Glycogen plays an important role in glucose homeostasis and contributes to key functions related to brain cancer cell survival in glioblastoma multiforme (GBM) disease progression. Such adaptive molecular mechanism is dependent on the glycogenolytic pathway and intracellular glucose-6-phosphate (G6P) sensing by brain cancer cells residing within those highly hypoxic tumors. The involvement of components of the glucose-6-phosphatase (G6Pase) system remains however elusive.We questioned the gene expression levels of components of the G6Pase system in GBM tissues and their functional impact in the control of the invasive and brain cancer stem cells (CSC) phenotypes.In silico analysis of transcript levels in GBM tumor tissues was done by GEPIA. Total RNA was extracted and gene expression of G6PC1-3 as well as of SLC37A1-4 members analyzed by qPCR in four human brain cancer cell lines and from clinically annotated brain tumor cDNA arrays. Transient siRNA-mediated gene silencing was used to assess the impact of TGF-β-induced epithelial-to-mesenchymal transition (EMT) and cell chemotaxis. Three-dimensional (3D) neurosphere cultures were generated to recapitulate the brain CSC phenotype.G6PC3, SLC37A2, and SLC37A4 was found in GBM tumor tissues in comparison to low-grade glioma and healthy tissue. The expression of these genes was also found elevated in established human U87, U251, U118, and U138 GBM cell models compared to human HepG2 hepatoma cells. SLC37A4/G6PC3, but not SLC37A2, levels were induced in 3D CD133/SOX2-positive U87 neurospheres when compared to 2D monolayers. Silencing of SLC37A4/G6PC3 altered TGF-β-induced EMT biomarker SNAIL and cell chemotaxis.Higher expression in Two members of the G6Pase system, G6PC3 and SLC37A4, associate with GBM disease progression and regulate the metabolic reprogramming of an invasive and CSC phenotype. Such molecular signature may support their role in cancer cell survival and chemoresistance and become future therapeutic targets.  The metabolic reprogramming of cancer cells is a key contributing factor to tumorigenesis enabling rapid proliferation, survival, invasion, metastasis, resistance to treatments, and other processes associated with carcinogenesis –3. Reproin vivo, which was especially pronounced after 2DG treatment and recovery. Mechanistically, G6PC silencing is believed to lead to glycogen accumulation and to result in reduced activation of AKT (protein kinase B)  can be sensed, in part, through components system of the glycogen metabolism which appear dysregulated in a wide variety of malignancies . Indeed,inase B) .High G6PC expression was found to be associated with poor overall and disease-free survival in ovarian cancer . Here agThe G6PC family consists of three members: G6PC1 (G6PCα), G6PC2, and G6PC3 (G6PCβ) . DespiteSLC37A4 transcriptional regulation under hypoxic culture conditions was also reported to require hypoxia inducible factor (HIF)-1α -1α . SLC37A4(HIF)-1α , 33.G6PC and SLC37A members were regulated in i) the transition from healthy brain to low-grade glioma, then to GBM tissues, and ii) in established human GBM cell line models. Moreover, their contribution in iii) the chemotactic response and the acquisition of a CSC phenotype were also explored. Understanding CSC biology and metabolic reprogramming involving the G6Pase components could become keys to optimizing anticancer treatments  and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Canada . Cell culture media was obtained from Life Technologies . Electrophoresis reagents were purchased from Bio-Rad . All other reagents were from Sigma-Aldrich Canada.http://gepia.cancer-pku.cn/detail.php, accessed on July 5th, 2023). One-way ANOVA was used for differential analysis of gene expression, using disease states  as variables for the box plots.A Gene Expression Profiling Interactive Analysis (GEPIA) web server was used to analyze the RNA sequencing expression data of glioblastoma tumors  vs. healthy tissue (n = 207), and of low-grade glioma  vs. healthy tissue (n = 207) from the TCGA and the normal brain tissue in Genotype-Tissue Expression (GTEx) databases . GEPIA pG6PC3, SLC37A2, and SLC37A4 factors in GBM patients was analyzed by GEPIA  to identify and build protein-protein interaction networks  at a density of 2x105 cells/mL in complete media for 24-72 hours. Then, the supernatant was removed, and serum-free EMEM supplemented with 10 ng/mL human basic fibroblast growth factor , 20 ng/mL human epidermal growth factor , 5 μg/mL insulin , and BSA , at 4% was carefully added to the dishes. Spheroids were defined as rounded aggregates of cells with a smooth surface and poor cell-to-cell definition. Perimeters of 30-70 spheroids/flask were assessed for each experimental condition performed in triplicate and derived from three independent experiments.The human U87 (HTB-14), U118, U138, and U251 glioblastoma cell lines, as well as the human HepG2 hepatoma cell line, were from American Type Culture Collection . They were all maintained in Eagle’s Minimum Essential Medium  containing 10% (v/v) calf serum , 2 mM glutamine, 1 mM sodium pyruvate , 100 units/ml penicillin and 100 mg/ml streptomycin . Cells were incubated at 37°C with 95% air and 5% COG6PC3, SLC37A2, and SLC37A4 gene expression levels according to the manufacturer’s recommendations. Tissue cDNAs in each array were synthesized from high-quality total RNAs of pathologist-verified tissues, normalized and validated with ß-actin in two sequential qPCR analyses, and accompanied by clinical information for 18 WHO grade I, 11 WHO grade II, 10 WHO grade III, and 2 WHO grade IV brain tumors.TissueScan™ cancer and normal tissue cDNA arrays were purchased from OriGene , covering 43 clinical samples of the four stages of brain cancer as well as normal tissues, and were used to assess vs. cycle number). The difference (ΔCT) between the mean values in the triplicate samples of the target gene and those of GAPDH and β-actin mRNAs were calculated by CFX manager Software version 2.1 (Bio-Rad) and the relative quantified value (RQV) was expressed as 2-ΔCT.Total RNA was extracted from cell monolayers using TriZol reagent . For cDNA synthesis, 2 μg of total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit . cDNA was stored at -80°C prior to PCR. Gene expression was quantified by real-time quantitative PCR using Sso Fast EvaGreen Supermix (Bio-Rad). DNA amplification was carried out using a CFX Connect Real-Time System (Bio-Rad) and product detection was performed by measuring the binding of the fluorescent dye EvaGreen to double-stranded DNA. The following QuantiTect primer sets were provided by QIAGEN: G6PC1 (Hs_G6PC_1_SG QT00031913), G6PC2 (Hs_G6PC2_va.1_SG QT01664152), G6PC3 (Hs_G6PC3_1_SG QT00033453), SLC37A1 (SLC37A1_1_SG QT00073094), SLC37A2 (Hs_SLC37A2_1_SG QT00056203), SLC37A3 (Hs_SLC37A3_1_SG QT00057148), SLC37A4 (Hs_SLC37A4_1_SG QT00024325), GAPDH (Hs_GAPDH_2_SG QT01192646), β-actin (Hs_Actb_2_SG QT01680476) and PPIA (Hs_PPIA_4_SG QT01866137). The relative quantities of target gene mRNA compared against two internal controls chosen from GAPDH, β-actin or PPIA RNA, were measured by following a ΔCT method employing an amplification plot . Gene silencing was performed using 20 nM siRNA against G6PC3 , SLC37A2 , SLC37A4 , or scrambled sequences . The above small interfering RNA and mismatch siRNA were all synthesized by QIAGEN and annealed to form duplexes. Gene silencing efficacy was assessed by RT-qPCR as described above.2 for 24 hours. Prior to cell seeding, the underside of each well in the upper chamber was coated with 0.15% gelatin in PBS and incubated for 1 hour at 37°C. The lower chamber was filled with serum-free medium. The upper chamber of each well was filled with 250,000 cells. After 30 min of adhesion, cell migration was monitored every 5 min for 20 hours. The impedance value was measured by the RTCA DP Instrument and expressed as an arbitrary unit called the Cell Index. Each experiment was performed in quadruplicate wells.Experiments were carried out using the Real-Time Cell Analyser (RTCA) Dual-Plate (DP) Instrument and the xCELLigence system , following the instructions of the supplier. U87 cells were transfected with 2 nM siRNAs (Control and G6PC3) as described above. After transfection, 25,000 cells per well were seeded in a CIM-plate 16 (Roche Diagnostics) and incubated at 37°C under a humidified atmosphere containing 5% COData are representative of three or more independent experiments. Statistical significance was assessed using Student’s unpaired t-test or 1-way ANOVA with a Dunnett post-test. Probability values of less than 0.05 were considered significant, and an asterisk (*) identifies such significance in the figures.in silico differential analysis of G6PC    and was used to predict and analyze potential or existing PPI. Indirect target proteins of G6PC3 were retrieved from STRING as described in the Methods section and predicted G6PC3 interrelationship with potential biomarkers involved in the G6Pase system, namely G6PC1 and SLC37A4 (A protein-protein interaction (PPI) network of G6PC3 was constructed by using the STRING database  and from one human hepatoma cell line (HepG2). Primers and RT-qPCR results were validated with agarose gel electrophoresis, which showed a single amplicon product for each of the genes amplified  markers FN1 and SNAIL were also increased in neurospheres compared to adherent cells (in vitro chemotherapy and therefore may relate G6PC3 (G6PCβ) and SLC37A4 (G6PT) to some chemoresistance and invasive molecular signature of GBM-derived CSC.Neurospheres culture conditions are known to recapitulate, in part, the cancer stem cells (CSC) phenotype , 39. U87G6PC3  and/or hydrolysing (G6PC3) activities.We next wished to address the potential crosstalk between the acquisition of CSC phenotype and the corresponding increase in G6PC3 and SLC37A4 expression upon neurospheres formation. U87 glioblastoma monolayer cells were transiently transfected with a scrambled sequence . Cells were then challenged with 10 nM TGF-β for 24 hours. Total RNA was extracted, followed by RT-qPCR assessment of SNAIL gene expression. TGF-β effectively induced SNAIL in siScrambled-transfected cells, and this was significantly inhibited upon G6PC3 and SLC37A4 gene silencing, but not in cells where SLC37A2 was silenced , a known potent EMT inducer in glioblastoma cells , 42, andSLC37A4 and the transition from low-grade glioma state to GBM, adding to their angiogenic and chemoresistance phenotype.Glioblastoma (GBM) is a hypoxic and aggressive brain tumor associated with poor patient prognosis and limited treatment options. Such clinical manifestation is, in part, attributable to the highly adaptive mechanisms of the brain cancer cells allowing their metabolic reprogramming within a low oxygen tension tumor microenvironment. In this study, we assessed the transcript levels and the specific roles of the G6P sensing components G6PC3, SLC37A2, and SLC37A4, all found increased in clinical GBM tissues Figure 1in vitro and in vivo (While the importance of glucose metabolism alterations in cancer development and progression is well recognized , the spe in vivo . These fin silico data analysis, functional experiments were performed using several established GBM-derived cell lines in which high expression of G6PC3, SLC37A2, and SLC37A4 in comparison to HepG2 hepatoma cells was observed. Specific knockdown of G6PC3 and SLC37A4 genes further altered the acquisition of a CSC phenotype, and G6PC3 as well as SLC37A4 silencing prevented the TGF-β signal transduction response that led to increased chemotaxis. TGF-β plays an important role in cell metabolism and immunity and can induce a shift in cell metabolism from oxidative phosphorylation to aerobic glycolysis, providing a favorable environment for tumor growth (G6PC3 and SLC37A4 gene silencing alters the crosstalk between G6P sensing and TGF-β signaling remains to be investigated.Consistent with our r growth . TGF-β ar growth , 47. TGFr growth . How G6PG6PC3 and SLC37A4 were found induced in neurospheres, a 3D cell culture model which recapitulates a stem cell-like phenotype associated with increased tumorigenicity and therapeutic resistance (G6PC3 and SLC37A4 levels may contribute to the chemoresistance and invasive molecular signature of GBM and of GBM-derived CSC. The knockdown of G6PC3 and SLC37A4 in neurospheres resulted in a significant reduction in the expression levels of CSC markers, including EMT biomarkers, all collectively associated with stemness, self-renewal capacity, and invasive properties of glioma stem cells (G6PC3 and SLC37A4 again confirm their implication in metabolic reprogramming and suggest their potential as future therapeutic targets to mitigate the aggressive behavior of GBM. Although glycolysis inhibitors are widely used to target such reprogramming, their efficacy in GBM remains unclear, especially within a hypoxic tumor microenvironment (Notably, the expression levels of sistance , 39, 40.em cells . The impironment –52. Inhiironment .KM hexokinases, which catalyze the first step in glycolysis in most tissues. Failure to eliminate 1,5AG6P appears to be the mechanism of neutrophil dysfunction and death in G6PC3-deficient mice (Transcriptomic-based studies revealed that upregulated levels of G6PC3 in GBM patients associated with a high-risk group which had significantly poorer survival results , 54–57. ent mice .In conclusion, our study highlights an original and underestimated potential of G6PC3 and SLC37A4 as prognostic markers and therapeutic targets in brain cancer. The upregulation of these genes in GBM tissues, and additionally in brain CSC models, further suggests their association with stemness properties and invasive characteristics. Whether G6PC3 and SLC37A4 may contribute, in part through G6P sensing processes, to the pro-angiogenic phenotype of GBM remains speculative, but would align with previous studies indicating the involvement of glucose metabolism alterations in angiogenesis regulation , 64. TarFurther studies are warranted to elucidate the precise molecular mechanisms underlying the functional implications of the G6Pase components G6PC3 and SLC37A4 at the protein level in GBM. The targeted delivery and immunoregulatory effects of chlorogenic acid, a potent SLC37A4 inhibitor , 65, werThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used.ST: Formal Analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. M-ER: Data curation, Formal Analysis, Investigation, Writing – review & editing. BA: Conceptualization, Formal Analysis, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing.


