While influenza and SARS-CoV-2 virus can be severe, co-infection with these viruses has the potential for worsening the clinical outcome. The combined infection of influenza A virus (IAV) and SARS-CoV-2 in animal models has also shown elevated SARS-CoV-2 viral load and pneumonia. Co-vaccination may provide a useful option in prevention of combined infection. In this study, a composite peptide vaccine comprised of highly conserved influenza neuraminidase (NA) and matrix protein (M2e) epitopes, in combination with either conserved spike protein (SP) or RNA polymerase (POL) epitopes of SARS-CoV-2 generated robust immune responses to both coronavirus and influenza virus.Mice were immunized with 20µg of Coronavirus Pep02  or Coronavirus Pep05  or Coronavirus Pep11 , formulated with AddaVax™. Serum antibody titers to both coronavirus and influenza peptides, and whole viruses were analyzed using ELISA. Antisera neutralizing activity against IAV was determined using microneutralization assay (MNA).CorPep05 immunization generated enhanced IgG1 and IgG2b antisera responses to the immunogens compared to CorPep02, starting at Day-21. Immunization with CorPep05 or CorPep11 induced strong IgG1 and IgG2b immune responses to the composite coronavirus peptides, influenza NA and M2e peptides, coronavirus POL and SP peptides. Antisera from CorPep05 and CorPep11 groups bound to both viruses (IAV and human coronavirus NL-63 (HCoV NL-63)) and demonstrated neutralizing activity against IAV, with titers >5000.A composite peptide vaccine comprised of both influenza (NA+M2e) and coronavirus (POL) epitopes generated enhanced immune responses in comparison to peptide containing only POL. Th1 and Th2 immune responses to coronavirus POL and SP were observed. Serum neutralizing activity was demonstrated against IAV. Studies are underway to examine neutralizing antibodies against HCoV NL-63 and multiple variants of SARS-CoV-2. Highly conserved epitopes in these composite peptide vaccines may provide an important strategy to prevent infections with IAV or SARS-CoV-2 and mitigate the threat of co-infection.All Authors: No reported disclosures.


EBSCO-host web, Pubmed and South Africa (SA) epublications, for the period of January 2012 to January 2022. Findings showed that a paucity of studies identified the link between agency, hope and adolescent SRH respectively. Our review included 12 articles and found no studies that focused on hope and its role in adolescent SRH or seeking SRH services. However, the literature revealed the complexities of adolescent SRH agency and autonomy where female adolescents had limited autonomy to make SRH decisions. Limited access to adolescent friendly SRH services was also found to restrict girls' agency to prevent unintended pregnancies or to take up SRH support. Given the paucity of research, empirical studies are needed to further understand the extent to which hope, agency and other subjective factors implicate adolescent SRH in the African context.Availability of and access to services that promote sexual and reproductive health (SRH) amongst adolescent girls have become a global priority. Yet, while researchers have explored factors that influence the uptake of SRH services in low-and-middle income countries, the roles that “agency” and “hope” play in adolescent SRH is less understood. To study this, this mini review systematically reviewed the literature across three databases,  Over the past two decades, there has been growing interest in the sexual and reproductive health (SRH) of adolescent girls and young women (AGYW) who have been identified as a particularly vulnerable group , 2. AdolWhile there have been remarkable improvements in the promotion of SRH knowledge and access to SRH services, globally, these gains have been inequitable in low-and-middle-income countries where the access, availability and quality of services have been inadequate . This isThis paper is concerned with the roles that agency and hope plays in the SRH of adolescents including accessing SRH services. Current SRH studies and programs have largely been directed towards enhancing AGYW's SRH knowledge and access to quality services, with a growing need to explore informed decision-making around their SRH . Agency Closely linked to agency, increasing attention has been given to the extent to which having hope shapes adolescent wellbeing –19. HopeWhile studies have examined the roles of hope and/or agency in adolescent health behaviour and outcomes, little is known about how these constructs intersect with adolescents' SRH practices in the African context. To study this, we conducted a systematic search of the literature, using set criteria, to identify published studies conducted in Africa on these topics. This mini review thus synthesises the identified literature to explore how these subjective factors influence adolescents' SRH and access to SRH services.n = 455), SA epublications (n = 25), and Pubmed (n = 36). The second keyword search were “hope” OR “hopefulness” OR “self efficacy” AND “adolescents” OR “teenagers” OR “teen” OR “youth” AND “sexual reproductive health” OR “sexual reproductive health services” AND “Africa”. This search produced the following, EBSCO-host web (n = 18), SA epublications (n = 8), and Pubmed (n = 124).This mini-review consulted three databases, namely, EBSCO-host web, SA epublications and Pubmed, making use of Boolean phrase options within databases. The first set of keywords used were “agency” OR “autonomy” OR “self determination” OR “self esteem” OR “self concept” AND “adolescents” OR “teenagers” OR “teen” OR “youth” AND “sexual reproductive health” OR “sexual reproductive health services” AND “Africa”. Apart from the term “Africa”, only articles with the respective keywords in the abstracts were deemed relevant. This strict criterion was applied to ensure that we identify articles that directly link to our interest area and, in this way, avoid articles that merely mention the respective keywords in the body of the text. This search produced the following, EBSCO-host web (n = 10) articles , only peer-reviewed articles, English language, and studies including empirical data. The three reviewers used these criteria, together with the primary objective that only articles that relate to the link between hope and SRH (including services) or agency and SRH (including services), to assess the relevance of each abstract and whether it was included or excluded. After the abstract assessment, 34 articles were selected for full text assessment. Full text articles were independently assessed by three authors for methodological rigor and to avoid bias see . The fincles See .Furthermore, we also conducted quality appraisals of the articles included in this paper using two frameworks. For papers that employed qualitative methods (including the mixed methods study), we used the Long and colleagues' revised critical appraisal skills programme (CASP) tool , while wn = 7), followed by 2020 (n = 1), 2019 (n = 1) and 2016 (n = 1). Although most papers were recently published, no studies focused directly on the COVID-19 pandemic. As shown in n = 5) (n = 4) (n = 1) (n = 1) used a randomized evaluation design, three (n = 3) used a randomized control trial (including the mixed methods study) and two (n = 2) used a cross-sectional design. In terms of context, the articles focused on Kenya (n = 1), Ghana (n = 1), South Africa (n = 4), Uganda (n = 1), Zambia (n = 2), and Liberia (n = 1).Most of the papers were published in 2021 (n n = 5) –33, foll (n = 4) –27, 34,  (n = 1) . Of the n = 2), and agency and autonomy (n = 3). Authors also evaluated programmes that generally aimed to enhance uptake of SRH services amongst adolescents (n = 5). No studies in our sample examined the association between hope and SRH amongst adolescents as framed by our inclusion criteria.Of the articles that were relevant, studies focused on self-efficacy and self- esteem  for adolescents across different African contexts. In South Africa, the Tutu Teen Truck (TTT) mobile health clinic was developed to improve adolescents access to SRH services . ServiceThe remaining three papers reported on the value of education programs to enhance youth SRH knowledge and facilitate positive SRH behaviours. This included the evaluation of the HealthyActions curriculum in Liberia where out of school youth were provided with SRH information and access to products and services . An incrThe final step in our review was to conduct a risk of bias assessment of the studies using two separate frameworks for papers employing qualitative methods and those using quantitative approaches, as outline previously. Summaries of these assessments are presented in The quantitative studies included in this review generally used structured assessment techniques. As indicated in In all, the quantitative studies offered less risks of bias, given the structured and objective approaches used to gather and analyze the data. The qualitative studies on the other hand, showed less room for such objectivity, which is not unexpected given the subjective nature of qualitative research. However, qualitative papers used established data collection approaches , and usually employed a group coding approach to assess the groundedness (strength) of codes and sub-themes. These approaches enhance the credibility of the findings which were also interpreted in relation to the literature.The aim of this review was to explore the literature on hope and agency in adolescents' SRH in the African context. While research has explored the factors that influence uptake of SRH services in low-and-middle income countries, our findings show that the role of hope and agency in accessing SRH services is less understood. Importantly, no studies in our sample examined the association between hope and adolescent SRH, highlighting a significant gap in the literature. While we also included the keyword “hopefulness” as part of our Boolean search, this too generated no studies that were directly linked to adolescent SRH. Still, enhanced hope has been associated with decreased participation in health-related risky behaviours such as alcohol use  while hoStudies also explored the link between self-efficacy, self-esteem, agency and autonomy, and adolescents SRH, respectively. While the influence of these constructs in adolescents seeking of SRH services was not a primary focus in some studies, self-efficacy was found to influence sexual behaviours either through an inability to negotiate condom use in sexual relationships  or an inMajority of the included articles discussed SRH programmes to improve adolescents access to SRH services. These included a mobile clinic to deliver SRH services , distribMulti-country investment is pertinent to identify contextually relevant and culturally sensitive interventions and programmes to enhance adolescent SRH and prevent, interrupt, and address the various barriers to accessing adolescent friendly SRH services and products. Contextual factors not only play significant roles in shaping the quality, availability, and accessibility of SRH services and programs for AGYW, but also regulates how young people make decisions or enact their agency and constructions of hope , 17, 37.This mini review highlights the importance of understanding the role of individual factors like hope, self-efficacy, agency, and autonomy in adolescent SRH, behaviours and access to services. Studies are necessary to also consider how these factors interact to either facilitate or compromise safe SRH practices and access to services amongst adolescents. Recognising the influence of the social environment in examining these interactions is key, given the important roles that culture, religion and traditional norms play in shaping risk and protective SRH behaviours in African contexts , 38–41. Moreover, this paper is not without limitations. The review included papers published within particular journals and databases, and within a set timeframe where we may have excluded other relevant. Importantly, access to full text articles were provided through our institutional subscriptions, which may have further restricted access to articles published in journals to which the institution does not subscribe. Our criteria also strictly included empirical studies while other systematic reviews (if available) or unpublished dissertations could offer additional insights into the relationships between hope or agency and adolescent SRH. Papers also focused only on studies that include adolescents within their samples and we therefore acknowledge that further understandings can be gleaned from the broader literature on adult youth SRH.


