasic tumor. Furthermore, it might be of interest to investigate the contribution of RNA editing in the increased expression of thelioma . The mRNIn conclusion, RNA related processes, and in particular RNA editing and by consequence RNA splicing, would deserve further investigations to precisely determine their contribution in the PM phenotype.n = 87) downloaded from the NCBI database of Genotypes and Phenotypes (dbGaP) in 2019, under phs000178.v10.p8; the Malignant Pleural Mesothelioma cohort from the Bueno study (n = 223), downloaded from the European Genome-phenome Archive (EGA) in 2020, under EGAS00001001563 (EGAD00001001915 and EGAD00001001916).Mesothelioma RNA-seq reads included in the analysis were: the TCGA- Mesothelioma cohort (n = 64) provided by Didier Jean’s team in 2022, for which RNA-Seq was performed as described in [https://gdc.cancer.gov/about-data/gdc-data-processing/gdc-reference-files, downloaded on 12 February 2020) using STAR (2.7.8a) with 2-pass mode. PCR duplicates were marked using Picard (2.22.8). Variants in the aligned, duplicate marked RNA-seq reads were identified using GATK (v3.8.1.0) following RNA-seq best practices workflows. In detail, mapping quality reassignment, splitting spliced aligned reads into multiple supplementary alignments and clipping mismatching overhangs were performed using “SplitNCigarReads” with options “-rf ReassignOneMappingQuality -RMQF 255 -RMQT 60 -U ALLOW_N_CIGAR_READS”. Base quality recalibration was performed using “BaseRecalibrator” with dbSNP release151_GRCh38p7 downloaded in 2018 as the true variant set. Variant calling was performed using “HaplotypeCaller” with options “-dontUseSoftClippedBases -stand_call_conf 20.0”. Called variants were filtered using “VariantFiltration” with the following options: -window 35 -cluster 3 -filterName FS -filter “FS > 30.0” -filterName QD -filter “QD < 2.0”. Variants known in dbSNP, and/or in genes encoding immunoglobulins were also filtered out using SnpSift (v4.3) and bedtools (v2.29.2), respectively. Filtered variants were annotated using SnpEff (v4.3) and GDC.h38 GENCODE v22 gene annotation. Percentages of A to G changes by genomic regions in the SnpEff csv summary file were extracted for sample clustering analysis in R (v4.1). In detail, pairwise Euclidean distances among samples were computed based on percentage values of A to G changes by genomic regions with R function “dist”.The method for the analysis of RNA editing clusters has been recently detailed . It is bribed in , was alsribed in  introducTo assign each primary PM sample to the molecular subtypes of the classification in two clusters (C1 and C2), a 3-gene predictor based on qRT-PCR measurements was used as previously described ,12.genecode.gene.info.v22.tsv”).ADAR1/2 gene expression values from aligned RNA-seq reads were computed using htseq-count (v1.99.2) with options “-a 10 -t exon -i gene_id -m intersection-nonempty”. For the stranded FunGeST dataset “-s reverse” was set, while “-s no” was used for all other non-stranded datasets. FPKM and FPKM-UQ values were computed using R (v4.1), where transcript length information was downloaded from GDC . Statistical analysis was performed using Prism 8 (Graphpad 8.0.0).


The COVID-19 pandemic could worsen adolescent sexual and reproductive health (ASRH). We sought evidence on the indirect impacts of previous infectious disease epidemics and the current COVID-19 pandemic on the uptake of ASRH in sub-Saharan Africa (SSA) to design relevant digital solutions.We undertook a literature scoping review to synthesize evidence on the indirect impacts of COVID-19 on ASRH in SSA per the Arksey and O’Malley framework and PRISMA reporting guidelines. We conducted the search on PubMed, Embase, Google Scholar, and ResearchGate in June and November 2020. We included all peer-reviewed, English-language primary studies on the indirect impacts of infectious disease epidemics on the uptake of sexual and reproductive health (SRH) in SSA.n = 13 [61.9%]) and family planning (n = 8 [38.1%]) outcomes. Although we sought out to assess all outbreaks, epidemics, and pandemics, the only relevant studies took place during the West African Ebola pandemic (n = 17 [80.9%]) and COVID-19 pandemic (n = 4 [19.0%]). One study (4.8%) highlighted adolescent-specific outcomes and condom use. Most studies found declined access to and utilization of facility delivery, antenatal care, family planning, and HIV care. One study noted an increase in adolescent pregnancies. However, other studies noted similar, or even increasing trends in access to and utilization of other SRH services  during epidemics. Barriers to SRH uptake included factors such as a reduced ability to pay for care due to lost income, travel restrictions, and fear of infection. Supply-side barriers included lack of open facilities, workers, commodities, and services. Community-based peer delivery systems, telemedicine, and transport services improved SRH uptake.We included 21 of 42 identified studies. Sixteen studies (76.2%) quantitatively assessed utilization and access to SRH during epidemics. Five studies  explored factors affecting SRH services. All studies focused on adult populations, most often on labor and delivery (Access to SRH services during epidemics among adolescents and young people in SSA is understudied. We found that no studies focused on SRH outcomes of abortion, emergency contraception, sexually transmitted infections, or cervical cancer. To improve access to and utilization of SRH during pandemics, we recommend the following; in terms of research, key standardized SRH indicators should be included in routine data collection, routine data should be disaggregated by age, gender, and geography to understand gaps in ASRH service delivery, and additional rigorous epidemiological and social-behavioral studies should be conducted. On implementation, community-based peer delivery systems and telemedicine, internet-based, and other technological solutions may better reach adolescent and young people in SSA.The online version contains supplementary material available at 10.1186/s13643-022-02035-x. Adolescents and young people face barriers to accessing sexual and reproductive health (SRH) services such as birth control, condoms, HIV/AIDS and sexually transmitted infection (STI) testing. Changes in health care, social policy, and household’s economic status due to infectious disease epidemics may further reduce access to SRH services by young people. We conducted a literature scoping review on the impacts of past epidemics on SRH to anticipate and mitigate the indirect impacts of COVID-19 on SRH among young people. We conducted a search of literature related to SRH services during infectious disease epidemics in sub-Saharan Africa (SSA) and found 21 studies. Included studies focused on adult populations with only one study specific to adolescents. Our review showed that utilization and access to labor, delivery, and antenatal services decreased dramatically during the Ebola outbreak with long-lasting detrimental effects. Barriers to care included increased costs of care, difficulty traveling distances due to lockdowns, fear of infection, and a lack of operating facilities, workers, supplies, and services. The evidence for adult populations suggests that adolescents and young people may face heightened challenges to accessing SRH services during epidemics which may lead to poor health outcomes. This review highlights key areas for future research programs and policies.Globally, adolescents and young people (AYP) bear a disproportionate burden of adverse sexual and reproductive health (SRH) outcomes , 2. Of tThe COVID-19 pandemic in Zambia and other LMICs, specifically prevention measures, are expected to exacerbate barriers to SRH services and contribute to poor health outcomes among AYP –13. PrioPrior observational and modeling studies suggest that essential health services may decline during the COVID-19 epidemic, resulting in larger negative impacts on morbidity and mortality . We undeWe conducted a scoping review driven to systematically map the literature on SRH services for AYP during public health crises. We aimed to describe gaps in research to guide further research, program, and policy opportunities during the ongoing COVID-19 pandemic . This reWe developed research questions to guide the scoping review. The objective of conducting this scoping review was to understand access and utilization of SRH services among AYP during health crises. However, due to lack of literature during initial searches, we expanded the study to include adult (≥ 25 years of age) populations. Our specific research questions were the following: (1) what is the landscape of access to, and utilization of, SRH services during COVID-19 and prior public health crises in SSA? (2) What factors have contributed to access to and utilization of SRH during COVID-19 and prior public health crises in SSA?We conducted a full systematic search of relevant indexed peer-reviewed publications from June 15 to 30, 2020. Given how quickly COVID-19 literature evolved, we conducted another search from November 24 to 30 in three academic databases: (1) PubMed/MEDLINE ; (2) EMBASE (Excerpta Medica dataBASE); (3) Google Scholar. In addition, we searched ResearchGate and reference lists of articles for additional relevant studies. Search terms included the following population: adolescents (10–19 years); young people (10–24); and the general population. SRH outcomes included sexual behavior; contraceptive use; pregnancy; labor and delivery; HIV/AIDS; STIs; and