In vivo or ex vivo inhibition of CSF1R profoundly changes the transcriptional profile of CD4+ cells and suppresses Th1 and Th2 differentiation in directionally stimulated and unstimulated cells and independently of microglia depletion. Given that T cells also contribute in CNS pathology, these effects may have practical implications in the interpretation of relevant experimental data.Colony-stimulating factor 1 receptor (CSF1R) inhibition has been proposed as a specific method for microglia depletion. However, recent work revealed that in addition to microglia, CSF1R inhibition also affects other innate immune cells, such as peripheral monocytes and tissue-resident macrophages of the lung, liver, spleen, and peritoneum. Here, we show that this effect is not restricted to innate immune cells only but extends to the adaptive immune compartment. CSF1R inhibition alters the transcriptional profile of bone marrow cells that control T helper cell activation.  However, it is still unknown if these off-target effects extend cells of the adaptive immune compartment. This is critical since recent studies have employed such inhibitors to investigate interactions between innate and adaptive immune cells and have demonstrated specific roles of T cells in CNS pathology, especially in models of autoimmune diseases and glaucoma9.We recently showed that colony stimulating factor 1 receptor (CSF1R) inhibition not only affects microglia but also alters the function of bone marrow-derived macrophagesin vivo and ex vivo models, along with targeted transcriptomics and Ingenuity Pathway Analysis (IPA), to investigate the role of CSF1R inhibition on the adaptive immune system. Here provides a brief report of the off-target effects of CSF1R inhibition, which extend to the adoptive immune system, and alert the community about the implications of these findings in interpreting relevant experimental data.Here, we adopt Hdac9, Cd1d, Lrmp, Pax5, Pecam1, Cd8A, Stim2, Blnk, B2m, Rbpj and repression of Spp1 genes in bone marrow cells , nuclear receptor co-activator 6 (Ncoa6) and CD14 . CD4+ cells were analyzed with targeted transcriptomics to assess T helper cell differentiation pathway. Administration of CSF1R inhibitor for 3 weeks in mice profoundly affected T helper cell differentiation, as evident by transcriptional profile changes, notably through suppression of interleukin 12 receptor subunit beta 2 (Il12rb2) and over expression of CSF2 , to show that CSF1R inhibition not only affects innate immune cells, especially microglia and macrophages, but also bone marrow processes that control T-cell activation and differentiation. Previous studies have assumed that CSF1R inhibition may only affects microglia13, however, our recent studies demonstrated that CSF1R inhibitors profoundly affect peripheral monocytes and the function of macrophages5. To this end, additional off-target effects in other immune compartments became the target of this investigation. Here, we provide insights into the role of CSF1R inhibition on the adaptive immune cells and show that it causes changes in the regulation CD4+ and T helper cell differentiation. Our findings are important considering that T cells have recently implicated in CNS pathologies, especially in autoimmune conditions and glaucoma, and such studies have derived their conclusions by employed small-molecule CSF1R inhibitors to deplete microglia. Future experiments using CSF1R inhibitors need to account for these off-target effects in the interpretation of relevant experimental data.Here, we adopt ad libitum for 3 weeks.Animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research, and the National Institutes of Health (NIH) Guidance for the Care and Use of Laboratory Animals. This study was approved by the Mass. Eye and Ear Animal Care Committee. Mice at 6–8 weeks old of both genders were used: C57BL/6J . PLX5622  was formulated into AIN-76A chow  at the dose of 1200 ppm and given + isolation, using the CD4+ T cell isolation Kit (Miltenyi). 0.5 ×106 CD4+ cells/mL were suspended in 10% FBS RPMI-1640 (ThermoFisher) supplemented with 5 ng/mL IL-2 (PeproTech) and 0.5 μg/mL anti-CD28 antibody  and seeded into 2 μg/mL anti-CD3 antibody  pre-coated 24-well plates. Th1 differentiation was achieved using 1 μg/mL anti-IL-4 antibody , 5 ng/mL IL-2 (PeproTech), and 10 ng/mL IL-12 (PeproTech) in the culture media. Th2 differentiation was achieved using 1 μg/mL anti-IFN-γ antibodies , 5 ng/mL IL-2(PeproTech), and 10 ng/mL IL-4 (PeproTech) in the culture media. Cells were incubated for 96 hours in 5% CO2 at 37°C. Cell Activation Cocktail with Brefeldin A (Biolegend) was added to the culture media 5 hours prior to harvesting and staining the cells. For PLX5622 treatment, different concentrations of PLX5622 were added to the culture media during the stage of differentiation to Th1/Th2.Spleen and lymph nodes from C57BL/6 mice were surgically removed and single cells were obtained mechanically using a 70μm cell strainer . Red blood cells were lysed by ACK lysis buffer (Lonza) before CD4+ T cells and ex vivo splenocytes were processed for flow cytometry. Anti-murine CD16/32  was used for blocking. Anti-CD3 (Clone: 17A2), anti-CD4 (Clone: GK1.5), anti-IL-4 (Clone: 11B11), anti-INF-γ (Clone: XMG1.2) were purchased from BioLegend. Data were acquired by LSRII cytometer (BD Biosciences) and analyzed by FLowJo V10 (Tree Star).Cultured CD4https://geneglobe.qiagen.com/us/analyze/) to generate a normalized expression profile. Further normalization was performed based on 2^ (- Delta Delta CT). Gene expression heat-map, 3D PCA plot, and scatter plot were generated using the normalized gene set in R-software version 1.14.4.A total of 1 μg of RNA was used for each array analysis. cDNA was synthesized by RT2 First Strand Kit  and then mixed with RT2 SYBR Green ROX qPCR Mastermix (Qiagen) before loading into the array well . qPCR was performed by using QuantStudio 3 (ThermoFisher) according to manufacturer’s manual. Data analysis was performed by using Qiagen’s data analysis web portal  was employed to assess how gene changes effect canonical pathways, upstream regulators, and disease/functions associatedData were analyzed with GraphPad  using two-tailed unpaired t-test and ordinary one-way ANOVA with Dunnet’s correction for multiple comparisons. Statistical significance was set at P < 0.05.