Deep learning, or artificial neural networks, is a type of machine learning algorithm that can decipher underlying relationships from large volumes of data and has been successfully applied to solve structural biology questions, such as RNA structure. RNA can fold into complex RNA structures by forming hydrogen bonds, thereby playing an essential role in biological processes. While experimental effort has enabled resolving RNA structure at the genome-wide scale, deep learning has been more recently introduced for studying RNA structure and its functionality. Here, we discuss successful applications of deep learning to solve RNA problems, including predictions of RNA structures, non-canonical G-quadruplex, RNA-protein interactions and RNA switches. Following these cases, we give a general guide to deep learning for solving RNA structure problems. As a data-driven algorithm, deep learning has shown promise with successful applications in biology, healthcare, and drug discovery . One of As a key in the central dogma, RNA is essential for gene expression. RNAs fold into RNA secondary structures by base pairing, which can fold further to form RNA tertiary structures. This RNA folding is extremely important for achieving RNA’s diverse and complex biological functions . For insIn this review, we have compiled examples from previous research whereby deep learning methods were adopted to solve RNA structure-related problems. Firstly, we introduce the brief process of deep learning modelling through a G-quadruplex classification question. Secondly, we propose that the availability of high-throughput sequencing data has facilitated deep learning modelling of RNA structure-related problems. Next, the experience of deep learning architecture design is presented through the examples of RNA-protein binding prediction and toehold-switch prediction models. Subsequently, we introduce classification and regression in supervised learning through RNA secondary structure prediction and RNA tertiary structure scoring. Lastly, we present several solutions for the interpretation of deep learning models as mentioned in studies. Our review provides an overview of deep learning modelling approaches from the perspective of RNA structure-related research, before providing suggestions for future efforts to address more questions in RNA structure by deep learning.via Hoogsteen hydrogen-bonded guanines)  . In tradFor deep learning, it is possible for modeling without feature extraction . This pain vivo G4 data (G4 ChIP-seq) and identified key DNA motifs associated with GQS region activity  in one single experiment, transforming the scale of RNA structure study to an unprecedented level  that disrupts RNA structures  and recurrent neurons network (RNN)  Figure . The feein silico predicted RNA structure. The close relationship between RNA sequence and structure was then captured by Bi-LSTM for an estimate of possible long-range dependencies  . SPOT-RNIn supervised learning, if the labels are continuous values, it becomes a regression question. Recently, the model for scoring RNA tertiary structures, ARES, is an example of a deep learning regression model developed with a small amount of training data . In compA deep learning model is typically thought of as a “black box” containing millions of weights that predict input data as output values. But for researchers in biology, the biological features that the model learns and the biological questions it can explain are more important than just predictions. Contrary to standard statistical models and machine learning methods based on features extraction, deep learning models are challenging to interpret .in silico mutations by algorithm (in silico mutations of the input data followed by re-prediction with the model to assess the impact of changes in the input on the output. For example, for a bi-classification model for GQS prediction, it is possible to simulate base-by-base mutations to alter the input sequence and predict its classification, thus evaluating which nucleotides affect GQS folding. During translation, ribosomes are known to actively unwind the RNA structure where a complex interaction between the ribosome and RNA structure occurs. DeepDRU is a deep learning model for predicting the unwinding state of RNA structures in vivo (in vivo than the sequence itself by simulating mutations of a feature while the rest of the features are fixed (The most straightforward interpretation means is to perform an lgorithm  (Zou et lgorithm . This ap in vivo . This rere fixed .For interpreting CNN models, the convolutional filters in the model can be visualised as heat maps or position weight matrices to extract the high-level patterns learned . In the The emergence of data-driven deep learning approaches integrates technological innovation, “Big Data” exploitation, and huge computational power to significantly transform the scale for studying RNA structures and their functions. We introduce the basic concepts of deep learning, the importance of data volumn, supervised learning, design of deep learning architectures and model interpretation by reviewing recent deep learning applications in deciphering different aspects of RNA structure studies and highlighting those that demonstrate the best potential for future development.Although deep learning has shown promise for application in the RNA structure field, there are still some issues that need to be addressed. Firstly overfitting is presently the major risk to deep learning models, especially when faced with limited data size. Advances in technology have led to the development of multi-layered, high-capacity models that can be applied to obtain features for more complex data structures. However, simultaneously, the risk of overfitting arises. In a recent study of RNA secondary structure prediction, it was suggested that E2Efold may suffer from overfitting and is therefore not suitable for predicting broader datasets . Hence iEncouragingly, with the dramatic increase of high throughput RNA structure data generated from different organisms under diverse conditions, deep learning will be increasingly appreciated by RNA structure researchers and be progressively used to deduce RNA structure information and associated functionality. As the rise of available deep learning models increases, it will become progressively easier for researchers to apply deep learning in their routine data analysis for studying RNA structures.


The innate immune receptor RIG‐I provides a first line of defense against viral infections. Viral RNAs are recognized by RIG‐I's C‐terminal domain (CTD), but the RNA must engage the helicase domain to release the signaling CARD (Caspase Activation and Recruitment Domain) domains from their autoinhibitory CARD2:Hel2i interactions. Because the helicase itself lacks RNA specificity, mechanisms to proofread RNAs entering the helicase domain must exist. Although such mechanisms would be crucial in preventing aberrant immune responses by non‐specific RNAs, they remain largely uncharacterized to date. This study reveals a previously unknown proofreading mechanism through which RIG‐I ensures that the helicase engages RNAs explicitly recognized by the CTD. A crucial part of this mechanism involves the intrinsically disordered CARDs‐Helicase Linker (CHL), which connects the CARDs to the helicase subdomain Hel1. CHL uses its negatively charged regions to antagonize incoming RNAs electrostatically. In addition to this RNA gating function, CHL is essential for stabilization of the CARD2:Hel2i interface. Overall, we uncover that the CHL and CARD2:Hel2i interface work together to establish a tunable gating mechanism that allows CTD‐chosen RNAs to bind the helicase domain, while at the same time blocking non‐specific RNAs. These findings also indicate that CHL could represent a novel target for RIG‐I‐based therapeutics. Specificity for RNA‐binding mediated activation of RIG‐I is ensured by a gating mechanism involving the CHL and CARD:Hel2i interface. No known functions were associated with the CHL, except that it tethers the CARDs to the helicase and that appropriately long CHL might be important for enabling intermolecular CARDs:CARDs interactions. Therefore, we were surprised to find that small deletions in CHL did not impair RIG‐I signaling and instead constitutively activated RIG‐I signaling without the addition of PAMP RNA. These results suggest that CHL is not a passive linker but a regulatory region that keeps RIG‐I in the autoinhibitory state in the absence of PAMP RNA. To understand the role of CHL, we carried out a detailed mechanistic study of the CHL mutants using a combination of cellular, biochemical, and biophysical methods, including hydrogen–deuterium exchange and mass spectrometry (HDX‐MS), equilibrium RNA binding, and stopped‐flow kinetics. These studies revealed two essential roles of the CHL. One role of the CHL is to stabilize the CARD2:Hel2i interface and maintain RIG‐I in an autoinhibitory conformation in the absence of PAMP RNA. The second role of CHL is to shield the helicase domain and antagonize non‐specific RNAs. Both functions of the CHL depend on its negatively charged amino acids acting as an electrostatic gate. The RNA proofreading function of the CHL is greatly enhanced by a stable CARD2:Hel2i interface. The two work synergistically to minimize erroneous RNA binding events, ensuring that RNAs chosen by the CTD are specifically loaded into the helicase domain. The newly discovered regulatory functions of the CHL can be potentially leveraged in RIG‐I‐based therapeutics.et al, et al, et al, The negatively charged CHL (186–241 aa) is predicted to be intrinsically disordered  response pathway, we measured the expression of several IFN response genes, including IFNB, MX1, ISG15, OAS1 using qRT–PCR in the absence and presence of 5’ppp ds39 PAMP RNA. The Δ190–200 RIG‐I activated the IFN response genes and pIRF3 expression in the absence of PAMP RNA, whereas WT RIG‐I required PAMP RNA for activation of these genes Fig . These cSmall deletions in CHL can increase the basal signaling activity of RIG‐I in two ways. First, the deletions may destabilize the autoinhibited conformation of RIG‐I and spontaneously expose the CARDs independently of RNA binding. Additionally, the deletions may dysregulate RNA binding, promoting CARDs activation through promiscuous binding of self RNAs.et al, To test the first possibility, we used differential HDX‐MS analysis, a powerful technique to monitor CARDs exposure in RIG‐I used previously , which is four times longer than the distance it needs to span between CARD2 and Hel1 (~40 Å). A 33 Å deletion is not long enough to shorten the linker and disrupt the interface if the CHL is truly unstructured, as the Alphfold structure prediction indicates Fig . Thus, CSequence analysis of several RIG‐I homologs from bony fish to mammals shows that CHL is highly negatively charged. Although the CHL sequence conservation is poor relative to the helicase domain, the negative charges in CHL are well conserved Fig . BetweenTo determine if CHL's negatively charged regions are involved in the autoinhibitory mechanism, we systematically introduced clusters of neutral or positively charged amino acids in the 190–220 region. We chose this region because deletions in the 190–220 region aberrantly activated RIG‐I signaling in above signaling experiments. The 190–200 segment