GCN5 mediates DNA-PKcs crotonylation for DNA double-strand break repair and determining cancer radiosensitivity.

BACKGROUND: DNA double-strand break (DSB) induction and repair are important events for determining cell survival and the outcome of cancer radiotherapy. The DNA-dependent protein kinase (DNA-PK) complex functions at the apex of DSBs repair, and its assembly and activity are strictly regulated by post-translation modifications (PTMs)-associated interactions. However, the PTMs of the catalytic subunit DNA-PKcs and how they affect DNA-PKcs's functions are not fully understood. METHODS: Mass spectrometry analyses were performed to identify the crotonylation sites of DNA-PKcs in response to γ-ray irradiation. Co-immunoprecipitation (Co-IP), western blotting, in vitro crotonylation assays, laser microirradiation assays, in vitro DNA binding assays, in vitro DNA-PK assembly assays and IF assays were employed to confirm the crotonylation, identify the crotonylase and decrotonylase, and elucidate how crotonylation regulates the activity and function of DNA-PKcs. Subcutaneous xenografts of human HeLa GCN5 WT or HeLa GCN5 siRNA cells in BALB/c nude mice were generated and utilized to assess tumor proliferation in vivo after radiotherapy. RESULTS: Here, we reveal that K525 is an important site of DNA-PKcs for crotonylation, and whose level is sharply increased by irradiation. The histone acetyltransferase GCN5 functions as the crotonylase for K525-Kcr, while HDAC3 serves as its dedicated decrotonylase. K525 crotonylation enhances DNA binding activity of DNA-PKcs, and facilitates assembly of the DNA-PK complex. Furthermore, GCN5-mediated K525 crotonylation is indispensable for DNA-PKcs autophosphorylation and the repair of double-strand breaks in the NHEJ pathway. GCN5 suppression significantly sensitizes xenograft tumors of mice to radiotherapy. CONCLUSIONS: Our study defines K525 crotonylation of DNA-PKcs is important for the DNA-PK complex assembly and DSBs repair activity via NHEJ pathway. Targeting GCN5-mediated K525 Kcr of DNA-PKcs may be a promising therapeutic strategy for improving the outcome of cancer radiotherapy.

PARP1 modulates METTL3 promoter chromatin accessibility and associated LPAR5 RNA m6A methylation to control cancer cell radiosensitivity.

Chromatin remodeling and N6-methyladenosine (m6A) modification are two critical layers in controlling gene expression and DNA damage signaling in most eukaryotic bioprocesses. Here, we report that poly(ADP-ribose) polymerase 1 (PARP1) controls the chromatin accessibility of METTL3 to regulate its transcription and subsequent m6A methylation of poly(A)+ RNA in response to DNA damage induced by radiation. The transcription factors nuclear factor I-C (NFIC) and TATA binding protein (TBP) are dependent on PARP1 to access the METTL3 promoter to activate METTL3 transcription. Upon irradiation or PARP1 inhibitor treatment, PARP1 disassociated from METTL3 promoter chromatin, which resulted in attenuated accessibility of NFIC and TBP and, consequently, suppressed METTL3 expression and RNA m6A methylation. Lysophosphatidic Acid Receptor 5 (LPAR5) mRNA was identified as a target of METTL3, and m6A methylation was located at A1881. The level of m6A methylation of LPAR5 significantly decreased, along with METTL3 depression, in cells after irradiation or PARP1 inhibition. Mutation of the LPAR5 A1881 locus in its 3' UTR results in loss of m6A methylation and, consequently, decreased stability of LPAR5 mRNA. METTL3-targeted small-molecule inhibitors depress murine xenograft tumor growth and exhibit a synergistic effect with radiotherapy in vivo. These findings advance our comprehensive understanding of PARP-related biological roles, which may have implications for developing valuable therapeutic strategies for PARP1 inhibitors in oncology.

Exposure to polystyrene nanoplastics induces abnormal activation of innate immunity via the cGAS-STING pathway.

Endogenous immune defenses provide an intrinsic barrier against external entity invasion. Microplastics in the environment, especially those at the nanoscale (nanoplastics or NPs), may pose latent health risks through direct exposure. While links between nanoplastics and inflammatory processes have been established, detailed insights into how they may perturb the innate immune mechanisms remain uncharted. Employing murine and macrophage (RAW264.7) cellular models subjected to polystyrene nanoplastics (PS-NPs), our investigative approach encompassed an array of techniques: Cell Counting Kit-8 assays, flow cytometric analysis, acridine orange/ethidium bromide (AO/EB) fluorescence staining, cell transfection, cell cycle scrutiny, genetic manipulation, messenger RNA expression profiling via quantitative real-time PCR, and protein expression evaluation through western blotting. The results showed that PS-NPs caused RAW264.7 cell apoptosis, leading to cell cycle arrest, and activated the cGAS-STING pathway. This resulted in NF-κB signaling activation and increased pro-inflammatory mediator expression. Importantly, PS-NPs-induced activation of NF-κB and its downstream inflammatory cascade were markedly diminished after the silencing of the STING gene. Our findings highlight the critical role of the cGAS-STING pathway in the immunotoxic effects induced by PS-NPs. We outline a new mechanism whereby nanoplastics may trigger dysregulated innate immune and inflammatory responses via the cGAS/STING pathway.

