Picornaviridae, Flaviviridae, Matonaviridae, Caliciviridae and Coronaviridae) showed a >2-fold base composition normalised excess of C->U transitions compared to U->C (range 2.1x–7.5x), with a consistently observed favoured 5’ U upstream context. The presence of genome scale RNA secondary structure (GORS) was the only other genomic or structural parameter significantly associated with C->U/U->C transition asymmetries by multivariable analysis (ANOVA), potentially reflecting RNA structure dependence of sites targeted for C->U mutations. Using the association index metric, C->U changes were specifically over-represented at phylogenetically uninformative sites, potentially paralleling extensive homoplasy of this transition reported in SARS-CoV-2. Although mechanisms remain to be functionally characterised, excess C->U substitutions accounted for 11–14% of standing sequence variability of structured viruses and may therefore represent a potent driver of their sequence diversification and longer-term evolution.The rapid evolution of RNA viruses has been long considered to result from a combination of high copying error frequencies during RNA replication, short generation times and the consequent extensive fixation of neutral or adaptive changes over short periods. While both the identities and sites of mutations are typically modelled as being random, recent investigations of sequence diversity of SARS coronavirus 2 (SARS-CoV-2) have identified a preponderance of C->U transitions, proposed to be driven by an APOBEC-like RNA editing process. The current study investigated whether this phenomenon could be observed in datasets of other RNA viruses. Using a 5% divergence filter to infer directionality, 18 from 36 datasets of aligned coding region sequences from a diverse range of mammalian RNA viruses (including  The rapid evolution of RNA viruses is thought to arise from high mutation frequencies during replication and the rapid accumulation of genetic changes over time in response to its changing environments. This study describes an additional potent factor that contributes to the evolution of RNA viruses infecting mammals, the occurrence of specific C->U mutations in a subset that possess intensely structured genomes. This mutational process substantially damages the virus’s ability to replicate and is potentially akin to “genome editing” of HIV-1 and other retrovirus genome sequences by APOBEC, one of the principal components of vertebrate antiretroviral defence mechanisms. This study however provides evidence for a wider mutational activity against several human and veterinary RNA viruses, including HCV and foot and mouth disease virus. The C->U mutational process accounted for 15–20% of standing sequence variability of these RNA viruses, representing a potent driver of their sequence diversification and longer-term evolution. The evolution of viruses is typically conceptualised as a combination of adaptive sequence change in response to a range of selection pressures in the environment and a process of random diversification in which neutral or near neutral nucleotide substitutions become fixed in virus populations –3. An exA further source of mutations in viruses arises from effects of several innate antiviral effector mechanisms in vertebrate cells that operate through viral genome editing. Of these, the best characterised are the interferon-inducible isoform of adenosine deaminase acting on RNA type 1 (ADAR1) that tarThe current study investigated this possibility through construction and analysis of sequences changes in alignments of mammalian RNA virus sequences from a wide range of families. Since APOBEC-mediated RNA editing has been proposed to occur in the context of RNA structure elements, virus datasets were additionally scanned for sequence-dependent internal RNA base-pairing ,20. ViruThe primary resource for the study were aligned sequences from a wide range of mammalian RNA viruses derived from several vertebrate virus families Tables  and S1. The previous analysis of the directionality of sequence changes in SARS-CoV-2 and the detection of an excess number of C->U changes was simplified by the minimal sequence diversity of the assembled post-pandemic sequences . SARS-CoSequence datasets was analysed using the program Sequence Change in the SSE package, which records the occurrences and sites of sequence changes from a majority rule alignment consensus sequence. Collectively, there was a significantly greater number of C->U changes compared to its reverse (U->C) and to other transitions in the RNA virus datasets at sites showing <5% heterogeneity although the degree of over-representation was highly variable between viruses . TransitTo more formally quantify the degree of over-representation of C->U transitions in each virus dataset, the ratio of each transition to its reverse . Similarly for HCV-1a, both U->C and C->U transition frequencies followed marginally skewed Poisson distributions (test statistics 1.5 [p = 0.03] and 2.5 [p < 0.001] respectively), but with a higher mean number of C->U transitions per sequence (12.1 / sequence) than the reverse (2.8 / sequence). The sequence datasets of HCV and EV-A71 sequences therefore showed no evidence for the occurrence of individual hypermutated sequences as described previously for HIV-1 [The estimation of relative transition frequencies was collectively based upon all sequences within each virus alignments. To investigate the degree of heterogeneity in C->U changes between sequences, numbers of this transition were computed individually and compared with those of the reverse mutation  in two oor HIV-1 –25; the p = 0.002). There was no association with MFE, representing the minimum free energy on RNA folding, a property primarily influenced by the G+C content of the sequence, which also showed no association with the C->U / U->C transition asymmetry. There were similarly no associations with extents of CpG or UpA dinucleotide suppression in RNA virus genomes, or base imbalances . As expected from the minimal differences from the null expectation, no association between G->A/A->G transition asymmetry values with any compositional or structural sequence attribute was detected.To investigate which genome features of RNA genomes were predictive of the C->U/U->C transition asymmetry, a range of compositional attributes , mean folding energies (MFEs) of consecutive 300 base fragments and differences of this value from sequence order randomised controls (MFEDs) were computed. The association of each with C->U/U->C and G->A/A->G transition asymmetry values was analysed by multivariate analysis . MFED vap values of 0.0004, 5 x 10−11 and 8 x 10−5 respectively by Mann Whitney U test). Effects of 3’ context were far more variable between viruses, but with evidence for favoured C->U transitions upstream of A in FMDV and to lesser extents in other RNA viruses.C->U transitions in SARS-CoV-2 genomes were influenced by the immediate 5’ and 3’ base contexts of the mutated site . Other RPrevious analyses demonstrated that a proportion of C->U mutations in SARS-CoV-2 failed to become genetically fixed in a population . The disThe program, Homoplasy Scan in the SSE package was developed to sequentially analyse each variable site in a virus sequence alignment; this recorded the degree of segregation of each base in a global tree constructed from 1200 base genome fragment that incorporates the interrogated bases . AssociaBroadly, the observed differences arose from different genetic structuring of sample populations; phylogenetic trees from alignments with predominantly informative sites showed a marked degree of structured internal branching , while vIrrespective of the actual distributions of AI values (and associated differences in tree topologies of the different RNA virus datasets), categorisation of sites based on AI value ranges allowed an investigation of whether C->U transition frequencies were specifically over-represented at phylogenetically uninformative sites as would be expected if these mutation were subject to homoplasy . AmongstThe extent to which the observed excess of potentially homoplastic and transient C->U mutations in certain viral datasets contributed to overall viral sequence diversity was calculated. The number of sites in a virus alignment showing a majority C->U change subtracted by those showing majority U->C changes (excess C->U) were expressed as proportion of the number of variable sites in the alignment, with totals sub-divided into sites showing different ranges of AI values . There wThis study provides evidence for an excess of C->U changes over the reverse (U->C) and complementary (G->A) transitions in a diverse range of RNA viruses, including HCV , FMDV, MNV, HPgV-1 and rubella virus. Fold excesses ranging from 1.9x – 3.6x  approachThe underlying mechanism(s) for the observed elevated frequencies of C->U changes in certain RNA viruses are functionally uncharacterised and conceivably may originate through several mechanisms. In the discussion, we will briefly review the evidence for or against transcriptional, RNA damage-associated and cellular RNA editing mechanisms that may create the excess C->U mutations observed in RNA viruses, taking into account their prominent (+)strand asymmetry, 5’ base context effect and their variable distributions in viruses with structured and unstructured genomes.A major source of mutations in viruses and other organisms are transcription errors by cellular RNA or DNA polymerases that are incorporated during replication. Different types of polymerases may show varied mutational profiles with separate propensities to mis-incorporate particular transitions or transversions. Very limited data exists on mutation frequencies associated with viral RNA dependent RNA polymerases (RdRps). However, biochemical assays of misincorporation kinetics on RNA templates by the poliovirus RdRp ,33 or reMutations introduced by RNA transcription would be expected to be symmetric in RNA viruses as their genomes derive from an equal number of positive and negative strand copyings by the same RdRp operating in the same cellular compartment. A tendency to misincorporate a U instead of a C would therefore be reflected in a parallel number of G->A mutations where it occurred on the minus strand. However, as observed previously for SARS-CoV-2 –14,26, tTo counter this, it could be argued that natural selection operates differently on the minus- and plus-RNA strands where the limited number of minus-strand templates produces a much larger number of genomic RNAs. Therefore there may be more stringent selection against mutations occurring in the negative-strand as these are invariably copied into the plus-strand, whereas plus-strands are relatively dispensable and mutations occurring during their synthesis will not overly