was replaced with a glycine‐serine‐threonine repeat to make the neu190–200 mutant, mimicking a more neutral, potentially less structured region. To create positively charged segments, the negative aspartate and glutamate residues were charged flipped to lysine in cf190–200, cf200–210, and cf210–220 Fig . The sigCell signaling assays in 293T RIG‐I KO cells showed a similarly high basal signaling response from the cf200–210 RIG‐I, confirming that endogenous RIG‐I activation was not responsible for the aberrant signaling in the absence of PAMP RNA Fig . The cf2D of CHL‐Hel‐CTD to Hel‐CTD allowed us to determine whether CHL regulates RNA binding independently of maintaining the CARD2:Hel2i interface. Furthermore, comparing CHL‐Hel‐CTD and cf190–210 CHL‐Hel‐CTD allowed us to determine whether CHL's function is related to its exceptional negative charge.Above results indicate that CHL inhibits stem RNA binding. Does CHL regulate stem RNA binding on its own, and if so, are the negatively charged amino acids involved in RNA regulation? To address these questions, we purified four RIG‐I constructs: WT RIG‐I, CHL‐Hel‐CTD (lacks CARDs but retains CHL), Hel‐CTD (lacks CARDs and CHL), and CHL‐Hel‐CTD cf190–210 (negatively charged residues between 190–210 mutated into lysines) Fig . CompariFluorescence‐intensity‐based titrations measured the affinity of the Cy3‐labeled ds26 stem RNA to the various RIG‐I constructs. Hel‐CTD binds the stem RNA with a high affinity, but CHL‐Hel‐CTD and WT RIG‐I showed no RNA binding under the same conditions Fig . The strD, like WT RIG‐I . Previous studies have shown that 5’ppp RNA binds to CTD with the highest affinity (0.5 nM), followed by 3' ovg RNA (240 nM), 5' ovg RNA (840 nM), and stem RNA has no affinity to CTD  mutants are constitutively activated by self RNAs  were a gift from Prof. Charles Rice, Rockefeller University USA. This cell line was described and used previously , Renilla luciferase reporter plasmid (pRL‐TK / 500 ng), and a plasmid carrying either the WT RIG‐I gene or mutant RIG‐I construct under the constitutively active CMV promoter (pcDNA 3.1 / 2 μg). The firefly luciferase gene is under the control of the interferon β promoter, and the Renilla luciferase plasmid is under the control of the constitutively active TK promoter. The plasmid transfections were carried out with X‐tremeGENE HP DNA Transfection Reagent (Roche). Cells were replated in 96‐well plates the next day at 2 × 104 cells/well density and transfected with either 50 nM 5’ppp ds39 RNA or a titration of 700 nM, 70 nM, and 7 nM 5’ppp ds39 RNA using Lyovec transfection reagent (InvivoGen). After 20 h, the activities of firefly and Renilla luciferases were measured sequentially with the Dual‐Luciferase reporter assay (Promega). Data was collected in sextuplicate sets, and relative luciferase activities were calculated. Error bars represent the standard error of the mean (SEM). Protein expression was tested using western Blots with a primary α‐Myc antibody and β‐actin as a normalization control.HEK293T and HEK293T RIG‐I KO cells were grown in at 5% COet al, et al, et al, et al, et al, Total RNA was extracted from transfected 293T cells using TRIzol reagent  following the manufacturer’s instructions , sonicated for 30 pulses, and then boiled for 10 min (Edwards Escherichia coli strain Rosetta (DE3) (Novagen). The proteins were purified using a series of chromatography columns, as published previously , then heating to 95°C, then finally cooling slowly to 4°C.The single‐stranded RNAs to make the ds27 PAMP and non‐PAMP RNA ligands, ds39 complementary RNA, and 5’ppp ds10 hairpin were chemically synthesized and HPLC purified by Biosynthesis, Dharmacon, and Trilink BioTechnologies. The 5’ppp 39‐mer RNA was Peptides were identified using tandem MS (MS/MS) with an Orbitrap mass spectrometer . Product ion spectra were acquired in data‐dependent mode with the top five most abundant ions selected for the product ion analysis per scan event. The MS/MS data files were submitted to Mascot (Matrix Science) for peptide identification. Peptides included in the HDX analysis peptide set had a MASCOT score greater than 20, and the MS/MS spectra were verified by manual inspection. The MASCOT search was repeated against a decoy (reverse) sequence and ambiguous identifications were ruled out and not included in the HDX peptide set.2, 2 mM DTT) were incubated with the RNA ligand at a 1:1.2 molar ratio for 1 hr (protein:ligand) before the HDX reactions at 4°C. Five‐microliter of protein/protein complex with ligand/peptide was diluted into 20 µl D2O (deuterium) in exchange buffer  and incubated for various HDX time points  at 4°C and quenched with 25 µl of ice‐cold 4 M guanidine hydrochloride, 1% trifluoroacetic acid. Dmax samples were incubated in D2O in exchange buffer containing 3 M guanidine hydrochloride  overnight at room temperature. The samples were immediately placed on dry ice after the quenching reactions until they were injected into the HDX platform. Upon injection, samples were passed through an immobilized pepsin column (2 mm × 2 cm) at 200 µl min−1, and the digested peptides were captured on a 2mm × 1cm C8 trap column (Agilent) and desalted. Peptides were separated across a 2.1mm × 5cm C18 column  with a linear gradient of 4% ‐ 40% CH3CN and 0.3% formic acid, over 5 min. Sample handling, protein digestion, and peptide separation were conducted at 4°C. Mass spectrometric data were acquired using an Orbitrap mass spectrometer  with a measured resolving power of 65,000 at m/z 400. HDX analyses were performed duplicate or triplicate, with single preparations of each protein‐ligand complex. The intensity weighted mean m/z centroid value of each peptide envelope was calculated and subsequently converted into a percentage of deuterium incorporation. In the absence of a fully deuterated control, corrections for back‐exchange were made by an estimated 70% deuterium recovery, and accounting for the known 80% deuterium content of the deuterium exchange buffer. When comparing the two samples, the perturbation %D is determined by calculating the difference between the two samples. HDX Workbench colors each peptide according to the smooth color gradient HDX perturbation key (D%) shown in each indicated figure. Differences in %D between −5% to 5% are considered non‐significant and are colored gray according to the HDX perturbation key. In addition, unpaired t‐tests were calculated to detect statistically significant (P < 0.05) differences between samples at each time point. At least one time point with a P‐value less than 0.05 was present for each peptide in the data set, confirming that the difference was significant . Cy3‐labeled ds26 stem RNA or 5’ppp ds27 RNA (15 nM) was titrated with increasing concentration of RIG‐I proteins, and the change in fluorescence intensity was measured at 570 nm after excitation at 547 nm. The observed fluorescence intensity change (F) from the initial fluorescence intensity (F0) is proportional to the amount of protein‐RNA complex (PR) and modified by a coefficient of complex formation (fc). This value was plotted as a function of protein concentration (P) and fitted to Equation KD) and Hill coefficient (n). The reported RNA KD values were consistently observed in titrations repeated two times.Fluorescence intensity measurements were carried out at 25°C using FluoroMax‐4 spectrofluorimeter (Horiba Jobin Yvon) in Buffer A . A mixture of Cy3‐labeled ds26 stem RNA mimic (100 nM) and protein (400 nM) in Buffer A (pre‐incubated at 25°C for 10 min) from syringe A was mixed with a 10‐fold excess of an RNA trap (unlabeled 5′ppp ds12 hairpin RNA) from syringe B. The Cy3 fluorescence emission was measured using a 570 nm band‐pass filter after excitation at 547 nm. The change in fluorescence intensity was plotted as a function of time, and the data were fit to a single exponential or sum of two to three exponentials to estimate the dissociation rates.The RNA association rates were measured at 25°C using the stopped‐flow instrument. A fixed concentration (100 nM) of Cy3‐labeled ds26 stem RNA from syringe A was rapidly mixed with the RIG‐I construct (250 nM) from syringe B. The fluorescence intensity was plotted as a function of time and fitted a single exponential equation or sum of two to three exponentials to obtain the observed rates of binding.).Fitting association and dissociation kinetics yielded the rate constant (k) and the population fraction (A) associated with each phase. Average association and dissociation times (t) were calculated by taking the inverse of the rate constant and weighting it by population fraction, as shown below in Equation 32P] ATP). The ATPase reaction was measured after 20, 40, 60 min or 30, 60, 90 min of reaction times in Buffer A at 25°C. Reactions were stopped at each time point using 4 N formic acid and analyzed by PEI‐Cellulose‐F TLC (Merck) developed in 0.4 M potassium phosphate buffer (pH 3.4). TLC plates were exposed to a phosphorimager plate, imaged on a Typhoon phosphor‐imager, and quantified using the ImageQuant software. The molar [Pi] generated during the reaction time intervals was plotted against time and fit to a linear equation. The slopes (ATPase rate) were plotted as a function of RNA concentration and fitted to the following equation to obtain the RNA KD,app values: slope = katpase × [PR]/[Pt]; where [Pt] is total protein concentration; [PR] is the amount of RIG‐I/RNA complex formed and [R] is the RNA concentration being titrated. The hyperbolic  and increasing RNA concentration (1 nM – 5 μM) in the presence of 1 mM ATP , a 5’ ppp ds27 RNA carrying a 3’ DY547 fluorophore and distal 5’ biotin (50 nM), and monovalent streptavidin (55 nM) in Buffer A for 60 min at 4°C. 2 mM ATP was added to each reaction, where applicable, 15 min before loading the sample into the gel. Loading buffer  was added to the samples and run on a 4–16% gradient Native PAGE gel (Invitrogen) at 4°C. Gels were scanned at 532 nM using a Typhoon FLA 9500 laser‐based scanner (GE).n) are noted in the corresponding figure legends.Luciferase signals in cell‐based signaling assays were measured using a luminometer (Tecan Spark) and quantified in Microsoft Excel. Steady‐state fluorescence intensity binding assays and ATP hydrolysis‐based binding assays were quantified and fitted using SigmaPlot v11.0 (Systat Software). Associated errors of the measurements and number of sample sets (Brandon D Schweibenz: Conceptualization; Data curation; Formal analysis; Investigation; Writing—original draft; Writing—review & editing. Swapnil C Devarkar: Conceptualization; Investigation; Writing—review & editing. Mihai Solotchi: Investigation. Candice Craig: Investigation. Jie Zheng: Investigation; Writing—review & editing. Bruce D Pascal: Investigation. Patrick R Griffin: Supervision; Writing—review & editing. Samantha Gokhale: Investigation. Ping Xie: Supervision; Investigation; Writing—review & editing. Smita S Patel: Conceptualization; Supervision; Funding acquisition; Writing—original draft; Project administration; Writing—review & editing.CRediT author contributions listed above, the contributions in detail are:In addition to the Conceptualization: BDS, SSP, SCD; Investigation: BDS, SCD, JZ, PX, SG, MS, CC, PRG, BDP; Writing—Original Draft: BDS, SSP; Writing—Review/Editing: All Authors; Funding Acquisition: SSP, PX.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.