ANP32 proteins, which act as influenza polymerase cofactors, vary between birds and mammals. In mammals, ANP32A and ANP32B have been reported to serve essential but redundant roles to support influenza polymerase activity. The well-known mammalian adaptation PB2-E627K enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbor this substitution. Here, we show that alternative PB2 adaptations, Q591R and D701N, also allow influenza polymerase to use mammalian ANP32 proteins, whereas other PB2 mutations, G158E, T271A, and D740N, increase polymerase activity in the presence of avian ANP32 proteins as well. Furthermore, PB2-E627K strongly favors use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs, and horses, where ANP32A proteins are the preferred cofactor. Using an experimental evolution approach, we show that the passage of viruses containing avian polymerases in human cells drove acquisition of PB2-E627K, but not in the absence of ANP32B. Finally, we show that the strong pro-viral support of ANP32B for PB2-E627K maps to the low-complexity acidic region (LCAR) tail of ANP32B.IMPORTANCE Influenza viruses naturally reside in wild aquatic birds. However, the high mutation rate of influenza viruses allows them to rapidly and frequently adapt to new hosts, including mammals. Viruses that succeed in these zoonotic jumps pose a pandemic threat whereby the virus adapts sufficiently to efficiently transmit human-to-human. The influenza virus polymerase is central to viral replication and restriction of polymerase activity is a major barrier to species jumps. ANP32 proteins are essential for influenza polymerase activity. In this study, we describe how avian influenza viruses can adapt in several different ways to use mammalian ANP32 proteins. We further show that differences between mammalian ANP32 proteins can select different adaptive changes and are responsible for some of the typical mutations that arise in mammalian-adapted influenza polymerases. These different adaptive mutations may determine the relative zoonotic potential of influenza viruses and thus help assess their pandemic risk. The natural host reservoir of influenza A viruses is wild aquatic birds. To efficiently replicate in mammalian hosts, avian influenza viruses need to overcome multiple barriers by adapting to several mammalian host factors . One maj5–Mutations in the heterotrimeric viral polymerase can enable efficient use of mammalian ANP32 proteins, of which PB2-E627K is the best characterized , 9. HoweAs well as differences in avian and mammalian ANP32 length, there are also differences in the level of redundancy to support influenza virus polymerase in different vertebrate hosts. ANP32A is the only ANP32 family member that efficiently supports influenza polymerase in birds, while in humans and most other mammalian influenza hosts ANP32A and ANP32B can both support polymerase activity to varying levels , 14, 15.In this study, we aimed to understand whether alternative mammalian PB2 adaptations other than PB2-E627K function by adapting the polymerase to utilize mammalian ANP32 proteins, or whether they achieve adaptation through an ANP32-independent mechanism. We found that alongside PB2-E627K, -Q591R and -D701N specifically adapt avian polymerases to use mammalian ANP32 proteins. Furthermore, while PB2-D701N allows the polymerase to efficiently use both ANP32A and B proteins, -E627K specifically favors the use of mammalian ANP32B proteins. To support this finding, we used bioinformatics to show that viruses adapting to hosts in which ANP32B is the more potent pro-viral factor, such as humans and mice, generally adapt via PB2-E627K; while viruses that emerge in pigs, horses, or dogs, hosts with potent pro-viral ANP32A proteins, more often gain PB2-D701N or -Q591R. Using an experimental evolution approach, we passaged a pair of viruses containing the polymerase from avian influenza viruses in human cells and observed the emergence of the PB2-E627K mutation. This adaptation was not seen during passage of the same avian virus in human cells lacking ANP32B (BKO). Finally, we mapped the difference in ANP32B preference of polymerases containing E627K to the ANP32B protein LCAR, the region that has recently been implied to directly interact with the 627 domain in the influenza polymerase/ANP32 co-structure , 19.We previously described a library of mammalian PB2 adaptations in an avian influenza virus polymerase backbone A/turkey/England/50-92/1991  . Here, wA range of host factors have been implicated in the mammalian adaptation of avian influenza virus polymerase, including α-importins, DDX17, and ANP32 proteins , 14. We As before the PB2 mutants displayed a differential phenotype which corresponded with that described in We then considered whether any other host specific factors which differed between avian and mammalian cells might affect the complementation of influenza polymerase by ANP32 proteins. We reconstituted each polymerase containing different PB2 mutations in chicken DF-1 cells in which chANP32A expression was ablated by CRISPR editing , then reThese results indicate that PB2 Q591R or D701N, but not the other substitutions tested here, act in a similar manner to E627K and specifically enable the viral polymerase to utilize human ANP32A or -B proteins as a cofactor.We next tested the ability of ANP32A and ANP32B proteins from different mammalian species relevant to influenza ecology to support influenza A polymerase activity. First, using a polymerase constellation from the 2010 pandemic H1N1 virus (A/England/687/2010) that is adapted to mammals via the PB2 mutation Q591R, we found that there was variation across species in the relative potencies of ANP32A and ANP32B. For example, in humans and mice, ANP32B was the more potent polymerase cofactor, while the ANP32A proteins of pigs, horses, dogs, seals, and bats more efficiently supported pandemic H1N1 polymerase activity  5, 15)..15). TheTo further investigate the compatibility between different polymerase constellations and different mammalian ANP32 proteins, we tested polymerase reconstituted with PB2 mutants from group 3 to see if they displayed any bias in ANP32 paralogue usage in human dKO or avian AKO cells  to I. ThThese data illustrate a bias of different PB2 polymerase adaptations for different mammalian ANP32A or B proteins, which is particularly evident for PB2-E627K and, to a lesser extent, -Q591R, which show a preference for ANP32B proteins.Although PB2 E627K is the key polymerase adaptation in human seasonal influenza virus and is commonly found in human zoonotic infections as well as in laboratory-adapted mouse-passaged avian influenza viruses, it is rarely found in viruses that have crossed from birds into swine, dogs, or horses , 21. InsTo test this hypothesis, we first performed bioinformatics analysis on 526 viruses, comparing the proportions of viruses with mammalian adaptions found at sites 591, 627, and 701 during zoonotic infections, laboratory mouse adaptation studies, or sustained transmission of avian-origin PB2 segments in different mammalian species . PB2 E62In viruses that have crossed from birds and sustainably circulated in mammalian hosts, we saw a clear difference in PB2 adaptations in humans, compared to viruses endemic in swine, horses, and dogs. The only sustained avian-origin PB2 in humans (which transmitted during the 1918 Spanish influenza pandemic and was donated to subsequent pandemic viruses in 1957 and 1968) possesses E627K, whereas none of the polymerases from viruses adapted in other species, including pandemic 2009 H1N1, show this adaptation. Instead, such viruses contain D701N or Q591R/K/L; or, in the case of canine H3N2 viruses, none of the currently described mammalian ANP32-specific adaptations .To investigate whether different ANP32 proteins drive different PB2 adapting mutations, we used an experimental evolution approach, serially passaging a virus with the polymerase gene segments derived from avian influenza viruses though human cell lines lacking either ANP32A (AKO) or ANP32B (BKO) .Six populations of a reassortant virus containing the Hemagglutinin (HA), Neuraminidase (NA) and Matrix (M) segments from the laboratory-adapted PR8 strain and the polymerase genes derived from avian influenza virus 50-92 (PB2-627E) were passaged 10 times through either control eHAP cells (which express both ANP32A and ANP32B) or cells that expressed human ANP32B only (AKO) or human ANP32A only (BKO). PB2 segments from each population were Sanger-sequenced at passages 2, 5, and 10. By passage 5, in both the control and AKO cells, three out of six (50%) populations had evolved PB2-E627K. This adaptation was not detected in the BKO cells, even by passage 10. Instead, in these cells, one virus population (17%) by passage 5 and two by passage 10 (33%), gained the D701N mutation . D701N wTo confirm that this phenotype was not specific to a single strain of avian influenza, we repeated this experiment with a recombinant virus bearing the polymerase gene set from an H7N9 virus, A/Anhui/1/2013 (Anhui). The Anhui isolate had naturally gained PB2-E627K during zoonosis, but this was reverted to 627E by reverse genetics for the purpose of the passage experiment. In a similar manner to the H5N1 50-92 polymerase-bearing virus, the Anhui PB2 gained a mixture of E627K or D701N, as well as Q591K, in the control cells or AKO cells by passage 5; whereas in the BKO cells, every population evolved D701N or Q591K, but E627K was not detected .Taken together, these observations suggest that the predominance for the PB2-E627K adaptive mutation seen in human cells is specifically driven by adaptation to utilize the strongly pro-viral host factor human ANP32B.To investigate the molecular basis of the superior ability of human ANP32B over ANP32A to complement two different avian influenza polymerase constellations bearing the mammalian-adapting PB2-E627K substitution, we generated human ANP32A/B chimeric ANP32 constructs. Because the LCAR is described as directly interacting with the 627 domain of PB2 , 19, 22,In this study, we showed that besides E627K, several other mutations in PB2 adapt the polymerase to utilize the shorter ANP32 proteins found in mammalian cells. However, PB2 E627K strongly biases polymerases toward reliance specifically on mammalian ANP32B. While ANP32A and ANP32B serve redundant pro-viral roles in many mammals, ANP32B is the dominant pro-viral factor in humans and mice, whereas in most other relevant mammalian hosts, such as pigs, horses, and dogs, ANP32A proteins is more potent , 17. ThiPrevious studies have shown that varying expression of ANP32A isoforms resulting from differential splicing of transcripts in different avian species results in a different propensity to drive mammalian-like polymerase adaptations  , 23. OurSurprisingly, we found that PB2 D701N was an efficient adaptation to short mammalian ANP32 family members, regardless of whether the assay was performed in human or chicken cells. Previously, PB2 D701N has been described as an adaptation to a different set of host factors, the importin-α family , 13. OurInfluenza virus polymerases co-opt a range of host factors for their replication and transcription . Our app2 while DF-1 cells were maintained at 39°C, 5% CO2.Human engineered-haploid cells  without gene knockout (Control) or with ANP32A (AKO), ANP32B (BKO), or both knocked out (dKO) by CRISPR-Cas9, as described previously , were maThe viruses and virus minigenome full strain names used through this study were A/duck/Bavaria/1/1977 , A/turkey/England/50-92/1992 , A/Anhui/1/2013 , A/England/687/2010 (pH1N1), A/Victoria/1975 (H3N2), and A/swine/England/453/2006 (H1N1). Viral minigenome expression plasmids were either generated previously or created using overlap extension PCR , 28. ANPAll virus work in this study was performed with A/turkey/England/50-92/1992  or A/Anhui/1/2013 (H7N9) (K627E); reassortant viruses were generated by rescuing the polymerase, NP, and NS segments of the homologous virus with the HA, NA, and M segments of A/Puerto Rico/1/1934  as previously described . Virus wRenilla luciferase, and 40 ng of polI vRNA-Firefly luciferase. DF-1 AKO cells were transfected in 12-well plates using double the amount of plasmid in eHAP cells, and polI vRNA-Firefly plasmids with a chicken polI site as described previously . All assays were performed on a minimum of two separate occasions with the representative data shown.eHAP dKO cells were transfected at around 50% confluence in 24-well plates using lipofectamine 3000 (Thermo Fisher) with the following mixture of plasmids: 100 ng of pCAGGs ANP32 or empty pCAGGs, 40 ng of pCAGGs PB2, 40 ng of pCAGGs PB1, 20 ng of pCAGGs PA, 80 ng of pCAGGs NP, 40 ng of pCAGGs To visualize protein expression during mini-genome assays, around 500,000 transfected cells were lysed in radioimmunoprecipitation assay (RIPA) buffer  supplemented with an EDTA-free protease inhibitor cocktail tablet (Roche).AB129002, Abcam), rabbit α-PB2 , and mouse α-NP . The following near-infrared (NIR) fluorescently tagged secondary antibodies were used: IRDye 680RD goat anti-rabbit (IgG) secondary antibody  and IRDye 800CW goat anti-mouse (IgG) secondary antibody . Western blots were visualized using an Odyssey Imaging System (LI-COR Biosciences).Proteins were detected with mouse α-FLAG , rabbit α-Vinculin , frozen down, and stored at −80°C. Each passage experiment was done with 6 concurrent populations.AGCGAAAGCAGG). Sequencing of PB2s was performed using two sets of primers with 5′-M13F or M13R primer sites . PA was sequenced similarly . PCR was performed using KOD Hot Start DNA Polymerase (Merck). PCR products were purified using the Monarch PCR and DNA Cleanup kit (New England Biolabs) and sequenced using the Sanger method with M13F or M13R primers.To sequence viruses, RNA was extracted from cell-free virus-containing supernatants using a viral RNA extraction minikit (Qiagen). cDNA synthesis was conducted using Superscript IV and the uni12-FluG primer (To assess the proportion of zoonotic influenza viruses with different mammalian ANP32 adaptations, the PB2 sequences of all non-H1, -H2, and -H3 human or swine influenza viruses were downloaded from the NCBI influenza virus database and aligned. Sequences which were determined to be of seasonal human influenza origin were identified by BLASTn and removed, and the proportions of viruses with adaptation at positions 591, 627, and 701 were calculated. For the mouse adaptation summary, we undertook an exhaustive literature search for any study taking an avian-origin virus without any prior mammalian adaptation and passaging it through mice in PubMed using the search terms “mouse,” “influenza,” and either “adaptation,” “adaption,” or “passage.” A list of the papers identified and included in this analysis is included in Table S1 in the supplemental material.For the timeline of stably circulating avian-origin mammalian influenza virus strains, viruses were chosen due to the strength of the evidence that they came directly from birds into said species and not from another mammalian species—hence, pH1N1 swine-origin H1N1 and equine-origin canine H3N8 are excluded. Swine H9N2 was included because there remains fairly good evidence that although this virus may have continued to co-circulate between poultry and pigs, it does show several mammalian adaptations and therefore probably constitutes a swine-adapted avian-origin virus.All studies of infectious agents were conducted within biosafety level 2 facilities approved by the UK Health and Safety Executive and in accordance with local rules at Imperial College London.The manuscript contains no data deposited in external repositories.


