The human adenovirus E1B-55K oncoprotein coordinates cell transformation through regulation of DNA-bound host transcription factors.

The multifunctional adenovirus E1B-55K oncoprotein can induce cell transformation in conjunction with adenovirus E1A gene products. Previous data from transient expression studies and in vitro experiments suggest that these growth-promoting activities correlate with E1B-55K-mediated transcriptional repression of p53-targeted genes. Here, we analyzed genome-wide occupancies and transcriptional consequences of species C5 and A12 E1B-55Ks in transformed mammalian cells by combinatory ChIP and RNA-seq analyses. E1B-55K-mediated repression correlates with tethering of the viral oncoprotein to p53-dependent promoters via DNA-bound p53. Moreover, we found that E1B-55K also interacts with and represses transcription of numerous p53-independent genes through interactions with transcription factors that play central roles in cancer and stress signaling. Our results demonstrate that E1B-55K oncoproteins function as promiscuous transcriptional repressors of both p53-dependent and -independent genes and further support the model that manipulation of cellular transcription is central to adenovirus-induced cell transformation and oncogenesis.

The APOBEC3B cytidine deaminase is an adenovirus restriction factor.

Human adenoviruses (HAdVs) are a large family of DNA viruses counting more than a hundred strains divided into seven species (A to G). HAdVs induce respiratory tract infections, gastroenteritis and conjunctivitis. APOBEC3B is a cytidine deaminase that restricts several DNA viruses. APOBEC3B is also implicated in numerous cancers where it is responsible for the introduction of clustered mutations into the cellular genome. In this study, we demonstrate that APOBEC3B is an adenovirus restriction factor acting through a deaminase-dependent mechanism. APOBEC3B introduces C-to-T clustered mutations into the adenovirus genome. APOBEC3B reduces the propagation of adenoviruses by limiting viral genome replication, progression to late phase, and production of infectious virions. APOBEC3B restriction efficiency varies between adenoviral strains, the A12 strain being more sensitive to APOBEC3B than the B3 or C2 strains. In A12-infected cells, APOBEC3B clusters in the viral replication centers. Importantly, we show that adenovirus infection leads to a reduction of the quantity and/or enzymatic activity of the APOBEC3B protein depending on the strains. The A12 strain seems less able to resist APOBEC3B than the B3 or C2 strains, a characteristic which could explain the strong depletion of the APOBEC3-targeted motifs in the A12 genome. These findings suggest that adenoviruses evolved different mechanisms to antagonize APOBEC3B. Elucidating these mechanisms could benefit the design of cancer treatments. This study also identifies adenoviruses as triggers of the APOBEC3B-mediated innate response. The involvement of certain adenoviral strains in the genesis of the APOBEC3 mutational signature observed in tumors deserves further study.

Targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells in vivo using the engineered AVID adenovirus vector platform.

Targeted delivery and cell-type-specific expression of gene-editing proteins in various cell types in vivo represent major challenges for all viral and non-viral delivery platforms developed to date. Here, we describe the development and analysis of artificial vectors for intravascular delivery (AVIDs), an engineered adenovirus-based gene delivery platform that allows for highly targeted, safe, and efficient gene delivery to human hematopoietic stem and progenitor cells (HSPCs) in vivo after intravenous vector administration. Due to a set of refined structural modifications, intravenous administration of AVIDs did not trigger cytokine storm, hepatotoxicity, or thrombocytopenia. Single intravenous administration of AVIDs to humanized mice, grafted with human CD34+ cells, led to up to 20% transduction of CD34+CD38-CD45RA- HSPC subsets in the bone marrow. Importantly, targeted in vivo transduction of CD34+CD38-CD45RA-CD90-CD49f+ subsets, highly enriched for human hematopoietic stem cells (HSCs), reached up to 19%, which represented a 1,900-fold selectivity in gene delivery to HSC-enriched over lineage-committed CD34-negative cell populations. Because the AVID platform allows for regulated, cell-type-specific expression of gene-editing technologies as well as expression of immunomodulatory proteins to ensure persistence of corrected HSCs in vivo, the HSC-targeted AVID platform may enable development of curative therapies through in vivo gene correction in human HSCs after a single intravenous administration.

