Prevotella sp. (56.50%), it is speculated to originate from an early linker between XynA and another domain. Besides, structure simulation showed that C60 enhances the molecular interactions between substrate and active residues to improve catalytic efficiency. Moreover, three truncated variants with different lengths of C-terminal regions were successfully generated in Escherichia coli. The specific activities of variants were 6.44–10.24 fold of that of XynA-Tr, and their optimal temperature and pH were the same as XynA-Tr. Three truncated variants released more xylooligosaccharides, especially xylobiose , than XynA-Tr (32.43%). These results are helpful to understand the molecular mechanism of C60, and also provide new insight to improve the yields of short xylooligosaccharides by molecular modification at the terminal of xylanases.Increasing the yields of short xylooligosaccharides by enzymatic production is efficient to improve prebiotic effects. Previously, C-terminal oligopeptide C60 was found to accelerate short xylooligosaccharides. Herein, in order to further understand the molecular mechanism of C60, the sequence analysis firstly showed that C60 displays typical properties of a linker . C60 shared the highest identity with the N-terminal region of esterase (98.33%) and high identity with the linker between xylanase and esterase from  Bifidobacteria and Lactobacillus spp., thus significantly exhibiting a variety of health-benefiting effects such as promoting calcium absorption, lowering cholesterol, and reducing symptoms of diseases like diabetes, atherosclerosis, and colon cancer [Prebiotic xylooligosaccharides (XOS) are sugar oligomers consisting of 2–7 xylosyl residues linked to each other by β-1,4 glycosidic bonds [n cancer .In view of the rising demand for XOS around the world, XOS are mainly produced from xylan in lignocellulosic biomass, since they exist in small amounts in fruits, vegetables, and dairy . CompareIt has been shown that XOS with a low degree of polymerization such as xylobiose (X2) have enhanced prebiotic effects conferring health benefits to humans and animals . Thus, iE. coli DH5α and BL21 (DE3) were cultured in Luria-Bertani (LB) medium  at 37 °C. Phusion DNA polymerase, restriction endonuclease, T4 DNA ligase, and alkaline phosphatase were purchased from ThermoFisher Scientific . A plasmid extraction kit and DNA purification kit were bought from Solarbio . Xylan from beechwood used as xylanase substrate, xylooligosaccharide standards including xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) as well as xylose (X1) were all obtained from Sigma . All other analytical grade chemicals were commercially available.The recombinant strains pET-XynA and pET-XynA-Tr were constructed previously and used as the template in this study . The exphttps://blast.ncbi.nlm.nih.gov/Blast.cgi, 1 February 2022). Multiple-protein sequence alignments were performed by ClustalW . The amino acid contents of different sequences were calculated using Protein Stats . The three-dimensional structural model of xylanases and C60 were predicted using SWISS-MODEL  and the protein-structure prediction algorithm AlphaFold2 . The protein structure and the molecular interaction were visualized by PyMOL software . The active-site networks between XynA and X5 or X6 are viewed after superimposition of XynA with XOS from the xylanase complex structures (PDB code: 1r87 and 4pmd).The identities of XynA or C60 with other homologous sequences were analyzed by BLASTP programs  vector and transformed into E. coli BL21 (DE3). The positive transformants harboring pET-XynA-Tr-C15, pET-XynA-Tr-C30, and pET-XynA-Tr-C45 were confirmed by DNA sequencing .To investigate the effects of length of the C-terminal region on catalytic activity, three variants with different C-terminal peptides named XynA-Tr-C15, XynA-Tr-C30, and XynA-Tr-C45 were generated using the corresponding primer pairs, respectively . After bAfter induction by IPTG (1 mM) at 25 °C for 16 h, the recombinant proteins were eluted with 10 to 500 mM imidazole buffer  using nickel-affinity chromatography . The collected recombinant proteins were further concentrated by ultrafiltration and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). A protein assay kit was used to determine the protein concentration .w/v) and 50 μL protein sample (2 μM) were mixed in each reaction system. A 3,5-dinitrosalicylic acid (DNS) assay was applied to determine xylanase activity by measuring the release of reducing sugar . A totalTo evaluate the effects of different C-terminal peptides on catalytic activities, the optimum temperature of all variants was determined at different temperatures of 30–60 °C at pH 6.0, and the optimal pH was measured using 0.2 M citric acid-Na2HPO4 buffer (pH 4.0–8.0) at the optimum temperature. A total of 1 unit (U) is the amount of xylanase needed to release 1 μmol of reducing sugar per minute at optimal reaction conditions (pH 6.0 and 45 °C). The increased folds of catalytic activity of the variants were calculated using the specific activity of XynA-Tr as control.®® centrifugal 3K devices . The released products of XynA, XynA-Tr, XynA-Tr-C15, XynA-Tr-C30, and XynA-Tr-C45 were detected by high-performance anion-exchange chromatography (HPAEC) eluted by buffer . A Dionex CarboPac PA100 (4 by 250 mm) column  and pulsed amperometric detection (PAD) were used. To identify hydrolysis products, xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) were used as controls.Each reaction system contained 900 μL of 0.5% (wt/vol) beechwood xylan and 100 μL of each enzyme sample  and was incubated at optimal conditions. After 12 h, the residual enzymes were removed from the reaction system using NanosepIn this study, the experiments were repeated three times, and the data were plotted using the SD (mean ± standard deviation) method using Graphpad Prism 7 software . Significance analysis was performed by t-test at a significance level of 0.05% using Prism 7.Our previous study reported that XynA, a predominant xylanase in a rumen competitive environment, contains a catalytic domain XynA-Tr and a C-terminal oligopeptide C60. C60 improved the hydrolysis efficiency of the catalytic domain by increasing the yields of xylobiose and xylose . HomologPrevotella sp. (GenBank accession number MBQ5981334), whereas the homologous sequences of C60 were not found in alignments by BLASTP analysis. Thereby, the 60 residues of C60 were aligned individually, and the results suggested that it shows the highest identity (98.33%) with the N-terminal region of an esterase from Prevotella sp. (GenBank accession No. MBQ6229348), which is annotated based on DNA sequencing from the ruminant gastrointestinal microbiome. It also shared a high identity (56.50%) with the linker region between xylanase and esterase from Prevotella sp. Besides, it is worth noting that the majority of xylanases sharing high identity with XynA-Tr were followed by another domain of enzymes like acetylxylan esterase , Bacteroides intestinalis (WP_021967563), and Bacteroides cellulosilyticus (WP_022209201). Acetylxylan esterase (EC 3.1.1.72) is an important enzyme for the hydrolysis of side chains in xylan degradation [Prevotella sp. , Bacteroides intestinalis (WP_021967563), and Bacteroides cellulosilyticus . The sequence length of these linkers in sequence alignment varies from 30 to 66 residues, which are similar to that of C60. Among these linkers, BLASTP analysis found that the amino acid sequence of C60 shares the highest identity (56.50%) with the linker sequence from Prevotella sp. (MBQ8453537).In this study, the key amino acid residues affecting C60 function were further investigated. Firstly, using an intact amino acid sequence of XynA as the query sequence, XynA-Tr exhibited above 80% sequence identity with endo-1,4-beta-xylanase from esterase , such aslis WP_0267563, anus WP_0229201. AceGeobacillus stearothermophilus and xylanase-X6 structure (PDB code: 4pmd) from Caldicellulosiruptor bescii, respectively. For X5, 21 hydrogen bonds were formed in the active-site networks of XynA   B, respecGeobacillus thermodenitrificans C5 [Utilizing protein engineering is an effective strategy for improving enzyme characteristics such as specific activity and stability. Generally, functional regions like the catalytic site are usually recognized as the typical target regions to modify wild-type enzymes. In addition to the catalytic domain, it has been well documented that N- and C-terminal regions are also crucial factors for enzyme properties . For exaicans C5 , the chaicans C5 , and theicans C5 . E. coli. After induction by IPTG for 12 h, the crude proteins of positive transformants showed xylanase activities. All purified recombinants of XynA-Tr-C15, XynA-Tr-C30, and XynA-Tr-C45 showed a single band of 39.09, 40.66, and 42.45 kDa by protein SDS-PAGE analysis  of variants XynA-Tr-C15, XynA-Tr-C30, and XynA-Tr-C45 were obviously enhanced. In particular, the ratios of producing X2 significantly increased to 46.33, 43.41, 49.60, and 45.82% from 32.43% (XynA-Tr), respectively. These products have a similar constitution to C15, C30, C45, and C60, clearly suggesting the first 15 residues are crucial to promoting xylan degradation, which is in accordance with the improvement of a specific activity. It is well known that XOS with a lower DP shows a higher prebiotic potential, and X2 has been reported to have the strongest prebiotic effects among XOS , and X4 . Thus, iMoreover, C-terminal regions were reported to be involved in the enzyme–substrate reaction, like the C-terminus segment of phospholipase A2 changes the substrate specificity and substrate binding . Since tIn the present study, it was found that the C-terminal region C60 exhibits typical characteristics of the linker region, improves the catalytic efficiency, and releases more XOS through increasing the molecular interaction between substrate and active residues. The critical residues of C60 were also identified by mutation analysis. This study provides new insight into the molecular mechanism of C60 and offers an efficient strategy to improve XOS production by molecular modification.