gender-based violence. In terms of context, the review included any studies that collected data during epidemics in SSA. The aim of this scoping review was to understand indirect impacts of public health crises, including outbreaks, epidemics, and pandemics. Direct relationships between public health crises and SRH were excluded from review. We have included the main search terms in Table Studies were included based on the inclusion and exclusion criteria highlighted in Table All relevant search results were exported into an electronic spreadsheet to manage and ensure completeness. Two reviewers (AM and MN) screened titles and abstracts with literature identification rules to ensure articles met the inclusion criteria. After screening, a review of full-text articles was conducted independently by both members to ensure the article met pre-determined criteria for inclusion. Discordant determinations were resolved through discussion and did not need a third reviewer. Reasons for not including an article were documented..The results of the search are reported below and presented in a Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) flow diagram in Fig. 1After articles were selected, the reviewers independently conducted data extraction of the following variables: year; author; abstract; country/region; population/sample; type of study; type of data collected; key findings on utilization and access to SRH; factors (barriers and facilitators) related to SRH; and other interesting findings related to the research questions.The team has summarized the findings below using the thematic areas that emerged through a priori themes informed by the research questions  and those arising from the literature .We identified 51 relevant studies across PubMed, Embase, ResearchGate, and Google Scholar platforms, which met the search criteria for assessing SRH services during epidemics. After removing duplicates, 42 records remained, of which 12 were excluded due to inaccessibility or lack of relevance. Of the 30 full-text articles reviewed, 9 did not meet the inclusion/exclusion criteria described in Table n = 17, 80.9%), and took place in West Africa . The most studied outcomes in quantitative studies (n = 16) were labor and delivery , family planning , antenatal care , and HIV . Study details including authors, the epidemic, location, population, setting, data collection, and outcomes are included in Table A summary of the included studies can be found in Table We have summarized the detailed results on access and utilization varied by SRH outcome in Supplementary Materials Of the 18 studies (16 quantitative and 2 mixed methods), many showed a decline in facility deliveries in the Ebola period compared to the pre-Ebola period , 35, 38.Similarly, mixed findings on maternal mortality emerged with two studies showing an increase , 35 and ANC services were dramatically reduced during the EVD epidemic compared to the pre-Ebola period , 31, 33.Between pre- and post-Ebola periods, new and continuing family planning visitations increased in health centers but decreased in hospitals . One stuTwo studies showed a decline in HIV-related facility visits in the Ebola period compared to the pre-Ebola period , 39. Howp < 0.03 [As aforementioned, we identified a gap in the literature in regard to adolescent-specific studies. One mixed-methods study revealed significant increase in the mean teenage pregnancies per chiefdom in Moyamba district of Sierra Leone during the Ebola outbreak (173 pregnancies) compared to the pre-Ebola phase (137 pregnancies), p < 0.03 . RespondSeven studies discussed barriers and facilitators affecting SRH utilization during epidemics. Table Across countries, the COVID-19 pandemic increased cost of medicines and supplies. A study noted that individuals working in the informal sector could not afford to buy medicine due to a lack of income after COVID-19 restrictions were imposed, while the health facility could not pay the higher costs of supplies . SeveralSeveral supply-side issues affected healthcare access and utilization including healthcare facilities closing and/or reducing hours during the EVD and COVID-19 epidemics , 32, 41.Some health system responses demonstrated promising facilitators for increasing access to SRH services during epidemics. During the COVID-19 outbreak in Kenya, phone consultations and an emergency phone number to access free taxi transfers at night addressed transportation difficulties for pregnant women . SimilarOverall, this comprehensive scoping review revealed the scarcity of literature on SRH services during epidemics in SSA. The studies covered two pandemics (Ebola and COVID-19) though there have been several other disease outbreaks, such as influenza, bubonic plague, cholera, yellow fever, meningitis, measles, rift valley fever, and polio in SSA . SimilarGlobally, governments are taking unprecedented measures to limit the spread of the COVID-19 virus, while health and social systems are struggling to cope with rising caseloads, supply chain bottlenecks, movement restrictions, and economic difficulties. In humanitarian/fragile settings and LMICs, where systems are already weak, the epidemic may cause more collateral and long-lasting damage without thoughtful and comprehensive SRH services. In a recent mathematical modeling study, Riley et al. 2020) estimated a 10% proportional decline in use of contraceptive methods in LMICs during the COVID-19 pandemic 20 estima.This review confirms that leaving SRH unaddressed amid a public health crisis impacts access and utilization during and after the epidemic. Many studies included in this scoping review showed that access to SRH services, notably facility delivery and antenatal care declined during the early and post Ebola outbreak phases in West Africa. However, some better-funded services such as HIV and family planning were more resilient. Methodological differences such as setting/sample , analysis techniques , and the number and types of SRH services create variability in observed magnitude and direction of impact. Health system context and temporality may also account for the observed differences. For instance, in Ethiopia, facility-level deliveries remained stable early in the epidemic, but gynecological emergency visits decreased; in Guinea health centers performing better than hospitals; and, in Sierra-Leone government facilities performed better than private, not-for-profit facilities during the peak, but worse during the slow-down of the Ebola epidemic , 26. FurThe findings from this scoping review led us to provide clear recommendations for SRH service delivery to AYP during pandemics as listed in Fig. Beyond data and research recommendations, we have outlined two key areas which may improve AYP health during epidemics. Firstly, we recommend leveraging existing community-based peer delivery systems to increase access to prophylactics, contraceptives, and ART. Our scoping review found that formal healthcare utilization decreased across several outcome areas, while simultaneously home-based services  increased. This was likely due to both demand-side issues , and supply-side issues . This finding is well-aligned with other calls for increased demand-generation and community-based activities alongside existing facility-based offerings, to improve AYP SRH access . SecondlSupport consistent documentation and representation of key SRH data elements with real-time feedback to make quality improvements Reduce methodological heterogeneity in assessing access and utilization of SRH services during epidemics, using “Strengthening the Reporting of Observational Studies in Epidemiology” (STROBE) statement Disaggregate routinely collected SRH data by age, sex, and geography to understand gaps in service delivery to sub-populations during pandemics , 53Additional studies should be rapidly implemented to capture information on SRH services that are not routinely recordedLeverage community-based peer delivery systems for prophylactics, contraceptives and ART/other chronic illnesses could increase access to essential services for AYP Telemedicine, internet-based and other technological solutions may be appropriate to reach AYP who may otherwise not have the means or the autonomy to access SRH services Figure This review had several limitations. We may have missed additional relevant studies through our inclusion of only peer-reviewed, English language, and full-text publications. For example, we did not have any studies related to the recent Ebola epidemic in DRC, which may have been a result of the peer-review inclusion criteria. Relatedly, though we searched for relevant articles several times throughout the study (in June and November 2020), some articles may not have been included in this analysis given how quickly and continuously the literature has evolved for COVID-19. Also, we did not assess the rigor or quality of these studies, indicating that this does not represent as rigorous of a process that would be expected for a systematic review. Most studies relied on routine facility-level data, which may have issues with data quality and completeness. Despite these limitations, our study highlights that SRH services will be disrupted and access to and utilization of services will decrease without deliberate efforts to address the needs of all seeking care, particularly AYP and adolescent females.Indirect effects of infectious disease public health crises can be long term. It is critical that support for access to and utilization of SRH services be maintained or, better still, improved during epidemics. Particularly, services which address the unique needs of AYP are markedly absent. Findings suggest that more data and research in SSA are needed to understand SRH access and utilization. Data should be disaggregated by age, sex, and urbanicity and account for methodological and cultural/contextual differences to quickly understand gaps and develop localized responses. Recommendations to improve AYP SRH access include leveraging existing community-based delivery systems and technological approaches to increase access, knowledge, and promote behavior change during epidemics. Without targeted efforts to improve access, adverse SRH outcomes will increase, reversing recent progress in SSA and LMICs.Additional file 1: Supplementary materials 1. Detailed quantitative Findings on Access and Utilization by SRH Outcome During Pandemics (N=18). Supplementary materials 2. Barriers and Facilitators Affecting Access and Utilization of SRH Services During Outbreaks, Epidemics and Pandemics