Long-term activation of anti-tumor immunity in pancreatic cancer by a p53-expressing telomerase-specific oncolytic adenovirus.

BACKGROUND: Pancreatic cancer is an aggressive, immunologically "cold" tumor. Oncolytic virotherapy is a promising treatment to overcome this problem. We developed a telomerase-specific oncolytic adenovirus armed with p53 gene (OBP-702). METHODS: We investigated the efficacy of OBP-702 for pancreatic cancer, focusing on its long-term effects via long-lived memory CD8 + T cells including tissue-resident memory T cells (TRMs) and effector memory T cells (TEMs) differentiated from effector memory precursor cells (TEMps). RESULTS: First, in vitro, OBP-702 significantly induced adenosine triphosphate (ATP), which is important for memory T cell establishment. Next, in vivo, OBP-702 local treatment to murine pancreatic PAN02 tumors increased TEMps via ATP induction from tumors and IL-15Rα induction from macrophages, leading to TRM and TEM induction. Activation of these memory T cells by OBP-702 was also maintained in combination with gemcitabine+nab-paclitaxel (GN) in a PAN02 bilateral tumor model, and GN + OBP-702 showed significant anti-tumor effects and increased TRMs in OBP-702-uninjected tumors. Finally, in a neoadjuvant model, in which PAN02 cells were re-inoculated after resection of treated-PAN02 tumors, GN + OBP-702 provided long-term anti-tumor effects even after tumor resection. CONCLUSION: OBP-702 can be a long-term immunostimulant with sustained anti-tumor effects on immunologically cold pancreatic cancer.

Enforced Dimerization of CD45 by the Adenovirus E3/49K Protein Inhibits T Cell Receptor Signaling.

Human adenoviruses (HAdVs) are widespread pathogens that generally cause mild infections in immunocompetent individuals but severe or even fatal diseases in immunocompromised patients. In order to counteract the host immune defenses, HAdVs encode various immunomodulatory proteins in the early transcription unit 3 (E3). The E3/49K protein is a highly glycosylated type I transmembrane protein uniquely expressed by species D HAdVs. Its N-terminal ectodomain sec49K is released by metalloprotease-mediated shedding at the cell surface and binds to the receptor-like protein tyrosine phosphatase CD45, a critical regulator of leukocyte activation and functions. It remained elusive which domains of CD45 and E3/49K are involved in the interaction and whether such an interaction can also occur on the cell surface with membrane-anchored full-length E3/49K. Here, we show that the two extracellular domains R1 and R2 of E3/49K bind to the same site in the domain d3 of CD45. This interaction enforces the dimerization of CD45, causing the inhibition of T cell receptor signaling. Intriguingly, the membrane-anchored E3/49K appears to be designed like a "molecular fishing rod" using an extended disordered region of E3/49K as a "fishing line" to bridge the distance between the plasma membrane of infected cells and the CD45 binding site on T cells to effectively position the domains R1 and R2 as baits for CD45 binding. This design strongly suggests that both secreted sec49K as well as membrane-anchored full-length E3/49K have immunomodulatory functions. The forced dimerization of CD45 may be applied as a therapeutic strategy in chronic inflammatory disorders and cancer. IMPORTANCE The battle between viruses and their hosts is an ongoing arms race. Whereas the host tries to detect and eliminate the virus, the latter counteracts such antiviral measures to replicate and spread. Adenoviruses have evolved various mechanisms to evade the human immune response. The E3/49K protein of species D adenoviruses mediates the inhibition of immune cell function via binding to the protein tyrosine phosphatase CD45. Here, we show that E3/49K triggers the dimerization of CD45 and thereby inhibits its phosphatase activity. Intriguingly, the membrane-anchored E3/49K seems to be designed like a "molecular fishing rod" with the two CD45 binding domains of E3/49K as baits positioned at the end of an extended disordered region reminiscent of a fishing line. The adenoviral strategy to inhibit CD45 activity by forced dimerization may be used for therapeutic intervention in autoimmune diseases or to prevent graft rejection after transplantation.