We estimated influenza vaccine effectiveness (IVE) in preventing outpatient and hospitalized cases in the 2022–2023 season. A test-negative design included a representative sample of outpatients and all hospitalized patients with influenza-like illness (ILI) from October 2022 to May 2023 in Navarre, Spain. ILI patients were tested by PCR for influenza virus. Influenza vaccination status was compared between confirmed influenza cases and test-negative controls. Among 3321 ILI patients tested, IVE to prevent influenza cases was 34% : 16 to 48) overall, 85% (95%CI: 63 to 94) against influenza B, and 28% (95%CI: 3 to 46) against A(H3N2). Among 558 outpatients, 222 (40%) were confirmed for influenza: 55% A(H3N2), 11% A(H1N1), and 31% B. Overall, IVE to prevent outpatient cases was 48% (95%CI: 8 to 70), 88% (95%CI: 3 to 98) against influenza B, and 50% (95%CI: −4 to 76) against A(H3N2). Of 2763 hospitalized patients, 349 (13%) were positive for influenza: 64% A(H3N2), 17% A(H1N1), and 8% B. IVE to prevent hospitalization was 24% (95%CI: −1 to 42) overall, 82% (95%CI: 49 to 93) against influenza B, and 16% (95%CI: −17 to 40) against A(H3N2). No IVE was observed in preventing influenza A(H1N1). IVE was high to prevent influenza B, moderate against A(H3N2) and null against A(H1N1). A lower proportion of influenza B cases may explain the smaller IVE in hospitalized patients than in outpatients. The null IVE against A(H1N1) was consistent with the observed antigenic drift and supports the new composition of the 2023–2024 influenza vaccine. The 2022–2023 influenza season was characterized by A(H3N2) influenza virus circulation until January, being displaced by influenza B/Victoria and AH1N1) since then N1 since ,5,6. As Most IVE studies evaluate only the effect in preventing primary care consultations or in preventing hospital admissions ,6,13,14.We estimated the IVE in preventing outpatient and hospitalized cases during the 2022–2023 influenza season in Navarre, Spain.The present study was performed in the region of Navarre, Spain, where IVE have been assessed on a population-based scheme since 2009 ,16. The Influenza surveillance relied on electronic reporting of influenza-like illness (ILI) cases by primary healthcare centers and hospitals. ILI was defined as the sudden onset of any general symptom  in addition to any respiratory symptom  . The pro2 and more), and month of sample collection was obtained from the electronic records of primary healthcare. The major chronic conditions were codified according to the International Classification of Primary Care, 2nd edition [The variables on age, sex, presence of major chronic conditions  with their 95% confidence intervals (CI) were calculated using logistic regression. Adjusted models included sex, age groups , presence of major chronic conditions, month of sample collection, and healthcare setting  used as categorical variables. The IVE was estimated as a percentage: (1 − OR) × 100.Characteristics of cases and controls were compared by χThe main analysis considered only influenza vaccination in the current season (yes/no). An alternate analysis evaluated combinations of influenza vaccination in the current and three previous seasons in the following categories: vaccination in the current season regardless of previous seasons, vaccination in previous seasons only, and unvaccinated in the current and previous seasons as the reference category .The interaction terms between the vaccination status and healthcare setting or age group were tested. Stratified analyses were carried out by age groups (9–64 and ≥65 years), for the target population for influenza vaccination, and for each virus (sub-)type . Only months with five or more cases of a given (sub-)type were included in the analysis for that outcome.A sensitivity analysis including COVID-19-confirmed patients in the control group was performed to evaluate the possible bias .p < 0.001)  attended in primary healthcare centers and 2763 (83%) admitted to hospitals, of which 222 (40%) and 349 (13%) were confirmed for influenza, respectively. Of the 571 confirmed cases, 348 (61%) were due to A(H3N2), 85 (15%) to A(H1N1), 95 (17%) to influenza B, and 43 to A non-subtyped virus. Among outpatient cases, 55% were A(H3N2), 11% A(H1N1), and 31% influenza B, while in hospitalized patients, the proportion of influenza A viruses was higher: 64% A(H3N2), 17% A(H1N1), and 8% influenza B (< 0.001) .p < 0.001) and had less presence of major chronic conditions . No statistically significant difference by sex was observed between influenza cases and test-negative controls. Among the confirmed influenza cases, 36% had received the current-season vaccine, and 44% had not been vaccinated in the current and in the three previous seasons in comparison to 56% and 25% of the test-negative controls, respectively (p < 0.001 for both comparisons)  .p < 0.001). Major chronic conditions were also more frequent in influenza A(H1N1) and A(H3N2) cases than in influenza B cases  (Only 16% of influenza B cases were older than 65 years compared to 54% of influenza A(H3N2) and 60% of A(H1N1) cases (< 0.001) .Sequence characterization of the product of amplification of the whole hemagglutinin gene or the HA1 fragment was available in 230 viruses. Of the 100 A(H3N2) strains characterized, 56 (56%) were A/Bangladesh/4005/2020-like (5% clade 2a.3 and 95% clade 2b), and 44 (44%) were A/Slovenia/8720/2022-like (77% clade 2a.1b and 23% clade 2a.1). Of the 68 A(H1N1) strains characterized, 58 (85%) were A/Sydney/5/2021-like (clade 5a.2a), and 10 (15%) were A/Norway/25089/2022-like (clade 5a.2a.1). All the 62 influenza B strains were B/Austria/1359417/2021(B/Victoria lineage)-like clade V1A.3a.2 .The phylogenetic trees of characterized A(H3N2) and B influenza viruses from Navarre are shown in The overall adjusted IVE estimate was 34% (95% CI: 16 to 48) when previous vaccination was not considered and was similar  when individuals vaccinated in any of the three previous seasons were excluded from the reference category. Individuals unvaccinated in the current season did not show a significant residual effect of previous doses  .interactionp- = 0.920). The estimate was also similar for the target population for influenza vaccination (35%). The IVE estimates were 85% (95% CI: 63 to 94) and 28% (95% CI: 3 to 46) in preventing influenza B and A(H3N2) cases, respectively. No effect was observed in preventing influenza A(H1N1) cases   found similar IVE estimates for current-season vaccination  .interactionp- = 0.028. No differences in IVE were observed between people aged 9 to 64 and those aged 65 years and older, in both primary healthcare  and hospital settings . Similar estimates were observed in analyses restricted to the target population for influenza vaccination. In both settings, no effect was observed in individuals unvaccinated in the current season who had been vaccinated in any previous season  vs. 24% (95% CI: −1 to 42), s season .The IVE estimates in preventing influenza outpatient cases were 50% (95% CI: −4 to 76), 7% (95% CI: −233 to 72), and 88% (95% CI: 3 to 98) against influenza A(H3N2), A(H1N1), and B, respectively, and to prevent hospitalizations were 16% (95% CI: −17 to 40), −53% (95% CI: −233 to 30), and 82% (95% CI: 49 to 93), respectively .The sensitivity analysis including the COVID-19-positive patients in the control group provided similar IVE estimates .The present study found a moderate IVE, on average, during the 2022–2023 influenza season in Navarre, Spain, although estimates against each specific circulating virus (sub-) type were very diverse. This influenza season was characterized by circulation of three (sub-)types of influenza viruses in different moments. The IVE was high in preventing influenza B cases (85%), moderate to low against influenza A(H3N2) (28%), and null against influenza A(H1N1). All the 62 influenza B strains were B/Austria/1359417/2021(B/Victoria lineage)-like, which was similar to the B/Victoria component included in the 2022–2023 influenza vaccine. While the A(H3N2) strain included in the vaccine (A/Darwin/9/2021) inhibited the clade 2a1b.2a.2 represented by Bangladesh-like viruses well [The IVE in preventing outpatient cases was moderate 48%), and the effect was similar in patients aged 9 to 64 years 45%) and 65 years and more (48%). These IVE estimates were consistent with interim results from a European Network, the United States, and Canada (40–54%) [8%, and t% and 65 The T135A substitution was only present in 10% of the characterized A(H3N2) influenza viruses from Navarre. Variation in position 135 has been associated with IVE [The null IVE observed in Navarre against A(H1N1) virus contrasted with an appreciable protection observed in other studies ,5,6,21. In the present study, the overall IVE estimate to prevent outpatient cases (48%) was higher in comparison to preventing inpatient cases (24%). However, no differences in the IVE estimates were observed between settings in the analysis stratified by virus (sub-) types. These findings may be explained by the lower proportion of influenza B cases in hospitalized patients than in outpatients and the high vaccine effectiveness found to prevent cases of this type of virus. A previous study described, in some seasons, similar IVE estimates in preventing outpatient cases and hospitalizations with confirmed influenza but, in other seasons, an additional effect of the influenza vaccination in reducing the risk of hospital admission among people in whom the vaccine failed to prevent influenza infection .The strengths of the present study include the test-negative design that is considered the recommended design for IVE assessment ,25,26,27In contrast with findings from previous influenza seasons ,12, the The inclusion of COVID-19-positive patients in the control group did not appreciably modify the results, which rules out the possible bias suggested by other authors .Some limitations may affect this study. This study was carried out in a region where vaccination was recommended for persons aged 60 years and older and those with risk conditions, and only a tetravalent egg-produced inactivated vaccine was used during the study period; therefore, caution should be taken in making generalizations of our results to other countries with different influenza vaccination indications, vaccination coverage, or where other vaccines brands were used. As the statistical power was reduced in some analyses, the estimates of IVE in the elderly population attended in primary healthcare or in the hospitalized young adults should be interpreted cautiously. This study included individuals with different chances for influenza vaccination due to their age or presence of comorbidities; therefore, analyses were adjusted for these variables to control for potential confounders ,29.In conclusion, the vaccine effectiveness during the 2022–2023 influenza season in Navarre was moderate on average, high to prevent influenza B, moderate to low against influenza A(H3N2), and null against influenza A(H1N1). A lower proportion of influenza B cases in hospitalized patients explains the lower IVE in preventing hospitalized influenza cases than outpatient cases. These results support the recommended composition of influenza vaccines to be used in the 2023–2024 influenza season and reinforce the annual recommendation of influenza vaccination.