To date women are still underrepresented in disciplines like mathematics, computer science, natural sciences and technology, despite numerous efforts to reduce the gender gap in the field . MoreoveMoringa oleifera miRNAs on lipid metabolism. Furthermore, one methods article reports advances in endogenous RNA pull-down. Finally, the Research Topic culminates in a review on intracellular and extracellular transport of RNA organelles in physiological conditions and neurodegenerative diseases.The papers published in this Research Topic highlight diverse areas of RNA research in which women play a central role. Three original articles describe experimental work on transcriptome changes in myotonic dystrophy type 1 (DM1), the role of extracellular vesicle miRNAs and tRNAs in synovial fibroblast senescence, and the effects of Wijesinghe et al. combine transcriptomic and proteomic approaches with computational analysis to identify differentially expressed sncRNAs and differentially abundant proteins in senescent EVs compared to non-senescent EVs in the context of osteoarthritis (OA). Several differentially expressed miRNAs and tRNAs are identified in EVs isolated from human synovial fibroblasts and pathway analysis unveils their involvement in senescence, fibrosis and ageing as well as osteoarthritis. Functionally, the authors show that senescent synovial fibroblasts can induce senescence in surrounding cells through the release of senescence associated EVs, and that treatment of OA synovial fibroblasts with senescent EVs may cause a transcriptional switch from arthritic synovial fibroblasts to senescent synovial fibroblasts that results in reduced synthesis of pro-inflammatory and extracellular matrix molecules, potentially through the downregulation of CRE and NF-κβ signalling pathways.Nearly two decades ago it was discovered that RNA is not only found in the nucleus and specialized regions of the cytoplasm of living cells, but also in extracellular vesicles (EVs) that are released by many cell types into the extracellular space . VariousRoglia et al. report a novel example of such cross-kingdom interactions, involving Moringa oleifera, a plant native to tropical forests of India and food additive, and two mammal species. Building on the knowledge that Moringa oleifera possesses lipid modulatory properties (Moringa oleifera miRNAs on lipid metabolism. Both treatment of a human liver cancer cell line (HepG2) with Moringa oleifera seed extract, as well as transfection of these cells with purified miRNAs appear to decrease intracellular lipid accumulation and induce apoptosis. Furthermore, in vivo administration of Moringa oleifera miRNAs in pre-obese mice prevent dysregulation of lipid metabolism. Further research to confirm that the miRNAs are the bioactive components of the Moringa oleifera extract is imperative if a new therapeutic approach based on plant miRNAs for the treatment of diseases such as obesity is to be considered.Exogenous miRNAs are increasingly being shown to regulate gene expression between different species . In partoperties , the autBraun et al. studied transcriptome changes in DM1. They found characteristic changes in gene expression with genes bearing CTG/CAG-repeats being differentially expressed between DM1 patients and controls. Furthermore, the pathophysiology of the disease is reflected and can be predicted by the differential gene expression across patients.The miRNA machinery is also involved in neurodegenerative diseases, for example, in DM1. DM1 is caused by pathogenic expansion of a CTG repeat in the DMPK gene. The expanded CUG repeat transcripts aberrantly recruit RNA-binding proteins culminating in cellular toxicity. These include proteins of the miRNA machinery, recruitment of which results in generation of aberrant small CUG RNAs. These small siRNA-like CUG-RNA molecules then can target CAG/CUG-containing RNAs. Here, Nabariya et al. that further elaborates on RNA dysregulation in CXG repeat expansion diseases. In greater detail, this review summarizes the role of intracellular RNA granules and extracellular vesicles such as exosomes in neurodegeneration. Intracellular RNA granules are important to fine-tune RNA localization, stability and translation and their aberrant regulation is connected to CXG repeat expansion diseases. RNA can further be transported between cells via exosomes, which can transfer pathogenic molecules of CXG repeat expansion diseases from cell to cell, thereby driving disease progression. The review further summarizes recent studies showing that cytosolic RNA granules and exosomes share common content. Therefore, intracellular RNA granules and exosome loading may be related.Besides the above-mentioned original articles, the research topic includes a review by Desideri et al. describe recent advances in endogenous RNA pull-down. While RNA pull-down is broadly used to study RNA-protein or RNA-RNA interactions in in vitro set-ups with in vitro transcribed transcripts as a bait, endogenous RNA pull-down assays are more relevant to identify intracellular binding partners of an RNA. Desideri et al. apply an endogenous RNA pull-down to a neuron-specific long non-coding RNA and show that both the yield of RNA-recovery and the specificity of detection of known interaction partners dramatically increase in the presence of Dextran Sulfate Sodium (DSS) salt. This modified experimental set-up may be valuable to study other RNA-RNA or RNA-protein interactions of biological significance. Furthermore, the review by Nabariya et al. provides a summary of established protocols for the isolation and characterization of exosomes and intracellular RNA granules.The research topic also contains information on different methods to study RNA interactions. We hope that further research topics will continue to celebrate the contributions of female researchers in the field of RNA research, promote their visibility and, in turn, help retain more women scientists and gender minorities in academia.


Here we report an additional, essential transfer factor of SGI1. This is a small RNA deriving from the 3′‐end of a primary RNA that can also serve as mRNA of ORF S022. The functional domain of this sRNA named sgm‐sRNA is encoded between the mpsA gene and the oriT of SGI1. Terminator‐like sequence near the promoter of the primary transcript possibly has a regulatory function in controlling the amount of full‐length primary RNA, which is converted to the active sgm‐sRNA through consecutive maturation steps influenced by the 5′‐end of the primary RNA. The mobilization module of SGI1 seems unique due to its atypical relaxase and the newly identified sgm‐sRNA, which is required for the horizontal transfer of the island but appears to act differently from classical regulatory sRNAs.The SGI1‐family elements that are specifically mobilized by the IncA‐ and IncC‐family plasmids are important vehicles of antibiotic resistance among enteric bacteria. Although SGI1 exploits many plasmid‐derived conjugation and regulatory functions, the basic mobilization module of the island is unrelated to that of IncC plasmids. This module contains the  oriT and two mobilization proteins, is unrelated to that of IncC plasmids. Here we report an additional, essential transfer factor encoded by SGI1, which is a small RNA. The mobilization module of SGI1 appears unique due to its atypical relaxase and the newly identified sRNA involved in the horizontal transfer of the island.The SGI1‐family mobilizable genomic islands and the IncA‐ and IncC‐family plasmids that specifically mobilize the SGI1‐like elements are major distributors of antibiotic resistance among enteric bacteria. The sophisticated interactions between SGI1 and its helper plasmid guarantee the efficient horizontal transfer and stability of SGI1 in the host cell. Interestingly, the basic mobilization module of SGI1, including  The most efficient distributors of resistance determinants are the conjugative plasmids and the integrative elements (IE), for example, the autonomous conjugative (ICE) and the nonautonomous mobilizable (IME) elements, which need a conjugative helper element (plasmid or ICE) for their horizontal transfer , followed by nicking either strand of the plasmid or IE DNA at the nic site of oriT by the relaxase. Subsequently, the relaxase and the covalently bound single‐stranded DNA are delivered to the type IV secretion system (T4SS) with the assistance of the membrane‐associated coupling protein (T4CP), which binds the cognate T4SS and the relaxosome complex. Finally, the transported DNA strand