Extracellular vesicles (EVs) are cell-released, nanometer-scaled, membrane-bound materials and contain diverse contents including proteins, small peptides, and nucleic acids. Once released, EVs can alter the microenvironment and regulate a myriad of cellular physiology components, including cell–cell communication, proliferation, differentiation, and immune responses against viral infection. Among the cargoes in the vesicles, small non-coding micro-RNAs (miRNAs) have received attention in that they can regulate the expression of a variety of human genes as well as external viral genes via binding to the complementary mRNAs. In this study, we tested the potential of EVs as therapeutic agents for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. First, we found that the mesenchymal stem-cell-derived EVs (MSC-EVs) enabled the rescue of the cytopathic effect of SARS-CoV-2 virus and the suppression of proinflammatory responses in the infected cells by inhibiting the viral replication. We found that these anti-viral responses were mediated by 17 miRNAs matching the rarely mutated, conserved 3′-untranslated regions (UTR) of the viral genome. The top five miRNAs highly expressed in the MSC-EVs, miR-92a-3p, miR-26a-5p, miR-23a-3p, miR-103a-3p, and miR-181a-5p, were tested. They were bound to the complemented sequence which led to the recovery of the cytopathic effects. These findings suggest that the MSC-EVs are a potential candidate for multiple variants of anti-SARS-CoV-2. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the positive sense single-stranded RNA virus that causes coronavirus 2019 (COVID-19). SARS-CoV-2 is a variant of the coronavirus SARS-CoV, which is associated with severe acute respiratory syndrome. SARS-CoV-2 is the seventh coronavirus known to infect humans; the others are 229E, NL63, OC43, HKU1, MERS-CoV, and the original SARS-CoV [The RNA genome has a high rate of mutation, making RNA viruses very heterogeneous . As in HMicroRNAs (miRNAs) are small non-coding RNAs of 18–25 nucleotides (nts); these small molecules are important regulators of gene expression because they suppress messenger (m)RNAs. miRNAs regulate approximately 30–70% of human gene expression by matching their “seed” sequence with complementary mRNA . More ofThe regulatory or regenerative functions of miRNAs contained in stem cell-derived EVs have attracted attention as a novel therapeutic approach . EVs secHere, we identified candidate therapeutic miRNAs with important roles in the biological functions of virus-infected host cells, and characterized the antiviral effects of miRNAs derived from placenta-derived MSC-EVs (pMSC-EVs) or placenta EVs, which exhibit potent regenerative and anti-inflammatory effects. Five selected miRNAs  blocked SARS-CoV-2 RNA replication and suppressed virus-mediated pro-inflammatory responses by human bronchial epithelial cells and lung fibroblasts, all of which express ACE2 receptors. These effects were also observed in human brain cells and brain immune cells. Interestingly, we found that ACE2 is expressed in human fetal brain stem cells, suggesting that the brain could be a target of SARS-CoV-2.Most importantly, we found that the five miRNAs bound to the 3′ UTR of SARS-CoV-2, the sequence of which is conserved among coronaviruses.Neuronal stem cells (NSCs) were generated from the central nervous system tissue of spontaneously aborted fetus from ectopic pregnancy during gestational week (GW) 8. Isolation method is described in Moon et al. . Normal 2, and 3% O2 in DMEM/F12 (Gibco) with B27 supplement (Gibco), 50 µg/mL gentamicin, human bFGF, human EGF 20 ng/mL , tocopherol, and tocopherol acetate . SK-N-BE(2)C cells were cultured in DMEM with 10% FBS, 1X P/S. The cells were pre-treated with EVs for 24 h and then stimulated with LPS  for either 6 or 1 h.Human lung fibroblast cell line LL24, human bronchial epithelial cell line Beas-2B, mouse microglial cell line BV2, and human neuroblastoma cell line SK-N-BE(2)C were obtained from ATCC . LL24 cells were grown in RPMI-1640 medium  with 10% fetal bovine serum  and 1× penicillin/streptomycin . Beas-2B cells were cultured in 1× defined keratinocyte serum-free media  with 5 µg/mL gentamycin (Gibco). BV2 cells were cultured in Dulbecco’s modified Eagle’s medium  with 10% FBS and 1% P/S. Neuronal stem cells (NSCs) were cultured at 37 °C/5% COg for 1 min in an Ultrafree 0.22 μm centrifugal filter device .EVs were isolated by size-exclusion chromatography (SEC) columns . Samples were overlaid on the qEV size exclusion column, followed by elution with particle-free phosphate-buffered saline (PBS). The flow was collected in 500 µL fractions, and EV-rich fractions 7 and 10 were combined for analysis. The combined fractions were centrifuged at 1000× For NTA analysis, EVs were diluted in PBS and examined under a ZetaView Nanoparticle Tracking Video Microscope . For each measurement, three cycles were performed by scanning 11 cell positions and capturing 60 frames per position. The following settings were used: focus, autofocus; camera sensitivity for all samples, 80.0; shutter, 100; scattering intensity, 1.2; cell temperature, 23 °C. After capture, the videos were analyzed using the built-in ZetaView Software 8.02.31  with the following parameters: maximum particle size, 1000; minimum particle size, 5.For fluorescence imaging, EVs were biotinylated using EZ-Link Sulfo-NHS-LC-Biotin . Biotinylated EVs were loaded onto Zeba spin desalting columns  to remove the remaining free biotin. Additionally, then, 20 μL of biotinylated EVs were placed in a streptavidin-coated glass slide . After 30 min, EVs were fixed with BD Fix Perm  and blocked with 0.2% BSA. Immunofluorescence staining was performed using anti-human CD63  for 2 h at room temperature (RT), followed by incubation with Alexa Fluor 488-conjugated secondary antibodies  for 1 h at RT. Additionally, then, these slides were mounted and analyzed by confocal fluorescence microscopy .For PKH26 staining, EVs were labeled with the PKH26 Red Fluorescent Cell Linker Kit for General Cell Membranes (Sigma-Aldrich).For TEM imaging, 5 µL aliquots of diluted sample containing 0.5 µg protein were dropped onto hydrophilic grids. After a few minutes, the grids were washed with distilled water and contrasted for 20 s with 2% uranyl acetate. Images were acquired on a JEM-1010 microscope  operating at 80 kV.EVs and cells were lysed in 10× RIPA buffer containing a protease inhibitor cocktail tablet  and phosphatase inhibitors II and III (Sigma-Aldrich). An aliquot of each sample was subjected to 10% SDS-PAGE, followed by Western blotting. Blots were blocked for 1 h in 10% skim milk. Antibodies specific to the following proteins were obtained from the indicated suppliers: CD81 , CD9 , CD63 , TSG101 , Vinculin , p65 , phospho-p65 , and GAPDH . Primary antibodies were incubated with blots overnight at 4 °C, followed by incubation with anti-rabbit or anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies  for 1 h. Immunoreactivity was detected using enhanced chemiluminescent HRP substrate .Cells were fixed with 4% paraformaldehyde (PFA) for 30 min. Cells were washed with PBS and blocked with 1% BSA for an additional 20 min at RT. Cells were then incubated with anti-NF-κB p65 subunit  for 24 h at 4 °C. Following washing with PBS to remove excess primary antibody, the cells were further incubated with Alexa Fluor 488-conjugated goat anti-mouse IgG  for 1 h at RT, and stained with Gold Antifade reagent containing 40,6-diamidino-2-phenylindole  for 5 min. Subsequently, the slides were coverslipped and visualized using a fluorescence microscope .Total RNA for qPCR was extracted from cells and EVs using the TRIzol reagent (Invitrogen). Next, cDNA was synthesized from 1 μg of total RNA using a cDNA kit . For qPCR analysis, reaction mixtures contained 0.5 μM primer mixture, SYBR-Green with low ROX , nuclease-free water , and cDNA. Reactions were performed on a StepOne Real-Time PCR instrument . Quantification of gene expression was based on the CT value for each sample.50). All experiments using the virus were performed in a Biosafety Level 3 (BSL-3) laboratory located in College of Medicine and Medical Research Institute, Chungbuk National University  and the College of Environmental and Bioresources, Jeonbuk National University . Cytotoxicity values of the EVs on Vero cells were determined using the methyl thiazolyl tetrazolium (MTT) assay.The African green monkey kidney epithelial cell line (Vero) was used as an appropriate cell line for SARS-CoV-2 propagation. The cells were cultured in DMEM (Gibco) with 10% FBS (Gibco). The antiviral activity assay was carried out using the NMC-nCoV02 strain of SARS-CoV-2. The virus was propagated in Vero cells and the titer of propagated viral stock was expressed as the tissue culture infective dose 50 . After 3 days, cells were fixed by 10% formaldehyde solution for 5 min and stained by 1% crystal violet. The wells were inspected for the presence of virus, as judged by the appearance of cytopathic effect (CPE). The virus endpoint titration (dilution required to infect 50% of the wells) was expressed as TCID50/mL. CPE (%) was calculated, along with the virus titer .For TCID1–102 TCID50 of SARS-CoV-2 and different concentrations  of EVs were added to each well. For the virus control, 101–103 TCID50 of virus plus the highest concentration of DMSO were added to six wells. Additionally, six wells were treated with DMSO alone (negative control). The plates were incubated and CPE monitored for up to 3 days post infection.The cytotoxicity inhibition assay was used to assess the antiviral activity of each EV. Briefly, Vero cells were grown in a 96-well plate until 80% confluent. Subsequently, the culture medium was removed from each well and 10Additionally, the immunofluorescence assay was used to assess the antiviral activity of EVs. Vero cells were cultured and confluent cells were infected with SARS-CoV-2 at