Our results show that HCV is folded extensively in the cellular context. By performing comprehensive structural analyses on both in vivo data and in vitro data, we identify compact and conserved secondary and tertiary structures throughout the genome. Genetic and evolutionary functional analyses demonstrate that many of these elements play important roles in the virus life cycle. In addition to providing a comprehensive map of RNA structures and riboregulatory elements in HCV, this work provides a resource for future studies aimed at identifying therapeutic targets and conducting further mechanistic studies on this important human pathogen.Hepatitis C virus (HCV) is a positive-strand RNA virus that remains one of the main contributors to chronic liver disease worldwide. Studies over the last 30 years have demonstrated that HCV contains a highly structured RNA genome and many of these structures play essential roles in the HCV life cycle. Despite the importance of riboregulation in this virus, most of the HCV RNA genome remains functionally unstudied. Here, we report a complete secondary structure map of the HCV RNA genome IMPORTANCE HCV has one of the most highly structured RNA genomes studied to date, and it is a valuable model system for studying the role of RNA structure in protein-coding genes. While previous studies have identified individual cases of regulatory RNA structures within the HCV genome, the full-length structure of the HCV genome has not been determined in vivo. Here, we present the complete secondary structure map of HCV determined both in cells and from corresponding transcripts generated in vitro. In addition to providing a comprehensive atlas of functional secondary structural elements throughout the genomic RNA, we identified a novel set of tertiary interactions and demonstrated their functional importance. In terms of broader implications, the pipeline developed in this study can be applied to other long RNAs, such as long noncoding RNAs. In addition, the RNA structural motifs characterized in this study broaden the repertoire of known riboregulatory elements. RNA viruses provide a unique source of information on RNA structure. Because their genomes are relatively compact, RNA viruses must maximize the utility of RNA, using both primary sequence and structure to convey information, often within the same sequence. This “code within code” strategy provides viruses with extra layers of regulation. However, we still have limited understanding of RNA structure-based regulation and its contribution to viral growth and host-pathogen interactions.Flaviviridae family  is a 9.6-kb positive-strand RNA virus in the e family . With mae family , 3, HCV e family –10. For  family –. The cor family –, 12, 13.ynthesis , 15. A dRiboregulatory structures, such as knots, pseudoknots and kissing loop interactions, are clearly important in the life cycle of other positive-strand viruses, such as flaviviruses , so it iin vitro . We show that the HCV RNA is highly structured both in vitro and in vivo, identifying 20 well-folded regions in the HCV genome. Relying on the analysis of synonymous mutation rates (dS), base pair content (BPC), and differential reactivities between the two experimental conditions, we found highly structured motifs with evolutionary support as well as potential protein binding sites. We also identified three novel pseudoknots in the HCV coding region and demonstrated with structure-disrupting and compensatory mutations that all three play important roles in HCV replication. Overall, we present a detailed map of HCV secondary and tertiary structure and show that the viral open reading frame (ORF) contains compact structures that are important for viral growth. The methods of analysis and quality control used in this study can be extended readily to the study of other RNA viruses and other long RNA transcripts.In this study, we generated a full-length HCV structure map, including pseudoknots, for both in vitro structure model that would be suitable for guiding mutational study and downstream functional analysis, we used a recently optimized semi-native purification method (in vitro transcription. The secondary structure of the purified RNA was then probed by using the SHAPE-MaP strategy (P < 0.0001), indicating that the HCV RNA was modified successfully. The Pearson’s correlation analysis showed that the results are reproducible among different biological replicates  . PseudokP < 0.0001). In contrast, when we separated the SHAPE reactivities of single-stranded and double-stranded regions from a previously published HCV genomic structure determined by using denatured and refolded RNA  in the NS5B, and the long-stem structure in NS4B(SL6038) , which is higher than that reported for the SARS-CoV-2 genome   . Inspect 0.0001)  and dengin vitro data; representative structural motifs are shown in in vivo probing studies conducted during the second phase of the study (see below).In order to determine the most compact, well-determined regions of the genome, we computed local median Shannon entropy values and plotted them, together with SHAPE reactivities, at each position along the HCV genome, thereby enabling us to correlate regions of low SHAPE reactivity with low Shannon entropy . Shannonin vitro structural map, it was important to obtain a parallel map that captured conformational states of the HCV genome in a cellular context. A comparison of the two maps provided information on highly stable secondary structures, and it also flagged potential protein binding sites and revealed RNA elements that sample multiple functional conformations.Despite the high quality of the in vivo SHAPE-MaP library, we used the well-established HCV subgenomic replicon (HCV-SGR) cell culture model , thereby demonstrating that the data were suitable for de novo structure prediction.To determine whether the in vivo SHAPE data set. Since the in vivo reactivity data matched well with structures of the three pseudoknots predicted earlier using the in vitro data (not shown), the same pseudoknots were used as hard constraints during subsequent in vivo RNA structure prediction, which was conducted by Superfold as described in Materials and Methods. The resulting in vivo structural model of the HCV-SGR demonstrated that almost all previously known functional RNA structures in the UTRs and ORF were correctly predicted and that they are stable elements that exist in a cellular context  analysis showed that both models have 60% BPC, indicating a similar level and type of secondary structure complexity in both cases. Since SHAPE reactivity directly reflects nucleotide flexibility, we then analyzed the reactivity distribution of the two data sets. As shown in in vivo SHAPE reactivity and the in vitro reactivity values fell within a similar range. However, the in vivo reactivity was slightly lower than that measured in vitro (P = 0.0013), suggesting that, despite comparable BPC, the RNA in vivo was somewhat less flexible than the RNA in vitro. As a second comparison, we compared the Shannon entropy values obtained from both in vivo and in vitro structural models and found that Shannon entropy values for the in vivo model were significantly lower than those for the in vitro model (P < 0.0001). Since a lower Shannon entropy value indicates that a smaller set of conformations are compatible with the measured SHAPE reactivities  that was cloned within the HCV-SGR RNA. The expression of the neomycin resistance gene and the HCV genes are regulated by the HCV IRES and EMCV IRES, respectively, and both type of IRES drive translation with comparable efficiency . They included a bulged stem in NS3 (SL4826), two branched stems in NS4B (SL5724), and NS5B (SL8431) (in vivo data and two regions (J7800 and SL8209)  , Table 1s in HCV , 33. Ther of ∼0.5) within a section encoding domains of NS5B (7877 nt to 8412 nt) , and negative ΔSHAPE values suggest enhanced SHAPE reactivity in cell (suggesting more dynamic structure in vivo). We found that the region of poor correlation between in vivo and in vitro maps (8086 nt to 8286 nt) corresponds to a highly positive ΔSHAPE region , suggesting that this region might contain a stable protein binding site . A codon was considered to be double stranded or single stranded depending on whether or not the third position of the codon was base paired.As a first step for conducting phylogenetic analysis, we created two multiple sequence alignments, as follows: (i) a pan-genotype alignment that includes 126 full-length HCV sequences from all 7 HCV genotypes and (ii) a genotype 2-specific alignment that includes 7 full-length HCV genotype 2 sequences. We then calculated dS using FUBAR , binningP < 0.0001)  . This reTo determine whether certain sections of the HCV ORF experience particularly strong evolutionary pressure to maintain specific secondary structures, we applied dS analysis to individual protein-coding domains within the ORF. Expanding on previously published results , we founin vitro and in vivo. Indeed, we found that several functional elements demonstrated previously to play important roles in the HCV life cycle were flagged as evolutionarily conserved in the pan-genotype alignment. Specifically, they included a well-folded region within the core-coding region  and three additional stems  .To further examine evolutionary support for individual RNA substructures, we applied the dS analysis to all 20 well-folded regions that were identified 5BSL3.3)  and E. T5BSL3.3)  and G, sGaussia princeps luciferase (Gluc) reporter was inserted between the IRES structure and EMCV IRES structure, thereby providing a quantitative readout for the efficiency of viral replication  . In desilication . A lethalication . BecauseThe resulting time course shows that HCV replication was severely impaired by mutants that unzip key helices within SL8431, resulting in an ∼5-fold defect at 48 h posttransfection . ImportaOur pseudoknot prediction pipeline revealed the presence of three novel pseudoknots in the HCV genome with potential regulatory function . Given the lack of knowledge about RNA tertiary structures in the ORF region, and the important role played by pseudoknots in the function of many different viruses, we decided to focus our functional analysis on the three novel pseudoknots. As described in the previous section, we introduced synonymous mutations to break or rescue the base pairs in the pseudoknot structures. The effects of these mutations on viral