Targeting Cell Cycle Facilitates E1A-Independent Adenoviral Replication.

DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus. Our analyses of this issue revealed that in particular inhibition of cyclin-dependent kinases 4/6 (CDK4/6i) increased E1-independent adenovirus E2-expression and viral DNA replication. Detailed analysis of the E2-expression in dl70-3 infected cells by RT-qPCR showed that the increase in E2-expression originated from the E2-early promoter. Mutations of the two E2F-binding sites in the E2-early promoter (pE2early-LucM) caused a significant reduction in E2-early promoter activity in trans-activation assays. Accordingly, mutations of the E2F-binding sites in the E2-early promoter in a virus named dl70-3/E2Fm completely abolished CDK4/6i induced viral DNA replication. Thus, our data show that E2F-binding sites in the E2-early promoter are crucial for E1A independent adenoviral DNA replication of E1-deleted vectors in cancer cells. IMPORTANCE E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells. Here, we report, that the two E2F-binding sites in the adenoviral E2-early promoter contribute substantially to the so-called E1A-like activity in tumor cells. With this finding, on the one hand, the safety profile of viral vaccine vectors can be increased and, on the other hand, the oncolytic property for cancer therapy might be improved through targeted manipulation of the host cell.

TBK1 is part of a galectin 8 dependent membrane damage recognition complex and drives autophagy upon Adenovirus endosomal escape.

Intracellular pathogens cause membrane distortion and damage as they enter host cells. Cells perceive these membrane alterations as danger signals and respond by activating autophagy. This response has primarily been studied during bacterial invasion, and only rarely in viral infections. Here, we investigate the cellular response to membrane damage during adenoviral entry. Adenoviruses and their vector derivatives, that are an important vaccine platform against SARS-CoV-2, enter the host cell by endocytosis followed by lysis of the endosomal membrane. We previously showed that cells mount a locally confined autophagy response at the site of endosomal membrane lysis. Here we describe the mechanism of autophagy induction: endosomal membrane damage activates the kinase TBK1 that accumulates in its phosphorylated form at the penetration site. Activation and recruitment of TBK1 require detection of membrane damage by galectin 8 but occur independently of classical autophagy receptors or functional autophagy. Instead, TBK1 itself promotes subsequent autophagy that adenoviruses need to take control of. Deletion of TBK1 reduces LC3 lipidation during adenovirus infection and restores the infectivity of an adenovirus mutant that is restricted by autophagy. By comparing adenovirus-induced membrane damage to sterile lysosomal damage, we implicate TBK1 in the response to a broader range of types of membrane damage. Our study thus highlights an important role for TBK1 in the cellular response to adenoviral endosome penetration and places TBK1 early in the pathway leading to autophagy in response to membrane damage.

Impact of MicroRNA Levels, Target-Site Complementarity, and Cooperativity on Competing Endogenous RNA-Regulated Gene Expression.

Expression changes of competing endogenous RNAs (ceRNAs) have been proposed to influence microRNA (miRNA) activity and thereby regulate other transcripts containing miRNA-binding sites. Here, we find that although miRNA levels define the extent of repression, they have little effect on the magnitude of the ceRNA expression change required to observe derepression. Canonical 6-nt sites, which typically mediate modest repression, can nonetheless compete for miRNA binding, with potency ∼20% of that observed for canonical 8-nt sites. In aggregate, low-affinity/background sites also contribute to competition. Sites with extensive additional complementarity can appear as more potent, but only because they induce miRNA degradation. Cooperative binding of proximal sites for the same or different miRNAs does increase potency. These results provide quantitative insights into the stoichiometric relationship between miRNAs and target abundance, target-site spacing, and affinity requirements for ceRNA-mediated gene regulation, and the unusual circumstances in which ceRNA-mediated gene regulation might be observed.