is recircularized and converted to the double‐strand form in the recipient cell  include HUH nucleases XK restriction enzymes, whereas the MOBT family enzymes belong to the Rep_trans nucleases , and TraI of the Neisseria genomic island GGI (MOBH) appear to be exceptional, which probably have adopted unique molecular mechanisms for DNA cleavage without forming covalent relaxase‐DNA intermediates , its variants, and related elements that form a large family of IMEs are often responsible for the MDR phenotype of human pathogens like Salmonella enterica serovars, Proteus mirabilis, Morganella morganii, Acinetobacter baumannii, Providencia stuartii, Enterobacter spp., Escherichia coli, or Klebsiella pneumoniae strains  and share a conserved backbone , the replication module     and several ORFs with unknown functions    , they are not essential for SGI1 transfer  have been identified  and the oriTSGI1, which is localized within the overlapping 3′ parts of S021 and S022  result was obtained as with SGI1‐CKOS021  deriving from pKK223‐3 and the resulting expression plasmid pJKI881 was used to complement the SGI1‐CKOS021 in a mating assay. For positive control, the entire mobSGI1 region was provided on the plasmid pMSZ949. In this assay, R55ΔTn6187, whose transfer functions are the same as that of the wt R55, was applied as a mobilization helper due to its reduced antibiotic resistance spectrum , which enabled us to apply the KmR complementing plasmids together with it. The results showed that SGI1‐CKOS021 could not be rescued by in trans expression of S021 protein, supporting the previous observations that S021 protein itself is not required for SGI1 transfer. In contrast, the entire mobSGI1 complemented the SGI1‐CKOS021 mutant, although its transfer frequency did not reach that of SGI1‐CWT  knocked out all the short ORFs that the KO S021 mutation might affect  had no deleterious effect as it caused only about twofold and fourfold reduction in the transfer rate of the complementing plasmid and SGI1‐CKOS021, respectively, compared with the positive control. This slight decrease was probably due to the partial withdrawal of MpsA and MpsB, which were now expressed only by SGI1 but utilized for the mobilization of both SGI1 and the approximately 15‐copy complementing plasmid. It is worth noting that this experiment proved again that the S021 KO mutation did not impair the expression of mpsAB operon as SGI1‐CKOS021 expressed MpsA and MpsB proteins in a sufficient amount for the transfer of both elements. Further extensive shortening of the 5′‐end of mobSGI1 fragment was not possible as deletion of mpsAB along with its 28 bp upstream sequence had no negative effect (pMNI71), but the removal of additional 34 bp (pMNI36) completely terminated the transfer of both the SGI1KOS021 and the complementing plasmid. On the other hand, longer DNA sequences could be deleted from the 3′‐end, which influenced the efficiency of complementation at various degrees. Removal of the last 169 bp of mobSGI1 had no significant effect (pMNI37), while larger deletions  abolished the mobilization of SGI1‐CKOS021, but the transfer of the complementing plasmids remained detectable (approximately 2 logs decrease in the transfer rate compared with that of pMNI37). The shortest fully active complementing segment (cloned in pMNI40) was deduced as 1,295–2,065 bp of mobSGI1. Its 5′‐end lies in the noncoding region (NCR) between mpsA and ORF S021 and the 3′‐end is 184 bp upstream of the START codon of S022 in the NCR between S022 and S023.The region of mob1 Figure . Since a2.3SGI1 fragment. Two promoter‐like motifs directed toward ORF S021 were found in the NCR upstream of S021 . In contrast, removal of the next promoter motif P5 caused almost 2 logs to decrease in the transfer rate, which could be fully restored by insertion of Pcat . Finally, the negative control plasmid pMNI41, which contained only the full‐length oriTSGI1, was not transferable. This confirmed again that MpsAB proteins along with the transfer apparatus of the helper plasmid are not enough for mobilization of oriTSGI1 and implied that the most important functional part of the required transfer factor is encoded in the region between mpsA and oriT. The results showed that the RNA factor is expressed mainly from P5 promoter, although P6 may also have a minor role. The 5′‐end of the fully active RNA lies in the sequence corresponding to the 1,966–2,012 bp region of mobSGI1; however, significant activity remains even after removing the large 5′ part of the RNA corresponding to the region between the promoter and oriT. In contrast, deletions affecting the sequences around the START of ORF S021  was inserted into a β‐galactosidase test plasmid pMSZ946 , while in SGI1‐C, the entire P5 was replaced with a cassette containing rrnBT1T2 terminators and the KmR gene (SGI1‐CΔP5)  Figure . The tra) Figure . The red) Figure  has a st2.5SGI1  or its 3′ truncated derivative lacking the downstream part of mobSGI1 from the 3′‐end of oriT (pJKI781). Both plasmids produced MpsAB proteins and contained intact ORF S021 (including the oriT), but in pJKI781, the P3–P6 region, the identified TSS and the sequence encoding the 5′‐end of the putative RNA was absent  or a truncated nonfunctional version of the presumptive RNA. The wt control pJKI1126 carried the mobSGI1 fragment identical to that of pMNI34, which proved previously transferable and could complement SGI1‐CKOS021, whereas pJKI1127 contained the slightly shorter fragment present in pMNI36, which was inactive in transfer and complementation . Every combination of the compatible test plasmids was introduced into the donor strain TG1Nal/R55ΔTn6187 and their transfer rate was measured in a mating assay. All plasmids (except c−) carried intact oriT, therefore, their transfer was detectable in the same mating. As expected, both plasmid partners were transferable (except c−), if the full‐length mobSGI1 was present in the p15A‐based plasmid pJKI780 (this part of the assay can be regarded as a positive control). The maximal transfer rate for pJKI780 was obtained in the presence of the nonmobilizable negative control (c−). This rate was slightly (but not significantly) reduced when pJKI780 was paired with pJKI1126 even though it could produce functional RNA. This observation can be explained by the competition between the two plasmids for MpsAB proteins and other components of the transfer apparatus that are provided by the low‐copy plasmid pJKI780 and the single‐copy helper plasmid, respectively. Titration of the mobilization proteins by oriT of the high‐copy plasmid leads to reduced levels of these components available for the low‐copy partner. Further decrease of pJKI780 transfer occurred when the high‐copy partner carried the 5′‐truncated nonfunctional mobSGI1 fragment (pJKI1127). In this case, competition exists not only for MpsAB and the transfer apparatus but also for the functional RNA produced only by the low‐copy pJKI780. The negative effect of the functional RNA deprivation was more obvious if the transfer of the high‐copy plasmids were compared: in the presence of pJKI780, the transfer rate of pJKI1127 producing the 3′‐truncated  RNA was about two orders of magnitude lower than that of pJKI1126  could only be mobilized if the longer, functional RNA was expressed by the high‐copy plasmid partner (pJKI1126). The transfer rate of pJKI781, however, did not reach that of wt mobSGI1‐bearing counterpart . These results indicated that deletions in the P5 promoter region and in the surroundings of the 5′‐end of S021 affect the same long RNA.The data accumulated so far not only excluded the participation of proteins in the establishment of the phenotype of S021 KO mutant but also explicitly suggested the existence of an RNA factor whose expression occurs mainly from P5 promoter located upstream of S022 Figures . The delt Figure . The pMBn Figure . The emp6 Figure . On the WT and the promoter mutant SGI1‐CΔP5 were compared. One of the probes was complementary to the 5′‐end of RNAs and hybridized near the TSS (5′‐probe), whereas the other overlapped the region affected by the KO S021 mutation and hybridized to RNAs that include sequences located at 470–720 bp from the TSS , confirming that the elimination of P5 promoter significantly decreased the transcription from this region. A 650–700‐base long RNA was detectable with both probes in the wt sample, supporting our results from the trans‐complementation test  can form a hair‐pin structure on the RNA and may be responsible for this effect.Therefore, the RNA species synthesized from P5 promoter region were analyzed by Northern hybridization, where the chromosomal SGI1‐Ct Figure . The 11‐repASGI1/SGI1‐CWT strain, and after joining the 5′‐ and 3′‐ends by T4 RNA ligase the circularized RNA was used as a template for reverse transcription. Then, the 5′–3′‐end junctions were amplified from the cDNA population with primers enclosing the ligation site , this assay suggested the existence of several alternative start sites. The 5′‐end of four RNAs . T4 RNA ligase can circularize only uncapped RNAs and the lack of 5′‐pyrophosphate cap makes RNA sensitive for degradation, which may explain the divergence observed in the 5′‐ends. The similar variance was detected in the other four clones, which started around the 1,957 bp position . The next group of transcripts started at or near 1,911 bp . The −10 box of the nearest predicted promoter P6 is located at 1,986–1,993 bp, thus it seems impossible that it could initiate transcription at a distance of 30 and 75 bp