affect the overall replication process. Indeed, full-length transcripts with plus-strand synthesis errors could be readily packaged into virions assembled from viral proteins synthesised from independently transcribed mRNAs.An alternative possibility is that mutations occurring during transcription of the minus strand are more rather than less likely to be fixed because minus strands are in a substantial minority of viral RNA sequences in the infected cell. Mutations arising from specific RdRp misincorporation biases occurring early in infection during synthesis of the minus-strand might be more likely to become fixed in the infected cell through a founder effect than mutations occurring from the 10s or 100s of plus-strands synthesised from that template. In contrast to the selection-based asymmetry hypothesis above, the observed excess of C->U mutations in the genomic (plus-)strand must therefore have originated from elevated frequencies of G->A mutations in the minus-strand rather than C->U.Neither hypothesis for a transcriptional origin of the C->U strand asymmetry recorded in the current study is supported by the observation of its variable presence in different RNA virus groups and association with genomic RNA secondary structure. While it is possible that RdRps from different RNA viruses may vary in their spectra of misincorporation frequencies, it is notable that C->U asymmetries cut across family divisions, with examples of picornaviruses and flaviviruses showing examples of viruses with both biased  and unbiased  C->U transition frequencies. The association of C->U asymmetry with the presence of genomic RNA secondary structure  thereforIt has been recently proposed that substitutions occurring in the SARS-CoV-2 genome, notably the G->U transversion may originate from mutational damage to viral RNA during periods of oxidative stress ,16. ROSsAn alternative source of mutations arises from the documented effects of several innate antiviral effector mechanisms in vertebrate cells that operate through a process of genome editing; these may potentially introduce mutations into RNA virus genomes during replication. Of these, the best characterised are the interferon-inducible isoform of adenosine deaminase acting on RNA type 1 (ADAR1) that tarMembers of the APOBEC family typically target single stranded DNA templates for mutagenesis of cytidine to thymidine during reverse transcription of retroviruses and hepatitis B virus (HBV) and in the genomes of small DNA viruses such as papillomaviruses ,38,39. FIf APOBEC was responsible for the over-representation of C->U changes in RNA virus genomes, there should be an increased C->U/U->C mutational asymmetry downstream of a U, as this is the target preference of most mammalian APOBEC paralogues. Consistently with findings of cellular mRNA editing by A3A ,42 and peg. the relative representation of UpC, ApC, GpC and CpC) to normalise for 5’ compositional effects. However, the problem with this approach is determining 3’ contexts since the CpG dinucleotide is substantially suppressed in mammalian +strand viruses, often to 15–20% of expected frequencies [The wide range of G+C contents of the viruses analysed in the current study  necessitquencies –46; atteThe favoured 5’ context of APOBEC-mediated mutations and consequent depletion of TpC/UpC dinucleotides has been exploited as a means to identify editing “footprints” in viral genomes in a previous bioinformatic analysis . DepletiApart from 5’ and 3’ base contexts, the only other compositional metric influencing the extent of C->U / U->C transition asymmetry was RNA structure formation , a genomThis association with structured RNA virus genomes potentially recapitulates the previous noted restriction of editing of human mRNA sequences by A3A to sites in defined stem-loop contexts . In an aWhile these associations are intriguing, the evidence is circumstantial without systematic functional studies to support a role of APOBEC-mediated RNA editing in restriction of RNA virus replication. Indeed, it was shown that antiviral effects of APOBEC occurred in the absence of any evidence for RNA editing of coronavirus HCoV-NL63 genomic sequences . It woulin vitro and a targeted gene deletion approach to functionally test editing abilities of individual APOBEC proteins on RNA templates would be important steps in such investigations.Inferences on mechanism based on bioinformatics analyses therefore must at this stage be indirect; they should additionally acknowledge that the currently recognised repertoire of RNA editing pathways and other mutational mechanisms in mammalian and other vertebrate cells is almost certainly incomplete. Viral RNA editing may indeed originate from an entirely different mechanism outside the current ADAR1 and APOBEC paradigm. As recently suggested, overlapping but nevertheless distinct C->U mutational contexts in SARS-CoV-2 and rubella genomes points towards the operation of more than one mutational mechanism ; the hetIrrespective of the underlying mutational mechanisms, the analyses performed in the study provided evidence that a substantial proportion of population variability in HCV and other structured viruses could be attributed to a marked over-representation of C->U transitions  at an individual sequence level approximated to a Poisson distribution , albeit While tangential to the primary focus of the study, the shape of phylogenetic trees constructed from different RNA viruses differed substantially Figs  and S2 ahttp://www.virus-evolution.org/Downloads/Software/) [ie. picornaviruses, flaviviruses) was based on coding regions only. Regions spanning the start of the first open reading frame (ORF) to the end of the last ORF were used for analysis of viruses with polycistronic genes . Alignments are available from the author on request.Alignments of sequences of HCV genotypes 1a, 1b, 2a and 3a, SARS-CoV-2 and other coronaviruses were derived from previous studies ,50. Furtftware/) . AnalysiCalculation of pairwise distances and nucleotide composition was performed using the SSE package version 1.4. Sequence changes were compiled using the program Sequence Changes with a variability threshold typically set at 5% heterogeneity, where heterogeneity was calculated as the cumulative frequency of all non-consensus bases. Multiple thresholds were used to analyse mutation representation at sites showing different levels of variability Figs .Normalised ratios (nC-U) of C->U and U->C transitions (and comparably for G->A and A->G) were calculated as:Normalised 5’ and 3’ context preferences for C->U/U->C transition asymmetries (nC->U) in specific 5’ and 3’ contexts, (5’x)nC->U and (3’x)nC->U  were calculated as:Computation of MFE and MFED values was carried out using the program Folding energy scan in the SSE package using sequential 300 base sequence fragments incrementing by 30 bases between fragments. The program call the RNAFold.exe program in the RNAFold package, version 2.4.2  with defet al.[et al. [AI values were calculated using the algorithm originally described by Wang et al. and Coch.[et al.  and implNeighbour joining trees were constructed from aligned sequences using the program MEGA7 . LineageAll statistical calculations and histogram constructions used SPSS version 26.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S3 Fig(DOCX)Click here for additional data file.


Pax5 mutant mice that converge upon a common set of mutations observed in human acute lymphoblastic leukemia (ALL). We also discuss reverse genetic models and screens that use CRISPR-Cas, ORFs and shRNAs to provide high throughput in vivo proof of oncogenic function, with an emphasis on models using adoptive transfer of ex vivo cultured cells. Finally, we summarize mouse models that offer temporal regulation of candidate genes in an in vivo setting to demonstrate the potential of their encoded proteins as therapeutic targets.Cancer genome sequencing has identified dozens of mutations with a putative role in lymphomagenesis and leukemogenesis. Validation of driver mutations responsible for B cell neoplasms is complicated by the volume of mutations worthy of investigation and by the complex ways that multiple mutations arising from different stages of B cell development can cooperate. Forward and reverse genetic strategies in mice can provide complementary validation of human driver genes and in some cases comparative genomics of these models with human tumors has directed the identification of new drivers in human malignancies. We review a collection of forward genetic screens performed using insertional mutagenesis, chemical mutagenesis and exome sequencing and discuss how the high coverage of subclonal mutations in insertional mutagenesis screens can identify cooperating mutations at rates not possible using human tumor genomes. We also compare a set of independently conducted screens from  B cell neoplasms can be categorized by their cell of origin, each subtype being representative of a discrete stage in differentiation with characteristic phenotypes and genetic lesions , 2]. AccIgH/BCL2 t which occurs early in B cell development during V(D)J recombination mediated by RAG in the primary lymphoid organs (BCL2 gene under control of the immunoglobulin heavy chain (IgH) enhancer and plays a causal role in germinal center derived malignancies. Though this translocation is commonly an initiating event in development of FL, it alone is not sufficient to initiate lymphomagenesis and similar translocations can be detected in the peripheral blood and lymph nodes of healthy individuals  or FL  or less Recent genomic studies of B cell malignancies implicate a handful of well-characterized, commonly mutated or translocated genes as well as a long tail of genes that have limited or no experimental evidence to support a role in disease. Since it is unlikely that all these mutations are driving oncogenesis there is need for validation methods such as functional genomic screens, forward genetic screens and comparative genomics to scrutinize driver mutations and better prioritize candidates for further study. The diverse mechanisms by which mutations contribute to disease means that many can only be tested within the context of specific developmental stages, predisposing mutations and optimal antigen receptor stimulation. For secondary/later stage mutations, oncogenic function and selection may only occur within a specific context of germline variants and/or early stage initiating mutations. This exponentially expanding set of parameters for validation experiments therefore requires prioritization by analysis of mutation profiles and higher throughput testing methods.In some respects, sequencing human tumors can be viewed as a special case of forward genetics, i.e. surveying genetic diversity under selection in order to identify