replication were then measured by using the luciferase reporter assay described above .PsK1 was located within the NS3-encoding region and contained two small stem-loops, which were linked with four consecutive G-C pseudoknotted base pairs . DisruptPsK2 was also located within the NS3-encoding region and consisted of a central loop and two neighboring stem-loops. The pseudoknotted region was composed of 7 consecutive base pairs between the central loop and the 5′ stem-loop , left. APsK3 was located at the 3′ terminus of the NS5B-coding region, with pseudoknot base pairs formed between nucleotides within the bulged stem and an upstream, single-stranded region . The bulGCCCGA-3′) intact  , and giv) intact . This pa) intact . We then) intact . The res) intact  and F.CCGAGC-3′ → 5′-GCGAGC-3′). We observed that PsK3-unzip3 lost the ability to replicate , we designed another set of unzip and compensatory mutants to specifically probe the PsK3 conformation . PsK3-uneplicate , which ceplicate , insets.Taken together, our ability to identify novel, functional pseudoknots in the HCV ORF not only underscores the power of our prediction pipeline but also highlights the existence and importance of three novel predicted pseudoknots, of which all appear to be important for viral replication.In this work, we present the complete secondary structure of the HCV RNA genome, revealing a multitude of complex architectural units that are found in all sections of the genome, throughout the UTRs and long open reading frame. The resulting secondary structural maps revealed a network of riboregulatory elements with the potential to modulate diverse aspects of the viral life cycle. These findings expand and build upon earlier mechanistic studies showing that specific long-range interactions within the HCV genome are critical for the regulation of replication and infectivity , 43–45. 43–cis  metric, which indicates regions that are not only thermodynamically stable but also likely to maintain a single conformation . By usincis , this RNin vitro versus in vivo (ΔSHAPE) suggested the presence of specific protein-binding sites in the viral genome  structures that contribute to diverse functions in viral biology . For exa52–cis for HCV replication  at the 3′ end of the NS5B ORF  in the cellular environment and that one can build artificial genes in which secondary structures will persist, which has important implications for the design of mRNA vaccines and RNA devices in the cell.One of the most significant aspects of this study is our finding that structural features within the HCV genome were largely independent of the environmental context; similar structural elements were observed for HCV RNA probed  in vivo , 55, 56. in vivo –31. RNA in vivo may be attributable to a lack of encoded secondary structure within these particular genes, which may not have been subjected to selective pressure to evolve a stable architecture. By design, our study contained an internal control that enabled us to directly test this idea; inserted within the ORF for the HCV replicon was a bacterial neomycin resistance gene and its structure was probed alongside the immediately adjacent viral genes. We observed that the neomycin ORF is almost devoid of secondary structure, while right next to it, the HCV genes adopted their typical elaborate architectures. Both segments of this RNA were exposed to the same action of ribosomes and other cytosolic factors, and yet only the HCV sequences folded. This result indicates that UTRs and ORFs can adopt discrete folded structures in vivo but only if this capability is encoded within the sequence. The HCV ORF has been selected to adopt a structured, folded architecture that persists in the cell, and it is an inherent attribute of its specific sequence, as originally hypothesized by Simmons  can be used to ascertain trends with potential relevance to function. An analysis of SHAPE-MaP data across the HCV genome revealed regions of various structural complexities. For example, segments encoding NS2, NS4B, NS5A, and the second half of NS5B have a high BPC (>60%), while regions encoding E2 and the first half of NS5B are almost single stranded (BPC of <25%).Previous studies on viral RNAs have shown that high BPC structures often serve as hubs for protein-binding sites , while low BPC regions help maintain overall flexibility of the RNA , 44. In The same probing and prediction methods employed in this study were also applied to another RNA virus, namely, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , providiBoth strategies may play specific roles in evading the innate immune system. The short stems in SARS-CoV-2 may help the virus escape from pattern recognition receptors, such as MDA5, which recognizes long RNA duplexes . In contThe distinct genome architectures of these two viruses indicate that they face different selective pressures and that, in response, they have developed different strategies for organizing their secondary structures. Although it is well accepted that a highly structured genome may provide protection from the innate immune system and an enhancement of RNA stability , differein vivo. We established the functional importance of individual RNA structural elements through evolutionary and genetic studies, identified a novel role for pseudoknots in viral ORFs, and analyzed correlations between architectural complexity and genomic context. Together, these data provide a functional atlas of the HCV genome structure and lay the foundation for additional mechanistic and pharmacological studies. Importantly, the significance of the study extends beyond its implications for molecular virology. The analysis pipeline can be applied to other studies of long RNA transcripts, such as long noncoding RNAs, and the study provides much-needed comparative data on the differences between RNA structure in the cell and RNA that has been purified and studied in vitro. The implications of the study therefore have broad implications for RNA science.In this study, we provided a full-length structural model of the HCV RNA genome and showed that it maintains a highly compact structure in vitro transcription and semi-native purification of the full-length HCV RNA genome  . RNA from the peak fraction was diluted to 250 ng/μL and was folded by incubating with 10 mM MgCl2 at 37°C for 30 min. A final concentration of 200 mM NAI or the same volume of DMSO was added to the RNA solution and incubated for 10 min at 37°C. RNA was then LiCl-precipitated, washed with 80% EtOH, and resuspended in RNA storage buffer .The ository)  were perository) . Briefly variant  and treaIn vitro-purified RNA (1 μg) was reverse transcribed by using 200 U SuperScript II under standard buffer conditions  with random hexamers (LifeTech). Second strand synthesis was performed by using NEBNext Ultra II nondirectional second strand synthesis module (New England BioLabs [NEB]). Double-stranded cDNA was purified with Monarch DNA cleanup kits (NEB) and diluted to 0.2 ng/μL. Sequencing libraries were generated by using a Nextera XT DNA library preparation kit (Illumina) according to the manufacturer’s instructions. Libraries were quantified by using a Qubit instrument (Life Technologies) and bioanalyzer (Agilent) and sequenced on a NextSeq 500/550 system (Illumina).in vivo SHAPE-MaP, Huh-7.5 cells constitutively expressing the HCV subgenomic replicon (SGR-neo-JFH-1)  was reverse transcribed with SuperScript II by using gene-specific primers. Following reverse transcription, the cDNA products were cleaned up by using Agencourt AMPure XP beads and amplified by using Q5 hot start polymerase (NEB). RT primers and PCR primers for the amplicons are available at the GitHub repository.To perform o-JFH-1)  were groin vitro SHAPE-MaP library was aligned to the sequence of HCV Jc1 (based on JF343782.1). The raw sequencing files (.fastq files) for the in vivo SHAPE-MaP were concatenated first and then mapped to HCV pSGR-JFH1 (based on AB114136.1). The sequence files of the HCV genome used in the SHAPE-MaP analysis are available at the GitHub repository. All libraries passed the three quality-control checks of ShapeMapper 2.All SHAPE-MaP libraries were analyzed by using ShapeMapper 2 . The in In vitro SHAPE reactivity replicate 1 data were used as a soft constraint with default slope and intercept parameters . Secondary structure (with pseudoknots) was predicted in 500-nt sliding windows with a 100-nt step size across the HCV genome. Using the well-characterized IRES-pseudoknot as a benchmark (ShapeKnots (implemented in RNAstructure v5.8 ) was useenchmark , we set in vitro or in vivo NAI SHAPE reactivities from replicate 1 were used as constraints using default slope and intercept parameters. The maximum base-pair distance was set to 500 nucleotides, as done in the previous studies . Scorerin vitro model and in vivo model are available at the GitHub repository.All structures and reactivities shown in this manuscript were drawn using StructureEditor . The fulBPC is a metric describing overall RNA secondary structural complexity, computed by calculating the percentage of double-strand character within a given transcript . In thisin vitro and in vivo data sets is shown in Shannon entropy for each nucleotide was derived from the Superfold output. The local median SHAPE reactivity and Shannon entropy values were calculated using a centered 50-nt sliding window (25 nt flanked on each side). Global median SHAPE reactivities and Shannon entropies were subtracted from the calculated local medians for the purpose of identifying low Shannon entropy and low SHAPE reactivity regions. Regions of 40 nucleotides or longer with local median SHAPE reactivities and Shannon entropies below the global median are considered well-folded. In certain cases, identified well-folded regions were expanded to include the complete structure (SL4826 and J7800). The complete collection of well-folded regions identified from in vivo and in vitro SHAPE analysis were used as the input. The region from 7877 to 8412  was included in the analysis. The standard deviation and Z-score threshold were set to default. The find cite threshold was set to 10,15. Smoothing window size was set to 10.Delta SHAPE analysis was performed using the delatSHAPE program . The .main vitro transcribed as described above and then purified by the Qiagen RNeasy kit. Then, RNA was resuspended in RNA storage buffer to a final concentration of 1 μg/μL. The luciferase activity assay was performed in 12-well plate format. HCV RNA was transfected with the MIR-2250 mRNA lipid transfection reagent (Mirus) according to the manufacturer’s protocol. At each time point, the medium was collected, cells were washed three times with phosphate-buffered saline, and fresh medium was added. The collected medium was clarified by centrifugation , placed into fresh tubes, mixed with one volume of 2× luciferase lysis buffer, and stored frozen(–80°C). Luminescence was measured on a Biotek Synergy H1 plate reader using 20 μL of the medium, 50 μL of Gaussia luciferase reagent , and a 5-s integration time.Synonymous mutations in the ORF region containing pseudoknots were incorporated using the Q5 site-directed mutagenesis kit (NEB). The template plasmid was linearized and in vitro structure model  was generated using Clustal W codon alignment (implemented in MEGA7 ). Sequenre model . The MSAhttps://github.com/pylelab/HCV_SHAPE_MaP_Structure).Sequence files, primer data, SHAPE-MaP outputs (.map files), structural prediction outputs (.ct files), multiple sequence alignment files, and high-resolution structural map figures are available at the GitHub repository (