multiplicity of infection (m.o.i) of 0.01. After 1 h absorption, virus inoculum was removed and fresh medium containing EVs  was added into the chamber slide. Sixteen hours later, cell culture medium was removed, and cells were used for immunofluorescence assay.Vero cells grown on 8-well chamber slides were fixed overnight at 4 °C with acetone and methanol mixture. Cells were washed with PBS, followed by blocking and permeabilization with 0.1% Triton-X 100 (Sigma-Aldrich) and 5% normal goat serum  in PBS for 1 h at RT. After washing with 0.1% Triton-X 100, cells were incubated with anti-SARS-CoV-2 nucleocapsid antibody  and Alexa 488-conjugated goat anti-mouse IgG . Nuclei were stained with DAPI (Thermo Fisher Scientific). Stained cells were mounted with anti-fade mounting solution  and observed by fluorescence microscopy A 208 bp fragment of the 3′ UTR of the SARS-CoV-2 genome was synthesized by Bionics. The 3′ UTR fragment was digested with Xba I and inserted downstream of the firefly luciferase gene in pGL3-control , yielding pGL3 SARS-CoV-2 3′ UTR-Luc. All constructs were verified by sequencing. The mutant 3′ UTR of the SARS-CoV-2 genome was generated by randomly designed sequence of the miRNA binding site and then inserted in pGL3 vector to make a pGL3 SARS-CoV-2 MUT-3′ UTR-Luc plasmid. The mutated sequence was as follows: 5′- TCTAGACAATCTTTAATCAGTGTGTAACATTAGGGAGGGTAGCTAAGAGCCACCACATTTTCACCGAGGCCACGCGGTTGCTACGTCCTAAGACAGTGAACAATGCTCACATTTTCACCGAGGCCACGCGGTTGCTACGTCCTAAGACAGTGAACAATGCTAGGGTATTGACGCTATATGGAAGAGCCCTAATGTGTAAAATTAATTTTAGTCCGAGCTACCTGCCTATTTTTTAATAGCTTCTTAGGAGAATGACTCTAGA-3′.5 cells/well) with antibiotic-free DMEM 1 day prior to transfection. Transfections were carried out with Lipofectamine 2000 (Invitrogen). To test particular miRNAs’ mediated suppression of luciferase activity, the cells were transfected with 0.2 µg of pGL3 SARS-CoV-2 3′ UTR-Luc and 0.3 µg of pRSV βgal as an internal control along with either 20 nM miRNA . Twenty-four hours after transfection, cells were lysed with 100 µL of lysis buffer (25 mM Tris-phosphate (pH 7.8), 2 mM DTT, 2 mM CDTA , 10% glycerol, and 1% Triton X-100), followed by addition of an equal volume of firefly luciferase substrate. Then, the luciferase activity was measured in a luminometer plate reader and normalized against beta-galactosidase activity. In order to examine whether miRNAs downregulate luciferase activity in a sequence-specific manner, SK-N-BE(2)C cells were transfected with either WT or MUT 3′ UTR vector along with pMSC-EVs , and then luciferase activity was analyzed.For transfection, SK-N-BE(2)C cells were cultured in 24-well plates  using BGISeq-500. Reads were aligned to a human reference genome (GRCh38) using subread aligner  and the The PITA tool  was usedTo investigate conserved regions within miRNA binding sites in SARS-CoV-2, the 3′ UTR sequences were obtained from the NCBI reference sequence database. The virus 3′ UTR sequences from the following coronaviruses were used: SARS coronavirus (NC_004718.3), SARS-CoV-2 (NC_045512.2), SARS coronavirus BJ01 (AY278488.2), Bat SARS coronavirus HKU3-1 (DQ022305.2), Bat SARS-like coronavirus SL-CoVZC45 (MG772933.1), Bat SARS-like coronavirus SL-CoVZXC21 (MG772934.1), and six complete genomes of SARS-CoV-2 from Korean patients . The 3′ UTR sequences of the coronaviruses were aligned using the MUSCLE tool . The def−∆Ct values. A graph of qPCR results for inflammation-associated genes shows fold change values. A heatmap of the log2-CPM of miRNAs in EVs was produced using the R package gplots. p values for GO term analysis and KEGG pathway analysis were corrected for multiple comparisons using the Benjamini–Hochberg method. Data are presented as the mean ± standard error of the mean. A p value or adjusted p value < 0.05 was considered significant.Statistical analysis was performed using either ANOVA, followed by Tukey’s HSD post hoc test, or a generalized linear model followed by a least square mean post hoc (R basic functions and lsmeans package). For the qPCR results, all statistics were based on 2To find the mechanism of EV’s antiviral effect, we first analyzed miRNAs within EVs. Based on the fact that the viral genome can be targeted by human miRNAs, we hypothesized that miRNAs within EVs directly interact with the SARS-CoV-2 genome. EVs obtained from eight MSCs under various cell culture methods and from six placenta derivatives were assessed by small RNA sequencing. Unassigned reads were most common, followed by miRNAs and tRNAs, indicating that miRNAs are the major cargo in EVs . The props < 0.05). Subsequently, we developed a 96-well plate assay in which live cells were stained with crystal violet, whereas cells showing virus-mediated CPE were not. To confirm the cytotoxic concentration of SARS-CoV-2, Vero cells were infected with the virus  and incubated for 3 days. The amount of cell death after 3 days was confirmed by examining CPE. Cell detachment was observed at doses of 101 to 103 TCID50/well  as measured by immunofluorescence assay. To further test whether miRNAs in EVs directly mediate antiviral effects, SARS-CoV-2-infected cells were treated with 50, 200, and 350 ng of total miRNAs isolated from EVs, which is equivalent to 250 ng, 1 μg, and 1.75 μg of EVs, respectively. Infection was significantly reduced in 200 ng and 350 ng (ps < 0.001). In order to understand the mechanism of antiviral effects mediated by either total EVs or miRNAs in EVs, miRNAs in EVs were analyzed and identified (ps < 0.001). Viruses infecting the respiratory system can invade cells expressing the ACE2 receptor, resulting in cell damage. Because ACE2 receptors are expressed primarily in the liver, kidney, male reproductive tissue, muscle, and the gastrointestinal tract (GI), these organs can be damaged by such viruses (ps < 0.05), of IL-1β, IL-6, and TNF-α in BV2 cells (ps < 0.01), and IL-1β and IL-6 in NSCs (ps < 0.05) fell significantly, whereas expression of IL-1β, IL-6, and TNF-α in cells not pre-treated with EVs increased after exposure to LPS. These results suggest that EV treatment may prevent cytokine storms caused by SARS-CoV-2 infection. NF-κB is a major transcription factor stimulated by LPS. Under normal conditions, NF-κB is located within the cytoplasm. Upon activation, the IKK complex phosphorylates IκBα, leading to translocation of NF-κB to the nucleus. Although NF-κB is an important regulator of immune responses, we found that expression of mRNA encoding NF-κB was not affected by EVs after LPS stimulation. Therefore, we examined the translocation of NF-κB protein. NF-κB was translocated into nucleus in LPS-treated Beas-2B cells, but was sequestered in the cytoplasm after EV treatment (EVs isolated from placenta stem cells (pMSC-EVs) were positive for the common EV marker CD63 . The hydentified . Among t viruses . Althoug viruses , clinica viruses  at level viruses . In addi viruses . This re viruses . Cells treatment I. In addreatment J,K. Thesps < 0.001). Specifically, relative luciferase activity was downregulated when 3′ UTR was co-transfected along with expression vectors containing each of the five miRNAs or a vector containing all five miRNAs. The results show a significant decrease in the luciferase activity mediated by the SARS-CoV-2 3′ UTR sequence , and IL-6 in LL-24 cells (p < 0.0005); the other four miRNAs did not reduce expression of pro-inflammatory factors. Interestingly, the increase in TNF-α induced by the inflammatory response was marginally significantly reduced by miR-181a-5p in Beas-2B (p < 0.01) and in LL-24 (p < 0.06). Furthermore, we found transfection of miR-181a-5p reduced NF-κB translocation to the nucleus markedly in Beas-2B cells  miRNA in EVs suppress SARS-CoV-2 by binding directly to the 3′ UTR, resulting in translational repression F. EVs prmiRNAs can regulate the replication and translation machinery involved in virus replication and inhibit viral infection by controlling inflammation and apoptosis, both of which promote the spread of assembled virus particles . BecauseTherapeutic agents based on miRNAs derived from EVs have several advantages as therapeutic agents. First, a major problem with conventional treatments for RNA-based viruses is that they have potentially fatal side effects . Due to MSCs have attracted attention as COVID-19 therapeutics because several lines of evidence show that they can significantly improve the pathological state of damaged lungs, including pneumonia; in addition, they could activate phagocytosis by macrophages, thereby preventing virus spread . FurtherMSC-EVs have several other advantages over MSCs. First, MSC-EVs are easier to manipulate and store . AdditioMore interestingly, some of the miRNAs in EVs are possible therapeutic agents for other RNA viruses B. MiraviAlthough MSC-EVs are expected to be effective for antiviral therapy and immune enhancement, one major obstacle to their widespread use is that they are difficult to mass-produce. To mass-produce EVs consistently, it will be necessary to establish reproducible culture conditions and scale up the system in an economically viable manner. Moreover, mass production of EVs is limited by the challenge of producing MSCs themselves. However, in this study, we showed that the miRNA profiles of MSC-EVs and placental derivatives EVs are very similar. Consequently, it should be possible to obtain more EVs than cells by isolating placenta-derived EVs. Alternatively, it might be possible to synthesize effective miRNAs and package them into EVs to increase treatment efficacy.To summarize, we demonstrated that EVs and five major miRNAs significantly inhibit SARS-CoV-2 replication and exert anti-inflammatory activity in vitro. Moreover, EVs regulate the pro-inflammatory environment induced by viral infection and suppress replication of SARS-CoV-2. In addition to the antiviral effect of EVs, EVs and miRNAs prevented spread of the virus when administered prior to a pro-inflammatory agent (LPS).