which genetic changes drive a biological process. Forward genetic screens can also be targeted to the somatic cells of mice using either chemical or insertional mutagens. In the context of mouse cancer models, reverse genetics is the introduction of variation known to be associated with cancer in order to study the function of that variation. This variation can be introduced to the germline or targeted to specific populations of somatic cells.in vivo in ways not yet possible with cultured cells or organoids.The benefit of using mouse models to study hematologic malignancies is that the major genes and biological processes driving hematopoiesis and immunity are sufficiently conserved such that human malignancies can be recapitulated by the introduction of equivalent mutations. Mice also allow the complexity of immune system biology to be recapitulated This review covers mouse models used to rapidly screen for and validate large numbers of candidate genes driving B cell malignancies and is aimed at readers seeking to understand various techniques for targeting different developmental stages and disease subtypes. We provide a history of forward and reverse genetic approaches in mice that balance the trade-offs between fidelity, precision, and throughput. In the first half we discuss how forward genetics in mice can independently validate the oncogenic function of rarely mutated genes from human studies, and in some cases is a tool leading to discovery of human cancer genes. In the second half we cover reverse genetic screens and validation experiments where throughput is increased through the use of transplantation of primary cells transduced with ORF, shRNA and CRISPR-Cas viral constructs. Throughout we also emphasize strategies to direct models toward specific malignancy subtypes by a combination of germline lesions, targeted cell populations and temporal control of gene expression.Many driver genes are difficult to identify because they are subject to deregulation by non-coding mutations, copy number changes or epigenetic modification. Comparative genomic analyses of human tumors with the findings from forward genetic screens in mouse models can narrow down the identity of these driver genes. Mice with germline mutations that sensitize them to a specific disease subtypes are subjected to mutagenesis of their somatic cells and the resultant tumors are sequenced. We discuss here the combined use of genetically modified mice, insertional mutagens and exome sequencing as tools to perform forward genetic screens in B cell neoplasms . Selection of mutations at these CIS loci indicates that they cause changes in expression levels or cause disruption/truncation of cancer drivers. There can be substantial phenotypic and genetic variability between malignancies of a single cohort and driving specificity toward B cells rather than T or myeloid lineages has been achieved through a combination of limiting mutagenesis to B lineages, screening mice with a predisposition toward B malignancy subtypes and curating uniform subsets of B lineage tumors from mixed cohorts.Slow transforming retroviruses have an extensive history of use as insertional mutagens in the hematopoietic compartment [reviewed in ]. When nThese viruses induce a range of hematologic malignancies. B cell tumor cohorts have been generated by choosing specific combinations of virus strain and host mouse strain. Sequences within the virus LTRs are responsible for cell type-specific expression and alterations can skew tumors toward B cell subtypes , 64. AkvBlm hypomorphic background  shows a statistically significant bias toward verified drivers of human B lymphoma and leukemia  in addition to dozens of other candidate loci  fusion transcript, seen frequently in B cell acute lymphoblastic leukemia (ALL). Mice expressing the ETV6/RUNX1 fusion and the SB transposase variant HSB5 from the endogenous Etv6 locus had a background of long latency hematologic malignancies similar to wild type controls i.e. the fusion itself caused only slightly higher rates of lymphomagenesis  agenesis . When thof B ALL , 76 whicSTAT5 activating mutations are observed in a small proportion of B ALL, however mice expressing a constitutively active form of STAT5B (STAT5-CA N642H) do not develop ALL (Cd79a-Cre (switching in developing B cells) induced progenitor B cell leukemias thereby verifying STAT5B N642H as an oncogene in B ALL. Integrations of 65 mice identified 12 CIS, the most commonly mutated genes suggesting three major mechanisms for STAT5 mediated transformation: disruption of B-cell development , enhanced JAK/STAT5 signaling (Jak1) and modification of the CDKN2A tumor-suppressor pathway .elop ALL . CombiniTCL1 transgenic model develops a disease that is phenotypically similar to CLL. The B cells of these mice were mutagenized using a conditional SB transposase switched by CD19-Cre (pan B cell) . NF-kappaB activating mutations are rare in the coding regions of human CLL however it has been shown that CLL cells are frequently dependent on extracellular signaling pathways that activate Nf-kappaB  , 59. Traf-kappaB  and sevef-kappaB –80.Cnp-Cre strain expresses Cre in the nervous system and splenic germinal centers  generated a mixture of solid tumors as well as lymphoid malignancies with an FL/DLBCL like phenotype. Integrations from 23 samples identified Bach2 as the most frequently mutated locus. Pten disrupting mutations were also identified and the authors verified Pten as a tumor suppressor in the Cnp-Cre driven model. These results are consistent with human germinal center B cell DLBCL where PTEN is mutated or deleted  at other loci , 103. ThThe expense of exome and whole genome sequencing is generally reserved for the study of human tumors however, an increasing number of mouse tumor exomes have also been sequenced, albeit with cohort sizes that are far smaller than their human counterparts. In most cases this sequencing is to verify that the model accurately recapitulates human disease, however, some studies also suggest or verify candidate drivers where coding mutations in human cohorts fail to reach statistical significance. Several examples are discussed below.Myd88 (L252P) and Rosa-26 expressed Bcl2 with germinal center specific Cγ1-Cre generated a human ABC-DLBCL like model  gave rise to Burkitt’s lymphoma (BL) like disease with recurrent mutations in Ccnd3, which is mutated in BL and known to regulate germinal center B cell proliferation  develop B cell lymphoma  translocation where human BCL2 expression from an Eμ enhancer is sporadically induced by RAG recombinase in pro/pre B cells  mouse. B cell subsets of the BCL2-transduced mice had between 111 and 2,565 nonsilent SNVs compared with 63 to 70 SNVs in the control mouse, despite the absence of overt morphological differences between hBCL2 and control animals. This model demonstrates a role for BCL2 in enhanced survival of cells that accumulate potentially oncogenic off target mutations of AID.Aside from driver detection, exome sequencing can also be a powerful tool to examine the mutagenic processes of early premalignant states under defined experimental conditions. Sungalee  B cells . Repeat PAX5 encodes a B cell development transcription factor that activates B cell specific genes and represses genes of alternative lineages. It is one of the most frequently altered genes in B ALL and a varied spectrum of somatic rearrangements, translocations, mutations and deletions have been observed at this locus  and focal deletions of the second allele of Pax5. Exome sequencing revealed B ALL samples from ENU treated mice had 5-fold more exonic mutations than MuLV infected mice and identified recurrent mutations in the human ALL mutated genes: Pax5, Jak3, Ptpn11, Jak1, and Nras. Pax5 mutations were almost exclusively within the DNA-binding paired domain and equivalent to mutations observed in human B ALL. Jak1 and Jak3 mutations were located in the pseudokinase domains of JAK1 and JAK3, and are known to induce cytokine-independent proliferation and activation of downstream signaling pathways sensitive to JAK inhibitors. MoMuLV CIS included an overlapping set of B ALL drivers including Myc, Stat5b, Zeb2, Jak1, Jak3, Ikzf1, Gsdmc, Ebf1 and Ptpn11.gnancies . Array-CCd19, Mb1(Cd79a) or Mx1) were used to induce conditional expression of a E2A/PBX1 fusion, resulting in B cell precursor ALLs, which arrested at the pro B/large pre B II stages in a manner similar to human E2A/PBX1 ALL -ALL  have well-characterized roles in B cell maturation. Increased ZNF423 expression is associated with ETV6/RUNX1+ B ALL and Pax5 or Ebf1 mutations synergize with STAT5 in B ALL. It is worth noting this mutation profile is more likely a function of Pax5 loss since a different model of ETV6/RUNX1 with wild type Pax5 primarily identified loss of function mutations in the KDM lysine demethylase family (The  section  was subsBCP)-ALL . Targetee family .Pax5 heterozygous mice do not develop leukemia when housed in specific pathogen free (SPF) conditions however they are prone to precursor B ALL when exposed to common pathogens and sequencing of these leukemias identified recurrent Jak3 mutations . Non-SPF mice treated with antibiotics also harbored recurrent mutations in Kit, Flt3, and Cbl that have not been observed in other Pax5+/– cohorts. This may suggest that selection of secondary mutations reflects differences in gut biome.B ALL models have also been used to study the relationship between gut microbiota and leukemia incidence , 55, 61.utations . In an iPAX5/ENL fusion identified mutations in Ptpn11, Kras, Pax5, and Jak3 genes  and four had Ptpn11 mutations. Collectively this work demonstrates how mutagenesis and exome sequencing of mouse models with defined lesions can reliably and reproducibly reveal cooperating mutations in independent cohorts, and that Pax5 heterozygosity in mice faithfully reproduces mutational profiles observed in human B ALL.These six independent studies using complementary experimental designs all identify mutations in Over the past decade the expanding number of candidate mutations associated with B cell malignancies has increased the requirement for medium to high throughput methods of validation. There is an extensive literature of transgenes, knockouts and conditional alleles for B cell lymphomas [reviewed in –121]. AlEarly transplantation-based B cell malignancy models used cell lines derived from spontaneous malignancies injected into syngeneic hosts and/or human malignant cell lines injected as xenografts into immunocompromised mouse strains [reviewed in ]. More rin vivo shRNA screens. In one study shRNAs against 1000 candidate cancer genes were selected to identify genes that are required for B lymphoma growth in vivo and this identified regulators of actin dynamics and cell motility  yielded B