Introduction: The oncogenic transformation is driven by genetic and epigenetic alterations influencing cancer cell fate. These alterations also result in metabolic reprogramming by modulating the expression of membrane Solute Carrier (SLC) transporters involved in biomolecules trafficking. SLCs act as tumor suppressors or promoters influencing cancer methylome, tumor growth, immune-escape, and chemoresistance.Methods: This in silico study aimed to identify the deregulated SLCs in various tumor types compared to normal tissues by analyzing the TCGA Target GTEx dataset. Furthermore, the relationship between SLCs expression and the most relevant tumor features was tackled along with their genetic regulation mediated by DNA methylation.Results: We identified 62 differentially expressed SLCs, including the downregulated SLC25A27 and SLC17A7, as well as the upregulated SLC27A2 and SLC12A8. Notably, SLC4A4 and SLC7A11 expression was associated with favorable and unfavorable outcome, respectively. Moreover, SLC6A14, SLC34A2, and SLC1A2 were linked to tumor immune responsiveness. Interestingly, SLC24A5 and SLC45A2 positively correlated with anti-MEK and anti-RAF sensitivity. The expression of relevant SLCs was correlated with hypo- and hyper-methylation of promoter and body region, showing an established DNA methylation pattern. Noteworthy, the positive association of cg06690548 (SLC7A11) methylation with cancer outcome suggests the independent predictive role of DNA methylation at a single nucleotide resolution.Discussion: Although our in silico overview revealed a wide heterogeneity depending on different SLCs functions and tumor types, we identified key SLCs and pointed out the role of DNA methylation as regulatory mechanism of their expression. Overall, these findings deserve further studies to identify novel cancer biomarkers and promising therapeutic targets. The multistep process of malignant transformation is driven by some genetic and epigenetic alterations leading to the aberrant function of key genes involved in the growth, survival, and antitumoral surveillance of normal cells. In the last few years, several studies have highlighted the involvement of DNA methylation in genetic reprogramming occurring during cancer development . DNA metOver the years, a growing number of studies have highlighted the involvement of SLCs in cancer development with tumor suppressor or promotive functions depending on cancer type. For instance, tumor-promoting SLCs have been described to be involved in angiogenesis, metastatic growth, immune escape, drug resistance, and Epithelial-Mesenchymal Transition (EMT). Conversely, the downregulation of several SLCs promotes tumor growth and proliferation, suggesting their role as tumor suppressor genes . NotablySLC2A family) and lactate transporters (SLC16A family), cystines (SLC7A11), glutamine (SLC1A5), and leucine (SLC7A5/SLC3A2) transporters, which provide the substrates for lactate dehydrogenase and enzymes of glycolytic and pentose-phosphate pathways , the TCGA Pan-Cancer , and Cancer Cell Line Encyclopedia (CCLE) cohorts (N = 1.076 RNAseq gene expression samples and N = 504 pharmacologic profiles for 24 anticancer drugs across 504 CCLE lines) were used to analyze the gene expression and DNA methylation status of 429 SLC genes (https://www.bioparadigms.org/slc/intro.htm—accessed on 27 May 2022). In this study, all SLC gene identities used in each Dataset were uniformed according to the “SLC Name” reported in https://xenabrowser.net/, accessed on September 2022) was used to retrieve gene expression and DNA methylation data of selected SLC genes to perform Pearson’s correlation and DNA methylation status analyses.In this study, the TCGA TARGET GTEx (LC genes . The lisThe abbreviation and extended full name of the 33 cancer types are reported in https://www.r-project.org/) to compute the log2FC as the difference between the log2 expression means of each tumor type and the pooled GTEx control group, whereas the p-value was calculated using two-tailed Student’s t-Test (see https://xenabrowser.net/) on TCGA TARGET GTEx cohort.Differential analysis between each TCGA tumor type and GTEx normal tissues, pooled all together as one control group, was performed to identify the SLC genes differentially expressed in cancer. This approach, based on the same global pool of GTEx tissues, allowed us to compare SLC Differentially Expressed Genes (DEGs) among all TCGA tumor types. Since the Xena Differential Gene Expression Analysis tool can analyze up to 2000 samples, the differential analysis was performed using R software version 4.2.0 (Test see  using thversus C4-C6 (immuno-quiet) and analyzed using XENA UCSC Limma-Voom pipeline. The immune-response and immune-quiet signatures were obtained from “Subtype_Immune_Model_Based” in analytic data type available on Xena UCSC  and Progression Free Interval (PFI) at 5 years separately, as well as the intratumoral immune state C2-C3 (immune-response) ena UCSC .2FC, whereas the p-value ≤0.05 was considered statistically significant.The difference between each comparison group was expressed as loghttps://www.r-project.org/)  and DNA methylation (Methylation 450 K) levels of selected SLC genes retrieved from the TCGA Target GTEx cohort were performed using cor_test function of rstatix package executed on R software version 4.2.0  and Down (lower median DNA methylation level) methylation groups according to the methylation levels of the cg06690548 (SLC7A11).Kaplan Meier survival analysis was performed using GraphPad Prism 8 (version 8.0.2) . Log-rank (Mantel-Cox) test was used to calculate the http://heatmapper.ca). Volcano plot was implemented using the publicly available tool VolcaNoseR (https://huygens.science.uva.nl/VolcaNoseR2/). Alluvial plot was drawn using the RAWGraphs tool (https://www.rawgraphs.io/). Correlation and DNA methylation graphs were adapted from GraphPad Prism (version 8.0.2)  scatter dot plots in which each dot indicates a Pearson’s correlation value or median CG probeset DNA methylation levels computed for each tumor type.Heatmap of SLC DEGs and clustering analyses were performed using Heatmapper  compared to the pooled GTEx control group. The analysis identified 331 DEGs in at least 1 tumor type   in different tumor types including LIHC, PCPG, UVM, THCA, LUAD, KICH, and KIRC. Conversely, SLC58A2, SLC26A10, SLCO2B1, SLC1A1, and SLC6A1 genes were strongly downregulated (log2FC ≤ −6) in LAML  . Interes in LAML .N ≥ 15) in which DEGs showed log2FC higher than 2, 4, and 6 or less than −2, −4, and −6 was calculated  were 33 including SLC25A27 (N = 29), SLC17A7 (N = 27), SLC26A10 (N = 26), SLC2A4 (N = 26), SLC42A1 (N = 26), and SLC6A1(N = 24) that showed the highest numbers of tumors  in more than 14 tumors reaching 20 for SLC27A2. Among these, SLC44A4, SLC34A2, and SLC6A14 showed log2FC ≥ 4 in at least 9 tumor types and log2FC ≥ 6 in more than 5 tumors  in PRAD, COAD, READ, PAAD, ESCA, and STAD. In particular, the highest values of log2FC were observed for SLC44A4 (9.53) in PRAD, SLC26A3 (8.30) in READ, and SLC6A14 (7.41) in PAAD  in LGG and GBM  in OV, followed by SLC18A2 and SLC18A1 in PCPG, and SLC34A2 in UCEC. Of note, SLC6A14 was strongly upregulated (log2FC ≥ 6) in 4 gastrointestinal tumors, including COAD, PAAD, READ, and STAD. Conversely, 91 SLCs were significantly downregulated (log2FC ≤ - 6) in more than 50% of tumors (N = 18). Notably, SLC4A1 (THYM and DLBC), SLC36A2 (SARC), SLC57A6, SLC15A1, and SLC5A1 (UVM), and SLC11A1 (THYM) showed the lowest log2FC (≤−10)  . Among t5 tumors . Both th5 tumors .2FC ≥ 1 or ≤ −1 (p ≤ 0.05) were represented in SLC7A11 was negatively associated with OS in 6 tumors . Similarly, SLC65A2 and SLC26A6 were significantly associated with poor prognosis in 3 tumor types including KICH and PRAD for both SLC genes, KIRP for SLC65A2, and ACC for SLC26A6. The highest log2FC was observed for SLC17A4 and SLC30A10, however both genes were negatively correlated with OS in one tumor   and PRAD (log2FC = −2.39). Interestingly, SLC30A2 and SLCO1A2 were strongly associated with favorable OS in ACC and THYM, respectively (2FC ≤ −1) or negatively (log2FC ≥ 1) associated with OS in all tumor types. The analysis revealed that the highest percentage of SCL genes negatively associated with OS was observed for UVM (16%), THYM (14%), ACC (13%), KIRP (12%), and MESO (11%). Conversely, the pro-survival SLC genes were mainly represented in ACC (17%), KIRC (13%), THYM (13%), and PCPG (11%)  . On the ectively . SupplemPG (11%) .SLC7A11, SLCO5A1, and SLC30A10 were the highest significant upregulated DEGs, which are predictive of a worse prognosis. In particular, SLC7A11 was an unfavorable factor in ACC, KICH, and KIRP  in tumors compared to pooled GTEx control group, except for SLC5A1  to C2-C3 signature . The IC50 of 24 anticancer drugs was retrieved along with the expression levels (RPKM) of SLC genes  with the IC50 of 13 drugs. Among these PLX4720, PD0332991, and L685458 compounds correlated with more than 10 SLC genes (N > 2) was observed for SLC43A1, SLC25A42, SLC49A4, SLC10A7, and SLC35A2. Of these only SLC49A4 and SLC35A2 were always positively correlated, while the other ones were negatively correlated , whereas SLC24A5, SLC45A2, SLC43A3, and SLC19A2 negatively correlated with both AZD6244 (MEK inhibitor) and PLX4720 (Raf inhibitor) (SLC24A5 and SLC45A2 were strongly upregulated (log2FC > 8) in SKCM and UVM when compared to both matched normal tissue and pooled GTEx control group, for which the elective pharmacological strategy is based on MAPK inhibitors treatment , while it was downregulated in MESO when performed differential analysis between each tumor type and matched normal tissue (2FC ≤ −2) in 5-year dead PRAD patients  . Remarkareatment . The ErlA14 gene  that wasA14 gene , as wellA14 gene . Moreovel tissue . Interespatients . Collectp ≤ 0.05) in more than half of Pan-Cancer tumor types    with the SLC24A5 expression only in SKCM  showed the highest number of correlation pairs in ≥7 tumor types. Notably, ACC, BLCA, ESCA, SARC, and UCEC showed the highest number of positive correlated CG probesets (N ≥ 18)  . As rega in PRAD . Interes(N = 17) . The cor in SKCM . Moreoveor types . Similar in TGCT . Surpris(N ≥ 18) .SLC4A4 CG probesets of the distal promoter region were hypomethylated  in all Pan-Cancer tumor types (SLC7A11 (from cg00361146 to cg00534274), SLC12A8 , and SLC27A2 (from cg02365220 to cg02726943)  . In addithylated . Interesd in UVM .SLC26A10, the median levels of CG probesets showed a broad distribution of beta values (from 0.01 to 0.96) among the analyzed tumor types, except for some CG probesets of body region (from cg02079831 to cg05157486) that were strongly hypomethylated , whereas BRCA, ESCA, KIRP, and CHOL showed ≥10 promoter CG probesets negatively correlated  with the SLC44A4 expression (SLC44A4 body region (from cg25560247 to cg20383155) and the 3′UTR CG probeset showed a cluster of positive correlated pairs , especially for