Crossing GC-B7E/7E mice with FGFR3G380R/G380R mice increased naso-anal and long (tibia and femur), but not cranial, bone length twice as much as crossing GC-B7E/7E mice with FGFR3WT/WT mice from 4 to 16 weeks of age. Consistent with increased GC-B activity rescuing ACH, long bones from the GC-B7E/7E/FGFR3G380R/G380R mice were not shorter than those from GC-BWT/WT/FGFR3WT/WT mice. At 2 weeks of age, male but not female FGFR3G380R/G380R mice had shorter long bones and smaller growth plate hypertrophic zones, whereas female but not male GC-B7E/7E mice had longer bones and larger hypertrophic zones. In 2-week-old males, crossing FGFR3G380R/G380R mice with GC-B7E/7E mice increased long bone length and hypertrophic zone area to levels observed in mice expressing WT versions of both receptors. We conclude that preventing GC-B dephosphorylation rescues reduced axial and appendicular skeleton growth in a mouse model of achondroplasia.Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) or inactivating mutations in guanylyl cyclase–B (GC-B), also known as NPR-B or Npr2, cause short-limbed dwarfism. FGFR3 activation causes dephosphorylation and inactivation of GC-B, but the contribution of GC-B dephosphorylation to achondroplasia (ACH) is unknown. GC-B Achondroplasia (ACH) is the most common form of human skeletal dwarfism, which affects about 1 in 20,000 live births worldwide . ACH is G374R/+Fgfr3 mice, these mice still developed an ACH-like phenotype (Fgfr3 gene was replaced with the human FGFR3-G380R cDNA to generate a humanized drug discovery model (G380R/G380R) is required for maximum skeletal reductions in these animals  is a membrane enzyme that produces cGMP when activated by C-type natriuretic peptide (CNP) –14. UndePlasma CNP is elevated in human ACH and AMDM patients –44. ImpoBoth FGFR3 and GC-B are highly expressed in proliferating chondrocytes, and reciprocal antagonism between the FGF-FGFR3 and CNP–GC-B pathways has been reported by multiple investigators , 49, 50.G380R/G380R GC-B7E/7E mice was greater than that for FGFR3G380R/G380R GC-BWT/WT mice. Importantly, we also observed that the hypertrophic zones of 2-week-old growth plates of the FGFR3G380R/G380R GC-B7E/7E male mice were the same size as those in WT male mice. Thus, prevention of GC-B dephosphorylation at an early age improved defective chondrocyte differentiation sufficiently to allow normal long bone growth.To test this hypothesis, we crossed mice where 1 allele of the FGFR3 gene was replaced with the human FGFR3-G380R gain-of-function gene  with mic7E/+ mice as originally described as Npr7E/+ by Shuhaibar et al.  or WT (FGFR3WT/WT) background resulted in greater long bone growth  was slightly greater than that for mice with 2 GC-B-WT alleles (23.00 mm) (P = 0.15)  , post hoWT/WT GC-BWT/WT, FGFR3WT/WT GC-B7E/7E, FGFR3G380R/G380R GC-BWT/WT, and FGFR3G380R/G380R GC-B7E/7E mice , as opposed to proliferation  compared with the FGFR3WT/WT mice . In fact, the hypertrophic zones were not significantly different between the FGFR3G380R/G380R GC-B7E/7E mice and the FGFR3WT/WT GC-BWT/WT mice (P = 0.80), which is consistent with GC-B7E/7E expression rescuing the hypertrophic zone defect in the FGFR3G380R/G380R mice.Analysis of growth plates from male mice of each genotype indicated that GC-B80R/G380 . HoweverG380R/G380R compared with FGFR3WT/WT mice at 2 weeks (P = 0.87). However, GC-B–7E expression increased hypertrophic zone areas in both FGFR3WT/WT  and FGFR3G380R/G380R  female mice , possibly because the FGFR3-G380R mutations failed to decrease long bone growth in the female mice at this early age. Together, these data indicate that the inhibitory effects of the FGFR3-G380R mutations occur earlier in male than female mice, whereas the positive effects of GC-B–7E expression on growth plate area occur earlier in female than male mice.In female mice, hypertrophic zone decreases were not observed in FGFR3ale mice . In contG380R/G380R mice by preventing the FGFR3-G380R–dependent decreases in growth plate hypertrophic zone area as graphically summarized in 7E/7E mice with the FGFR3G380R/G380R mice had a greater effect on naso-anal and long bone length than crossing the GC-B7E/7E mice with FGFR3WT/WT mice.In this report, skeletal phenotypes of mice expressing GC-B–7E alleles and FGFR3-G380R alleles were analyzed and described as a function of age. Our results demonstrate that blocking dephosphorylation-dependent inactivation of GC-B rescues the disproportionate dwarfism of the FGFR3G380R/G380R mice. In fact, there was no difference in the size of the growth plates from the FGFR3 G380R/G380R/GC-B7E/7E mice compared with mice expressing WT versions of both receptors, which is consistent with a rescue of the reduced hypertrophic zone by GC-B–7E expression. Sex-specific effects of ACH on limb lengthening and bone age delay in children have been described, but whether these effects are related to our results in mice is currently unknown  injections increase long bone growth in animal models of ACH , 62–66. 7E/7E mice  were genotyped and maintained as previously described on a C57BL/6J background . The freehand selection tool in ImageJ determined the number of square millimeters of the identified hypertrophic region from each section of bone as indicated by the blue outline in the images shown in P value was less than 0.05.Statistics and graphs were generated with Prism 8 software (GraphPad Software). For the naso-anal growth curves, a mixed-model 2-way ANOVA with repeated measures was applied using a Geisser-Greenhouse correction for sphericity and Tukey’s correction for multiple comparisons. Therefore, the naso-anal length values described under results were derived from repetitive measurements on the same animal at multiple ages. The remaining analyses including the growth plate measurements were performed using a standard, 1-way ANOVA assuming Gaussian distribution and equal SD with Tukey’s correction for multiple comparisons. Except for the naso-anal growth curves, each symbol in a figure represents a measurement from an individual mouse. The longer horizontal bar within each group of symbols represents the mean, and the shorter bars above and below the mean represent the SEM. For the naso-anal growth curves, each point represents the mean length measurement at the indicated age, and bars above and below the mean indicate SEM. Differences were considered significantly different when the Animal care was compliant with the University of Minnesota IACUC–approved protocol no. 1806A-35995.Study design was contributed by JWR, LLM, and LRP. Naso-anal length measurements were contributed by JWR and BMW. Tibia and femur measurements were contributed by JWR and BMW. Histological data were contributed by NK. Hypertrophic zone measurement was contributed by JWR, BMW, and LLM. Analysis of data was contributed by BMW, JWR, LLM, and LRP. Drafting the manuscript was contributed by JWR, BMW, and LRP. Revising manuscript content was contributed by BMW, JWR, LRP, and LLM. YWL and YCL provided the ACH mouse line, and YCL edited the paper. All authors approved the final version of the manuscript.


NL–FApp) or also Arctic mutation (NL–G–FApp) show neuropathology and hippocampus-dependent cognitive impairments pertinent to Alzheimer’s disease (AD) in mouse models at 18 and 6 months of age, respectively. Apolipoprotein E, involved in cholesterol metabolism, plays an important role in maintaining the brain. There are three human apolipoprotein E isoforms: E2, E3, and E4. Compared to E3, E4 increases while E2 protects against AD risk. At 6 months of age, prior to the onset of plaque pathology, E3, but not E4, protected against hAPP/Aβ-induced impairments in spatial memory retention in the Morris water maze. However, these earlier studies were limited as hapoE was not expressed outside the brain and E3 or E4 was not expressed under control of an apoE promotor, E2 was often not included, hAPP was transgenically overexpressed and both mouse and hAPP were present. Therefore, to determine whether apoE has isoform-dependent effects on hAPP/Aβ-induced behavioral alterations and cognitive impairments in adult female and male mice at 6 and 18 months of age, we crossed NL–G–FApp and NL–FApp mice with E2, E3, and E4 mice. To distinguish whether genotype differences seen at either time point were due to main effects of hAPP, hapoE, or hAPP × hapoE genetic interactions, we also behavioral and cognitively tested E2, E3, and E4 female and male mice at 6 and 18 months of age. We also compared behavioral and cognitive performance of 18-month-old NL–G–FApp and NL–FApp female and male mice on a murine apoE background along with that of age—and sex-matched C57BL/6J wild-type mice. For many behavioral measures at both time points there were APP × APOE interactions, supporting that apoE has isoform-dependent effects on hAPP/Aβ-induced behavioral and cognitive performance. NL-G-F/E3, but not NL-G-F/E2, mice had lower cortical insoluble Aβ42 levels than NL-G-F/E4 mice. NL-F/E3 and NL-F/E2 mice had lower cortical insoluble Aβ42 levels than NL-F/E4 mice. These results demonstrate that there are apoE isoform-dependent effects on hAPP/Aβ-induced behavioral alterations and cognitive impairments and cortical insoluble Aβ42 levels in mouse models containing only human APP and apoE.Mice expressing human amyloid precursor protein (APP) containing the dominant Swedish and Iberian mutations ( NL–FApp) and shows neuropathology and hippocampus-dependent cognitive impairments at 18 months of age (NL–G–FApp) and shows neuropathology, hippocampus-dependent cognitive impairments, and impaired long-term potentiation (LTP) in the hippocampus and cortex at 6 months of age  . The amys of age , 2016. As of age . Recentlan males , consistan males .Apolipoprotein E is involved in cholesterol metabolism and plays an important role in development, maintenance, and repair of the brain following injury . There aWe previously reported that at 6 months of age, prior to the onset of plaque pathology, E3, but not E4, was able to protect against hAPP/Aβ-induced impairments in spatial memory retention in the water maze . This anNL–G–FApp and NL–FApp female and male mice on a murine apoE background along with that of age-and sex-matched C57BL/6J wild-type mice. In addition, in a subset of hAPP mice we analyzed the cortical Aβ levels.To determine whether apoE has isoform-dependent effects on hAPP/Aβ-induced behavioral alterations and cognitive impairments in adult female and male mice at 6 months of age, we crossed APP NL-G-F mice with E2, E3, and E4 mice to generate NL-G-F/E2, NL-G-F/E3, and NL-G-F/E4 mice. To determine whether apoE has isoform-dependent effects on hAPP/Aβ-induced behavioral alterations and cognitive impairments in aged female and male mice at 18 months of age, we crossed APP NL-F KI with E2, E3, and E4 mice to generate NL-F/E2, NL-F/E3, and NL-F/E4 mice. To distinguish whether genotype differences seen at either time point were due to main effects of hAPP, hapoE, or hAPP × hapoE genetic interactions, we also behaviorally and cognitively tested E2, E3, and E4 female and male mice at 6 and 18 months of age. Separately, we compared behavioral and cognitive performance of 18-month-old NL–FApp) or the Arctic mutation included as third mutation (NL–G–FApp) generated by Dr. Saido (We started to cross hAPP knock-in (KI) mice containing the Swedish and Iberian mutations  50°C. The PCR products were digested with the BsaBI restriction enzyme:Digestion of WT APP produces 110 bp and 93 bp fragments. The presence of the Iberian mutation