BRCA1/2 mutations), the introduction of poly ADP-ribose polymerase inhibitors (PARPi) has dramatically improved outcomes for women with HR defective HGSOC. By blocking the repair of single-stranded DNA damage in cancer cells already lacking high-fidelity HR pathways, PARPi causes the accumulation of double-stranded DNA breaks, leading to cell death. Thus, this synthetic lethality results in PARPi selectively targeting cancer cells, resulting in impressive efficacy. Despite this, resistance to PARPi commonly develops through diverse mechanisms, such as the acquisition of secondary BRCA1/2 mutations. Perhaps less well documented is that PARPi can impact both the tumour microenvironment and the immune response, through upregulation of the stimulator of interferon genes (STING) pathway, upregulation of immune checkpoints such as PD-L1, and by stimulating the production of pro-inflammatory cytokines. Whilst targeted immunotherapies have not yet found their place in the clinic for HGSOC, the evidence above, as well as ongoing studies exploring the synergistic effects of PARPi with immune agents, including immune checkpoint inhibitors, suggests potential for targeting the immune response in HGSOC. Additionally, combining PARPi with epigenetic-modulating drugs may improve PARPi efficacy, by inducing a BRCA-defective phenotype to sensitise resistant cancer cells to PARPi. Finally, invigorating an immune response during PARPi therapy may engage anti-cancer immune responses that potentiate efficacy and mitigate the development of PARPi resistance. Here, we will review the emerging PARPi literature with a focus on PARPi effects on the immune response in HGSOC, as well as the potential of epigenetic combination therapies. We highlight the potential of transforming HGSOC from a lethal to a chronic disease and increasing the likelihood of cure.High-grade serous ovarian carcinoma (HGSOC) is a genomically unstable malignancy responsible for over 70% of all deaths due to ovarian cancer. With roughly 50% of all HGSOC harboring defects in the homologous recombination (HR) DNA repair pathway (e.g.,  Even with years of research and the development of a new effective therapy, high-grade serous ovarian carcinoma (HGSOC) remains, to this day, one of the most lethal gynaecological malignancies. HGSOC belongs to the type II class of epithelial ovarian cancers (EOC) and mostly develops from fallopian tube secretory cells into aggressive high-grade tumours with early metastatic potential . In contWomen who have received first-line therapy can be stratified into having platinum-resistant or platinum-sensitive HGSOC/OC (defined as women whose cancer progresses within six months or after six months respectively) . HoweverBRCA1/2-mutant HGSOC , PAOLA-1/ENGOT-ov25 (NCT02477644), PRIMA/ENGOT-OV26/GOG-3012 trial (NCT026555016) and VELIA/GOG-3005 (NCT02470585) ) led to PARPi’s more recent use as a front-line maintenance therapeutic for women with mutant BRCA1/2 (both germline and somatic) and then in the setting of platinum-responsive or HR defective (HRD) HGSOC (HRD status determined through tests such as the Myriad MyChoice™ test)  polymerase inhibitors (PARPi) was approved for the treatment of recurrent, advanced nt HGSOC . Within (BRCA1/2 . This lee™ test) .This was a major advance, as PARPi was the first targeted treatment approved for women with HGSOC which was dependent on certain genetic mutations being present in the cancer itself. However, the presence of specific HRD gene mutations have been concluded by recent phase III trials to not be essential, rather to predict which women will benefit from experiencing the strongest responses to PARPi therapy and summarised in a meta-analysis of a trial in relapsed OC . TreatmeTP53 and BRCA1/BRCA2, driver mutations in other tumour suppressor or oncogenes are less common  in DNA    in DNA . In norm changes . G (Bowt changes . Apart fctively) . The remnd EMSY) . Loss ofnd EMSY) . These end EMSY) . This HRthesised . This HRMUC16/CA125 and MUC1 , C2 (immunoreactive), C4 (differentiated), and C5 (proliferative) . Each suand MUC1 . Thus, cand MUC1 . The C5  markers . C1, C2  markers . The C5  markers .BRCA-mutant HGSOC have improved prognoses compared with their HR proficient counterparts, especially BRCA2-mutant HGSOC , and increased expression of immune pathway genes  is associated with a better prognosis (Different lymphocyte subsets present in the tumour microenvironment (TME) can affect prognosis and tumour progression . Most nosurvival . Particurognosis . The mecrognosis . Neoantirognosis . HR-defirognosis . An addirognosis . These prognosis .BRCA1 mutant HGSOC are prone to maintaining an obligatory inflammatory state through the upregulation of cGAS/STING signalling and producing an abundance of ctDNA fragments ation (PARylation)  . PARylatRylation . The PARRylation .+, inhibiting PARylation, effectively disrupting recruitment of DNA repair proteins and PARP dissociation, thereby “trapping” PARP-1/2 on damaged DNA, and further reviewed in  and European Medicines Agency (EMA) approval for use in OC in the clinic . A key fAdditionally, another aspect of PARPi to consider is substrate selectivity and specificity. Most PARPi are highly selective toward PARP-1/2 although, computational in-silico analyses have uncovered 58 potential interactions with kinases of which only 10 were previously known . SupportBRCA1/2 mutations or platinum-sensitive HGSOC respectively . Strikingly, at 5-year of follow-up, the PFS for the placebo arm was 13.8 months compared with the olaparib arm, on which women had achieved an unprecedented 56.0 months PFS, a 4-fold improvement, and 48% of women on olaparib remained disease free at this time, compared with only 20.5% of women on the placebo arm (BRCA mutation and/or HRD status (p < 0.001). Roughly 50% of women were classified as having HGSOC with HRD and the greatest benefit derived from niraparib was seen in the subset of these with BRCA-mutations ; followed by that observed in the non-BRCA HRD HGSOC subset ; lastly the remaining ∼50% of women had HGSOC which was HR proficient and responded the least well to PARPi   . As seenRegardless of the efficacy of PARPi as a monotherapy, a growing concern is the development of resistance with the prolonged use of PARPi. There are five main classes of resistance that have been characterised; drug efflux, changes in PAR metabolism, mutational changes of binding sites or target proteins, rewiring of stalled fork replication and restoration of the HR pathways . SeveralBRCA1/2 mutations. However, since then, the field of PARPi has expanded to include the roles PARP-1 has in chromatin structure, gene expression, and innate and adaptive immune responses.Studies of PARPi initially focused on DNA damage repair and via PARylation of the histones. In its latent state, PARP-1 is found bound to linker DNA and/or histone proteins, resulting in the condensed structure of chromatin called heterochromatin. In this state, no transcription machinery can access the DNA, repressing gene transcription. In the presence of DNA damage however, PARP-1 becomes active . PARG counteracts the action of PARP-1 by cleaving the PAR on PARylated PARP-1, rendering it inactive . AmplifiAnother natural inhibitor of PARP-1 activity is macroH2A1.1 which binds to autoPARylated PARP-1 to prevent PAR hydrolysis, which can promote chromatin recondensation to interfere with transcriptional processes . MacroH2BRCA1 or RAD51C, occurring as an early clonal event, contributing to the development of OC cases , correlates with PARPi resistance  in eukaryotic genomes. The methylated cytosine (5 mC) is induced and maintained by DNA methyltransferase (DNMT). Promoter hypermethylation commonly promotes gene silencing. This epigenetic silencing has been observed in HR genes, including OC cases  BRCA1 cass in OC . Homozygsistance . Similarto PARPi .PARP-1 interaction with DNMT1 contributes to the regulation of DNA methylation. PARylation has been shown to maintain unmethylated CpG at specific sites of the genome, while blockade of PARylation increases DNA methylation levels in the genome . InteresIn BRCA1/2 defective cancer cells, when the backup DNA repair pathways are disrupted, PARPi can induce synthetic lethality. Thus, inducing the complete loss of DNA repair capability has been strategized to kill BRCA-proficient cancer cells, by combining PARPi with epigenetic drugs that can induce a BRCA defective-like phenotype . CombiniDNMT1 promoter from being methylated, and also by non-covalently interacting with DNMT1 to promote its methylating activity  in combination with PARPi to target HR pathways in BRCA-proficient triple-negative breast cancer, ovarian cancer, acute myeloid leukemia, and non-small cell lung cancer . DNMTi aactivity . A combiactivity . AdditioA genome-wide RNAi screen by in vitro studies show an augmented efficacy of PARPi at targeting HR pathways when combined with HDACi (Another type of epigenetic drug that could augment the effect of PARPi is the histone deacetylase inhibitors (HDACi). HDAC removes acetyl groups from the lysine residues of histone tails and has been shown to play various roles in cancer initiation, progression, metastasis and angiogenesis, thus it has emerged as anticancer drug . Severalth HDACi . Here, tth HDACi . Since ath HDACi . The ind+ T-cell