CESC, COAD, DLBC, ESCA, KIRP, PAAD, and READ (≥10 GC probesets per tumor type)  and the 3′UTR CG probeset that were mainly unmethylated or partially methylated (For the last group of the selected SLCs listed in SLC34A2  and SLC4(SLC44A4  showed apression . Concomior type) . Correlarobesets . Similarr region . Convers20494450 . The metr tumors . Interesor types . A similthylated .The SLCs strictly related to DNA methylation, their biological functions and involvement in main cancer features are summarized in SLC7A11 gene, which was found to be strictly associated with OS and PFI in several tumor types (p ≤ 0.05) with SLC7A11 expression in 17 tumor types  analysis was performed in all TCGA Pan-cancer tumor types stratifying the patients according to the cg06690548 methylation levels of the or types . Of noteor types . The KM or types . In addior types .in silico study was performed to identify the role of all annotated SLC genes in tumorigenesis, survival, tumor immune profile, and drug response in main cancer types. The analyses were conducted using the gene expression data from TCGA Pan-cancer tumor and GTEx normal samples, whereas DNA methylation data were obtained from the TCGA Pan-cancer cohort. In particular, differential analysis was executed to assess the association of each SLC gene with the different clinic-pathological features of cancer types analyzed in this study. The filtering procedure allowed to identify the most relevant SLC genes (N = 25) strictly related to cancer for which DNA methylation analysis was performed. Specifically, the median levels of DNA methylation were evaluated for each CG probeset relative to the selected SLCs in each TCGA Pan-cancer tumor type. In addition, correlation analysis between DNA methylation levels of these CG probesets and gene expression of related SLCs was performed to identify the SLCs strictly modulated by DNA methylation across different cancer types  signaling  scavenger . This gene is a sodium-dependent nicotinamide mononucleotide (NMN) symporter that ensures the Nicotinamide Adenine Dinucleotide (NAD+) cell reservoir essential for energy metabolism  compared to immuno-quiet (C4-C6) signatures in tumor samples, suggesting its involvement into immunity activation. However, further studies should be performed to better clarify the underlying mechanism.d kidney . Our comelopment . Howeverpatients . InteresSLC43A1, also known as LAT3, belongs to a SLC family involved in the transport of neutral AAs,  suggesting its involvement in mTOR leucine-dependent activation , Crizotinib (PF2341066), and γ-secretase inhibitor (L685458). These results do not match the literature, which only reported SLC43A1 cellular susceptibility to paclitaxel in lymphoblastoid B-cell lines  signatures, it could also play a key role in the activation of immunity. Although SLC44A4 belongs to choline transporter-like family, it is associated to the transport and synthesis of acetylcholine, as well as the uptake of thiamine pyrophosphate  and AZD6244 (anti-MEK). As reported by César-Razquin and colleagues, this relationship could be due to the direct influx/efflux mediated by SLCs transporters or indirect effects, including the metabolic reprogramming of cancer cells  activity  suggested that single-nucleotide resolution analysis of DNA methylation status may represent an independent prognostic factor in different tumors. Notably, the methylation status of this CG probeset was analyzed in several diseases, including fatty liver disease, hepatic steatosis, and Parkinson’s disease, however, no evidence was observed in tumors (Among the most significant SLCs, our DNA methylation analysis revealed that promoter hypomethylation of gulation . Previouarcinoma . Howevern tumors . Therefoin silico study provides an overview on the expression of SLC genes in the main tumor types focusing on their involvement in tumor growth, cancer metabolism, drug and immune response. However, it was not possible to achieve a comprehensive analysis of interplay between the most relevant SLCs due to the large number of analyzed SLCs, their multiple functions, as well as the high number of considered tumors. The obtained data suggested that the expression of several SLC genes is fine regulated to satisfy the molecules demand of cancer cells for metabolism and bimolecular synthesis. Since DNA methylation emerges as a key genetic regulation mechanism of these transporters, further studies should be addressed to investigate its regulatory role within tumor-specific context, as well as the SLCs dysregulation in cancer cells. In conclusion, our findings pave the way to a deeper understanding of the SLCs role in tumorigenesis to identify potential cancer-related biomarkers and targets for novel therapeutic strategies, including the selective targeting of SLCs and DNA methylation modulation.Overall, this


S100A16 is a member of the S100 protein family. S100A16 is expressed in a variety of human tissues, although at varying levels. S100A16 expression is especially high in tissues rich in epithelial cells. mRNA and protein levels of S100A16 have been reported to be differentially expressed in the majority of human cancers. Functionally, S100A16 has been linked to several aspects of tumorigenesis, for example, cell proliferation, differentiation, migration, invasion, and epithelial-mesenchymal transition (EMT). Accordingly, S100A16 has been suggested to have both tumour-promoting and suppressive roles in human cancers. S100A16-mediated cellular functions are suggested to be mediated by the regulation of various signaling pathways/proteins including EMT-related proteins E-cadherin and Vimentin, PI3K-AKT, p53, MMP1-1, MMP-2, MMP-9, JNK/p38, etc. In addition to the functional roles, expression of S100A16 has been suggested to have prognostic potential in various cancer types. The aims of this review are to summarise the expression profile, identify common molecular partners and functional roles, and explore the prognostic potential of S100A16 in human cancers. S100A16 gene was originally isolated from an astrocytoma cell line. The S100A16 protein is a small acidic protein comprising 103 amino acids with a molecular weight of 11,801.4 Da and the isoelectric point (pI) of 6.28 [2+ and Zn2+ binding sites of S100A6 protein. S100A16 binds to Zn2+ with a relatively low affinity at a site different from the Ca2+ binding site on the S100A16 protein [2+ binding site in the C-terminal EF-hand, composed of 12 amino acids per protein monomer [2+ binding site at the N-terminal region [S100A16 is one of the members of the S100 family of calcium-binding proteins. The S100 protein family consists of 25 known members [Unlike the majority of the other the S100 proteins, S100A16 has been suggested to undergoe only minor conformational changes after calcium binding. S100A16 mostly occurs as a dimer through interaction between helices I, I’, IV, and IV’, forming an X-type helix bundle in both the calcium bound and unbound form. The EF-hand motif does not move to open conformation upon calcium binding. The results of 3D structural analysis suggested that the largest angle changes due to calcium binding occurred between helices II and III which shifted from 157 ± 5° to 144 ± 4°. This shift was very small compared to the other S100 proteins. In addition, the angle shift between helices III and IV upon calcium binding was from 148 ± 3° to 150 ± 4°. The angles between helices I and I’ and helices IV and IV’ is similar on both calcium bound and unbound forms [The diverse cellular functions of S100A16 could possibly be explained by interactions with other proteins. Relatively few direct interactions have been identified, largely through high-throughput screening . An invehttps://www.proteinatlas.org/ENSG00000188643-S100A16/tissue (accessed on 14 March 2023). Expression of S100A16 has been extensively examined both in normal and malignant tissues ,5,9. S10https://www.proteinatlas.org/ENSG00000188643-S100A16/tissue (accessed on 14 March 2023)). Similar expression patterns can be observed in single-cell transcriptomic data from HPA, where epithelial cells were found to express higher levels of S100A16 mRNA (https://www.proteinatlas.org/ENSG00000188643-S100A16/single+cell+type (accessed on 17 March 2023)). Interestingly, within epithelial compartments, the expression of S100A16 was found to be higher in the supra-basal epithelial layers as compared to the basal layer in oral mucosa [S100A16 mRNA expression in suprabasal keratinocytes as compared to basal keratinocytes (https://www.proteinatlas.org/ENSG00000188643-S100A16/single+cell+type (accessed on 17 March 2023)). Taken together, the heterogeneous expression pattern of S100A16 with respect to the type and compartment of normal human tissues indicates that S100A16 might have a tissue- and context-specific expression and function.Similar to S100A14, another S100 protein member, the expression of S100A16 was also found to be enriched in anatomical sites with epithelial lining as compared to tissues with a mesenchymal-stromal phenotype ). The sections below will provide a comprehensive review of the expression pattern, possible functional roles, and prognostic significance of S100A16 in major human malignancies.These observations indicate a functional link between S100A16 and human malignancies. Indeed, several studies have linked S100A16 to the regulation of various cellular functions related to tumorigenesis, as summarised in S100A16 in LUAD using TCGA data and reported relative hypomethylation of CpG sites in LUAD lesions compared to the control specimens. These data indicate that the methylation status of S100A16 might be important for the increased expression of S100A16 mRNA in LUAD tissues [The expression and functional role of S100A16 are widely studied in different histologic types of lung cancer (LC), predominantly in the non-small cell lung cancer subtypes lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). Saito K et al. reported S100A16 as one of the enriched proteins in LUAD cell lines using proteomic analysis . Several tissues .S100A16 mRNA have been shown to be associated with poor prognosis in non-small cell lung cancer [High expression levels of g cancer ,16,37. Ig cancer .Functionally, S100A16 appears to be important in the metastasis of small-cell lung cancer cells to the brain. Xu et al. showed that the expression of S100A16 was upregulated in small cell lung cancer metastases in the brain as compared to the primary tumour lesions, both in humans and in mice . At the S100A16 mRNA in gastric cancer (GC) tissue as compared to normal gastric tissue [Using publicly available transcriptome datasets such as GEPIA and UALCAN, You and co-authors reported a significantly increased expression of c tissue . In linec tissue . Similarc tissue . Functionally, overexpression of S100A16 was reported to increase proliferation and migration of GC cell lines, while opposite effects were reported with knockdown of S100A16. Similar to the in vitro results, S100A16 