downstream. Even though the sequence of these clones supported the existence of two additional TSSs, promoters could not be found by in silico methods at the appropriate positions. Thus, these putative “TSSs” are formed rather by cleavage of longer transcripts at specific sites susceptible to the attack of endo‐RNases. Interestingly, two RNAs started upstream of the P5 TSS suggesting that weak upstream promoters could also initiate transcription with lower frequency. Nevertheless, the results clearly indicated that many transcripts initiated in the upstream region of S022 terminate in the critical region surrounding the START of ORF S021. These results also suggested that the primary transcripts undergo maturation steps, by which the biologically active transfer factor, called sgm‐sRNA , may be formed. It is worthy of note that the long primary RNA transcripts include the entire S022 ORF, which has an unambiguous SD‐box making its translation possible. Consequently, these RNAs can also serve as mRNAs for S022. Although the production and function of S022 protein were not directly examined, the involvement of this protein in SGI1 transfer was excluded , can be deleted from or replaced in the sgm‐sRNA without negative effects on SGI1 transfer  completely abolished pMNI41 transfer. In contrast, deletions directed from oriT toward the TSS did not, suggesting that the same sgm‐sRNA remained functional in these cases. SGI1‐CΔ7 containing the largest deletion, which removed the oriT, the entire S022, and its 5′‐UTR, was also active in complementation. However, it was about one to three orders of magnitude and less efficient compared with that of SGI1‐CΔoriT or SGI1‐CΔ2 . Considering these and the previous results  of the primary transcript. Mobilization of the oriT‐bearing pMNI41 by the inner deletion mutants of SGI1‐C  shows the best match to the σs Figure . This wa5 Figure , and the Figures  and S5.  Figures . Thus, wSGI1—that are per se not transferable—indicated that the deletion mutations affecting the 5′ and 3′ parts of mobSGI1 are in linkage, which anticipated an RNA spanning the downstream part of mobSGI1 . This sequence motif can form a stem‐loop structure on the RNA and may act as a termination signal or a cleavage site for endo‐RNases  have no serious effect on SGI1 transfer  terminates about 70–80 base from TSS of P5 Figure  and is pHybridization with the 5′‐probe revealed the presence of several further RNA species, whose length ranges between approximately 350 and 600 base and appear similar or less abundant than the 650–700 base long primary transcript. These derivatives can be formed also by early termination or site‐specific RNA cleavage causing 3′ truncation of the transcript and leading to the partial or entire removal of the core region required for the activity of sgm‐sRNA. Thus, most of these RNAs are probably not functional. In contrast, the 3′‐probe covering the core domain of sgm‐sRNA  noncoding RNAs synthesized in intergenic regions from their own promoters, but many of them are generated from 5′‐ or 3′‐UTRs or even from the coding regions of mRNAs by transcription or posttranscriptional processing  of the full‐length primary transcript possibly via posttranscriptional maturation, that is, RNase cleavages  was not necessary for the function, removal of the 1,877–1,966 bp of mob region caused a decrease in SGI1 transfer . This is consistent with the importance of sgm‐sRNA in the mobilization of SGI1‐like elements. SGI1‐B2 from Proteus mirabilis strain PmSC17 carries a large IS26‐based transposon inserted at the proximal end of the core domain of sgm‐sRNA , for example, between the 1,502 and 1,572 bp positions, where the 5′‐end of sgm‐sRNA was localized . The SmR/SpRSulRApSCmSFloSTcS segregant of ST1375, named ST21S/1 emerged during a passage experiment  according to  of donor and recipient strains grown in LB under selection for the chromosomal resistance markers and markers of all additional components  present in the donor strains were mixed, centrifuged for 1 min, washed with 0.5 ml 0.9% NaCl, and spread onto LB agar plates, which were incubated for 6 hr at 37°C. For compensation of the lower fitness of the donors (mainly based on the incompatibility of SGI1 and IncC helper plasmid and the genetic load due to the plasmid content), 3:1 donor/recipient ratio was applied in the matings. At the end of incubation, the bacterial lawn was suspended in 4 ml 0.9% NaCl solution, and 5 μl of serial dilutions was dropped onto selective LB plates to determine the titers of donor, recipient, and transconjugant cells. In cases of low transfer frequency , rare transconjugants were detected by spreading 100 μl (instead of dropping 5 μl) of undiluted bacterial suspension obtained from the mating LB plates. In cases where no transconjugants were detected, the detection limit was calculated as 1 transconjugant/ml divided by the donor or the recipient titer and this fraction was regarded as a threshold value of the detectable minimal transfer frequency in that assay. Titers of recipients and donors were determined by selection for their sole chromosomal marker  except in case of using the E. coli strain Tuner . The transconjugants were selected for the chromosomal marker of the recipient and the transferred marker of SGI1‐C or the test plasmid (TcSp or TcKm). The transfer frequencies were calculated as ratios of transconjugant/recipient and transconjugant/donor CFUs from at least four independent parallels (n ≥ 4).In the mating assays, r Figure  where, i4.3SGI1 in pFOL1372—delS021for‐delS021rev; SGI1‐CΔ2—deloriTfor‐delS022rev; SGI1‐CΔ5—delRNA3for‐deloriTrev; SGI1‐CΔ6—delRNA4for‐deloriTrev; and SGI1‐CΔ7—deloriTfor‐delS022upstream_rev  followed by religation in the case of pFOL1372 (resulting in pJKI773). In the KO mutant ORFs S019–S022, 83‐bp sequences near the 5′‐end of each ORF were replaced with an 84‐bp sequence deriving from the PCR template plasmid pKD3 after Flp‐induced deletion of the resistance marker.All KO mutagenesis were carried out according to the one‐step gene inactivation method . Then, SGI1‐CΔP5 was mobilized by R55ΔTn6187 into TG1Nal::repASGI1 host strain through the TcR TG90 strain in a two‐step mating process.For the generation of TG1Nal::SGI1‐C4.4E. coli TG1 harboring pMSZ946 or pJKI990 (as a negative control) using RNeasy Mini Kit (QIAgen) according to the manufacturer's recommendations. For Northern analysis, RNA was isolated from strain TG1Nal::repASGI1  was used for the extension, while Sequenase version 2.0 DNA sequencing kit (USB) was applied to generate sequence ladder for the test plasmid pMSZ946. Both kits were used as recommended by the manufacturers. The primers lacZoutE and pUCfor24 used for both reactions were labeled as described by Murányi et al. . Product4.6Five micrograms of total RNA samples were separated on a 5% or 8% denaturing TBE‐polyacrylamide gel containing 8 M urea 200 V (~12 mA). Gels were stained for 20 min in TBE buffer containing 0.5 μg/ml ethidium bromide, photographed next to a gel ruler under UV light, then electro‐transferred  and crosslinked onto Hybond‐N+ nylon membranes (Amersham) using the LKB 2117 Multiphore electroblotting unit and Amersham ultraviolet crosslinker, respectively. The template DNA for generation of the 3′‐ and 5′‐end hybridization probes were amplified using primer pairs T3_SGI_17741for–T7_SGI_17994rev and T3_SGI_18219for–T7_SGI_18628rev, respectively. The RNA probes were synthesized with T3 polymerase and the blots were hybridized and developed using the DIG Northern Starter Kit (Roche) according to the manufacturer's protocol.4.7repASGI1 , 40 units of RNase OUT (Invitrogen) in 1× T4 Ligase buffer (Thermo Fisher) in a final volume of 25 μl at 37°C for 1 hr. The enzymes were inactivated at 92°C for 10 min and the RNA was precipitated by 96% ethanol and dissolved in 10 μl nuclease‐free water. For reverse transcription, 20 pmole of SGI_S022promseq primer was added to 8 μl of the circularized RNA and annealed at 70°C for 5 min, then the mixture was cooled on ice for 1 min and used as a template for reverse transcription. The reaction was carried out in a final volume of 20 μl using the RevertAid H Minus First‐Strand cDNA Synthesis kit (Fermentas) according to the manufacturer's protocol. For the amplification of the 5′–3′‐end junctions, S022_promfor_Nc and S021promseq primers were used. The reaction mix contained 1 μl of cc. or 10× diluted reaction mix from reverse transcription or, as a negative control, 1 μl of the 4× diluted circularized RNA sample, 1μl dNTP mix (10 mM each), 10 pmol of primers, 1× DreamTaq reaction buffer (ThermoFisher), and 1.5 u DreamTaq polymerase supplemented to 2.5 mM MgCl2 in a final volume of 25 μl. The cycling conditions were as follows: initial denaturation at 94°C for 2 min, amplification in 35 cycles at 94°C for 20 s, 55°C for 30 s, 72°C for 40 s, and a final synthesis at 72°C for 7 min. The 250–300 bp range of the PCR product was isolated from the gel, digested with NcoI, and ligated into the NcoI‐HincII‐digested pGEM‐5Zf(+) (Promega). Individual clones were sequenced on ABI Prism 3100 Genetic Analyzer (Perkin–Elmer).This experiment was based on the method described by Kuhn and Binder . Five mi4.8Promoter motifs were predicted by BPROM (Solovyev & Salamov, J.K. conceived the project. I.N., J.K., M.S., and A.H. designed and carried out the experiments and analyzed the data. J.K. prepared the figures and wrote the paper and all authors reviewed the manuscript.Supplementary MaterialClick here for additional data file.Fig S8Click here for additional data file.