cell lymphomas that could be accelerated by loss of tumor suppressors in the germline  and by retroviral expression of cooperating ORFs  (Bcor (a tumor suppressor of Burkitt lymphoma) -/-Puma-/- in order to identify targets of p53 that were responsible for its tumor suppressor role independently of Cdkn1a or Puma   and Bcorymphoma) . The Eμ-g Rad17 . A more  or Puma . This idMyc transgenics, Trp53 mutants and Cdkn2a/ARFp19 mutants however many other strains have also been used to probe the cooperative or epistatic relationships of transduced genes with germline alleles  isoform , that expresses the  isoform , 170. My isoform , 170.BCR/ABL1 retrovirus typically gives rise to CML, however loss of both ARFCdkn2a/p19 or only ARFp19 in donor cells gives rise to B ALL   , 165. Geeukemias .BCR/ABL1 driven disease in different lineages might suggest a separate cell of origin for B cell and myeloid disease, however using purified HSCs as donor cells suggests both CML and B ALL can arise from a long-term HSC cell of origin  resulted in delayed onset of B ALL compared with wild type donors however this delay was not seen for the onset of CML derived using 5-fluorouracil treated donors  but phenotypically distinct diseases.Collectively the above literature of in vitro expansion of B cell progenitors  mice. The recipients develop an immature B cell lymphoma/leukemia which infiltrated the lymph nodes, spleen, thymus and bone marrow. Ex vivo cultured pro B cells transduced with Bcl2 and Myc were also transduced with a cDNA library and selected for continued growth in the absence of IL-7 and ST-2 stromal cells. CCND3 and NRAS both rendered these cultures independent of IL-7 and ST-2 cells and accelerated Myc/Bcl2 driven disease when transplanted into SCID mice.One disadvantage of using HSCs or mixed populations of bone marrow as donor cells is their potential to develop a wide spectrum of malignancy lineages from undefined developmental stages. Culture systems for genitors , 195 havgenitors  and moregenitors . These sMyc in an in vivo setting  pre BI cells were differentiated to immature B1 cells in vitro, and timed CpG stimulation generated either pre BII-like or mature B1-cell lines and IgM-secreting B1 cells. Introduction of these pre BI cells into Rag1-/- mice gives rise to plasmablast and plasma cell hyperplasia that was reversible when doxycycline was removed  were validated as cooperating with Myc and the authors proposed the mechanism is a function of global inhibition of mRNA degradation.This model was also used to screen a library of cDNAs in pre B cell lines to identify genes that cooperate with Myc . Cells win vitro. Propagating naïve splenic B cells on a layer of 3T3 fibroblasts expressing both CD40L and BAFF causes them to proliferate into germinal center B cell like cells called in-vitro-induced germinal center B (iGB) cells  cells . When iG disease .in vivo library based screens of B cell biology need not be strictly limited to readouts of malignancy. Various models of B cell development and positive/negative selection of B cells are also relevant to malignancy. The IgMb-macroself mouse strain ubiquitously expresses a superantigen that causes deletion of immature B cells through reaction of surface IgM with a “self” superantigen. Transduction of bone marrow HSPCs with retroviruses encoding a miRNA expression library, and reconstitution of macroself recipients led to selection of cells expressing miR-148a, which was then verified to prevent deletion of self-reactive B cells (It is worth noting that  B cells .hi transgenic strain has an increased affinity to the 4-Hydroxy-3-nitrophenylacetyl (NP) hapten after immunization and this model has been used to screen for modifiers of germinal center formation. Splenic B cells from B1-8hi transgenic mice were stimulated with anti-CD180 antibody, transduced with a library of shRNAs and transplanted into wild-type C57BL/6 mice  and Cas endonucleases have been adapted to targeted editing of mammalian genomes . Since tThe short targeting sequence of CRISPR-Cas endonucleases facilitates functional genomics experiments with a similar throughput to shRNA vectors, with the additional benefit of editing target alleles permanently rather than changing gene expression by lowering mRNA levels. Like shRNAs, sgRNAs can vary in their efficiency and off target effects. Double stranded DNA breaks created by SpCas9 are primarily repaired by nonhomologous end joining (NHEJ) which is an error prone process. Consequently, some earlier versions of CRISPR-Cas editing have been shown to create imprecise lesions. Depending on the delivery system used, a subset of target sites may generate deletions of thousands of bases, complex rearrangements and crossover events –207. NonRosa26 locus were transduced with sgRNA viruses, achieving up to 80% knockout efficiency in cultured primary B cells  (Rfx7 and Phip (combining a sgRNA vector with a Cas9 transgene)  , 108 andansgene) . Eμ-Myc me assay .in vivo growth of primary Eμ-Myc;ARF-/-p19 lymphoma and have been used to conduct an in vivo screen in Bcr-Abl driven B ALL  or inhibit transcription (referred to as CRISPRi) . CRISPRien B ALL . From a EML4/ALK translocations in a lung cancer model  fusion and is found in acute leukemias of B, T and myeloid lineages. The t translocation produces the MLL/AF9 (KMT2A/MLLT3) fusion and is primarily observed in childhood AML but also in a subset of ALL in children below 1 year of age. Lineage-depleted human umbilical cord blood infected with MLL/ENL virus gave rise to B precursor ALL when injected into sublethally irradiated NOD/LtSz-scid/scid mice  translocation produces the cid mice . An MLL/The lineage restriction of tumor initiating and tumor propagating cells was also investigated. The use of myeloid promoting suspension culture protocols skewed both transgenes toward a myeloid leukemia when injected. Transplantation of limiting cell numbers of primary tumors into secondary recipients indicated self-renewing leukemia cells were rare. Southern blots of retroviral vector integration and immunoglobulin heavy chain rearrangement suggested that leukemia initiating stem cells with germline immunoglobulin can differentiate into tumor propagating B lineage cell type having undergone immunoglobulin rearrangement. Lineage switching between B and myeloid lineages was also observed in culture.MLL/AF9 (KMT2A/MLLT3) translocation in cultured human cord blood HSCs. Transplanting these cells into sublethally irradiated NSG mice gave rise to B ALL, acute myeloid leukemia or mixed phenotype acute leukemia, as well as mice with a mixture of AML/ALL (MLL(KMT2A) and ENL(MLLT1) in human HSCs. When these cells were introduced to NSG mice, myeloid disease was most commonly observed however secondary outgrowths of B ALL were also identified  mice  mice . NSG micSG) mice .Historically, developing models using new germline alleles is time consuming. Traditional transgenesis by pronuclear injection of fertilized zygotes leads to unpredictable integration sites of unkown copy number. Gene targetting by homologous recombination in ES cells requires constructs with long homology arms and extensive screening and characterization of clones prior to blastocyst injection. Founder animals must then be crossed to verify germline transmission and to combine multiple alleles in sufficient animals to form experimental cohorts.Higher throughput strategies to generating these models and cohorts have now been developed using various combinations of CRISPR-Cas, recombinase mediated cassette exchange and routine derivation of new ES cell lines from mice bearing multiple germline alleles [reviewed in ]. SuperfTet1 and Tet2 have been achieved as inactivating mutations  and 4-hydroxytamoxifen  . Early rromoters , 249 whipression . StudiesPax5 tumor suppressor by shRNA in combination with constitutively activated STAT5b causes a B ALL like disease  is fused with the VP64 transactivation domain (i.e. four copies of the herpesvirus VP16 domain), the RELA p65 transactivation domain and the reverse tetracycline activation domain . This fuHuman populations are highly polymorphic and this, in combination with the mutator phenotype generated by many tumours, can make it difficult to subtract the background noise of unselected passenger mutations from the evidence for driver mutations. The growing volume of human cancer genome sequencing data has created an exponentially expanding number of potential validation experiments. This in turn creates a prioritization bottleneck deciding which combinations of mutations should be tested and the developmental stages in which they should be modeled.Forward genetics in mice can aid this prioritization by cross species comparative genomics i.e. identifying the overlap of mutation spectra identified between human and mouse cohorts. There is a tradeoff between the extent to which forward genetic screens recapitulate human disease and the throughput of mutation detection. Exome sequencing arguably better mimics human tumor exomes, however insertional mutagenesis screens better represent the effects of translocation/copy number/non-coding mutations and are relatively lower cost and higher yield in terms of the number of driver mutations identified per mouse. This cost benefit analysis becomes relevant when considering the statistical limits of proving selection of non-coding point mutations from WGS of human cohorts. Insertional mutagenesis models also have greater statistical power to identify cooperating mutations, especially when sequencing coverage is sufficient to identify subclonal mutations.Forward genetic screens in mice also have limitations. In some cases, the expected homologs of human drivers are not identified because functionally equivalent mutations that are specific to mice are identified instead. Another limitation of both forward and reverse genetic models is that the time frame for development of malignancies in mice is far shorter than observed in human lifespans. Consequently, predisposing mutations must be introduced and this in turn increases the chance of multiple clones arising independently in a single animal. Furthermore, Cre expressing strains used in some studies (such as Cd19 and Aicda) may replace the endogenous gene thereby altering the dosage of crucial B cell development genes.Since the emphasis of this review is experimental approaches with higher throughput to identify and validate large numbers of candidate genes, for the most part we have not covered reverse genetic models that exclusively employ cohorts generated by germline alleles, which is a vast literature in and of itself. Rapid model prototyping using transplantation of virus transduced cells reduces the commitment of resources for initial testing of candidate genes. This allows more genes to be tested before robust