prevents digestion. ApoE genotyping was performed as described .NL–G–FApp and NL–FApp female and male mice on a murine apoE background along with that of age—and sex-matched C57BL/6J wild-type mice we reported in an earlier study  and were consistent with the ARRIVE guidelines.Mice were tested at approximately 6 or 18 months of age. Mice were group housed in standard vivarium conditions until the start of the study. The animals were then singly housed for circadian home cage activity monitoring, as described below, and remained singly housed for the duration of the experiment. The vivarium was maintained at 20–21°C and food  was available Behavioral and cognitive tests were conducted in the order illustrated in Home cage activity was monitored using infrared home-cage activity sensors , as previously described . Mice weNest building was measured at two time points, once after the conclusion of activity monitoring and again after the conclusion of novel object recognition testing. The protocol described in Measures of anxiety were assessed in the elevated zero maze as previously described . The eleA combination of motor function, balance, endurance, and muscle strength was assessed using the wire hang task, adopting the “falls and reaches” method described by NL–G–FApp and NL–FApp mice  has raised sides  with plastic, opaque gray arms (37.98 cm length) at a 120° angle from each other. This vendor was selected as the same Y-maze was used in the original study of the L–F mice  and our L–F mice . Mice weExploratory activity and measures of anxiety were assessed in the open field test. The open field consisted of a well-lit square (L 40.6 × W 40.6 × H 40.6 cm) with a central light intensity of 100 lux. Mice were allowed to explore the open field for 5 min during three consecutive days. On day 4, mice were exposed to the open field containing two identical objects for a 15-min trial. The objects were placed 10 cm apart and 15 cm from the adjacent walls of the arena. On day 5, one object was replaced with a novel object and mice were allowed to explore the open field for 15 min. Object exploration was measured manually as time when the mouse’s nose point was within 2 cm of the object. The enclosures were cleaned with 0.5% acetic acid between trials. Performance of mice was tracked using Ethovision 15 XT software. Time spent in the center of the open field was analyzed to assess measures of anxiety. Percent time spent with the novel object was calculated as the number of seconds spent exploring the novel object divided by the total number of seconds spent exploring the novel and familiar objects combined.Fear conditioning was assessed over the course of three consecutive days using a Med Associates mouse fear conditioning system  and Med Associates VideoFreeze automated scoring system. Mice were placed inside the fear conditioning chamber, where chamber lights were turned on at the beginning of the trial. The first day of testing consisted of a 5-min habituation phase during which no tone or shock was administered. Day two consisted of the acquisition phase, which lasted for a total of 5 min per trial. Following a 120-s baseline habituation period, two 30-s tones (80 dB) were presented, separated by 60-s inter-stimulus-intervals (ISIs). There was also a 60-s interval after the second tone-shock period. During the last 2 s of each tone, a 0.30 mA foot shock was administered. Twenty-four hours later the hippocampus-dependent contextual fear memory was assessed by placing the animals into the same chamber as in the acquisition phase for a 5-min trial. The chamber lights were on, but no tones or shocks were presented. Between habituation, acquisition and context trials, the chambers were thoroughly cleaned with 0.5% acetic acid.Two hours following the contextual fear memory test, hippocampus-independent cued fear memory was assessed. Mice were placed in a novel environment, containing a novel floor texture, angled walls and vanilla extract soaked nestlet fixed to the outside of the wall. Each cued memory trial lasted a total of 6 min, consisting of a 180-s baseline period followed by a 180-s period during which the same tone used during training was presented. No shocks were administered during this phase of testing. Between cued trials, the chambers were thoroughly cleaned with 10% isopropanol.n = 4 samples/sex/genotype/time point) were processed for analyses of soluble and insoluble Aβ40 and Aβ42 levels using Invitrogen ELISA kits , according to the recommended guidelines in the production information sheets. To a thawed cortical tissue sample (both hemispheres), 400 μl of buffer A  was added. The tissue was homogenized using a Polytron for 10 s and subsequent a sonicator, and centrifuged at 45,000 rpm for 20 min at 4°C. The supernatant was collected as the soluble fraction. The same volume of buffer A was used to loosen the pellet. The sample was centrifuged again at 45,000 rpm for 5 min at 4°C. After removing the supernatant in a separate tube. the pellet was dissolved in Buffer B (containing 6 M Guanidine H-Cl and 50 mM Tris and filtered before use) and incubated at room temperature for 1 h. After this incubation, the sample was sonicated for 20 s and the extracted pellet was centrifuged at 45,000 rpm for 20 min at 4°C. The supernatant was collected as the insoluble fraction. Pilot experiments were performed to determine the optimal sample dilution. For the analyses of insoluble Aβ levels in 6-month-old mice, the tissue samples were diluted 1:4,000. For analysis of the soluble Aβ levels in 6-month-old mice, undiluted tissues samples were used. Standard curves were generated with the same buffer dilution as the samples. For the analyses of insoluble Aβ levels in 18-month-old mice, the tissue samples were diluted 1:20,000. For analysis of the soluble Aβ levels in 18-month-old mice, tissues samples were diluted 1:50. The ELISAs were read at 450 nm using a SpectraMax iD5 Multi-Mode Microplate Reader . The standard curves were generated and the levels in the samples determine using GraphPad Prism software, San Diego, CA, United States). Total protein amounts in the samples were determined by BCA protein assay kit  and reading the samples at 562 nm using the iD5 Reader.Cortices of a subset of 6-month-old, NL-G-F/E2, NL-G-F/E3, NL-G-F/E4, NL-G-F, and NL-F and 18-month-old NL-F/E2, NL-F/E3, NL-F/E4, NL-G-F, and NL-F mice . All figures were generated using GraphPad Prism software .The results of the statistical analyses of the behavioral and cognitive performance at the 6-month time point are summarized in For body weights, there were effects of APOE  and APP For quality of nest building, there were effects of APOE, APP, and an APP × APOE interaction in males  and femaWhen circadian activity levels were assessed in the home cage, there were trends toward APP × APOE interaction for activity during the dark period in males  and femaNext, measures of anxiety were assessed in the elevated zero maze. There was an effect of APP on the percent time spent in the more anxiety-provoking open areas in males  and femaWhen entries into the open areas were analyzed, there was an APP × APOE interaction in the males  and effeThere was an effect of APOE on fall scores in males  and femaWhen activity levels were analyzed during three subsequent days of open field testing, there was an effect of APOE in males  and femaThe time spent in the more anxiety-provoking center area of the open field was assessed as well. In males, there were effects of APOE and APP and an APP × APOE interaction . NL-G-F/When spontaneous alternation was analyzed in the Y maze, there were no effects of APP, APOE, or APP × APOE interactions in males  or femalWhen we analyzed the activity levels prior to the first tone during fear learning (baseline motion), there were no effects in males . HoweverNext we analyzed the freezing between the tone-shock intervals, a measure of fear learning. In males, there were effects of APP, APOE, and an APP × APOE interaction . In femaThe day after fear learning, we assessed hippocampus-dependent contextual fear memory. In males, there was an APP × APOE interaction . NL-G-E2Finally, we analyzed hippocampus-independent cued fear memory. When the freezing during the tone only was analyzed, there was an effect of APOE in males  but not F = 13. 83, p = 0.0002). Insoluble Aβ42 levels were lower in NL-G-F/E3 than NL-G-F/E2 (p = 0.0170) and NL-G-F/E4 (p < 0.0001) levels (–5 pg/μg protein) and 2 NL-G-F/E4 mice (0.000201 and 0.000387 pg/μg protein).There was an effect of genotype on insoluble Aβ42 levels  levels . In addi) levels  or the i) levels . SolubleThe results of the statistical analyses of the behavioral and cognitive performance at the 18-month time point are summarized in For body weights, there were effects of APOE, APP, and an APP × APOE interaction in males . Body weThere was an effect of APOE on nest building in males . Nest buThere was an effect of APOE on activity levels in the dark period in females  but not When measures of anxiety were assessed in the elevated zero maze in males, there were effects of APOE, APP, and an APP × APOE interaction for the percent time spent in the open areas . E2 maleThere was an effect of APP and an APP × APOE interaction for fall scores in the males . Fall scWhen activity levels were assessed in the open field, there was an APP × apoE interaction in males . In the Next measures of anxiety were assessed in the open field by analyzing the time spent in the more anxiety-provoking center of the open field. There was no effect of APP or apoE on percent time spent in the center of the open field in males  but in fThere was no effect of APP or apoE on spontaneous alternation in males . HoweverWhen activity levels were analyzed in the Y maze, there was an effect of APOE on the number of arm entries in males  and femaThere were no effects of APP or apoE on baseline motion in males . There wNext we analyzed freezing during the tones. There was an effect of APOE in males  and femaWhen freezing between the tone-shock intervals was analyzed, there was an effect of APOE, APP, and an APP × APOE interaction in males  and femaIn males, there was a trend toward an effect of APP on freezing in the contextual fear memory test but it did not reach significance . In femaIn the cued fear memory test, when freezing during the tone only was analyzed, there was an effect of APOE in males  and a trF = 5.873, p = 0.0090). Insoluble Aβ42 levels were higher in NL-F/E4 than NL-F/E2 (p = 0.0106) and NL-F/E3 (p = 0.0074) mice , with a trend toward higher soluble Aβ42 levels in NL-F/E4 than NL-F/E3 mice  mice . Insolubt = 7.321, p < 0.0001, 2-sided)   . Insolub2-sided) . The ins2-sided) . SolubleThere was an effect of APP on body weights in males, with lower body weights in NL-F than NL-G-F males . This APThere was no effect of APP on nest scores or in the change in nest building scores in females or males.There was a trend toward an effect of APP on activity during the dark period in males, with NL-G-F males being more active than WT males but it did not reach significance . This trThere was no effect of APP on measures of anxiety in the elevated zero maze in males or females. There was an effect of APP on entries into the open areas in the elevated zero maze in males and females. NL-F males entered the open areas more than WT males. There was also a trend toward NL-G-F males entering the open areas more than WT males but it did not reach significance. In females, NL-G-F mice entered the open areas more than NL-F and WT mice.There