Escherichia coli cells. Nsp7 and nsp8 of the functional RdRp holoenzyme were generated in E. coli. An in vitro RdRp activity assay has been established with a reconstituted nsp12/nsp7/nsp8 complex and biotin-labeled self-priming RNAs, and the activity of the RdRp complex was determined by detecting binding and extension of RNAs. Moreover, to meet the needs of high-throughput drug screening, we developed a fluorometric approach based on dsRNA quantification to assess the catalytic activity of the RdRp complex, which is also suitable for testing in 96-well plates. We demonstrated that the active triphosphate form of remdesivir (RTP) and several reported non-nucleotide analog viral polymerase inhibitors blocked the RdRp in the in vitro RdRp activity assay and high-throughput screening model. This high-throughput screening model has been applied to a custom synthetic chemical and natural product library of thousands of compounds for screening SARS-CoV-2 RdRp inhibitors. Our efficient RdRp inhibitor discovery system provides a powerful platform for the screening, validation, and evaluation of novel antiviral molecules targeting SARS-CoV-2 RdRp, particularly for non-nucleotide antivirals drugs (NNAs).SARS-CoV-2 (severe acute respiratory syndrome coronavirus‐2), a member of the coronavirus family, appeared in 2019 and has caused the largest global public health and economic emergency in recent history, affecting almost all sectors of society. SARS-CoV-2 is a single-stranded positive-sense RNA virus that relies on RNA‐dependent RNA polymerase (RdRp) activity in viral transcription and replication. Due to its high sequence and structural conservation in coronavirus and new SARS-CoV-2 variants, RdRp has been recognized as the key therapeutic target to design novel antiviral strategies. Nucleotide analogs (NAs), such as remdesivir, is the most promising class of RdRp inhibitors to be used in the treatment of COVID-19. However, the presence of exonucleases in SARS-CoV-2 caused a great challenge to NAs; the excision of incorporated NAs will lead to viral resistance to this group of inhibitors. Here, we expressed active RdRp protein in both a eukaryotic expression system of baculovirus-infected insect cells and a prokaryotic expression system of  In the past two decades, there have been three major coronavirus infection events worldwide. SARS-CoV-2 (severe acute respiratory syndrome coronavirus‐2), as the most highly pathogenic human coronavirus compared to SARS-CoV and MERS-CoV , has cauSARS-CoV-2 is a positive-sense single-strand RNA virus with a ~30-Kb genome, including 14 open reading frames (ORFs), encoding 29 proteins. Replication and transcription of viral genome depend on the replication–transcription complex composed of an RNA-dependent RNA polymerase (RdRp) complex, nsp13 helicase , and sevEscherichia coli cells. With this reconstituted RdRp holoenzyme, we established RdRp activity assay in vitro to detect the binding and extension activity of RdRp complex to RNA primers for discovery of novel RdRp inhibitors. Moreover, we developed a fluorometric approach based on dsRNA quantification to assess the catalytic activity of the RdRp complex suitable for high-throughput screening (HTS). To investigate the reproducibility and reliability of this assay for expanded application in HTS, a pilot screen was conducted and the inhibitory effect of various RdRp inhibitors including nucleotide analogs and non-nucleotide analogs was evaluated. The results suggested that the newly established RdRp activity assay is suitable for the rapid and accurate screening of specific SARS-CoV-2 RdRp inhibitors.Here, we describe the expression and purification of active SARS-CoV-2 RdRp holoenzyme (nsp12/nsp7/nsp8) in both eukaryotic expression system of baculovirus-infected insect Sf9 cells and in prokaryotic system of Spodoptera frugiperda (Sf9) cells. After being amplified twice in Sf9 cells, 10 ml of recombinant baculovirus was used to infect 1 L of Sf9 cells at a density of 2–3×106 cells per ml in Sf-900 II SFM media (GenScript) and incubated at 27°C in a humidified chamber for 48–72 h. Cells were collected and suspended in binding buffer  at 4°C, and then lysed by sonication on ice. Cell lysate was centrifuged for 30 min at 13,000 rpm, and the supernatant and the pellet were collected, respectively. The supernatant of cell lysate was incubated with a Ni-NTA column (L00250/L00250-C) for 2 h. Then, the Ni-NTA column was washed with binding buffer until a baseline absorbance was achieved and protein was eluted with elution buffer . Fractions were analyzed by SDS-PAGE and Western blot. The high-purity fractions were pooled followed by 0.22-μm filter sterilization and stored at −80°C. Nsp12 gene was also cloned into a pGEX-6P-1 plasmid and expressed in E. coli fused to an additional GST tag at the N-terminus. The GST-nsp12 was co-expressed with molecular chaperone pg-Tf2 in LBBS medium . Bacterial cultures grew at 37°C in LB medium containing 100 μg/ml ampicillin and 20 μg/ml chloromycetin overnight and then were seeded in LBBS medium growing to an OD600 of 0.4. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (0.1 mM) and tetracycline (5 ng/ml) were added to induce protein expression at 16°C for 60 h. Cell pellets were collected and resuspended in GST binding buffer , lysed by MP FastPrep-24 5G . Lysate was cleared by centrifugation of 4,700 rpm for 30 min and filtered with a 0.45-μm membrane. After being bound to GSTrap™HP , protein was eluted with GST elution buffer . Eluent was desalted by PD-10 Desalting Columns  in protein storage solution . The purified protein was packaged in Ultra-clean workbench and then stored at −80°C.The gene sequence of the SARS-CoV-2 nsp12 was optimized for expression in different host cells and then cloned into a modified pFastBac baculovirus expression vector containing a 5’ ATG starting sequence followed by a His tag coding sequence . Recombinant bacmid was transfected in 6-nsp8 were expressed in E. coli (Ec). The full-length genes coding nsp7, nsp8, and nsp7-His6-nsp8 were synthesized  and cloned into pET-22b (+) or pET-21a (+) vector, respectively. For protein expression, the vectors were transformed into E. coli BL21 (DE3). Bacterial cultures were grown to an OD600 of 0.6 at 37°C, and then the expression of protein was induced with a final concentration of 0.1 mM of IPTG at 16°C for 20–22 h. Cells were suspended in binding buffer  and lysed by MP FastPrep-24 5G . Lysate was cleared by centrifugation of 4,700 rpm for 30 min and filtered with a 0.45-μm membrane. The filtrate was incubated with HisTrap™HP . After washing with binding buffer, the protein was eluted with elution buffer . Eluent was desalted by PD-10 Desalting Columns  in protein storage solution and stored at −80°C.Nsp7, nsp8, and nsp7-HisCells were lysed by FastPrep-24 5G  and centrifuged at 13,000 rpm for 15 min. Lysate was separated on 8% (nsp12) or 12%  sodium dodecyl sulfate-polyacrylamide gels and stained directly with Coomassie G-250 according to standard protocols. As for Western blot assay, lysates were separated on SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes . The membranes were blocked with 5% skim milk TBST for 120 min at room temperature, rinsed with TBST, and stained with antibodies against His tag  or GST tag  at 4°C for 12 h. After three rinses with TBST, membranes were incubated with HRP-conjugated secondary antibodies  for 120 min, and detected by Immobilon Western Chemiluminescent HRP Substrate  using the ChemiDoc™ Imaging System (Bio-Rad Laboratories).*/S2 RNA primer/template consisted of two short RNA oligonucleotides. One short RNA oligonucleotide with a sequence of 5’-bio-GCUAUGUGAGAUUAAGAAU U-3’ was used as the primer strand and another RNA oligonucleotide with a sequence of 5’-UUUUUUUUUUAAUUC UUA AUCUCACAUAGC-3’ was used as template strand. To anneal the RNA duplex, two oligonucleotides were mixed at equal molar ratio in annealing buffer , denatured by heating to 94°C for 5 min and then slowly cooled to room temperature. A long self-priming RNA contains a 20-nucleotide (nt) self-complementary base pairing region and a 20-nt RNA template (Ux20) with a sequence of 5’-Biotin-UUUUUUUUUUUUUUUUUUUUUUUUUUUUUUAACAGGUUCUAGAACCUGUU-3’ was used as the RdRp elongation assay template  for 30 min at 30°C. Samples were resolved on 6% native polyacrylamide gels running in 0.5×TBE buffer at 120 V for 45 min in a 4°C room and then transferred to nylon membrane (Amersham Biosciences) running in 0.5×TBE buffer at 15 V for 40 min. The biotin-labeled RNA was detected by LightShift™ EMSA Optimization& Control Kit  according to the manufacturer’s protocol and imaged by the ChemiDoc™ Imaging System (Bio-Rad Laboratories).To verify the binding activity of the RdRp complex, an electrophoretic mobility shift assay was performed. The RdRp complex was preincubated at 4°C overnight and then incubated with S12, 10 mM KCl, and 0.01% Triton-X 100) for 60 min at 37°C. The reactions were stopped by 40 μl of quench buffer . The reaction products were loaded into a 10% Urea-PAGE denatured gel, running at 120 V for 1.5 h. After electrophoretic transfer to nylon membrane (Amersham Biosciences), the labeled RNA was detected by LightShift™ EMSA Optimization & Control