germline models are designed and, in a handful of instances, allows for high throughput screens of dozens or even hundreds of candidate genes within a single cohort. This approach does, however, lack the precision of inducible targeted germline alleles and the dosage of transduced constructs can be highly variable and potentially reach expression levels not observed in human cells. Many cancer genes have physiological roles that are highly sensitive to dosage, and non-physiological expression levels from vectors may block or drive differentiation, sensitize cells to apoptosis or alter proliferation in a manner not typically observed as a result of somatic mutations.in vivo. In vivo functional genomic screens are arguably a uniquely powerful application of adoptive transfer protocols, however in writing this review we only identified a limited number of studies using libraries of ORFs, shRNAs or gRNAs in B cell malignancy models. This would suggest that there is unrealized potential for more screens and/or that there are technical barriers to the reproducible identification of constructs selected in parallel. Functional genomic screens are a tradeoff between increasing the number of constructs screened whilst controlling for the stochasticity of selection of each construct relative to others in the library. B cell survival, migration, selection, differentiation and expansion are highly stochastic processes that may obscure selection in complex libraries. One approach to controlling for this stochasticity employed by several researchers is screening smaller pools of limited numbers of constructs to make selection of individual constructs easier to observe. Another approach to increasing confidence in the set of candidates identified in a screen is performing parallel screens using both shRNAs and sgRNAs to find genes that are selected by methodologies . Transplantation models could easily be diversified through the use of different germline lesions or culturing and transplanting later stages of B cell development. Many of the studies we discuss have successfully cultured and transduced primary B cells with high efficiency, though currently only a handful have used transplantation of transduced B cells as the basis of lymphoma models.In aggregate, the studies discussed in this review suggest several lines of research that are ripe for further innovation. The literature of transplantation-based models is dominated by the use of Eμ-Another promising direction for future development is the use of humanized models of human HSCs transplanted into immunodeficient mice. Immunodeficient host strains vary considerably in their engraftment potential, and although most are missing later stages of B cell development, they can recapitulate earlier stages of lymphoid development and have formed the basis of useful models for lymphoblastic leukemias. Immunodeficient mouse strains have various defects in the hematopoietic microenvironment and/or stromal cell proteins that are incompatible with their human targets and this can inhibit engraftment. For instance, IL2 receptor common gamma chain null mice (including NSG mice) lack lymphoid tissue inducer cells and innate lymphoid cells and do not express HLA molecules on thymic epithelia thereby preventing MHC restriction of T cells. Some models also develop graft-versus-host disease. There now is a growing literature of newer humanized models that can better recapitulate all aspects of human hematopoiesis –268. NewH.pylori infection in MALT lymphoma (Modelling the antigenic context of B cell malignancies is also rarely addressed in the literature. The role of antigenic stimulation or tolerization by commensal flora is frequently overlooked and SPF or germ free conditions may not accurately represent the microbiome of human cancer patients, particularly where disease is influenced by a combination of normal gut flora and infectious agents, as is the case with lymphoma .Identification of human cancer drivers and the cell types that are most responsible for tumor initiation and propagation are critical foundations for the discovery of novel targeted therapies. The “post-genomic era” with seemingly limitless genomic data to interrogate has brought its own challenges of how to prioritize the use of this data. Modeling large numbers of mutations over the many stages of B cell development is a vast undertaking, particularly when differentiating between genes responsible for tumor initiation and maintenance and when testing potential targets across diverse genetic contexts of a single disease subtype. As such even the highest throughput experimental approaches discussed here can benefit from the parallel use of complementary methods.All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.JD and AU were supported by MRC programme grant MC_A652_5PZ20.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In children lacking influenza-specific adaptive immunity, upper respiratory tract innate immune responses may influence viral replication and disease outcome. We use trivalent live attenuated influenza vaccine (LAIV) as a surrogate challenge model in children aged 24–59 months to identify pre-infection mucosal transcriptomic signatures associated with subsequent viral shedding. Upregulation of interferon signaling pathways prior to LAIV is significantly associated with lower strain-specific viral loads (VLs) at days 2 and 7. Several interferon-stimulated genes are differentially expressed in children with pre-LAIV asymptomatic respiratory viral infections and negatively correlated with LAIV VLs. Upregulation of genes enriched in macrophages, neutrophils, and eosinophils is associated with lower VLs and found more commonly in children with asymptomatic viral infections. Variability in pre-infection mucosal interferon gene expression in children may impact the course of subsequent influenza infections. This variability may be due to frequent respiratory viral infections, demonstrating the potential importance of mucosal virus-virus interactions in children.  •Upregulated mucosal interferon prior to vaccination leads to reduced LAIV shedding•Asymptomatic respiratory viral infections were seen in 42% of children•Other respiratory viral infections contribute to higher interferon gene expression Costa-Martins et al. show that in children seronegative to influenza vaccine strains, upregulated nasopharyngeal interferon genes prior to LAIV receipt associates with reduced vaccine strain shedding. Asymptomatic respiratory viral infections are common and result in higher interferon gene expression, highlighting the importance of virus-virus interactions in the upper respiratory tract. Influenza results in significant pediatric morbidity and mortality worldwide, with the largest burden in low- and middle-income countries.,Unlike in adults, ethical and feasibility constraints of conducting influenza challenge studies in children limit the ability to use this controlled experimental approach to gain insight into such aspects of protective immunity. We hypothesized that live attenuated influenza vaccines (LAIVs) could be used as a surrogate challenge model in children, with shedding of vaccine strains as the primary endpoint of interest. LAIVs contain influenza strains that replicate in the cooler URT only, showing induction of interferon-stimulated genes (ISGs) and replication kinetics similar to that of wild-type strains.Using LAIV challenge in children with no detectable serum HAI titers to vaccine strains, we sought to define pre-challenge transcriptome signatures in the nasopharyngeal mucosa that were associated with the degree of viral shedding several days later; thus, mimicking what may determine the outcome of URT influenza infections in influenza-naive children. We show that many children have upregulated interferon pathways at the time of vaccination that correlate with limited influenza strain shedding and that upregulated ISG signatures may, in part, be explained by asymptomatic infection with respiratory viruses such as human rhinoviruses.Influenza vaccine-naive children aged 24–59 months were given one dose of trivalent LAIV  as part of a previously described open-label, prospective, observational, phase 4 immunogenicity study in The Gambia .Nasopharyngeal viral loads at both day 2 and day 7 were significantly lower in seropositive than seronegative children for a given influenza strain B and 1C.Asymptomatic respiratory viral infections at baseline were detected in 41.8% (33/79) of children with valid multiplex RT-PCR assay results D. TwentyGene set enrichment analysis (GSEA) was conducted to identify mucosal transcriptional profiles from nasopharyngeal swabs taken prior to LAIV challenge that may influence replication of LAIV strains. Correlation coefficients were generated between mucosal gene expression at baseline and viral loads at days 2 and 7 for each strain. Negative normalized enrichment scores (NESs) from the GSEA indicate pathways enriched for genes whose expression inversely correlates with viral loads and vice versa. Five pathways were significantly enriched  for genes associated with lower viral loads for all three strains at day 2, and of these five pathways, two were also significant for all three strains at day 7 . A protein-protein interaction network constructed using these overlapping genes is shown in To see whether the presence of asymptomatic respiratory viral infection could explain upregulated genes from interferon signaling pathways in some children, differentially expressed genes (DEGs) between children with and without viral infections at baseline were identified  was confirmed by a deconvolution analysis based on single-cell data from healthy human airways , with concomitant reduction in those gene signatures from ciliated epithelial cells, goblet cells, and major histocompatibility complex (MHC) class II high goblet cells . The red airways .8,9FigurTo determine the relationship between enrichment for cell-type-specific signatures prior to LAIV challenge and subsequent viral load at days 2 and 7, GSEA was repeated using gene viral load correlation coefficients as rank and the cell-specific gene list as the reference set.Using live attenuated influenza strains as a surrogate controlled influenza challenge model in young children, we demonstrate that pre-existing upregulated interferon pathways in the URT mucosa may limit the replication of influenza viruses in seronegative children. Furthermore, we show that these changes may in part be due to asymptomatic infection with other respiratory viruses, highlighting the complex interplay between the large burden of viruses that children are exposed to. Our findings have implications not only for understanding the reasons for the heterogenous outcomes from pediatric influenza infectionsin vitro data from a