Adenovirus prevents dsRNA formation by promoting efficient splicing of viral RNA.

Eukaryotic cells recognize intracellular pathogens through pattern recognition receptors, including sensors of aberrant nucleic acid structures. Sensors of double-stranded RNA (dsRNA) are known to detect replication intermediates of RNA viruses. It has long been suggested that annealing of mRNA from symmetrical transcription of both top and bottom strands of DNA virus genomes can produce dsRNA during infection. Supporting this hypothesis, nearly all DNA viruses encode inhibitors of dsRNA-recognition pathways. However, direct evidence that DNA viruses produce dsRNA is lacking. Contrary to dogma, we show that the nuclear-replicating DNA virus adenovirus (AdV) does not produce detectable levels of dsRNA during infection. In contrast, abundant dsRNA is detected within the nucleus of cells infected with AdV mutants defective for viral RNA processing. In the presence of nuclear dsRNA, the cytoplasmic dsRNA sensor PKR is relocalized and activated within the nucleus. Accumulation of viral dsRNA occurs in the late phase of infection, when unspliced viral transcripts form intron/exon base pairs between top and bottom strand transcripts. We propose that DNA viruses actively limit dsRNA formation by promoting efficient splicing and mRNA processing, thus avoiding detection and restriction by host innate immune sensors of pathogenic nucleic acids.

Adenovirus-vectored vaccine containing multidimensionally conserved parts of the HIV proteome is immunogenic in rhesus macaques.

An effective vaccine that can protect against HIV infection does not exist. A major reason why a vaccine is not available is the high mutability of the virus, which enables it to evolve mutations that can evade human immune responses. This challenge is exacerbated by the ability of the virus to evolve compensatory mutations that can partially restore the fitness cost of immune-evading mutations. Based on the fitness landscapes of HIV proteins that account for the effects of coupled mutations, we designed a single long peptide immunogen comprising parts of the HIV proteome wherein mutations are likely to be deleterious regardless of the sequence of the rest of the viral protein. This immunogen was then stably expressed in adenovirus vectors that are currently in clinical development. Macaques immunized with these vaccine constructs exhibited T-cell responses that were comparable in magnitude to animals immunized with adenovirus vectors with whole HIV protein inserts. Moreover, the T-cell responses in immunized macaques strongly targeted regions contained in our immunogen. These results suggest that further studies aimed toward using our vaccine construct for HIV prophylaxis and cure are warranted.

Adenovirus-mediated Overexpression of FcγRIIB Attenuates Pulmonary Inflammation and Fibrosis.

Progressive fibrosing interstitial lung diseases (PF-ILDs) result in high mortality and lack effective therapies. The pathogenesis of PF-ILDs involves macrophages driving inflammation and irreversible fibrosis. Fc-γ receptors (FcγRs) regulate macrophages and inflammation, but their roles in PF-ILDs remain unclear. We characterized the expression of FcγRs and found upregulated FcγRIIB in human and mouse lungs after exposure to silica. FcγRIIB deficiency aggravated lung dysfunction, inflammation, and fibrosis in silica-exposed mice. Using single-cell transcriptomics and in vitro experiments, FcγRIIB was found in alveolar macrophages, where it regulated the expression of fibrosis-related genes Spp1 and Ctss. In mice with macrophage-specific overexpression of FcγRIIB and in mice treated with adenovirus by intratracheal instillation to upregulate FcγRIIB, silica-induced functional and histological changes were ameliorated. Our data from three genetic models and a therapeutic model suggest that FcγRIIB plays a protective role that can be enhanced by adenoviral overexpression, representing a potential therapeutic strategy for PF-ILDs.

Adenovirus entry: Stability, uncoating, and nuclear import.