differentiation  cells are effector lymphocytes utilized in the innate immune response against “non-self” cells and “self” cells undergoing stress in the form of infections or malignant transformations . When acH1 immune responses, however, maintaining a constant level of DNA damage and subsequent chronic inflammatory responses encourages the infiltration of immune-suppressive cells  cell lines and tumours samples after treatment with veliparib for three weeks. Upregulation of hallmark inflammatory TNFα pathways were observed after treatment with PARPi specifically in the BRCA1-deficient cells, and the mechanistic basis for this upregulation was through the cGAS/STING pathways. Separately, a study using genetically engineered mouse models (GEMM) of HGSOC investigated the effects of olaparib in BRCA1 deficient and BRCA1 wildtype settings. Treatment with olaparib elicited an increase in CD4+ and CD8+ T-cells as well as a pronounced increase in IFNγ and TNFα  in cells when treated with PARPi . Programoduction . The roloduction . All of ruitment . Additioruitment . Due to Currently, the most promising immunotherapy for HGSOC are immune checkpoint inhibitors (ICI), which are monoclonal antibodies that disrupt signalling that would promote effector T-cell deactivation. Most common ICI for HGSOC are antibodies that target PD-1 and Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), as a monotherapy they have had some efficacy in patients but few durable responses . The pha+ T cells were associated with an improved response  investigating the combination of niraparib/pembrolizumab (anti-PD-1) in recurrent OC and TNBC, an ORR of 25% was achieved in the general cohort which was an improvement compared to response rates of PARPi or IC as a monotherapy . Additioresponse . The pharesponse . The comresponse . HoweverBRCA1 mutant HGSOC and its inhibition increases hypoxia and subsequent downregulation of HR genes  as a result (p < 0.001) , followed by the HR-deficient subgroup with a PFS of 11.9 vs. 4.1 months  (Bevacizumab (BV) is an anti-VEGFR antibody already in use for the treatment of HGSOC in combination with standard chemotherapies. It targets the cytokine VEGF-A which is secreted by tumour cells and binds to VEGFR-1 and VEGFR-2 receptors, promoting angiogenesis  and metastasis. VEGF-A has been shown to be overexpressed in a result . This dea result . It has a result . The pha< 0.001) . The BRC17–0.58) . Overallp = 0.0125]. The biggest benefit gained was in the BRCA-mutant cohort where the median PFS has not been reached versus 45 months for placebo and BV . The next best group to benefit was the HRD cohort at 50.3 versus 35.3 months for placebo  comparatively the HRD negative cohort performed the worst at 24.4 vs. 26.4 months   investigating rucaparib and BV in a maintenance setting in HRD and HR proficient newly diagnosed HGSOC and endometrioid patients is currently recruiting and results may confirm the benefit of this combination in the HR proficient setting.In phase III PAOLA-1 clinical trial , the olaparib and BV combination was studied in a maintenance setting after first-line chemotherapy in HGSOC patients. In the overall cohort, the combination of BV and olaparib resulted in a median PFS of 36.5 months compared to the 32.6 months in the placebo and BV combination [HR 0.78, CI (0.64–0.95), 77–1.42) . The comp = 0.006) (p = 0.11), and similarly the BRCA-mutant cohort did not have a significant difference in PFS  or OS  between combination and olaparib only arms. Comparatively, women in the BRCA-wildtype cohort had a significant improvement in the combination arm versus the olaparib only with PFS at 23.7 versus 5.7 months . The OS for this cohort was also significantly improved at 37.8 versus 23.0 months   . The ove= 0.047) . This php < 0.0001). When compared across treatment arms the cediranib and olaparib combination extended PFS to 20.4 months  compared to 12.3 months in the chemotherapy arm and 13.1 months in olaparib only arm . There was no difference between treatments in the HR proficient cohort, with a median PFS of 8.5 months in the cediranib and olaparib combination arm , and 9.0 months in the chemotherapy arm and 6.4 months in the olaparib only arm . This study also looked at LOH as a prognostic factor and found it was not predictive of response to olaparib, combination cediranib/olaparib or chemotherapy possibly suggesting that the HRD assay used was not sufficiently discriminatory  comparing patients with platinum sensitive recurrent high-grade serous or high-grade endometrioid ovarian, primary peritoneal, or fallopian tube cancers. Patients were screened for HR and LOH status with the BROCA-HR targeted next generation sequencing assay on germline and tumour DNA in 491 of 565 patients and compared across treatment arms of olaparib only, chemotherapy only and combination cediranib and olaparib. The HR-deficient cohort did the best compared to the HR proficient cohort, with a median PFS of 13.7 vs. 8.3 months , 50% of PR patients (26.9–92.9) and 36% of PE patients (15.6–82.8) reached the 16-weeks PFS with OS at 1 year 81.8% (61.9–100) in PS, 64.8% (39.3–100) in PR and 39.1% (14.7–100) in PE , PARPi resistant (PR) and in patients who had chemotherapy post-PARPi progression (PE). A total of 34 patients were enrolled, with 9/11 PS, 8/10 PR and 7/13 PE patients with 0) in PE . This stOverall, the results from these clinical trials establish that there may be an OS benefit to be gained from the combination of cediranib and PARPi in the treatment of HGSOC, however results are inconclusive and are being investigated further in the phase III ICON9 trial assessing cediranib and olaparib vs. olaparib alone as a maintenance therapy in platinum sensitive recurrent OC currently enrolling and the phase II/III GY005 platinum-resistant relapsed OC cediranib PARPi combination therapy clinical trial (NCT02502266) .The triple combination of PARPi, bevacizumab and anti-PD-L1 therapy has also been trialed in the clinic. The phase II open-label study (NCT03574779) of dostarlimab, BV and niraparib in platinum-resistant recurrent EOC resulted in a median PFS of 7.6 months, disease control rate (DCR) of 76.9% with 23 patients with stable disease, seven patients with PRs and no CRs . The cliIn recent years, studies have demonstrated platinum compounds can act as immune modulators effectively inducing immunogenic cell death alongside their DNA-damaging characteristics . Platinup = 0.0012). The combination was well tolerated with only 15% reporting adverse events in the combinational group versus the 21% in the chemotherapy group alone. Most benefit was assumed derived from the maintenance phase of olaparib and specifically in the BRCA1/2 mutant cohort. Regardless, the ORR was 64% versus 58% between the different treatment arms.A phase I study (NCT00516724) trialled olaparib with paclitaxel or carboplatin or carboplatin or the paclitaxel (CP) combination in advanced solid tumours refractory to standard treatments . PatientBRCA-mutant and 55% noted as HRD. Overall, the addition of Veliparib significantly improved PFS with median PFS of 23.5 months vs. 17.3  and 31.9 months vs. 20.5 months  respectively. Veliparib has currently not been approved for use in the treatment of OC.Veliparib is a relatively weaker PARP trapper therefore potentially better tolerated for combination studies. Veliparib has been trialed with the standard carboplatin and paclitaxel (CP) chemotherapy combination in the phase III VELIA study in women with newly diagnosed HGSOC . PatientBRCA-mutant OC and HGSOC , it is a potent immunostimulant when delivered at low doses and is well tolerated, eliciting clinically beneficial responses in roughly 44% of recurrent OC cases . Studiesnd HGSOC . The addnd HGSOC . This trp = 0.024). Combining LDCy with PARPi and CI could elicit similar responses, targeting cancer cells and invigorating immune responses, particularly in platinum-sensitive/HRD patients that derive the most benefit from these drugs.The NCT02853318 phase II clinical trial observed the effects of pembrolizumab (anti-PD1), bevacizumab and LDCy in recurrent OC . The trigBRCAm subset, the unconfirmed objective RR was 48% and 64% respectively.The combination of olaparib and LDCy has been examined in recurrent OC and triple negative breast cancer to determine its safety and tolerability . A tolerable regimen was identified and in HGSOC and the The treatment of HGSOC provides an ongoing challenge, due to the heterogenous and metastatic nature of this disease rendering women susceptible to disease relapse. Extensive research performed to characterise the disease has led to a better understanding of which characteristics correlate with clinical benefit from current therapeutic regimens. However, monotherapies do not effectively target the multiple aspects of HGSOC tumours that can occur simultaneously, in the vast majority of cases. The development of drug resistance is a growing concern, especially following treatment with PARPi and thus the use of combination regimens has garnered increasing interest. The characterisation of PARPi has elucidated its myriad roles in DNA repair and regulation, including roles in chromatin remodelling and methylation. Additionally, effects of PARPi on immune cells and immune responses offer alternative pathways for therapeutic exploitation. To date we have seen PARPi combined with chemotherapy, angiogenesis agents, immune checkpoint inhibitors and more recently novel therapeutics including epigenetic drugs and other DNA repair inhibitors, with some trials investigating triple combination treatments. The benefit of these therapies for women with platinum-resistant and HR proficient OC are still unclear when compared to their platinum-sensitive and HRD OC counterparts. One of the most challenging aspects of combination therapy is tolerability. Investigating alternative treatment sequencing and scheduling could result in triple-combination therapies becoming more tolerable and in prolongation of survival if successfully matched to the molecular characteristics of the HGSOC. Additionally, clinical trials involving correlative studies to investigate outcomes are essential to establish ideal biospecimen cohorts to enable the most complete understanding of a trial outcome and for further research. Understanding the characteristics that drive responses could improve strategies for driving prolonged remissions and ultimately improve the survival outcomes for women with HGSOC.