co-culture model showing inhibition of pdm09H1N1 influenza virus replication by preceding human rhinovirus infection via ISG induction.,The ability of interferon to inhibit influenza virus replication in humans through the induction of ISGs is well described.Although inferred from transcriptional signatures alone, our data also suggest that the composition of cell types in the nasopharyngeal mucosa at the time of LAIV infection can impact viral replication in the first 2 days. Children with baseline gene signatures enriched in macrophages, neutrophils, and eosinophils were significantly more likely to have lower viral loads at day 2 for all three influenza strains. Macrophages, in particular, are known to play a key role in interferon induction and control of influenza infections.DYNC2H1, DYNC2LI1, IFT20, IFT88, IFT122, IFT172, KIF3A, TTC26, WDR35). These findings warrant further in vitro exploration to understand the role of ciliary function genes in facilitating influenza replication.Interestingly, we observed upregulation of pathways associated with ciliary function  at baseline were associated with higher LAIV viral loads. In addition, pre-challenge enrichment for a gene set representative of ciliated epithelial cells was significantly associated with higher viral loads at later time points. Asymptomatic viral infection also resulted in reduction in cell-type expression scores for several epithelial cells, but notably for ciliated epithelial cells. This could simply represent a scenario where loss of epithelial cells due to preceding viral infection results in a reduction in preferential target cells for influenza infection. Children with greater proportions of ciliated epithelial cells at the time of infection may end up having higher influenza viral loads. It is also possible that the infiltration of other cells during viral infection leads to a lower proportion of epithelial cells within the total cells sampled or that variability in nasopharyngeal swabbing may have contributed to our observed result. However, previous data from human airway epithelial cells have shown that human rhinovirus infection downregulates several genes associated with cilium movement and morphogenesis,Our findings would benefit from being replicated in other studies. Little is known about the variability in mucosal gene expression in children classified as healthy from diverse geographical, environmental, and socioeconomic settings. The burden of asymptomatic respiratory viruses in children will vary seasonally in the same location as well as between countries. Nevertheless, it is biologically plausible that a similar relationship will be seen between upregulated interferon pathways and impaired replication of LAIV strains regardless of where children reside. Reassuringly, we did not see any negative impact of asymptomatic viral infection or upregulated interferon gene expression pre-vaccination on antibody and T cell responses to LAIV. While we have shown that shedding of LAIV strains at day 7 is significantly associated with higher HAI and CD4+ T cell responses to LAIV,We present proof of concept that LAIV could be used as a surrogate influenza challenge model in young children to understand host-virus and virus-virus interactions in the upper respiratory tract. Mucosal transcriptomic studies in pediatric influenza infection to date are limited to sampling after acute presentation, where pre-infection factors cannot be determined. Our methodology can also be applied to future pediatric studies to obtain detailed nasopharyngeal host and pathogen data from minimally invasive sampling. Integrating such methods into household viral transmission studies with systematic sampling over time should be used to expand our experimental findings with LAIV to other respiratory viruses. This could be particularly relevant in exploring why children appear to be less susceptible to symptomatic SARS-CoV-2 infection and determining whether mucosal factors such as upregulated ISGs prior to exposure impact acquisition, severity of infection and onward transmission.in vitro than antigen-matched seasonal influenza viruses, which may contribute to the attenuated phenotype observed.The major limitation of our study is that only transcriptional signatures were assessed rather than protein and cytokine measurement, or cellular characterization via flow cytometry. Challenges to sampling in young children limit the ability to undertake extensive investigation, but it would be important to validate our findings in future studies. Although we documented asymptomatic viral infections at baseline by PCR in more than 40% of children, it is difficult to know when these children were infected as viral nucleic acid can be detected for prolonged periods after acute infection. This may in part explain the heterogeneity in interferon gene upregulation seen in children with asymptomatic respiratory viral infections. It is also not known whether the attenuating mutations in LAIV strains impact their susceptibility to antiviral interferons, which may affect to what degree our findings are generalizable to wild-type influenza strains. Despite producing significantly less infectious virus, LAIV strains have been shown to induce greater antiviral innate responses t.desilva@sheffield.ac.uk).Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Thushan de Silva .•Healthy male or female child at least 24 months of age and less than 60 months of age at the time of study entry.•Resident in the study area and with no plans to travel outside the study area during the period of subject participation.•Informed consent for the study participation obtained from a parent .•Willingness and capacity to comply with the study protocol as judged by a member of the clinical trial team.•Serious, active, medical condition, including but not limited to:•chronic disease of any body system•severe protein-energy malnutrition (weight-for-height Z-score of less than −3)•known genetic disorders, such as Down syndrome or other cytogenetic disorder•Active wheezing•History of documented hypersensitivity to eggs or other components of the vaccine , or with life-threatening reactions to previous influenza vaccinations.•History of documented hypersensitivity to macrolide antibiotics•History of Guillain-Barré syndrome.•Receipt of aspirin therapy or aspirin-containing therapy within the two weeks before planned study vaccination.•Any suspected or confirmed congenital or acquired state of immune deficiency including but not limited to primary immunodeficiencies including thymus disorders, HIV/AIDS, hematological or lymphoid malignancies.•Any current immunosuppressive/immunomodulatory treatment or receipt of any such treatment within the six months preceding trial enrolment  of greater than 2mg/kg/day for one week or 1mg/kg/day for one month. The use of topical corticosteroids is not an exclusion criterion.•The use of inhaled corticosteroids within the last one month.•Receipt of an influenza vaccine within the past 12 months.•Has any condition determined by investigator as likely to interfere with evaluation of the vaccine or be a significant potential health risk to the child or make it unlikely that the child would complete the study?•Any significant signs or symptoms of an acute illness or infection including:•an axillary temperature of 38.0°C or above or documented fever of 38°C or above in the preceding 14 days.•Any acute respiratory infection within 14 days of enrolment visit.50)/ml) were 1x107.7 for pH1N1 (A/17/New York/15/5364), 1x107.6 for H3N2 (A/17/Hong Kong/2014/8296) and 1x107.3 for the influenza B Victoria-lineage strain (B/Texas/02/2013).Only children given the 2017-18 northern hemisphere LAIV formulation were included in the current analysis (administered Feb – April 2018), as this vaccine included three influenza strains with good replicative ability following replacement of the A/California/07/2009-like pH1N1 strain from previous years.Nasopharyngeal swabs (NPS) were taken pre-LAIV challenge, and at day 2 (D2) and day 7 (D7), using flocked swabs (Copan FloQSwabs™) collected directly into RNAprotect Cell Reagent (QIAGEN). Samples were transported at 2-8°C and stored at −70°C within 4 hours of collection. Whole blood was collected pre-LAIV, serum separated and stored at −70°C prior to further processing.The study was approved by the Gambia Government/MRC Joint Ethics Committee (SCC1502) and the Medicines Control Agency of The Gambia, and conducted according to ICH-GCP standards. Parents provided written or thumbprinted informed consent for their children to participate.Haemagglutinin inhibition (HAI) assays were performed according to standard methods, using vaccine haemagglutinin (HA) and neuraminidase-matched viruses.Total nucleic acid was extracted from 180μL of RNAprotect using the QIAamp Cador Pathogen mini kit  as per manufacturer’s instructions. All samples were spiked with Equine Arteritis Virus as an extraction control. A previously described multiplex reverse-transcriptase polymerase-chain reaction (RT-PCR) was performed to detect influenza A, influenza B, Respiratory Syncytial Virus (RSV) A, RSV B, human parainfluenza viruses (HPIV) 1 to 4, human metapneumovirus, adenovirus, seasonal coronaviruses  and human rhinovirus.Total nucleic acid was extracted from 270μL of RNAprotect as described above. LAIV strain viral loads were quantified as previously described, with monoplex HA-specific RT-PCRs using a standard curve with known log10 50% EID/ml.RNA was extracted from 400μL of RNAprotect using the SPLIT RNA Extraction Kit (Lexogen) as per manufacturer’s instructions. Samples with at least 100ng of RNA were taken forward for QuantSeq 3′ messenger RNA sequencing (Lexogen).http://broadinstitute.github.io/picard/). Reads were counted at gene level using the Rsubread R package.Raw data quality control was performed using FastQC (Bioinformatics). Mapping to the human reference genome (GRCh38/hg38) was done using DART . PCR duplicates were removed using umi_tools dedup in Python 3.7.2 and optical duplicates removed using the Picard MarkDuplicates tool  were conducted using the fgsea Bioconductor package.P-values for each gene. A log2 fold-change of ± 0.5 and a stringent adjusted P-value of 0.001 were used to select DEG for further consideration.DESeq2 Bioconductor was used to detect differentially expressed genes (DEG) between children with and without asymptomatic viral infections prior to LAIV challenge.https://cytoscape.org/).Networks were constructed with NetworkAnalyst 3.0 using InnateDB protein-protein interaction data and the minimum network option.Comparisons of continuous data between groups were performed using the Mann Whitney U test. All correlations were estimated using Spearman’s rank correlation coefficients. Analyses were conducted in R software version 3.6.