Adenoviruses (AdVs) are widespread in vertebrates. They infect the respiratory and gastrointestinal tracts, the eyes, heart, liver, and kidney, and are lethal to immunosuppressed people. Mastadenoviruses infecting mammals comprise several hundred different types, and many specifically infect humans. Human adenoviruses are the most widely used vectors in clinical applications, including cancer treatment and COVID-19 vaccination. AdV vectors are physically and genetically stable and generally safe in humans. The particles have an icosahedral coat and a nucleoprotein core with a DNA genome. We describe the concept of AdV cell entry and highlight recent advances in cytoplasmic transport, uncoating, and nuclear import of the viral DNA. We highlight a recently discovered "linchpin" function of the virion protein V ensuring cytoplasmic particle stability, which is relaxed at the nuclear pore complex by cues from the E3 ubiquitin ligase Mind bomb 1 (MIB1) and the proteasome triggering disruption. Capsid disruption by kinesin motor proteins and microtubules exposes the linchpin and renders protein V a target for MIB1 ubiquitination, which dissociates V from viral DNA and enhances DNA nuclear import. These advances uncover mechanisms controlling capsid stability and premature uncoating and provide insight into nuclear transport of nucleic acids.

Adenovirus-mediated effects of Wnt and Notch signalling pathways on hair cell regeneration in mice.

Some genes are delivered to cochleae by adenoviruses to restore partial hearing function. This provides promising prospects for gene therapies for hearing loss from hair cell damage. To study the adenovirus (AD)-mediated effect of the Wnt and Notch signalling pathways on hair cell regeneration in the mouse cochlea, we constructed a ß-catenin-adenovirus (ß-catenin-AD) to increase the activity of the Wnt signalling pathway and a NICD (intracellular domain of Notch1)-RNAi-adenovirus to decrease the activity of the Notch signalling pathway (NICD-RNAi-AD). Our study indicated that approximately 40% of supporting cells in the cochleae damaged by gentamicin were infected with the adenoviruses. Following the ß-catenin-AD-mediated increase in Wnt signalling pathway activity, mitotic regeneration was increased, while direct transdifferentiation was increased after the NICD-RNAi-AD-mediated decrease in Notch signalling pathway activity. The expected synergistic interaction on hair cell regeneration was not obtained after coinfection of ß-catenin-AD and NICD-RNAi-AD into the damaged cochleae, which might be due to the low cotransfection efficiency to supporting cells. Our study indicated that it may be possible to develop AD mediated gene therapies for hearing loss that act by regulating the Wnt and Notch signalling pathways.

Mouse Adenovirus Type 1 E4orf6 Induces PKR Degradation.

Protein kinase R (PKR) is a cellular kinase involved in the antiviral response. The inactivation or inhibition of this protein is a conserved activity in DNA and RNA virus infections. In contrast to human adenovirus type 5, mouse adenovirus type 1 (MAV-1) inhibits PKR activity through proteasome-dependent degradation. However, the molecular mechanism by which this process takes place is not fully understood. We investigated whether ubiquitination, MAV-1 early region 1B 55k (E1B 55k), and early region 4 orf6 (E4orf6) play a role in PKR degradation in MAV-1 infection, because the enzyme 3 (E3) ubiquitin ligase activity with these viral proteins is conserved among the Adenoviridae family. We provide evidence that E4orf6 is sufficient to induce mouse PKR degradation and that proteasome pathway inhibition blocks PKR degradation. Inhibition of neddylation of cullin, a component of E3 ubiquitin ligase complex, blocked efficient PKR degradation in MAV-1-infected cells. Finally, we demonstrated that MAV-1 degradation of PKR is specific for mouse PKR. These results indicate that counteracting PKR is mechanistically different in two species of adenoviruses. IMPORTANCE Viruses have evolved to counteract the immune system to successfully replicate in the host. Downregulation of several antiviral proteins is important for productive viral infection. Protein kinase R (PKR) is an antiviral protein that belongs to the first line of defense of the host. Because PKR senses dsRNA and blocks the cellular translation process during viral infections, it is not surprising that many viruses counteract this antiviral activity. We previously reported PKR degradation during mouse adenovirus type 1 (MAV-1) infection; however, the molecular mechanism of this activity was not fully known. This work provides evidence about the MAV-1 protein that induces PKR degradation and expands knowledge about involvement of the proteasome pathway.