Kit  according to the manufacturer’s protocol. The inhibition assays of RdRp inhibitors are similar to the above for the RdRp enzymatic assays, except inhibitors were added to reactions for 30 min before the addition of 10 mM ATP.The SARS-CoV-2 RdRp complex  was incubated with 3.0 nM biotin labeled self-priming RNA, 2 U/μl RNase inhibitor, and 10 mM ATP in reaction buffer . Formation of dsRNA is assessed by the QuantiFluor dsRNA system (Promega). This is followed by the addition of 200 μl of QuantiFluor dsRNA dye mix and incubation for 5 min at room temperature according to the manufacturer’s recommendation. Increased fluorescence was detected using 2030 Multilabel Reader (PerkinElmer) at an excitation of 504 nm and an emission of 531 nm.A long self-priming RNA with a sequence of 5’-Biotin-UUUUUUUUUUUUUUUUUUUUUUUUUUUUUUAACAGGUUCUAGAACCUGUU-3’ was used as the fluorometric RdRp assay template. The reaction system of RdRp enzymatic activity assay was modified for high-throughput assay. After adding compounds for 30 min, 2.0 μM SARS-CoV-2 RdRp was incubated for 60 min with 1.0 μM self-priming RNA and 10 mM ATP in the presence of 2 U/μl RNase inhibitor in reaction buffer  of SARS-CoV-2 RdRp. A for each compound is normalized against the DMSO controls in each plate as follows:The concentration of total dsRNA was calculated using a linear regression model for each compound as the activity (A_DMSO) is the average of activity values of DMSO controls in a particular platewhere mean , without ATP, treated with 0.1% DMSO for 30 min; (2) positive group (n = 21 wells), treated with 0.1% DMSO for 30 min; and (3) inhibitor group (n = 21 wells), treated with 25 µM C646 for 30 min before adding ATP.To assess the reliability and reproducibility of the developed assay, g et al.  First, wZ-factor was calculated using the following equation:Z’-factor was calculated as follows:p-value < 0.05 was considered significant. Specific tests to determine statistical significance are noted in each figure legend.The data were presented as the mean ± SEM. All statistical data analyses were performed in GraphPad Prism 8. Statistical significance for each endpoint was determined with specific statistical tests. For each test, a E. coli cells, respectively. In the eukaryotic expression system, nsp12 with C-terminus tagged with 6xHis was produced and purified using immobilized metal affinity chromatography to ∼90% purity  for further experiments  primer S1 to the 30-nt template S2, while the long self-priming RNA contains a 20-nt self-complementary base pairing region and a 20-nt RNA template (Ux20). The short primer/template is more suitable for the binding test of RNA and protein (*/S2 RNA primer/template in electrophoretic mobility shift assays (EMSAs)  (mol:mol = 1:2) was incubated at 4°C overnight to form the RdRp complex first. After incubation of the RdRp complex with self-priming RNA substrate and ATP, extension RNA products were separated by Urea-PAGE denatured gel. We found that the RdRp complex was able to extend the self-priming RNA substrate to generate duplex RNA in a dose-dependent manner, confirming RNA-dependent RNA polymerization activity  within cells. We checked inhibition activity of RTP to RdRp catalytic activity in vitro based on quantification of dsRNA by a fluorometric approach  and in insect cells (His-tagged nsp12) is closely similar , % CV, S/B, and S/N met the criteria required for HTS assays  nsp14/nsp10 complex. Nsp14 is a bifunctional protein with two distinct activities, an N-terminal 3’-to-5’ ExoN and a C-terminal N7-methyltransferase (N7-MTase), both critical for coronavirus life cycle. Nsp14 ExoN activity is activated and stabilized through the interaction with the nsp10 protein. The proofreading function of the nsp14/nsp10 complex has been shown to critically decrease CoV sensitivity to chain terminating and mutagenic nucleotides , 37–39.  thought –42. Unli thought , and hum in vivo –46. Therin vitro inhibitors of SARS-CoV-2 RdRp with no typical chemical structures of nucleoside analogs. C646 is a competitive inhibitor of the histone acetyltransferase p300 , the Beijing Municipal Natural Science Foundation (7214286), and the CAMS Innovation Fund for Medical Sciences (2021-I2M-1-030 and 2020-I2M-2-010).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Coccidioides immitis and Coccidioides posadasii are causative agents of coccidioidomycosis, commonly known as Valley Fever. The increasing Valley Fever cases in the past decades, the expansion of endemic regions, and the rising azole drug-resistant strains have underscored an urgent need for a better understanding of Coccidioides biology and new antifungal strategies. Transporters play essential roles in pathogen survival, growth, infection, and adaptation, and are considered as potential drug targets. However, the composition and roles of transport machinery in Coccidioides remain largely unknown. In this study, genomic data mining revealed an abundant, uncharacterized repertoire of transporters in Coccidioides genomes. The catalog included 1288 and 1235 transporter homologs in C. immitis and C. posadasii, respectively. They were further annotated to class, subclass, family, subfamily and range of substrates based on the Transport Classification (TC) system. They may play diverse roles in nutrient uptake, metabolite secretion, ion homeostasis, drug efflux, or signaling. This study represents an initial effort for a systems-level characterization of the transport machinery in these understudied fungal pathogens. Coccidioides immitis and Coccidioides posadasii, are causative agents of coccidioidomycosis, commonly known as Valley Fever .,17.C. imhttps://www.tcdb.org/ (accessed on 20 July 2021), was used as the knowledge database for the identification and classification of transporters in Coccidioides [The transporter classification database TCDB, idioides ,28. TCDBC. immitis RS strain and C. posadasii SOWgp strain against the TCDB database was conducted to identify Coccidioides proteins that were homologs to known or predicted transporters [−20 and greatest positive percentage >50. The annotation of Coccidioides transporters was based on the hits in the TCDB with the lowest E-value, and the highest similarity score.The BLASTP query of all the proteins in sporters . The cutCoccidioides transporters were further classified into families and subfamilies based on the 5-letter Transport Classification (TC) system [The predicted ) system . Similar) system .Coccidioides transporter sequences were identified by searching against the Pfam 35.0 database [Coccidioides transporters were predicted based on the Chemical Entities of Biological Interest (ChEBI), an ontology and dictionary focused on small chemical compounds [Conserved domains/motifs in predicted database , which idatabase . The TMHdatabase . The subompounds .Multiple sequence alignments were obtained using the MUSCLE program ,35. PhylCoccidioides, the protein sequences in the C. immitis RS strain and C. posadasii SOWgp strain were subjected to an exhaustive search against the TCDB database, which has a catalog and a structure-, mechanic-, and phylogeny-based classification of transporters. Stringent threshold of E-value < 10−20 and positive percentage >50 were adopted to ensure the high coverage with low false-positives. A total of 1288 and 1235 transporter homologs were identified in C. immitis and C. posadasii, respectively, which account for 13.0% and 17.1% of their respective proteome , consisting of uniporters, symporters, and antiporters [Candida albicans [C. glabrata [C. tropicalis [Aspergillus fumigatus [C. immitis and C. posadasii, respectively. See The most abundant class of transporters is electrochemical potential-driven transporters (Class 2) . These 3iporters ,41. The albicans ,43,44,45glabrata ,47, C. topicalis , and Aspumigatus . We idenC. immitis and C. posadasii. This class of transporters uses a primary source of energy such as ATP to transport solutes across a membrane against their electrochemical gradient. The members of this class are major players in the uptake and excursion of diverse solutes. Most notably, a superfamily of ATP-binding cassette (ABC) transporters are known to confer efflux-mediated antifungal resistance in pathogenic fungi [C. immitis and C. posadasii, respectively. The genomic and phylogenetic analyses of these ABC transporters are seen in A. fumigatus, A. nidulans, A. oryzae, C. neoformans, Neurospora crassa, Saccharomyces cerevisiae, and Schizosaccharomyces pombe [C. immitis and C. posadasii genomes contain 20 and 18 P-type ATPases, respectively, including ATPases that translocate calcium, magnesium, copper, phospholipid, and potassium-sodium (The second most abundant class is primary active transporters (Class 3) . They reic fungi . Our anaes pombe . These aes pombe . C. immim-sodium .C. immitis and C. posadasii fall within this class. These transporters facilitate diffusion of solutes in an energy-independent mode. The two largest subclasses in Class 1 present in Coccidioides are α-type channels (TC Subclass 1.A) [Coccidioides possess various cation channels, including calcium, potassium, and transient receptor potential channels with potential roles in cellular signaling and