was an effect of APP on fall scores in males  and femaThere was an effect of APP on reach scores in males  but not There was no effect of APP on activity levels during the 3 days of open field testing in males  and femaThere was an effect of APP on time spent in the center of the open field in males but not females. In males, the WT mice spent less time in the center of the open field than NL-G-F and NL-F mice.There was an effect of APP on baseline activity (prior to the first tone-shock pairing) in males , with hiThere were no effects of APP on percent freezing during the tones, response to the shock, or freezing between the tone-shock periods in males or females.There was an effect of APP on percent freezing of males  and femaThere was also an effect of APP on percent freezing during the cued fear memory test in males  and femat = 2.828, p = 0.0116, 2-sided) t = 2.767, p = 0.0171, 2-sided) (Insoluble Aβ42 levels were higher in NL-F than NL-G-F mice (2-sided) .t = 8.649, p < 0.0001, 2-sided)  (Soluble Aβ42 levels were higher in NL-F than NL-G-F mice (2-sided) . There w2-sided) . There w2-sided) .r = −0.8494, p = 0.0076, n = 8 XY pairs, r = −0.7394, p = 0.036, n = 8 XY pairs, Finally, we assessed whether insoluble cortical Aβ levels correlated with behavioral measures in 6-month-old NL-G-F mice and 18-month-old NL-F mice. At 6 months of age, insoluble cortical Aβ40 levels were negatively correlated with activity levels on the first day of open field testing  showed APP × APOE interactions in males and females. Other measures showed sex-dependent APP × APOE interactions at 6 months of age. Change in nest score, entries in the open areas of the elevated zero maze, and contextual fear memory showed APP × APOE interactions in males. In contrast, average body weight, average nest score, activity during the light period, percent time in the open areas of the elevated zero maze, activity levels in the elevated zero maze, reach scores in the wire hang test, and baseline motion in the fear conditioning test showed APP × APOE interactions in females. At 18 months of age, average motion during the shocks during fear learning, and percent freezing during the ISIs showed APP × APOE interactions in males and females. Thus, at both 6 and 18 months an APP × APOE interaction is seen for fear learning in both sexes. Other measures showed sex-dependent APP × APOE interactions at 18 months of age. Average body weight, percent time in the open areas of the elevated zero maze, wire hang fall score, activity levels in the open field, showed APP × APOE interactions in males. In contrast, percent time in the center of the open field, percent spontaneous alternation in the Y maze, and percent freezing during the tones in the fear conditioning test showed APP × APOE interactions in females. While in males, measures of anxiety in the open field revealed an APP × APOE interaction at the 6-month time point, measures of anxiety in the elevated zero maze revealed an APP × APOE interaction at the 18-month time point. The opposite pattern was seen in females. In males, measures of anxiety in the elevated zero maze revealed an APP × APOE interaction at the 6-month time point, measures of anxiety in the open field revealed an APP × APOE interaction at the 18-month time point. Thus, although the measures of anxiety in the elevated zero maze and open field are distinct, they revealed an APP × APOE interaction at 6 and 18 months in males and females and highlight the importance of including more than one test to assess measures of anxiety. We recognize that age-related changes in performance in the behavioral tests used in this study, and the differential effects of the NL-G-F and NL-F mutation might have contributed to the distinct behavioral measures revealing an APP × APOE interaction at these two time points. The fact that at both 6 and 18 months an APP × APOE interaction is seen for fear learning in both sexes is remarkable considering age-related changes in hearing and responsiveness to aversive challenges and age-related changes in fear learning in fear conditioning paradigms in humans  and rodeFor body weights of females at 6 months age, NL-G-F/E4 mice were heavier than E4 mice. In contrast, NL-G-F/E2 and NL-G-F/E3 mice were lighter than E2 and E3 mice, respectively. For body weights of males at 18 months of age, NL-F/E2 mice were heavier than E2 mice and no differences in body weight were seen in the E3 and E4 background. The observation that these changes occurred in females at 6 months of age but in males at 18 months of age is consistent with the human data showing that while changes in body mass index within 2 years increased AD risk in women, changes in body mass over 4 years were required to increase AD risk in men . With reAt 6 months age, for average nest scores in males and females and for change in nest scores in males, NL-G-F/E2 mice had higher scores than E2 mice. This was genotype-dependent and not seen in the E3 or E4 background. As nest scores might reflect activities of daily living which are negatively impacted in AD  and incrWhen percent time spent in the anxiety-provoking areas of the elevated zero maze were analyzed in females at 6 months of age, measures of anxiety were lower in NL-G-F/E2 than E2 mice. This effect was genotype-dependent and not seen in the E3 and E4 background. At the 18-month time point, NL-F/E3 male mice were less anxious than E3 male mice and there was a trend toward lower measures of anxiety in NL-F/E2 than E2 male mice. These data suggest a beneficial effects of NL-F in the E2 and E3 backgrounds. In addition, NL-F/E3 males showed lower measures of anxiety than NL-F/E4 males in the elevated zero maze and NL-F/E3 females showed lower measures of anxiety than NL-F/E4 females in the open field. These data suggest a beneficial effect of E3 in the NL-F background. These effects are translationally relevant, as increased anxiety levels are observed in patients with mild cognitive impairment, mild dementia, or early-onset AD, and can promote progression or conversion to AD .At the 6-month time point, there was less fear learning in NL-G-F/E4 than E4 males. There was a trend toward such effect in the E3 background. However, this was not seen in the E2 background. In addition, E2 males showed better fear learning than E3 or E4 males. Contextual fear memory was also stronger in NL-G-F/E2 than NL-G-F/E3 mice. Finally, NL-G-F/E3 males showed less contextual fear memory than E3 males. These effects are consistent with a protective effect of E2 against a NL-G-F-induced learning deficit in males. In females, this effect was less clear; only during the second ISI was there less fear learning in NL-G-F/E4 than E4 females. In contrast to what was seen at the 6-month time point in E4, at the 18-month time point NL-F/E4 female mice showed better fear learning than E4 female mice and NL-F/E4 male and female mice showed more fear learning than sex-matched NL-F/E3 or NL-F/E2 mice. In addition, NL-F/E4 female mice froze more during the tones during the training day than NL-F/E3 and NL-F/E2 female mice. These data indicate that NL-F enhances fear learning in a sex-dependent fashion.For spontaneous alternation in Y maze at the 18-month time point, we did not see a detrimental effect of NL-F or E4. In females, E4 mice showed more spontaneous alternation than E3 mice. In addition, NL-F/E3 females showed more spontaneous alternation than E3 females and no effects of NL-F on spontaneous alternation of E2 or E4 females. We did not see an effect of APP on spontaneous alternation in NL-G-F, NL-F, and WT male or female mice at the 18-month time point either. At the 6-month time point, we did report a genotype effect of APP on spontaneous alternation in females with lower spontaneous alternation in NL-G-F than WT females . In contFor activity levels on second and third day in the open field and the 18-month time point, NL-F/E2 males moved more than E2 males. These data suggest reduced hippocampus-dependent spatial habituation learning in the NL-F background. E2 males moved more than E3 males on all days, and E3 males moved than E4 males on days 1 and 3. Based on the association of low levels of physical activity with AD risk and the beneficial effects of physical exercise on the prevention and treatment of AD , these rNL–G–FApp and NL–FApp female and male mice on a murine apoE background along with that of age—and sex-matched C57BL/6J wild-type mice. Consistent with a worse cognitive phenotype of NL-G-F than NL-F mice, NL-G-F females showed less contextual fear memory than WT or NL-F females. This cognitive impairment was not restricted to females or contextual fear memory. NL-G-F males showed less cued fear memory than NL-F or WT males and NL-G-F females showed less cued fear memory than NL-F females. These effects did not seem related to possible differences in baseline activity levels. NL-F males showed higher baseline activity levels (prior to the first tone-shock pairing) than NL-G-F and WT males. In the wire hang test, NL-G-F males had lower fall scores than NL-F and WT males. NL-G-F males also had lower reach scores than NL-F males. NL-G-F females had lowed fall scores than NL-F and WT females. These effects did not seem related to possible differences in anxiety levels either. In the elevated zero maze, NL-F males entered the open areas more often than WT males and there was a trend toward NL-G-F males entering the open areas more often than WT males. In addition, NL-G-F and NL-F males spent more time in the center of the open field than WT males. Similarly, NL-G-F females entered the open areas of the elevated zero maze more often the NL-F or WT females.Finally, we compared behavioral and cognitive performance of 18-month-old In females, there was an effect of APP on activity during the light period, with a trend toward higher activity levels in NL-G-F and NL-F than WT females. This might be related to sleep disturbances in AD  and the At 6 months of age, prior to the onset of plaque pathology, E3, but not E4, was able to protect against hAPP/Aβ-induced impairments in spatial memory retention in the water maze in a mouse model in which hapoE was not expressed outside the brain and E3 or E4 was not expressed under control of an apoE promotor in the brain and expressed at similar levels . SimilarSW/PS1dE9), containing both human and murine APP, there was a greater load of Aβ pathology in hAPP/E4 mice than APP/E2 mice at 6–10 months of age (APOE ε2 or one copy of APOE ε2 and one copy of APOE ε3 (APOE3 Christchurch (R136S) mutation reported in an individual who carried a PS1 mutation  with exon 9 deletion overexpressing mice (APPs of age  or 11 mos of age . These d APOE ε3  and the mutation . Considemutation , increasWhile at 6 months of age, insoluble cortical Aβ42 and Aβ40 levels were higher NL-G-F than NL-F mice, at 18 months of age insoluble and soluble cortical Aβ42 and the insoluble cortical Aβ42/40 ratio were higher in NL-F than NL-G-F mice. Remarkably, in 6-month-old NL-G-F mice insoluble cortical Aβ40 levels were negatively related to activity in the open field in the absence and presence of objects. In contrast, at 18 months of age, insoluble cortical Aβ42 levels in NL-F mice were positively correlated with cognitive performance in the Y maze. These data suggest that the Aβ species and age might modulate the relationship between Aβ pathology and behavioral measures, consistent with the independent association of Aβ pathology and hippocampal atrophy with memory seen in cognitively healthy elderly  and the In summary, these data support that there are apoE isoform-dependent effects on hAPP/Aβ-induced behavioral alterations and cognitive impairments and Aβ pathology in mouse models containing only human APP and apoE and not the murine counterparts. Future efforts are warranted to identify and compare the mechanisms underlying these apoE isoform-dependent effects in adulthood and aging.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by OHSU IACUC.JR designed the experiments. ET and SH supervised the breeding and the related genotyping. DG developed the genotyping protocols. DG, DK, and SH genotyped the samples. PK and SH performed the behavioral testing and related analyses. SH performed the statistical analyses for the behavioral data and helped with the related tables. RS helped with organizing and graphing of all the behavioral data. JR and SH processed the cortical samples for the Aβ ELISAs and analyzed the related data. All authors contributed to the writing of this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