The RGD-binding integrins αvß6 and αvß8 are receptors for mouse adenovirus-1 and -3 infection.

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvß6 and αvß8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mß6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvß6-positive CMT-93 cells, whereas mß8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvß8-positive M000216 cells. Soluble integrin αvß6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvß6/αvß8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvß6/ß8, where the distal leucine residue dips into a hydrophobic pocket of ß6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvß6/ß8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvß6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission.

Fowl adenovirus serotype 4 enters leghorn male hepatocellular cells via the clathrin-mediated endocytosis pathway.

Hepatitis-hydropericardium syndrome (HHS) induced by fowl adenovirus serotype-4 (FAdV-4) has caused large economic losses to the world poultry industry in recent years. HHS is characterized by pericardial effusion and hepatitis, manifesting as a swollen liver with focal necroses and petechial haemorrhage. However, the process of FAdV-4 entry into hepatic cells remains largely unknown. In this paper, we present a comprehensive study on the entry mechanism of FAdV-4 into leghorn male hepatocellular (LMH) cells. We first observed that FAdV-4 internalization was inhibited by chlorpromazine and clathrin heavy chain (CHC) knockdown, suggesting that FAdV-4 entry into LMH cells depended on clathrin. By using the inhibitor dynasore, we showed that dynamin was required for FAdV-4 entry. In addition, we found that FAdV-4 entry was dependent on membrane cholesterol, while neither the knockdown of caveolin nor the inhibition of a tyrosine kinase-based signalling cascade affected FAdV-4 infection. These results suggested that FAdV-4 entry required cholesterol but not caveolae. We also found that macropinocytosis played a role, and phosphatidylinositol 3-kinase (PI3K) was required for FAdV-4 internalization. However, inhibitors of endosomal acidification did not prevent FAdV-4 entry. Taken together, our findings demonstrate that FAdV-4 enters LMH cells through dynamin- and cholesterol-dependent clathrin-mediated endocytosis, accompanied by the involvement of macropinocytosis requiring PI3K. Our work potentially provides insight into the entry mechanisms of other avian adenoviruses.

Adenovirus-mediated SIRT1 protects cochlear strial marginal cells in a D-gal-induced senescent model in vitro.

BACKGROUND: A primary obstacle in age-related hearing loss (ARHL) study is the lack of accelerated senescent models in vitro that explore the precise underlying mechanism in different types of ARHL. The damage to strial marginal cells (SMCs) is a subset of strial presbycusis-associated pathological changes. We aimed to establish a D-galactose (D-gal)-induced SMCs senescent model and study the effect of deacetylase sirtuin 1 (SIRT1) on presbycusis in vitro. METHODS: SMCs from C57BL/6J neonatal mice were cultured and treated with D-gal to establish accelerated senescent models. And then D-gal-induced SMCs were transfected with adenovirus (Ad)-SIRT1-GFP or Ad-GFP. Oxidative stress and mitochondrial DNA (mtDNA) damage were determined by histological analysis or RT-PCR. Western blotting (WB) and RT-PCR were used to evaluate protein and mRNA levels of superoxide dismutase 2 (SOD2) and SIRT1, respectively. Additionally, apoptosis was investigated by WB and TUNEL staining. RESULTS: D-gal-induced SMCs exhibited several characteristics of senescence, including increased the level of 8-hydroxy-2'-deoxyguanosine, which is a marker of DNA oxidative damage, and elevated the amount of mtDNA 3860-bp deletion, which is a common type of mtDNA damage in the auditory system of mice. SIRT1 overexpression effectively inhibited these changes by upregulating the level of SOD2, thereby inhibiting cytochrome c translocation from mitochondria to cytoplasm, inhibiting cell apoptosis, and ultimately delaying aging in the D-gal-induced senescent SMCs. CONCLUSIONS: Altogether, the evidence suggests that the D-gal-induced SMCs accelerated aging model is successfully established, and SIRT1 overexpression protects SMCs against oxidative stress by enhancing SOD2 expression in ARHL.