was found to promote the growth of tumour xenografts in BALB/C nude mice . At the molecular level, S100A16 expression was found to be inversely correlated with Zona occludens-2  expression in GC specimens and GC cells. Further, S100A16 was found to suppress the expression of ZO-2 through ubiquitylation and degradation, thereby contributing to epithelial-mesenchymal transition (EMT) and the invasion of GC cells . SimilarIn addition to having a key functional role, S100A16 has been suggested to be a prognostic marker in GC ,40. For S100A16 mRNA in human colorectal cancer (CRC) specimens as compared to normal controls. Similar results were also reported by analysing CRC and corresponding normal controls using immunohistochemistry [Analysing a publicly available transcriptome dataset, Ou and coworkers found significant down-regulation of hemistry . Furtherhemistry . In linehemistry . The authemistry . S100A16 mRNA and protein levels in pancreatic cancer specimens. S100A16 mRNA and protein levels were found to be highly expressed in pancreatic adenocarcinoma as compared to normal tissues [Pancreatic adenocarcinoma, the most common form of pancreatic cancer, is a malignancy with a significantly poor patient survival rate. Several studies have investigated the expression of  tissues ,23,24,25 tissues .Functionally, S100A16 overexpression has been shown to promote cell proliferation and induce the EMT phenotype and invasive potential of pancreatic adenocarcinoma cell lines both in vitro and in vivo . These fAnalysis of publicly available transcriptomic datasets has further indicated a possible association between S100A16 and immune infiltrates in the tumour microenvironment. The expression of S100A16 was found to be negatively associated with CD8+T cells ,25. ThesS100A16 mRNA and protein levels in breast cancer (BC) specimens and cell lines as compared to the corresponding normal controls [Zhou and co-workers reported upregulation of controls . At the controls . Higher controls . Functionally, overexpression of S100A16 was found to promote proliferation, colony formation, migration, and invasion capabilities of breast cancer cells as compared to control cells . In lineS100A14 mRNA and protein levels, on the other hand, were found not to be dependent on the expression of S100A16 [Employing a yeast two-hybrid screen, followed by validation with co-immunoprecipitation and reverse co-immunoprecipitation assays, S100A16 was reported to bind with S100A14 in oral squamous cell carcinoma (OSCC) cells. The study found predominantly membranous co-localization of S100A16 and S100A14 in normal oral mucosa and OSCC specimens . Further S100A16 . S100A16 mRNA expression in suprabasal keratinocytes as compared to basal keratinocytes  epithelial cell layers and negative or weak staining in the basal cell layer (stem cell compartment) in normal oral mucosal tissues suggested a possible link between S100A16 and keratinocyte differentiation . Indeed,inocytes C. Furthesurvival . The assIn addition to the possible role in differentiation, S100A16 was further found to suppress the cell proliferation, colony formation, and invasion abilities of OSCC cells in vitro. In parallel, expression of S100A16 was found to suppress tumorigenesis of OSCC cells in a mouse xenograft model, and the resulting tumour xenografts demonstrated features of increased differentiation and reduced proliferation/self-renewal. At the molecular level, the tumour suppressive functions of S10016 might be mediated by down-regulation of proliferation/self-renewal markers (Bmi-1 and Oct4A) and MMP-1 and MMP-9 .S100A16 mRNA and protein levels in PC specimens and cell-lines as compared to the control specimens [S100A16 mRNA levels in PC specimens [Both up- and down-regulated expression of S100A16 has been reported in prostate cancer (PC) specimens as compared to the normal control ,29. Usinpecimens . On the Functionally, employing S100A16 over-expression and silencing strategies, S100A16 was suggested to promote proliferation, migration, and invasion of PC cells. These functions were suggested to be mediated by activation of AKT and ERK signalling pathways and down-regulation of cell cycle inhibitors such as p21 and p27 in PC cells . S100 gene members in urinary bladder cancer (UBC) specimens from humans, rats, and mice as compared to the respective normal controls. The authors identified S100A16 mRNA as being overexpressed in UBC specimens from humans, rats, and mice [S100A16 mRNA expression levels were suggested to be associated with a poor prognosis in UBC patients. Functionally, down-regulation of S100A16 was reported to suppress migration, invasion, and EMT of UBC cells. At the molecular level, knockdown of S100A16 was shown to reduce the expression of N-cadherin, vimentin, and slug, and increase the expression of E-cadherin in UBC cells [Yao R. and co-workers investigated the mRNA expression of several and mice . Using eand mice . High S1BC cells . Wang et al. investigated the role of S100A16 in chemoresistance in UBC cells. The authors identified S100A16 as a significantly up-regulated protein in a Mitomycin-C-resistant UBC cell line as compared to the parent cell line . Knock-dS100A16 mRNA to be significantly up-regulated in renal cell carcinoma (RCC) specimens as compared to noncancerous tissues [S100A16 mRNA in RCC specimens was suggested to be associated with poor overall survival, progression-free interval, and disease-specific survival. GO and KEGG analyses further identified VEGF/VEGFR2, PI3K-Akt, and p53/cell cycle as relevant pathways modulated by S100A16. The authors further demonstrated that knockdown of S100A16 suppressed cell proliferation and invasion abilities in RCC cells. These functional effects were suggested to be associated with inhibition of VEGF, VEGFR2, and pAkt in RCC cells with S100A16 knockdown [Using the TCGA transcriptome dataset, Wang et al. identified  tissues . High exnockdown . S100A16 mRNA has been reported in cervical cancer (CC) types such as cervical squamous cell carcinoma and endocervical adenocarcinoma specimens as compared to the control specimens [Analysing the TCGA transcriptome dataset using the GEPIA tool, significant up-regulation of pecimens . Tomiyampecimens . In the same line, Zhang et al. reported that S100A16 promoted cell proliferation and invasion properties and the EMT phenotype of the HeLa cell line (CC cell line), probably through the activation of PI3K/Akt signalling pathways . S100A16 mRNA in ovarian cancer (OC) as compared to the corresponding control tissues [S100A16 mRNA expression appeared to predict poor patient prognosis in grade II, stage II, and OC patients with TP53 mutations [Xu et al., analysing external transcriptome datasets, reported upregulation of  tissues . High S1utations . Bai Y eutations .A heterogeneous expression of S100A16 was found in different normal human tissues. Expression of S100A16 was altered in several human malignancies, indicating a possible role for S100A16 in cancer. With the exception of OSCC and CRC, S100A16 was overexpressed in several malignancies, leading to increased proliferation, invasion, and metastasis through a variety of molecular pathways, including PI3K-Akt, MAPK-ERK, JNK/p38, and EMT-signalling. The differential expression pattern of S100A16 and its involvement in key signalling pathways regulating various cellular functions in human cancers suggest that S100A16 might be a promising prognostic marker and a therapeutic target in human cancers.


KD26_E2LOM was obtained by transfecting the cDNA pACKD26_E2LOM into PK-15 cells. KD26_E2LOM chimeric pestivirus proliferated to titers of 106.5 TCID50/mL and 108.0 TCID50/mL at 96 h post-inoculation into PK-15 cells or MDBK cells, respectively. It also reacted with antibodies specific for CSFV E2 and BVDV Erns, but not with an anti-BVDV E2 antibody. Piglets (55–60 days old) inoculated with a high dose (107.0 TCID50/mL) of KD26_E2LOM produced high levels of CSFV E2 antibodies. In addition, no co-habiting pigs were infected with KD26_E2LOM; however, some inoculated pigs excreted the virus, and the virus was detected in some organs. When pregnant sows were inoculated during the first trimester (55–60 days) with a high dose (107.0 TCID50/mL) of KD26_E2LOM, anti-CSFV E2 antibodies were produced at high levels; chimeric pestivirus was detected in one fetus and in the ileum of one sow. When 5-day-old calves that did not consume colostrum received a high dose (107.0 TCID50/mL) of KD26_E2LOM, one calf secreted the virus in both feces and nasal fluid on Day 2. A high dose of KD26_E2LOM does not induce specific clinical signs in most animals, does not spread from animal to animal, and generates CSFV E2 antibodies with DVIA functions. Therefore, chimeric pestivirus KD26_E2LOM is a potential CSFV live marker vaccine.A chimeric  New pestiviruses include Pestivirus antilocaprae, Pestivirus australiaense, Pestivirus aydinense, Pestivirus brazilense, Pestivirus ratti, Pestivirus scrofae, Pestivirus L, Pestivirus M, Pestivirus N, Pestivirus O, Pestivirus P, Pestivirus Q, Pestivirus R, and Pestivirus S. These changes in pestivirus taxonomy have been approved and ratified by the International Committee on Taxonomy of Viruses (ICTV) in March 2022 .Bovine viral diarrhea virus  that is translated into 12 viral polypeptides; the genome is flanked by untranslated regions (UTRs) at the 5′- and 3′-ends ) of pregnant sows was recorded, and qRT-PCR was used to confirm the presence of KD26_E2LOM in the fetuses.Three sows in the middle stages of pregnancy (55–60 days) were inoculated with KD26_E2LOM  with BVDV type 1 , and Group 3  was used as a negative control. Blood, nasal, and fecal samples were collected before (−4 and 0 days) and after  inoculation with KD26-E2LOM or BVD26. Samples were also collected from calves in control Group 3.Nine calves (5 days old) that did not consume colostrum were divided into three groups prior to the safety experiments. Group 1  was inoculated with 10® CSFV AB ELISA kit, which is an E2 protein-based ELISA . The test serum samples were validated by comparing their OD values with those of the control . Results were interpreted according to the sample to positive ratio (S/P ratio), calculated as follows: /. Test samples with an S/P ratio ≥ 0.14 are considered positive for CSFV E2-specific antibodies, and those with an S/P ratio < 0.14 are considered negative. ELISAs measuring antibodies specific for BVDV E2 in serum of pigs were performed using VDPro® BVDV AB ELISA  kits, respectively. The BVDV AB ELISA is a blocking ELISA designed to detect neutralizing BVDV E2 antibodies. Test samples were validated by comparison with the control OD values . Interpretation involves calculating the S/P ratio as above. Samples with a value ≤ 0.7 were BVDV E2 positive and those with a value > 0.7 were negative.Serological differential diagnosis in animals inoculated with the KD26_E2LOM vaccine strain is possible using the DIVA function, which determines CSFV E2 antibodies as