homeostasis. Homologs of these cation channels are present in S. cerevisiae [C. albicans [Aspergillus spp. [C. neoformans [Coccidioides also possess Nuclear Pore Complex (NPC) transporters (TC family 1.I.1) that bidirectionally transfer macromolecules between the cytoplasm and the nucleus. Besides facilitating nucleocytoplasmic trafficking, the homologs of these porins were shown to be involved in chromatin organization, and gene expression regulation in fungi [Class 1 channel/pore proteins are ubiquitous in all living organisms. Over 19% of the transporter homologs in ass 1.A) , and memass 1.A) . Coccidirevisiae ,55, C. aalbicans , Aspergilus spp. , and C. oformans . Althougoformans . In addiin fungi ,60,61.C. immitis and C. posadasii. The physiological roles of these putative transporters are largely unknown.Taking a systems perspective, the TC system also groups various accessory factors that facilitate the transport, but do not directly transport solutes into Class 8. Over 140 predicted homologs fall into Class 8, representing 11% of the total transporter homologs in Coccidioides life cycle, for example, the ABC transporter superfamily (TC 3.A.1) and the major facilitator superfamily (MFS) (TC 2.A.1). Examples of potentially important transporters in Coccidioides and their homologs in other pathogenic fungi are shown in Among the 269 families of predicted transporters, undoubtedly some of the uncharacterized transporters perform important functions in the Coccidioides, however, remains to be defined.The ABC transporter superfamily is one of the largest protein families that are ubiquitous in all three domains of life ,65,66,67C. immitis and C. posadasii, respectively. Phylogenetic analysis revealed that these ABC transporters could be divided into five distinct efflux groups , which plays an important role in susceptibility to multiple antifungal drugs [ABCB, the largest group, includes three families. The Multidrug Resistance Exporter Family (TC 3.A.1.201) has been widely studied in other fungi species such as oformans . Four holofungin   The Pleiotropic Drug Resistance (PDR) Family (TC 3.A.1.205). In oformans ; two gen network . (2) Therevisiae .Coccidioides sequences in this family are homologous to various drug transporters, for instance, the YOR1 gene in S. cerevisiae conferring resistance to oligomycin, rhodamine B, tetracycline, verapamil, eosin Y and ethidium bromide [YCF1 gene in S. cerevisiae involved in vacuolar metal resistance and drug detoxification [BAT1 gene in S. cerevisiae, and the EcdL gene in A. fumigatus conferring resistance to antifungal Echnocandin B [The ABCC group includes the Drug Conjugate Transporter (DCT) Family (TC 3.A.1.208). The  bromide , the YCFfication , the bilcandin B .C. immitis and C. posadasii each possesses two paralogs of P-FAT genes, which may be involved in the fatty acid transport across the peroxisomal membrane [The ABCD group includes the Peroxysomal Fatty Acyl CoA Transporter (P-FAT) Family (TC 3.A.1.203). membrane .C. immitis and C. posadasii, which may mediate the efflux of cellular cholesterol and phospholipids [Cholesterol/Phospholipid/Retinal (CPR) Flippase Family (TC 3.A.1.211) is the single member of the ABCA group in holipids .Coccidioides lineage, indicating potential significance of MFS in Coccidioides adaptation [Coccidioides remains minimum.MFS (TC 2.A.1) constitutes a large and diverse superfamily of secondary active transporters. Widespread across all three domains of living organisms, MFS transporters move a broad spectrum of small molecules across membranes to maintain important physiological function of cells ,108,109.aptation , our knoC. immitis and C. posadasii, respectively, representing nearly 11% of the predicted transporters in their genomes. They belonged to 18 families. Of particular interest, abundant members were found in the Drug:H+ Antiporter-1 (DHA1) Family and the DHA2 Family. DHA1 and DHA2 transporters have demonstrated roles in antifungal resistance in fungal species in the genus of Saccharomyces, Candida, Cryptococcus and Aspergillus [We identified 140 and 135 MFS transporters in ergillus ,114.Coccidioides are distributed into two clusters, which is consistent with their distinct structural properties  includes 29 and 28 members in er fungi . For exa cations . Notablyuinidine ,92,118.  immitis .C. immitis and C. posadasii, respectively. Two paralogs of aflT genes are in the Coccidioides genomes; AflT is an efflux pump in the aflatoxin pathway in filamentous fungus Aspergillus parasiticus [Coccidioides genomes, closely related to the YOR378W gene in S. cerevisiae, which is involved in boron stress tolerance [C. immitis and C. posadasii, respectively, with high homology to MFS1 genes, which is related to the antifungal resistance in the wheat fungal pathogen Zymoseptoria tritici [The DHA2 cluster (TC 2.A.1.3) includes 24 and 21 members in asiticus . Four paolerance . Abundan tritici .Coccidioides transporters also includes novel transporters with broad implications in fungal physiology. For example, we found members in the Nucleobase: Cation Symporter-1 (NCS1) Family (TC 2.A.39) and in the Nucleobase/Ascorbate Transporter (NAT) or Nucleobase: Cation Symporter-2 (NCS2) Family (TC 2.A.40) [Coccidioides. TRK1 and TRK2 were found to participate in potassium uptake and response to internal and external signals in S. cerevisiae and C. albicans [Our catalog of  2.A.40) ,120,121, 2.A.40) . Two paralbicans ,123.Coccidioides, we performed TMHMM analysis [Coccidioides genomes. The number of TMSs range from 0 to 22  are important structural components for transporters to translocate solutes across membranes. The topology of a transporter is specified by the number of the TMSs, and its overall orientation in the membrane . The numanalysis . Similar 0 to 22 . While tCoccidioides transporters were predicted to be responsible for the translocation of extraordinarily diverse substrates, including inorganic molecules, carbon sources, drugs, toxins, electrons, macromolecules, amino acids and derivatives, nucleic acids, vitamins, and accessory factors  and PSP1  in C. posadasii [Coccidioides lineage [C. immitis [Transporters are essential components for the survival of living organisms. The roles of transporters have been demonstrated in a variety of fungi. Our knowledge of the transport machinery in osadasii ,17. To dosadasii , a trans immitis .Coccidioides biology, here, for the first time, we present a catalog of 1288 and 1235 putative transporters in C. immitis and C. posadasii, based on exhaustive homology search and comparative genomic analysis. These transporters fall into seven classes, 25 subclasses, and 269 families, with diverse transmembrane topologies and a wide array of substrates. Our hypothesis that Coccidioides fungi possess a rich and powerful transport machinery is justified.To fill in the critical knowledge gaps in Coccidioides is likely an attribute of their adaptation to the complex soil ecosystem and the alien mammalian host system. Soil represents one of most challenging natural environments in which microorganisms scavenge nutrients, produce toxins to competing organisms and resist the effects of such cytotoxic substances. A similarly high content of transporters was observed in soil microbials, especially in the genus of Streptomyces [Coccidioides have undergone extensive genomic evolution to adapt to the animal host niche [Coccidioides transporters shows that a large number of transporters use proteins, amino acids and derivatives as substrates  laboratory containment and the special expertise needed to manage the aerosol risk posed by the large amounts of spores, and the severity of coccidioidomycosis. Experimental assays standard for other organisms can often be time-consuming or cost-prohibitive. Thanks to the advent of the genomic era, the in silico approach is becoming a cost-effective and efficient approach to identify and prioritize genes for wetlab characterization, especially suitable for non-model organisms such as Coccidioides.We are bearing in mind that the catalog presented in this study are in silico predictions that await experimental validation. Historically, Coccidioides transporters in physiology, pathogenesis, and stress response are yet to be fully investigated, this study represents an initial attempt to a systems-level understanding of the mechanisms underlying Coccidioides survival and infection. Transporters on the catalog are likely members of the cellular networks associated with nutrient uptake, ion balance, drug excursion, signaling, and regulation. With the availability of high throughput assays, it is possible to integrate various types of omic data to interrogate the expression profiles and associations of transporter genes and their upstream regulators and downstream substrates/effectors in a network perspective [Coccidioides.While the roles of spective ,132,133.In addition to providing a catalog of Coccidioides transporters for characterization, this genomic study also raises an important unanswered question: what are the content and diversity of transporters among other pathogenic fungal species? Published work to date mostly focuses on specific transporters or transporter families. For example, Costa et al.  surveyed