P = 0.023) and metastatic status (P = 0.024). Univariate and multivariate analyses revealed that patients with low HPRT expression have shorter overall survival times . Furthermore, high IMPDH2/HPRT ratios were similarly associated with shorter overall survival times . Levels of the enzymes were also examined in post-chemotherapy tissues. The results showed that high IMPDH2 expression was associated with shorter metastasis-free survival . These results suggest a prognostic value of expression patterns of purine-metabolizing enzymes for the pre- and post-chemotherapy period of osteosarcoma treatment.Osteosarcoma is one of the most aggressive bone tumors in children and adolescents. Development of effective therapeutic options is still lacking due to the complexity of the genomic background. In previous work, we applied a proteomics-guided drug repurposing to explore potential treatments for osteosarcoma. Our follow-up study revealed an FDA-approved immunosuppressant drug, mycophenolate mofetil (MMF) targeting inosine-5′-phosphate dehydrogenase (IMPDH) enzymes, has an anti-tumor effect that appeared promising for further investigation and clinical trials. Profiling of IMPDH2 and hypoxanthine–guanine phosphoribosyltransferase (HPRT), key purine-metabolizing enzymes, could deepen understanding of the importance of purine metabolism in osteosarcoma and provide evidence for expanded use of MMF in the clinic. In the present study, we investigated levels of IMPDH2, and HPRT in biopsy of 127 cases and post-chemotherapy tissues in 20 cases of high-grade osteosarcoma patients using immunohistochemical (IHC) analysis. Cox regression analyses were performed to determine prognostic significance of all enzymes. The results indicated that low levels of HPRT were significantly associated with a high Enneking stage ( However, the five-year survival rate of patients not responding to chemotherapy is remains 30%4. Our previous study reported 37.9% of the 5-year overall survival rates in Northern Thailand5. This finding is comparable with studies conducted within Asia–Pacific region reporting 27–48% of the 5-year overall survival rates that were much lower in comparison to western regions with rates between 60 and 70%6. Major factors of the poor clinical outcomes were presence of metastasis and not completing treatment regarding patients’ non-compliance to treatment regime.Osteosarcoma is a malignant bone cancer that is more common in children and adolescents. Currently standard treatment includes a combination of chemotherapy and surgery. Since the introduction of chemotherapy in combination with surgery in 1970, the survival rate of osteosarcoma cancer patients with localized disease has increased to 70%8. There is also evidence of epigenetic changes resulting from histone modifications and DNA methylation9. These abnormalities result in changes in protein levels in both oncogenic proteins and tumor suppressors.The main reason treatment for osteosarcoma has not improved the survival rate over the past 30 years is due to an inability to specify the key mechanisms involve in the occurrence and spread of osteosarcoma. Information obtained from whole genome and exome sequencing has indicated that osteosarcoma has a highly chaotic genetic background including chromosome aberrations, chromothripsis, kataegis, and genome instability with a high rate of copy number alterations (CNAs)10. Cancer cells require both energy metabolism and increased nucleotide biosynthetic pathways. Purine nucleotides are the most important source of cellular energy and are involved in diverse signaling pathways11. Biosynthesis of purine nucleotides occur via two mechanisms, the “salvage pathway” that reuses biomolecules derived from DNA and RNA degradation and the “de novo pathway”, synthesis of purines from small molecules derived from cellular biochemical processes13. The de novo pathway produces greater amounts of purine nucleotides than the salvage pathway, but it uses more energy and involves more complex reactions14. It has been found that purine nucleotides are produced under many conditions, mainly through the de novo pathway. The most obvious example is the activation of the de novo pathway in active B and T lymphocytes15, as well as in various types of cancer16. Nucleotide metabolism in cancer cells has been studied for several decades. In 1975, a study by Jackson et al. first demonstrated the importance of the de novo pathway in nucleotide synthesis in liver cancer cells17. They reported higher activity of glutamine PRPP amidotransferase enzyme , an enzyme regulating synthesis of inosine monophosphate (IMP) in liver cancer cells, with significantly lower IMP degradation. They also found increasing activity of inosine-5′-phosphate (IMP) dehydrogenase (IMPDH), the enzyme with the highest activity level in the fastest dividing liver cancer cells tested in the experiment. IMPDH is an enzyme that is important in the process of guanine nucleotide synthesis through the de novo pathway. It accelerates the conversion of IMP to xanthosine 5′-phosphate (XMP), the rate-limiting step in the guanine nucleotide synthesis process. Proliferation of cancer cells depends largely on the amount of nucleotides within the cell as well as the activity of key enzymes regulating the rate-limiting step of the nucleotide synthesis process. Furthermore, cancer cells strongly rely on the glycolysis and the TCA cycle to obtain enough ribose-5-phosphate, a primary precursor of de novo nucleotide synthesis, to boost their intracellular nucleotide content. Ribose-5-phosphate is synthesized from the pentose phosphate pathway (PPP) through the nonoxidative branch, in which cancer cells vigorously stimulate the PPP through glycolytic flux by oncogenic signaling of the PI3K-Akt pathway18. Taken together, this makes the de novo purine nucleotide synthesis pathway an attractive target for cancer treatment.Cancer cells divide rapidly with a significantly higher rate of DNA and RNA replication. Cell division is controlled by many types of transcription factors and involves changes in volume, energy, and anabolic metabolism to pass through the S-phase in the cell cycle19. Inhibiting IMPDH results in decreased cellular guanine nucleotide pools and subsequently modulates the synthesis of DNA and RNA which is lethal for cells. Human IMPDH consists of 2 isoforms, IMPDH1 and IMPDH2, that are encoded from different genes. Both IMPDH isoforms share a similar amino acid sequence (84%), structure (95%), kinetic properties, and substrate binding site20. Expression of IMPDH2 is enhanced significantly in rapidly proliferating cells including various types of cancers including colorectal, bladder, prostate, kidney, nasopharyngeal carcinoma and leukemia24. Decades ago, researchers explored a link between Myc oncogene and production of nucleotides to support a high growth rate of cancer cells. Oncogenic signaling through the Myc pathway directly controls glutamine uptake and increases expression of various rate-limiting enzymes in de novo nucleotide synthesis including, but not limited to, IMPDH1 and IMPDH2 genes25.Inosine-5′-phosphate (IMP) dehydrogenase (IMPDH) is a well-known target for antileukemic and immunosuppressive therapy26. To maintain cellular GTP level in somatic cells, HPRT is reliably expressed in low level27. Unlike in normal cells, recent study showed that HPRT expression is highly variable in various cancer tissues including lung, breast, colon, prostate and pancreas27. HPRT activity was also detected in human osteosarcoma xenografts, in which the activity ranging from 0.97 to 4.06 nmol/min/mg of protein at pH 7.428. Further study of human osteosarcoma tissues showed that all samples contained substantial activities of HPRT enzyme within the range of xenografts of human osteosarcoma29.Hypoxanthine guanine phosphoribosyltransferase (HPRT) is a key enzyme in salvage pathway responsible for the formation of GMP and IMP by recycling guanine and inosine from degraded DNA and RNA, respectively30. MMF is a prodrug of mycophenolic acid (MPA), an inhibitor of IMPDH31. The main reason MPA is a potent immunosuppressive agent is that activated T- and B-lymphocytes are highly divided based on de novo purine nucleotide synthesis30. A previous study by the authors unveiled the potency of MPA, a pan-IMPDH inhibitor, as an anti-tumor agent for osteosarcoma32. That study showed MPA effectively inhibits osteosarcoma cell viability and also reduces tumor mass in animal models at clinically achievable doses32. The study also demonstrated an effect of MPA in the modulation of pulmonary metastasis of osteosarcoma.Mycophenolate mofetil (MMF) is an immunosuppressive agent approved for use in patients receiving allogeneic renal, cardiac or hepatic transplants to prevent organ rejection14. Integrative analysis of IMPDH2 and HPRT will provide valuable data on an association of rewired GTP synthesis system in osteosarcoma and clinical outcomes of the patients.To enhance understanding of guanine nucleotide synthesis at population levels, this longitudinal study investigated expression patterns of individual key enzymes regulating de novo and salvage guanine nucleotide pathways in osteosarcoma samples. Furthermore, accumulating studies have reported that cancer cells have an increase in the activation of nucleotide anabolism and a lower rate of nucleotide catabolismThe present study recruited 127 osteosarcoma patients who had been diagnosed and treated at Maharaj Nakorn Chiang Mai Hospital, Thailand, between 1999 and 2018. Patients were followed-up for survival and metastasis-free survival until 11 January 2020. Clinicopathological parameters, including date of diagnosis, date of metastasis, Enneking staging, location of tumor, and metastatic status were retrieved from hospital records and pathology reports  and relevant guidelines.9. The percentage of immunoreactive cells was estimated and scored as follows: negative = 0, positive staining < 10% = 1, positive staining ≥ 10 and < 33% = 2, positive staining ≥ 33% and < 66% = 3, positive staining ≥ 66% = 4. Intensity of staining was scored on a scale of 0–3: no color reaction = 0, weak staining = 1, moderate staining = 2, and strong staining = 3. Immunoreactive score (IRS) was derived by multiplying immunoreactive cell scores and intensity of staining scores to compute an immunoreactive score ranging from 0 to 12 defined as negative; IRS = 0, weak; IRS > 0–4, moderate; IRS > 4–8, and strong; IRS > 8–12.Immunohistochemistry was performed on formalin-fixed paraffin-embedded (FFPE) tissues from archival biopsy and post-chemotherapy paraffin blocks at the Department of Pathology, Faculty of Medicine, Chiang Mai University. We applied the Ventana automated staining system and an Ultraview Universal DAB Detection Kit  to detect primary antibodies including anti-IMPDH2  and anti-HPRT  . Cytoplasmic staining of IMPDH and HPRT was independently evaluated by two observers (by PC and JS), blind to patient outcome data, and scored by using a semi-quantitative immunoreactive scoring systemP values < 0.05 were considered to be statistically significant.Statistical analysis was performed using STATA version 11  and GraphPad Prism version 8.4.2  . The statistical significance of correlations between expressions of IMPDH and HPRT and clinicopathologic data were determined by Fisher’s exact and Chi-square test. Receiver operating characteristic (ROC) curve analysis was applied to define the optimal cutoff points for IMPDH2 