CNV values > 5.8 indicate a significantly increased risk for dSCC, while a reduced risk can be assumed for GS with CNV < 4.7. CNV values between 4.7 and 5.8 appear as a grey area. In summary, including the germline mutation (KITLG CNV) in breeding decisions will be complex, but this diagnostic test may help to assess the individual risk for dSCC and to sensitise owners of black GS accordingly if the CNV value is high.It is well known that dark-coated dogs, especially schnauzers, have a predisposition for digital squamous cell carcinoma (dSCC). Molecular genetic studies by other researchers indicate a pathogenetic role of copy number variations (CNVs) of the KIT ligand (KITLG) gene. Our study aimed to use droplet digital PCR of blood samples to investigate whether the copy number has a predictive value for the disease in schnauzer breeds. We showed that most giant (GS), standard (SS), and miniature schnauzers (MS) had more than four and up to seven copies of this gene segment. Furthermore, the copy number in black GS with dSCC was significantly higher than in the black control GS (p = 0.02). CN values > 5.8 indicate a significantly increased risk for dSCC, while CN values < 4.7 suggest a reduced risk for dSCC (grey area: 4.7–5.8). Diagnostic testing for KITLG CNV may sensitise owners to the individual risk of their black GS for dSCC. Further studies should investigate the relevance of KITLG CNV in SS and the protective effects in MS, who rarely suffer from dSCC.Copy number variations (CNVs) of the KITLG gene seem to be involved in the oncogenesis of digital squamous cell carcinoma (dSCC). The aims of this study were (1) to investigate KITLG CNV in giant (GS), standard (SS), and miniature (MS) schnauzers and (2) to compare KITLG CNV between black GS with and without dSCC. Blood samples from black GS (22 with and 17 without dSCC), black SS , and 50 MS (unknown dSSC status and coat colour) were analysed by digital droplet PCR. The results are that (1) most dogs had a copy number (CN) value > 4 (range 2.5–7.6) with no significant differences between GS, SS, and MS, and (2) the CN value in black GS with dSCC was significantly higher than in those without dSCC ( Diseases of the canine toe can be of non-neoplastic 43%), neoplastic 52%), or tumour-like (5%) origin . Squamou%, or tum%, neoplaSquamous cell carcinomas of the toes originate in the stratum spinosum of the claws or the sole epidermis. Clinically, they may initially look like inflammation of the nail bed. There may be lameness, thickening of the toes, and ulceration of the digital tissue. The nail material often appears softened and frayed . Most coAmputation of the toe is the treatment of choice in dogs with dSCC . The hisDark coat colour is correlated to the development and aggressiveness of squamous cell carcinoma ,6,13,16.Cutaneous squamous cell carcinomas in man, which are mainly induced by ultraviolet light, have a different and more complex mutational landscape, e.g., with NOTCH1, NOTCH2, TP53, and RAS mutations . Non-digTyrosine kinase receptors (TKR) comprise more than 60 molecules that play an essential role in the molecular pathways of cell survival and differentiation . The KITThe ligand for this KIT receptor is the stem cell factor (SCF), which is encoded by the c-KIT ligand (KITLG) gene. This ligand seems to play a role in canine mast cell tumours , melanogCopy number variations (CNVs) are defined as genomic regions that vary in number by amplification or deletion of DNA sequences. They play an important role in the genetic diversity necessary for evolution but are also responsible for hereditary and somatic human diseases, such as cancer . Small CA genome-wide association study (GWAS) on standard poodle DNA found an increased risk of developing digital SCC when an increased number of specific copies (>4 copies) was expressed at the KITLG locus . FurtherThe aims of this study were (1) to investigate KITLG CNV in miniature, standard, and giant schnauzers and (2) to compare the CNV of KITLG between black giant schnauzers with and without digital squamous cell carcinomas and to evaluate its possible predictive value in this breed.Blood samples from 39 giant, 27 standard, and 50 miniature schnauzers were selected from the archive material stored after routine diagnostic examinations at LABOKLIN GmbH & Co. KG, Bad Kissingen, Germany (2014–2021). Inclusion criteria were that breed, age, and sex of the dogs were indicated on the submission form.n = 39) included 13 male, 8 neutered male, 11 female, and 7 neutered female dogs aged 5 to 14 years (median 10 years). The group of standard schnauzers (n = 27) included 7 male, 7 neutered male, 5 female, and 8 neutered female dogs at the age of 5 to 13 years (median 10 years).Samples from giant and standard schnauzers were obtained from pre-operative blood samples from animals with digital masses before amputation as well as from dogs with geriatric blood screening. The group of giant schnauzers  and/or by telephone calls to the veterinarians. It was recorded whether the respective dogs had already suffered from SCC of the digit and which coat colour  it had: All standard schnauzers and giant schnauzers in studies 1 and 2 were black.n = 50) included 24 male and 26 female dogs between 1 and 9 years of age (median 1 year). Information about clinical findings or coat colour was not available.Samples from miniature schnauzers mainly came from breeding animals (young and not neutered) that were routinely screened for relevant genetic diseases. The group of miniature schnauzers n = 11): Blood samples from black giant schnauzers aged >10 years  that remained free of digital SCC until the end of the study were used. There were 4 male dogs, 2 neutered males, 4 females, and 1 neutered female. Six younger dogs without dSCC from study 1 were excluded;Control group ((2)dSCC group: Blood samples from 22 black giant schnauzers aged 6–13 years (median 10) with squamous cell carcinoma of the digit were diagnosed by different pathology laboratories. This group included 7 males, 6 neutered males, 6 females, and 3 neutered female dogs.Two groups of black giant schnauzers were selected from the cohorts of study 1:Clinical questionnaires for the standard schnauzers identified 18 black standard schnauzers with dSCC and 4 without dSCC. No data about dSCC were available in five cases; thus, they had to be excluded. As the control group of SS was too small, these data were not useful for a valid statistical analysis. We therefore did not conduct another study with these data.SCC of the digit is not expected in the young miniature schnauzers of cohort 1. Furthermore, clinical data and coat colour were not available. Thus, no further statistical analyses were performed.® probes and primers specific for the KITLG sequence and proto-oncogene 1 (ETS1) as reference gene based on the paper of Bannasch et al. (2021) and as performed previously [To determine the copy number of the relevant gene segments of KITLG, molecular genetic analyses were performed on blood samples as follows: Genomic DNA was isolated from EDTA blood with the MagNA Pure 96 system using DNA Tissue Lysis Buffer and viral NA Small RNA kit  according to the manufacturer’s instructions. Copy number quantification of the KITLG was performed by digital droplet PCR (ddPCR) using TaqManeviously ,37. Measp < 0.05 (weakly significant), p < 0.01 (significant), and p < 0.001 (strongly significant).All statistical analyses and visualisations were carried out with the statistical framework R version 4.2 . To compThe copy numbers of KITLG in the cohort of study 1  ranged from 2.5 to 7.6. There were no significant differences between the KITLG CNV values of the giant, standard, and miniature schnauzer groups tested by Kruskal–Wallis . MultivaThe CN values form 18 standard schnauzers with dSCC ranged from 3.8 to 6.7 (median 5.4). The copy number values in 22 giant schnauzers with dSCC varied between 2.5 and 6.9 (median 5.7). There was no statistical significance between these two breeds .n = 3), II (n = 7), III (n = 2), IV (n = 2), and V (n = 8). The other six GS without dSCC were younger than 10 years and were excluded from further statistical analyses because it was too uncertain whether they would develop dSCC later in life. Further medical history of neoplasia other than dSCC was available in only three dogs: One 13-year-old male GS never had any tumour. One 13-year-old female neutered GS died of cardiac insufficiency and suffered from diabetes insipidus. One 12-year-old female GS developed a non-specified mammary tumour after the end of this study.Of the 39 giant schnauzers of study 1, we selected 11 black giant schnauzers that had no SCC of the digit until the end of this study (controls) and 22 black giant schnauzers with digital SCC. In the dSCC group, 19 front legs and 3 hind legs were affected. Squamous cell carcinoma was diagnosed on all toes and was distributed as follows: toe I  factor (The number of KITLG gene copies in the control dogs ranged from 4.5 to 6.5 (median 5.2). The copy number in dogs with dSCC varied between 2.5 and 6.9 (median 5.7). The quality of the classification in giant schnauzers with and without SCC was analysed with a multiple logistic regression approach. It was identified that the copy number was significantly higher in the giant schnauzers of the dSCC group compared to the controls  factor  and was An ROC curve was used to determine the specificity (true negative rate) and sensitivity (true positive rate) for predicting whether the sample of a giant schnauzer is classified as “control” or “dSCC”. In general, values of the area under the curve (AUC) can range from 0.5 (random noise) to 1 . The AUC value in the present analysis of copy number testing of the KITLG locus in black giant schnauzers was 0.88 .To estimate the prediction quality on new samples, we applied leave-one-out cross-validation. This method yields an expected accuracy (percentage of correct predictions) of 81.25%. The results identified a 50% probability threshold of 5.2 copies with 95% confidence intervals for 10-year-old giant schnauzers as shown in Previous publications have identified that there is a basic risk of developing digital SCC in canine breeds with dark coats, such as standard and giant schnauzers ,6,17. InA KITLG copy number value >4 was found to be correlated with a predisposition to dSCC in black standard poodles compared with light-coloured standard poodles . In a rehttps://psk-projekt.jimdo.com/unsere-rassen, accessed on 5 February 2023), German giant and standard schnauzers are mainly black . In contrast, the coat colour of miniature schnauzers varies between black, pepper and salt, black-silver, and white (18.04.2007/EN FCI-Standard N° 183). The coat colour of the puppies per year is: 1 white to 1.3 black-silver to 3.4 black to 4.7 pepper and salt .The present study was the first to compare the three size variants of the schnauzer breed for their genetic profile with regard to the copy numbers of the KITLG locus. Our data did not find any significant differences in CN values among the three breed variants we examined. The results of our study showed that all but one of the schnauzers had a copy number value >4 and are thus probably at-risk animals, as it has been defined for black poodles . HoweverAs we only had black SS and GS in our study, we investigated whether there are any differences in KITLG copy numbers between black giant schnauzers with or without dSCC during their life. The number of standard schnauzers with reliable clinical information was too small for a valid statistical analysis.p = 0.02). One limitation of our GS control group is that it is based on the assumption that these animals have not developed squamous cell carcinoma of the digit even when older than 10 years. However, we have designed this group according to the control group of the standard poodles (older than 8 years) in the initial study mentioned above [Yet, for the first time, a predictive statement could be made about the probability of SSC occurring in giant schnauzers. We found that in black giant schnauzers, more copies of the KITLG locus were significantly associated with the probability of developing dSCC  with dSCC that had a CNV of 2.5. In this case, spontaneous dSCC can be assumed, which is independent of factors of breed predisposition.However, the copy numbers in black standard and giant schnauzers with dSCC did not vary significantly. As we were not able to collect enough samples from control standard schnauzers by the end of this study, further investigations are necessary to determine similar diagnostic thresholds for this breed. Furthermore, this phenomenon should be analysed comparing black and pepper and salt standard schnauzers as well.In contrast, miniature schnauzers very rarely develop digital SCC  but alsoThus far, only very few studies have investigated the KITLG (stem cell factor) expression level on mRNA or protein levels in human cells , mouse mIn summary, this is the first time that a prognostically relevant germline mutation has been detected for a specific canine tumour (dSCC in black giant schnauzers). However, the method of copy number analysis of KITLG does not say anything about the distribution of the chromosome sets. Thus, including CNV analysis of the KITLG locus in breeding decisions is complex, and the prevention of squamous cell carcinoma of the digit in giant schnauzers remains challenging for breeders. Nevertheless, this diagnostic test can help to assess the individual risk of developing the disease and to sensitise owners accordingly if the CN value is high. It may encourage owners to screen their dogs for neoplasms if they understand that there is a well-known breed predisposition. Thus, a regular medical check-up  of the toes is recommended especially in such high-risk dogs, and early surgery may prevent metastases.In summary, detecting the copy number variation of the KITLG locus in black giant schnauzers is a promising new tool to predict the individual risk of developing squamous cell carcinoma of the digit. Further research is necessary to make comparable statements in standard schnauzers and to understand the protective mechanisms in miniature schnauzers.