Adenovirus-IL-10 relieves chronic rejection after mouse heart transplantation by inhibiting miR-155 and activating SOCS5.

Objective: Chronic rejection remains the main factor that influence long-term survival of patients after heart transplantation. Interleukin-10 (IL-10) play critical role in macrophages-mediated transplant immune responses. We investigated the mechanism of IL-10 in macrophage related chronic rejection after mouse heart transplantation. Methods: Mouse heart transplant chronic rejection model was established to evaluate pathological changes in the allograft. Myocardial interstitial fibrosis, apoptosis, and inflammatory factor levels were detected in ad-IL-10-treated mice. The positive iNOS+ and Arg-1+ expressions, macrophage subset changes, and the proportion of regulatory T-cells (Tregs) and TIGIT+ Tregs were quantified by flow. In in vitro experiments, ad-IL-10 was transfected into macrophages followed by detection of apoptosis, phagocytosis, and CD163, CD16/32, and CD206 expression. The expression and relationships between IL-10, miR-155, and SOCS5 were also detected and verified. A rescue experiment was performed to evaluate macrophage function through the combined treatment of ad-IL-10 and overexpression of miR-155. Results: Significantly decreased IL-10 expression in chronic rejection during mouse heart transplantation was observed. Ad-IL-10-treated mice showed decreased pathological injury, perivascular fibrosis, apoptosis, inflammation, and iNOS+ and CD16/32+ expression, and increased Treg/TIGIT+ Treg cell, Arg-1+ and CD206+ cell proportion. Ad-IL-10-treated macrophages in vitro showed reduced apoptosis, improved phagocytosis, and M2 polarization. Mechanically, IL-10 negatively regulated miR-155 to activate SOCS5. Overexpression of miR-155 reversed IL-10 mediated-positive regulation of macrophage function. Conclusion: IL-10 downregulated miR-155 and activated SOCS5, thereby promoting macrophage M2 polarization to relieve chronic rejection after heart transplantation.

Efficacy of Adenovirus-mediated, miR-199a Nanoparticles on Expression of HSP27, HSP70, and Soluble Glycoprotein in Rats with Heart Failure.

Context: Heart failure (HF) refers to abnormal changes in the function of the body's heart pump under the action of a variety of pathogenic factors. Due to the complex etiology and course of HF, current research on its etiology and pathogenesis hasn't yet reached a clear conclusion. So, there are many manifestations of heart failure in patients, and there are also many changes in the treatment. Objectives: The study intended to evaluate the efficacy of adenovirus-mediated miR-199a nanoparticles (NPs) for heart failure (HF). Design: The research team performed an animal study. Setting: The study took place at Shanghai Pudong Hospital at Fudan University Pudong Medical Center in Shanghai, China. Animals: The animals were 40 healthy, adult, male, Sprague-Dawley (SD) rats. They were specific pathogen-free (SPF) grade SD rats, all weighing about 280 g and aged 7-8 weeks. Intervention: The research team: (1) induced HF using coronary artery ligation and established different HF models and (2) randomly divided the rats into two groups with 20 rats in each group-an experimental group, which received high-dose, microR-199a (miR-199a) NPs, and a control group, which received low-dose miR-199a NPs. The treatments occurred for seven days after the induction of HF. Outcome Measures: At baseline and postintervention, the research team measured the left ventricular ejection fraction (LVEF), left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), diastolic and systolic left ventricular anterior wall (LVAW) thickness, left ventricular posterior wall (LVPW) thickness, and expression of heat shock protein 27 (HSP27), HSP70, soluble glycoprotein 130 (SGP130). The team analyzed and studied the effects of the adenovirus-mediated miR-199a NP on that expression, based on the above indicators. Results: The miR-199a prepared with NPs had good specificity through observation. The expression of HSP27, SGP130 was significantly downregulated in the experimental group as compared to the control group (P < .05) and HSP70 was upregulated in the experimental group as compared to the control group (P < .05). The expression decreased, or increased, with an increase in the cardiac-function classification, with substantial differences between the control and experimental groups. Expression levels of HSP27, HSP70, and SGP in the experimental group were negatively correlated with those of controls and negatively correlated with the left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), and left ventricular ejection fraction (LVEF). Conclusions: NP had good specificity. The miR-199a NP downregulated levels of HSP, which had a certain protective effect against HF and had a high clinical-adoption and promotion value.