and HPRT expression levels as well as IMPDH2/HPRT ratio. Associations of IMPDH, HPRT and IMPDH2/HPRT ratio with overall survival and metastasis-free survival of osteosarcoma patients were evaluated by the Kaplan–Meier method with the log-rank test. Multivariate survival analysis was determined using Cox regression of proportional hazards to probe for significance at the 95% confidence interval (CI). To examine expression profiles and staining patterns of IMPDH2 and HPRT enzymes, we conducted immunohistochemical analysis of formalin-fixed paraffin-embedded tissues from biopsy samples of 127 osteosarcoma cases and 20 post-chemotherapy samples. We found IMPDH2 and HPRT expressed mainly in the cytosol of the osteosarcoma cells  as well as metastatic status (P = 0.024).Characteristics of 127 osteosarcoma patients in the cohort are shown in Table In the survival analysis, we excluded data from 8 osteosarcoma patients who were lost to follow-up. Overall, survival time of 119 patients ranged from 1 to 209 months after initial diagnosis (median = 18 months). Seventy percent (84 cases) of the patients died after 1 to 102 months (median = 13 months). The 1-year and 5-year survival rates were 70% and 32%, respectively.P = 0.0196), but not for IMPDH2 results , not completing treatment (P < 0.0001) and the presence of metastasis (P < 0.0001).Immunoreactive score (IRS) cutoff points for each of the purine metabolizing enzymes were defined by receiver operating characteristic (ROC) curves: IMPDH2, IRS = 8; HPRT, IRS = 2. Then, univariate survival analysis of individual enzymes (IMPDH2 and HPRT) was conducted using Kaplan–Meier survival analysis and log rank tests. We found that low expression levels of HPRT were associated with shorter overall survival times  and advanced Enneking stage (P < 0.001).In this study, we assessed expression of IMPDH2 and HPRT enzymes in biopsies as well as in post-operative chemotherapy tissues of 20 osteosarcoma patients. The results demonstrated that expression of IMPDH2 and HPRT was maintained at the same level in post-operative chemotherapy tissues as in biopsy samples Fig. A. We fur30] Fig. , 7, wher30] Fig. , 7, 8, 933. Nucleotide biosynthesis is formed in two major ways (1) by a de novo biosynthetic pathway through biochemical reactions of simple metabolites including glucose, amino acids, glycine, CO2, and one-carbon units derived from the serine-glycine pathway, and (2) nucleotide salvage pathway that utilizes free bases recycled from the degradation of DNA and RNA or from exogenous dietary intake34.Unrestricted proliferation of cancer cells is limited by the abundance of total nucleotide pools as well as the level and the activity of rate-limiting enzymes of nucleotide synthesis35. Sasaki et al. demonstrated that IMPDH2 was upregulated in glioblastoma tissues, while HPRT1 were reciprocally downregulated showing significant correlation with IMPDH2 levels35. Our results also revealed that IMPDH2/HPRT ratio is an independent prognostic marker, where a high IMPDH2/HPRT ratio associated with shorter survival of the patient. However, the result from multivariate analysis revealed a significant association between HPRT levels and survival rate of osteosarcoma patients, but not for IMPDH2 expression results , an enzyme that catalyzes the amination of xylitol monophosphate (XMP) to guanosine monophosphate (GMP) in de novo guanine nucleotide synthesis pathways in metastatic human melanoma samples41. GMPS inhibitor could also potentially modulate melanoma cell invasion in vitro.Our findings also suggest that high levels of IMPDH2 in post-chemotherapy tissues are significantly associated with shorter metastasis-free survival. Multivariate analysis showed the independent prognostic power of IMPDH2 expression. Several reports have previously demonstrated the role of IMPDH and de novo guanine nucleotide synthesis in cancer metastasis and progression. A study by Zhou et al30. The present findings on an association of low HPRT level and unfavorable clinical outcomes suggest a potential metabolic vulnerability of poor prognostic patients. Currently, we are running phase II multi-center clinical trial to evaluate the efficacy and safety of mycophenolate mofetil (MMF) in patients with high-grade locally advanced or metastatic osteosarcoma (ESMMO) 42. A total of 27 high-grade locally advanced or metastatic osteosarcoma patients have been recruited into the study from collaborating hospitals in Thailand.The main reason MPA, an IMPDH inhibitor, is a potent immunosuppressant agent is that lymphocytes are lacking salvage capability to restore level of GTP pool in the cellsIn summary, our findings strengthen the concept that nucleotide metabolic switches and de novo guanine synthesis in tumorigenesis and osteosarcoma progression could be used as prognostic markers and to indicate actionable targets for osteosarcoma treatment.


A cooperative solvation/surface engineering approach is reported to achieve the reversible Mg plating/stripping in conventional carbonate electrolytes.2+ can easily overcome the reduced desolvation barrier and penetrate the Mg2+-conducting polymer coating, deposited on the Mg metal anode successfully, promoting the application of magnesium anode in carbonate electrolytes toward high-energy rechargeable batteries.Benefitting from the strategy, MgThe online version contains supplementary material available at 10.1007/s40820-021-00716-1. 2 additive of electrolyte impairs the Mg…O = C interaction to reduce the Mg2+ desolvation barrier for accelerated redox kinetics, while the Mg2+-conducting polymer coating on the Mg surface ensures the facile Mg2+ migration and the effective isolation of electrolytes. As a result, reversible plating and stripping of Mg is demonstrated with a low overpotential of 0.7 V up to 2000 cycles. Moreover, benefitting from the wide electrochemical window of carbonate electrolytes, high-voltage (> 2.0 V) rechargeable magnesium batteries are achieved through assembling the electrode couple of Mg metal anode and Prussian blue-based cathodes. The present work provides a cooperative engineering strategy to promote the application of magnesium anode in carbonate electrolytes toward high energy rechargeable batteries.Magnesium metal anode holds great potentials toward future high energy and safe rechargeable magnesium battery technology due to its divalent redox and dendrite-free nature. Electrolytes based on Lewis acid chemistry enable the reversible Mg plating/stripping, while they fail to match most cathode materials toward high-voltage magnesium batteries. Herein, reversible Mg plating/stripping is achieved in conventional carbonate electrolytes enabled by the cooperative solvation/surface engineering. Strongly electronegative Cl from the MgClThe online version contains supplementary material available at 10.1007/s40820-021-00716-1. Net Zero’ further promotes the application of LIBs in the emerging market of energy storage ..37]. As ic force . The hig2 additive, the chosen electrolyte solutions were analyzed by Raman spectroscopy as shown in Fig. −1 are associated with the stretching vibration of the carbonyl group [−1) and antisymmetrical vibration (1792.41 cm−1) [−1) when Mg(TFSI)2 is added, illustrating the strong Mg…O = C interaction [2 additive, the two peaks shift back to low frequency (1772.38 and 1795.05 cm−1), implying the interaction of Mg…O = C has been weakened. To study the feasibility of MgCl2 additive in regulating the solvation structure, DFT calculations were carried out to compare the interaction energy among Mg…Cl, Mg…PC, and Mg…EC systems  . These t2 can greatly reduce the overpotential of Mg chemistry especially in the initial cycles but become invalidate in the subsequent cycles due to the passivation caused by the decomposition of the electrolyte. On the other hand, engineering the Mg2+-conducting polymer coating on Mg metal anode can elongate and stabilize the cycling with a still relatively large overpotential. Combined with the above cooperative strategy including the solvation and interface regulation strategy, we eventually obtained a reversible Mg anode/electrolyte system in carbonate electrolytes with a low overpotential. The modified cells exhibit a decreased resistance in EIS test, confirming the accelerated ion migration on the interface . The improved kinetics originated from the cooperative solvation/interface engineering is further verified by galvanostatic cycling test of symmetric cells, which shows the reduced overpotential particularly at high current density .Galvanostatic tests on different symmetric cells were carried out to identify the role of the cooperative engineering strategy, as shown in Fig. −1 in bare carbonate electrolyte. In contrast, the cycling stability of the NiHCF/coated-Mg in the MgCl2-added carbonate electrolyte was greatly promoted with a much decreased overpotential. The subsequent capacity decay might be attributed to the unavoidable water impurities in PBAs [2-added carbonate electrolyte achieves improved capacity (154 mAh g−1) and reduced overpotential compared with MnHCF/bare-Mg cell. To further prove the Mg2+ extraction and insertion behavior in MnHCF, ex situ XRD analysis was carried out to investigate the structural evolution of MnHCF with variation in the charge/discharge states. The primary (200) peak shifts to lower angle after magnesiation, indicating that inter-planar spacing is increased accompanied by the Mg2+ insertion. Upon demagnesiation, (200) peak moves back to its original angle demonstrating the reversible extraction of Mg2+. The successful demonstration of the cooperative strategy provides a comprehensive perspective for practical use of high-voltage cathode in rechargeable Mg battery.We then envisage the potential of cooperative strategy in practical application, by assembling the full cells with Prussian blue analogs (PBAs), also called as HCFs, as cathode materials. Although PBAs have been well known for the capability of magnesium storage, the prototype cells with PBAs cathode and Mg metal anode have never been reported elsewhere, possibly due to the limitation in the voltage window of the state-of-the-art electrolytes –46. The  in PBAs . MnHCF p in PBAs . The MnH2+-conducting coating provides tunnels for Mg2+ migration and facilitates the following deposition processes, while its electronic insulation nature prevents the reduction of electrolytes, ensuring the good reversibility of Mg plating/stripping. In addition, Cl-contained electrolyte additive is capable of capturing electrons from the solvent molecular and accelerating the release of Mg2+ from the solvated structure. Consequently, reversible Mg plating/stripping with low overpotential and long-term stale cycling is achieved in carbonate electrolytes. Benefitting from such cooperative approach, we also construct full cell by using PBAs as cathode for the first time, which exhibit superior electrochemical performance, demonstrating the potential of high-voltage Mg batteries based on carbonate electrolytes. This reversible Mg anode/electrolyte system enabled by the described cooperative engineering strategy has shown great promises to build high-voltage cathodes toward high energy magnesium rechargeable batteries.In summary, we report a cooperative engineering strategy of solvation and surface to resolve the dilemma faced by Mg batteries in carbonate electrolytes. The polymeric MgSupplementary file1 (PDF 731 kb)Below is the link to the electronic supplementary material.