positive and BVDV E2 antibodies as negative. Additionally, in the case of BVDV infection, the results were determined as BVDV E2 positive and CSFV E2 negative, and in the case of CSFV infection, the results were the opposite of those of BVDV infection. Serological ELISA analysis to determine specific antibody levels in serum was performed using commercially available kits. Levels of anti-CSFV E2 antibodies in pig serum were measured using the VDPro® CSFV qRT-PCR kit  designed to detect the 5′-UTR of CSFV [Viral RNA was extracted from blood, organs, and swab samples using the RNeasy Mini kit  and used for quantitative real-time PCR (qRT-PCR) to measure the CSFV RNA copy number. The qRT-PCR analysis was performed using a VDx of CSFV . The PCR50 of the LOM virus for 1 h at 37 °C. The neutralized serum containing virus was inoculated into PK-15 cells (porcine kidney cells) and cultured for 3 days in a 37 °C incubator. The cells were then fixed in chilled 80% acetone and reacted with a commercial anti-LOM mAb . The cells were then stained using a VECTASTAIN® ABC-HRP kit  and an ImmPACT DAB Peroxidase (HRP) substrate . Neutralizing antibody titers were expressed as the reciprocal of the highest dilution that yielded 50% neutralization and determined to ≥10-fold positive antibodies according to the WOAH criteria.Anti-CSF-neutralizing antibodies were detected by a neutralizing peroxidase-linked assay (NPLA) according to the standards manual of the World Organization for Animal Health (WOAH) . Heat-int-test, and a p-value < 0.05 was considered significant.All statistical analyses were performed using GraphPad Prism 6. Statistical significance was evaluated by Student’s SrfI recognition site was inserted at the end of the 3′-UTR to facilitate plasmid linearization  at either end of the E2 region using PCR  and purified using an Exprep™ plasmid SV mini kit . The sequence was confirmed by an ABI Prism 3730xl DNA sequencer . Subsequently, the cDNA construct was linearized by digestion with SrfI and in vitro transcribed to viral RNA using a MEGAscript™ T7 transcription kit . The resulting in vitro-synthesized RNA was transfected into porcine kidney 15 (PK-15) cells and cultured for 3 days. The recovered KD26_E2LOM virus was selected by detecting positive cells by immunofluorescent LOM E2-specific mAbs. The virus was then serially passaged in PK-15 cells and Madin–Darby bovine kidney (MDBK) cells.The LOM-E2 fragment, including a portion of E1 (40 bp at the end of LOM-E1), was amplified by PCR using LOM virus cDNA as a template. To replace the LOM-E2 encoding region with that of KD26, the LOM-E2 fragment was modified by inserting appropriate restriction sites ; however, only four genes harbored amino acid changes in Nand NS4B .6.7 TCID50/mL) at the 11th passage (7.9 TCID50/mL) at 96 h, but the LOM strain yielded only low (105.7 TCID50/mL) titers at this time (7.9 TCID50/mL) at 96 h when inoculated into PK-15 cells, but the LOM strain showed a low titer (105.7 TCID50/mL) (8.0 TCID50/mL) at 96 h  B. Howeve at 96 h C.2), with the highest antibody (8.0 log2) titer maintained between Days 28 and 42  on Day 14 and 0.76 (log2) on Day 21, respectively  of KD26_E2LOM, virus shedding was confirmed in one animal  at 7 dpi, and the virus was detected in blood samples taken on 1, 4, and 7 dpi (7.0 TCID50/mL) during the early stages (55–60 days) of pregnancy, we did not detect KD26_E2LOM RNA in the nasal or blood samples (After inoculation of growing pigs with a high dose (10nd 7 dpi . We attend 7 dpi . However samples .10) and spleen (4.5 log10), and the KD26_E2LOM virus from these two organs was isolated successfully in PK-15 cells  from sow A-9  of calves . These fPestivirus Vaccines and Recombinant Deletion or Mutation DIVA Vaccines [pestivirus have been conducted, and two vaccines have been licensed to date. The first is a chimeric pestivirus named “CP7_E2alf” , which was licensed as a DIVA vaccine in Europe in 2014. The second is Flc-LOM-BErns, designated as a chimeric pestivirus and licensed in Korea in 2017. The chimeric pestivirus “CP7_E2alf” DIVA Vaccine is an infectious cDNA clone of the cytopathic BVDV strain “CP7”, in which the E2 gene was replaced by that of the CSFV variant “Alfort/187” [pestivirus vaccine, licensed as Flc-LOM-BErns, is based on the attenuated CSFV vaccine strain LOM [rns vaccine protected fetuses from transplacental infection with CSFV strain YC11WB during gestation . However, since this chimera vaccine was first used in Korea only in 2020, it is argued that it is difficult to evaluate its safety, or the robustness of the DIVA function, because there are insufficient data.Since the early 1990s, researchers have tried to design new vaccines that can easily and accurately distinguish vaccinated individuals from those infected with CSF . One of Vaccines . HoweverVaccines ,29,30,31ort/187” ,33. The ort/187” . Anotherrain LOM . This chrns-specific mAb, which demonstrated DIVA function; however, it did not react with a BVDV E2-specific mAb. In addition, the proliferative capacity of the KD26_E2LOM virus differed according to cell type. MDBK cells contained the most KD26-E2LOM strain (average 108.0 TCID50/mL) at 96 h. A pestivirus chimeric study revealed amino acid mutations in rC/bUTRs-tE2 after serial passage in PK15 cells: M834K and M979K in the E2 gene at passage 30 [4.5±0.3 TCID50/mL) and rC/bUTRs-tE2 the lowest (103.1±0.2 TCID50/mL) titers [6.0 TCID50/mL) to the Alfort 187 virus in PK15 cells [The KD26_E2LOM virus rescued in this study harbored mutations in four amino acid positions  when compared with the original KD26 strain and the E2 gene of CSFV (LOM strain). The rescued KD26_E2LOM virus was detected specifically by an anti-CSFV E2-specific mAb and a BVDV Essage 30 . A compa) titers . Another) titers . After t) titers . However15 cells .rns sequences from Norwegian mouse and Pronghorn pestiviruses, or combinations of both [5.3 TCID50/mL), whereas “Ra” chimeras replicate less well (104.4 TCID50/mL) [Chimeric viruses “Ra”, “Pro”, and “RaPro” contain E of both . In pig ID50/mL) . ConfirmID50/mL) .Here, we found that inoculating the target animal  with KD26_E2LOM generated anti-CSF-E2 antibodies and CSF-neutralizing antibodies, but not anti-BVDV E2 antibodies, which is suggestive of DIVA function. Pigs inoculated with KD26_E2LOM generated anti-CSF E2 antibodies from Day 10, with the highest level maintained between Day 28 and Day 42. In addition, the ELISA S/P ratio for CSFV E2-antibodies in pregnant sows inoculated with KD26_E2LOM was positive from Day 14. After overdose-inoculation of KD26_E2LOM into growing pigs, we detected some viral shedding, and we isolated KD26_E2LOM from PK-15 cells. However, KD26_E2LOM RNA was not detected in the nasal cavity of sows inoculated with KD26_E2LOM during early pregnancy (Days 55–60). At 28 dpi, KD26_E2LOM RNA was detected in various organs harvested from five of seven pigs inoculated with KD26_E2LOM. The KD26_E2LOM virus was isolated from PK-15 cells cultured with positive samples.rns was replaced with a heterologous Erns from Bungowannah , and was attenuated by a deletion within the type I interferon inhibitor Npro protein-encoding sequence [pro_Erns Bungo_E1E2BVDV-2 CS vaccine, which is based on the BVDV-1b_synCP7_DNpro_Erns Bungo vaccine, harbors the gene sequences of glycoproteins E1 and E2 from BVDV-2 strain CS8644 (CS) [6 TCID50/mL, were safe for inoculation into cattle, and showed serological DIVA function [rns sequences from Norwegian mouse and Pronghorn pestiviruses, or a combination of the two [rns antibody ELISA [rns antigens of distantly related pestivirus could act as a strong serological negative marker in chimeric pestivirus [rns of GPE-vaccine strains to Erns of non-CSF pestiviruses, Pronghorn pestiviruses (PAPeV or Pocoena pestiviruses) [rns and GPE-/PhoPeV Erns) [rns or vGPE-/PhoPeV Erns at a dose of 104.0 TCID50 resulted in immunogenicity and safety profiles comparable with those of vGPE-vaccine strains [A previous study developed an efficient marker, or DIVA, vaccine to help eradicate success in regions with a high prevalence of BVDV to prevent fetuses . In the sequence . The BVD644 (CS) . Both vafunction . A recen the two . All vacdy ELISA . For a nstivirus . In anotviruses) . High vieV Erns) . In vivo strains .In the present study, among the three pregnant sows inoculated with the KD26_E2LOM strain, one was pregnant with five fetuses, which was lower than that of the other pregnant sows. However, no mummified fetuses were found in the pregnant sow with five fetuses, and the KD26_E2LOM strain was not detected in any of these five fetuses. Therefore, it seems likely that the reduced fetus count in this pregnant sow was due to low fertility that was not caused by inoculation with the KD26_E2LOM strain.Virus from the caecum of pregnant sows inoculated with KD26_E2LOM was isolated from the PK-15 cell cultures, and the virus was also isolated from one fetus. This suggests a partial vertical infection by the KD26_E2LOM strain. We confirmed a partial placental passage of the KD2_E2LOM strain in a small-scale experiment performed within 3 weeks of mid-gestation. However, during the second trimester of pregnancy, if the fetus had been infected with the KD26_E2LOM strain for more than 3–4 weeks, the possibility of antibody formation would be expected to be higher than that of the formation of the KD26_E2LOM antigen. Although it is difficult to draw a clear conclusion due to the small number of experiments in the second trimester, we recognize that the developed KD2_E2LOM strain partially passed into the placenta. However, to address this issue fully, future experiments will need to be conducted with a larger number of pregnant sows in the early, middle, and late stages of pregnancy. When 5-day-old calves were inoculated with KD26_E2LOM, antigens were detected in the blood and feces, and the viruses were isolated from the fecal samples. This suggests the possibility of a horizontal infection when cattle are inoculated with the KD26_E2LOM strain. However, when high-titer KD26_E2LOM vaccine was inoculated into pigs and cattle, clinical symptoms similar to those in animals infected with CSFV or BVDV were not observed. Nevertheless, the safety of the KD26_E2LOM strain needs to be clearly confirmed through additional animal testing.The KD26_E2LOM virus, which was created by inserting the CSFV LOM E2 gene into the BVDV backbone, is a DIVA vaccine capable of distinguishing between CSF E2 and BVD E2 antibodies. Pigs generated anti-CSF E2 antibodies from 10 dpi, with high antibody titers detected between 28 and 42 dpi. However, when inoculated with high doses, all calves and growing pigs shed the virus, which was isolated from positive samples using PK-15 cells. The KD26_E2LOM virus also suggested vertical infection from one pregnant sow to the fetus. Therefore, developed KD26_E2LOM shows DIVA function, but additional studies are required to assess its safety and ability to prevent infection by virulent CSFV.