[An Important Problem in AdV-36 Seropositive Obesity Patients: Leptin Resistance]. / AdV-36 Seropozitif Olan Obez Hastalarda Önemli Bir Sorun: Leptin Direnci.

Adenoviruses are naked viruses with an icosahedral nucleocapsid containing a 36 kb linear double-stranded DNA genome that encodes 30-40 proteins. The word "obesity" in Latin means "because of feeding". Obesity is an energy metabolism pathology that paves the way for physical and psychological problems with excessive fat accumulation that can impair health. Body mass index (BMI), unaffected by gender and age, is the most useful indicator of overweight and obesity at the population level. The concept of infectobesity was first introduced in 1978 after the data showed that viruses might also play a role in obesity cases. In the same year, adenovirus 36 (AdV-36) was isolated from the stool of a six-year-old girl with diabetes who was admitted to the hospital with the complaint of enteritis. One of the adipokines important for obesity is leptin. Leptin regulates food intake and energy metabolism by having a "negative feedback" effect on the hypothalamus. Leptin acts as a sensor that acutely regulates energy metabolism by creating hunger and satiety signals and it also regulates the amount of body fat and the required weight of the person by adjusting its concentration in the plasma according to the nutritional status. Changes in body weight and metabolic status are often associated with acute or chronic inflammatory processes. Human cells infected by AdV-36 showed greater differentiation and higher lipid accumulation than uninfected control cells, which increases the prevalence of obesity. There are two fractions of serum leptin, protein-bound and free form. The balance between these two fractions depends on serum leptin and soluble leptin receptor (sLR) plasma concentration, which is adversely affected by BMI. AdV-36 infection reduces norepinephrine and leptin levels. These two effects contribute to obesity by increasing appetite and food intake. In this study, it was aimed to determine the presence of immunoglobulin G against AdV-36 in the blood serum of obesity patients (BMI≥ 30) and healthy weight individuals (18.5≤ BMI≤ 25), and also aimed to determine and compare the leptin and soluble leptin receptor levels of these individuals. In this study, 10 ml of blood was collected on an empty stomach from obese individuals (n= 101; BMI≥ 30) and healthy individuals (n= 96; 18.5≤ BMI≤ 25) between the ages of 18-55. All participants consisted of who were not taking any medication and were not immunosuppressed. Blood samples separated into their serum were analyzed for AdV-36 IgG, leptin, and soluble leptin receptor levels. Mean, standard deviation, and percentage values were calculated by descriptive statistical analysis. The data with normal distribution were evaluated with the paired and independent sample t-test and data with abnormal distribution were evaluated with the paired and independent sample Mann-Whitney U test. Findings with a p-value less than 0.05 were considered statistically significant. In conclusion, leptin levels in obese individuals who were not infected with Adv-36 were found to be low, in line with the principle that "insufficient leptin synthesis has a role in the pathophysiology of obesity". When AdV-36 infection is added to the obesity picture, it may be due to the fact that it increases leptin synthesis in patients and the level of soluble leptin receptors increases in response to the increased leptin level, that AdV-36 suppresses the binding of the leptin molecule to its receptor, which leads to leptin resistance.