TCL1A) is a proto-oncogene that is mainly expressed in embryonic and fetal tissues, as well as in some lymphatic cells. It is frequently overexpressed in a variety of T- and B-cell lymphomas and in some solid tumors. In chronic lymphocytic leukemia and in T-prolymphocytic leukemia, TCL1A has been implicated in the pathogenesis of these conditions, and high-level TCL1A expression correlates with more aggressive disease characteristics and poorer patient survival. Despite the modes of TCL1A (dys)regulation still being incompletely understood, there are recent advances in understanding its (post)transcriptional regulation. This review summarizes the current concepts of TCL1A’s multi-faceted modes of regulation. Understanding how TCL1A is deregulated and how this can lead to tumor initiation and sustenance can help in future approaches to interfere in its oncogenic actions.T-cell leukemia/lymphoma 1A regulation. This review presents a comprehensive overview of TCL1A expression in tumors and the current understanding of its (dys)regulation via genomic aberrations, epigenetic modifications, or deregulation of TCL1A-targeting micro RNAs. We also summarize triggers that act through such transcriptional and translational regulation, i.e., altered signals by the tumor microenvironment. A refined mechanistic understanding of these modes of dysregulations together with improved concepts of TCL1A-associated malignant transformation can benefit future approaches to specifically interfere in TCL1A-initiated or -driven tumorigenesis.Incomplete biological concepts in lymphoid neoplasms still dictate to a large extent the limited availability of efficient targeted treatments, which entertains the mostly unsatisfactory clinical outcomes. Aberrant expression of the embryonal and lymphatic TCL1 family of oncogenes, i.e., the paradigmatic  TCL1A) was first described as a proto-oncogene in hematological neoplasms between 1989 and 1994 [TCL1B and mature T-cell proliferation 1 (MTCP1) [T-cell leukemia/lymphoma 1A  ,6 and co (MTCP1) .TCL1A locus. In these tumors, TCL1A expression parallels its regulation in non-neoplastic B cells [Physiologically, the expression of TCL1A is restricted to embryonic tissues and to pre-mature B cells and T cells, suggesting its role in reproduction and adaptive immunity, which could be corroborated in sub-total knockout mice ,9. Its a B cells .prLck-promoter (Lck-TCL1A) or in B cells under the VH-promoter/IgHµ enhancer (Eµ-TCL1A), the mice develop a disease closely resembling human T-PLL or chronic lymphocytic leukemia (CLL), respectively. A related model, pEμ-B29-TCL1A mice, produces germinal center (GC)-derived B-cell tumors that resemble Burkitt lymphoma (BL), follicular lymphoma (FL), and diffuse large B-cell lymphoma (DLBCL), and in one founder line also a T-PLL-like disease [Eµ-TCL1A model for CLL, have been crossed with a variety of other alleles  mice ,14,15,16iewed in ,18). Thi CD44−/− ; Eµ-TCL1R4C1013G ) or sign pkcβ−/− ; Eµ-TCL1fl-Akt-C ), as welAtmfl/fl ).In T-PLL and CLL patients, the expression of tumor-associated TCL1A mRNA and protein shows considerable inter-patient variability. Notably, high levels of TCL1A correlate with aggressive clinical features , high-risk cytogenetics, poorer responses to chemo-immunotherapies, and inferior clinical outcomes ,27,28,29Given TCL1A’s important role in tumor initiation, progression, and maintenance, investigating the modes of its oncogenic function and dysregulation can help to better understand the pathogenesis of these neoplasms and to contribute to the identification of possible new treatment targets. This review summarizes the current knowledge on the spectrum of TCL1A’s modes of upstream regulations in normal and transformed lymphocytes, as well as in stem cells and solid tumors.The expression of TCL1A is normally restricted to embryonic tissues and pre-mature B cells and T cells and is conserved to some mammals . Its expTRA locus rearrangements, which exchange the pre-TCR to the TCR at that DP stage, erroneous reassemblies of TRA regions juxtapose the TCL1A locus under control of regulatory elements of TRA/D genes, causing aberrant TCL1A expression toward T-PLL [T cells lose TCL1A expression starting at the CD4/CD8 double-positive (DP) thymocyte stage, and a role for TCL1A in pre-TCR signaling appears attractive to speculate; post-thymic T cells with matured TCRs no longer express TCL1A . It is brd T-PLL .In B cells, TCL1A experiences a drastic downregulation starting at the entry of the highly TCL1A-positive mantle-zone B cell into the GC environment. TCL1A is completely silenced in terminally differentiated B cells, such as plasma cells ,35.TCL1A gene was identified in several families with an autosomal dominant myeloid neoplasm predisposition syndrome. However, TCL1A’s role in its pathogenesis is still under investigation [Overexpression of TCL1A in the neoplastic context was first identified in T-PLL, where translocations or inversions juxtapose the gene locus at 14q32 to highly active regulatory elements of T-cell receptor (TCR) genes [tigation ,37.IGHV) region gene unmutated subtype of pre-GC origin shows higher TCL1A levels than the IGHV-mutated TCL1Alow CLL subset [In B-cell tumors, TCL1A expression mostly parallels its regulation in non-neoplastic B cells. Those of pre-GC derivation, such as B-cell acute lymphoblastic leukemia/lymphoma (ALL/LBL) and mantle cell lymphoma (MCL), are highly TCL1A positive . HodgkinL subset . BL showL subset ,38. FL sL subset .Recent years have seen an increasing number of publications that also implicate TCL1A in stemness programs of non-hematopoietic cancers. It was unexpected to find TCL1A to be expressed in epithelial solid tumors such as breast or colorectal cancers (CRC) . InteresGiven the apparently histogenetically fixed expression of TCL1A in lymphoid tumors, the expression of TCL1A harbors important diagnostic information. Due to its specific expression in T-PLL among other mature T-cell lymphomas (MTCL) with prominent peripheral blood (PB) presentation, TCL1A was established as a first-order marker of high specificity ,55. It i+ CD56+ blastic tumors of skin (previously thought to be of NK origin), helped to reclassify them as a blastic plasmacytoid dendritic cell neoplasm (BPDCN) of (pre)-pDC origin. TCL1A is now a core marker in its differential diagnosis, which has drastic prognostic and therapeutic implications [Importantly, detection of TCL1A expression in the former category of CD4ications . As the ications .In addition to diagnostic value, TCL1A mRNA and protein expression carry prognostic information in several leukemias/lymphomas. In CLL and T-PLL, higher TCL1A levels correlate with more aggressive disease features, such as higher white blood cell (WBC) counts and faster tumor cell doubling, as well as a shorter overall/progression-free survival ,26,27. FAcross non-Hodgkin lymphomas, a gene set enrichment analysis associated high TCL1A levels with important pathways controlling B-cell lymphomagenesis, including, e.g., B-cell receptor, NF-κB signaling, cell death, and MAP kinase, implicating a central role of elevated TCL1A expression in their pathogenesis and aggressiveness . In lineAdditionally, in some solid tumors, TCL1A can be utilized as a prognostic marker. In CRC, high TCL1A correlates with tumor differentiation and clinical stage and is an independent factor for CRC-specific and disease-free survival. Furthermore, it predicts the outcome of stage II/III patients who receive standard adjuvant chemotherapy . In hepaThe expression of TCL1A in embryonic tissues, as well as its recurrent overexpression and prognostic/predictive value in different malignancies, implicates an important function of TCL1A in key signaling pathways mediating stemness and survival .Generally, the 14 kDa TCL1A protein lacks kinase activity and a DNA-binding motif. Instead, its eight-stranded β-barrel with a hydrophobic core suggests its binding to small hydrophobic ligands . Its cur−/−Tcl1a female mice was ascribed to a frequent block of blastomere proliferation beyond the eight-cell stage, despite displaying normal major differentiative traits [There is increasing evidence for the role of TCL1A in embryonic development. The reduced fertility observed in e traits . Furthere traits ,69. An ee traits ,71. In ce traits . Furthere traits . Fittinge traits . In conte traits .PTPRO) [The adverse clinical outcomes in some solid tumors and leukemias in association with high TCL1A expression are likely to a considerable part the result of the TCL1A-mediated augmented activity of AKT contributing to enhanced proliferation and multi-nodal resistance ,35,75,76PTPRO) ,80.There is also evidence for TCL1A to form a functional synergism with hypomorphic ATM toward a prominent phenotype of deficient DNA damage responses in T-PLL . OverexpEµ-TCL1A mice show more hypomethylated regions than age-matched wild-type cells [TCL1A was also shown to contribute to epigenetic reprogramming via interacting with the de novo DNA methyltransferase 3A (DNMT3A) and reducing its enzymatic activity. Accordingly, B cells from pe cells . In sevepe cells , therefoIn HCC, TCL1A overexpression is implicated in mediating metabolic alterations. TCL1A enhances the pre-mRNA splicing and thereby protein expression of the glucose-6-phosphate dehydrogenase (G6PD) by interacting with the heterogeneous nuclear ribonucleoprotein (hnRNPK), leading to an increased pentose phosphate pathway flux and glucose consumption .In summary, based on the heterotypic functions of TCL1A on different signaling branches, its transforming impact is most likely a synergistic net effect of several dysregulated pathways.The marked and safeguarding silencing of TCL1A during embryogenesis, as well as during T-cell and B-cell differentiation, is tightly regulated. As outlined, dysregulation of this machinery is associated with carcinogenesis. Understanding TCL1A’s transcriptional and translational regulation is, therefore, highly important. In the following, we present a comprehensive overview of modes of TCL1A (dys)regulation, including genomic aberrations, epigenetic modifications, dysregulation of TCL1A-targeting microRNAs (miRs), as well as modulations via altered signals by the microenvironment, which are likely mediated through some of these relays .Tcl1a was shown to be part of an embryonic expression signature important for self-renewal, also involving POU class 5 homeobox 1 , Nanog homeobox (Nanog), SRY-box transcription factor 2 (Sox2), and the Myc proto-oncogene (Myc) [Tcl1a was identified as a direct transcriptional target of Oct3/4 by binding of the transcription factor (TF) to a sequence 410 bp upstream of the Tcl1a gene [Tcl1a promoter region in mESCs [Tcl1a expression, suggesting their direct regulation of Tcl1a transcription [Most of the evidence on TCL1A’s expression and transcriptional regulation in embryonic tissues derives from murine cells. ne (Myc) ,82. In al1a gene . Furtherin mESCs . Knockdocription ,71,82. Tcription ,42,45. Scription .TCL1A transcription, its 5′-promoter region contains a TATA box with cis-regulatory elements for several TFs expressed in somatic cells [Next to stem-cell factors regulating ic cells . These iic cells .TCL1A core promoter by binding to three sites within its first 150 bp [TCL1A expression does not seem to be dependent on mechanisms involving methylation of SP1 sites, as these were consistently un- or hypo-methylated in B-lymphoma cell lines of TCL1A-negative status. Furthermore, no differences in SP1 expression were seen in TCL1A-positive vs. negative cell lines [TCL1A gene expression [SP1 mediates transactivation of the t 150 bp . Howeverll lines . Therefopression ,84.TCL1A promoter. Overall, three discernable patterns of CpG DNA methylation in the promoter in TCL1A-silenced B-cell lines were noted, being methylations of only the CpGs in 5′-flanking regions, methylations of CpGs in 5′- and 3′-regions, and methylations spanning the whole promoter. Treatment with an inhibitor of DNA methylation, 5-azacytidine, was able to restore the expression of TCL1A in these cell lines, arguing for epigenetic regulation of TCL1A promoter repression [One explanation could be a staged progression in CpG methylation of the pression .TCL1A’s transcriptional start site, but most of them are implicated by circumstantial evidence from associative data. Examples are FOXO3 and p53 [TCL1A promoter, resulting in repression of TCL1A transcription [There are also repressive relationships of TFs predicted to bind to  and p53 ,93. A necription . This imTCL1A, with a safeguarding significance during the GC reaction of B cells [TCL1A promoter was identified and its activation was independent of the phosphorylation of CREB but depended on the CREB-regulated transcription coactivator 2 . Interestingly, GC-associated stimulation via CD40 ligand (CD40L)/interleukin 4 (IL4) or via BCR engagement resulted in phosphorylation of CRTC2, leading to its nuclear exclusion and subsequent partial TCL1A repression, while other pCREB/E1A binding protein p300 (EP300)-dependent genes were activated via CREB phosphorylation and EP300 recruitment. However, a reduction in TCL1A levels of only 40% at more than 95% CRTC2 repression implicates that the control of the TCL1A gene in the GC B-cell involves other regulatory levels as well [Next to this negative feedback loop, there is additional B-cell activation-induced repression of  B cells . In fact B cells . A CREB  as well .TCL1A promoter via ChIP-seq analyses [TCL1A, suggesting a positive regulation by this TF [TCL1A and expression of the ETS Variant TF 6 (ETV6) in BPDCN and in B-cell acute lymphoblastic leukemia (B-ALL) implicates an additional mode of TCL1A transcriptional regulation [Next to lymphocytes, TCL1A is also highly expressed in pDCs and in the derived BPDCN . The TF analyses ,96. Knocgulation .TCL1A. A genome-wide association study in women treated with aromatase inhibitors (AIs) for early breast cancer identified the SNP rs11849538 close to the 3′ end of TCL1A that generates an estrogen response element. In cell lines carrying this SNP, an estrogen-dependent expression of TCL1A was suggested [Notably, there is also evidence for the role of single nucleotide polymorphisms (SNPs) in the regulation of uggested ,99. Thisuggested ; howeveruggested .TCL1A with its preserved 3′ and 5′ untranslated regions (UTRs) [Eμ-TCL1A model, where only the human TCL1A open reading frame was overexpressed [Eμ-TCL1A tg mouse [Evidence on the relevance of the contribution of miRs to TCL1A suppression derives from a tg mouse model of full-length s (UTRs) . In contxpressed , this motg mouse .ATM) [TP53) [Several miRs were identified to negatively regulate TCL1A at the posttranscriptional level. MiR-29b-3p and miR-181b-5p, shown to repress TCL1A, inversely correlated in their expression with TCL1A levels across CLL subsets defined by features of clinical aggressiveness . GenomicATM)  and 17p ) [TP53)  and tRNA) [TP53) , respect) [TP53) .MECOM and TCL1A expression in a large cohort of CLL. TCL1A and MECOM showed a strong interactive clinical hazard prediction in prospectively treated patients, suggesting a contribution of the described regulatory circuit to an aggressive cellular and clinical phenotype in CLL [In addition, we identified miR-484 to target the 3′-UTR of TCL1A in CLL . MiR-484e in CLL .TCL1A, NANOG, was also identified to induce transcription of HSP90, thereby mediating a bimodal regulation of TCL1A at the gene and protein level [Evidence on the regulation by posttranslational modifications of the TCL1A protein is sparse. However, one site in TCL1A has been identified that, when phosphorylated potentially by the glycogen synthase kinase-3β (GSK3β), decreases the interaction of TCL1A with its client protein hnRNPK . Furtherin level .+ proliferating paraimmunoblasts [What triggers these molecular modes of TCL1A (dys)regulation as histogenetically driven (incl. differentiation-associated) programs? Several publications suggest the role of micromilieu-derived stimuli in regulating TCL1A levels in B-cell tumors. In sections of CLL and other B-cell tumors in lymph nodes, spleens, and bone marrow, and as mimicked in stimulated suspension cultures, strong TCL1A expression in the resting cells was paralleled by near-complete losses of TCL1A protein in the fraction of Ki67noblasts ,28. Thisnoblasts ,85. BaseTCL1A was among the top genes upregulated in CLL cells by cocultures on BMSC. Stroma-mediated increases in TCL1A were also associated with decreased levels of TCL1A-repressive miRs  [As another source of TCL1A-regulating milieu-derived stimuli, direct cell–cell contact of leukemia with bone marrow stromal cells (BMSCs) was identified . In contmiR-484) . These fThe regulation of TCL1A seems perturbed also in the context of B-cellular Epstein–Barr virus (EBV) infection. The majority of EBV-infected B-cell non-Hodgkin lymphomas (B-NHL) appear to be positive for TCL1A to a higher degree than their EBV-negative counterparts ,107. In However, there is also contradicting data for a repressive influence of EBV on TCL1A expression. EBV infection of DLBCL cell lines in vitro reduced TCL1A levels in an EBNA2-dependent manner . FurtherEBNA1, besides some non-coding genes. In contrast, proteins negatively regulating TCL1A expression are expressed in latency II and III profiles. Accordingly, most cell lines that showed an upregulation of TCL1A after infection in vitro expressed a type I latency. Second, the cell of origin might determine to which extend the expression of TCL1A can be manipulated by EBV infection. As discussed above, epigenetic methylation of the TCL1A promoter can be a mechanism by which the TCL1A gene is silenced during B-cell differentiation. EBV infection may be unable to counteract a strong epigenetic silencing in some cell types. This could explain why post-GC derived, TCL1A-negative primary effusion lymphomas (PEL) did not upregulate TCL1A upon EBV infection [Several possible reasons might, at least in part, explain these contradicting results, besides the general limitations of artificial EBV introduction into cell lines. First, the expression of certain EBV gene products is restricted to a specific latency pattern of EBV infection, which, in turn, is strongly impacted by the host immune competence. Cells with a latency I profile only express nfection , whereasnfection ,88,106. From the data on the impact of EBV on TCL1A levels, we postulate that generally, TCL1A expression is also influenced by severe immune dysfunction. In alignment with the findings of a TCL1A-repressive impact by T-cell-derived CD40L/IL4 stimuli or by BCR signals see  12,28,8,828,84, The adaptor protein TCL1A has vital functions in reproduction, development, and adaptive immunity. Identification of its overexpression and oncogenic role in lymphatic and in part in other tumors, particularly in T-PLL, CLL, and BPDCN, has established its diagnostic and prognostic marker properties ,114. In TCL1A gene, dysregulation of TCL1A-targeting miRs, but also chaperone-mediated protection from protein degradation were identified. Additionally, potential phosphorylation sites have been identified in murine and human TCL1A; however, their role in the function and regulation of the protein is still uncertain [This review provides a comprehensive overview of the different modes of (dys)regulation of this prototypic member of the TCL1 oncogene family. Tight regulation of TCL1A’s timely silencing is important, given the multiple oncogenic functions of TCL1A in mature lymphocytes and likely in other cell lineages as well. Mechanistic models of pathogenic TCL1A upregulation were first restricted to genomic translocations that involve its gene locus, as shown in T-PLL. However, to date, multi-faceted ways of TCL1A (dys)regulation have been identified, mostly by data from B-cell malignancies. In addition to promoter hypomethylation of the ncertain ,116.Exogeneous triggers of such TCL1A (dys)regulation in malignant B cells originate from the microenvironment. TCL1A suppressive signals from T-cell-derived  factors or upregulation of TCL1A in CLL cells in the bone marrow niche via cell–cell contacts with BMSC represent relevant influxes. However, the (disturbed) homeostasis between these suppressive vs. activating impacts on TCL1A levels is still inadequately addressed. A better characterization of these specific sources of TCL1A regulation and their molecular executions within a comprehensive regulatory network of TCL1A could be of benefit considering the increasing application of inhibitors disrupting the crosstalk of lymphatic tumor cells with their microenvironment.NANOG, OCT3/4, MYC, etc., during epithelial cell transformation. Although there is some evidence for a prognostic impact of TCL1A expression [In solid tumors, overexpression of TCL1A might originate from the reactivation of an embryonic program, including expression of pression , its funTCL1A rearrangements in T-PLL [TCL1A-tg mouse models [For several lymphatic tumors, TCL1A’s transforming capacity is firmly established by the highly recurrent genomic in T-PLL ,46 and be models ,16. HoweAs high TCL1A expression correlates with an inferior clinical outcome, an effect of TCL1A on tumor sustenance is likely, and its impact on several different oncogenic pathways makes it an appealing target. However, as a small adaptor molecule without catalytic domains, its pharmaceutical targeting is very challenging. New avenues in such specific therapeutic interventions would include the profiling for TCL1A dimerization inhibitors or non-peptide chemotypes that intercept in TCL1A-complex formation. It is also highly intriguing that TCL1A peptide sequences (TCL1A71-78 LLPIMWQL) were identified as an HLA-A*0201 binding T-cell epitope . TCL1A71In this review, we summarized the current knowledge on the oncogenic function and transcriptional and translational regulation of the TCL1A proto-oncogene, resulting in a mechanistic concept of its context-dependent transforming capacities and multiple modes of (dys)regulation . This im