RNA N6-Methyladenosine Modification in DNA Damage Response and Cancer Radiotherapy.

The N6-methyladenosine (M6A) modification is the most common internal chemical modification of RNA molecules in eukaryotes. This modification can affect mRNA metabolism, regulate RNA transcription, nuclear export, splicing, degradation, and translation, and significantly impact various aspects of physiology and pathobiology. Radiotherapy is the most common method of tumor treatment. Different intrinsic cellular mechanisms affect the response of cells to ionizing radiation (IR) and the effectiveness of cancer radiotherapy. In this review, we summarize and discuss recent advances in understanding the roles and mechanisms of RNA M6A methylation in cellular responses to radiation-induced DNA damage and in determining the outcomes of cancer radiotherapy. Insights into RNA M6A methylation in radiation biology may facilitate the improvement of therapeutic strategies for cancer radiotherapy and radioprotection of normal tissues.

Cytosolic Release of Mitochondrial DNA and Associated cGAS Signaling Mediates Radiation-Induced Hematopoietic Injury of Mice.

Mitochondrion is an important organelle of eukaryotic cells and a critical target of ionizing radiation (IR) outside the nucleus. The biological significance and mechanism of the non-target effect originating from mitochondria have received much attention in the field of radiation biology and protection. In this study, we investigated the effect, role, and radioprotective significance of cytosolic mitochondrial DNA (mtDNA) and its associated cGAS signaling on hematopoietic injury induced by IR in vitro culture cells and in vivo total body irradiated mice in this study. The results demonstrated that γ-ray exposure increases the release of mtDNA into the cytosol to activate cGAS signaling pathway, and the voltage-dependent anion channel (VDAC) may contribute to IR-induced mtDNA release. VDAC1 inhibitor DIDS and cGAS synthetase inhibitor can alleviate bone marrow injury and ameliorate hematopoietic suppression induced by IR via protecting hematopoietic stem cells and adjusting subtype distribution of bone marrow cells, such as attenuating the increase of the F4/80+ macrophage proportion in bone marrow cells. The present study provides a new mechanistic explanation for the radiation non-target effect and an alternative technical strategy for the prevention and treatment of hematopoietic acute radiation syndrome.

LPAR5 confers radioresistance to cancer cells associated with EMT activation via the ERK/Snail pathway.

BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is a critical event contributing to more aggressive phenotypes in cancer cells. EMT is frequently activated in radiation-targeted cells during the course of radiotherapy, which often endows cancers with acquired radioresistance. However, the upstream molecules driving the signaling pathways of radiation-induced EMT have not been fully delineated. METHODS: In this study, RNA-seq-based transcriptome analysis was performed to identify the early responsive genes of HeLa cells to γ-ray irradiation. EMT-associated genes were knocked down by siRNA technology or overexpressed in HeLa cells and A549 cells, and the resulting changes in phenotypes of EMT and radiosensitivity were assessed using qPCR and Western blotting analyses, migration assays, colony-forming ability and apoptosis of flow cytometer assays. RESULTS: Through RNA-seq-based transcriptome analysis, we found that LPAR5 is downregulated in the early response of HeLa cells to γ-ray irradiation. Radiation-induced alterations in LPAR5 expression were further revealed to be a bidirectional dynamic process in HeLa and A549 cells, i.e., the early downregulating phase at 2 ~ 4 h and the late upregulating phase at 24 h post-irradiation. Overexpression of LPAR5 prompts EMT programing and migration of cancer cells. Moreover, increased expression of LPAR5 is significantly associated with IR-induced EMT and confers radioresistance to cancer cells. Knockdown of LPAR5 suppressed IR-induced EMT by attenuating the activation of ERK signaling and downstream Snail, MMP1, and MMP9 expression. CONCLUSIONS: LPAR5 is an important upstream regulator of IR-induced EMT that modulates the ERK/Snail pathway. This study provides further insights into understanding the mechanism of radiation-induced EMT and identifies promising targets for improving the effectiveness of cancer radiation therapy.

MiRNA-155-5p inhibits epithelium-to-mesenchymal transition (EMT) by targeting GSK-3ß during radiation-induced pulmonary fibrosis.

Radiation-induced pulmonary fibrosis (RIPF) is a major lung complication in using radiotherapy to treat thoracic diseases. MicroRNAs (miRNAs) are reported to be the therapeutic targets for many diseases. However, the miRNAs involved in the pathogenesis of RIPF are rarely studied as potential therapeutic targets. Alveolar epithelial cells participate in RIPF formation by undergoing epithelial-mesenchymal transition (EMT). Here we demonstrated the critical role of miR-155-5p in radiation-induced EMT and RIPF. Using the previously established EMT cell model, we found that miR-155-5p was significantly down-regulated through high-throughput sequencing. Irradiation could decrease the expression of miR-155-5p in intro and in vivo, and it was inversely correlated to RIPF formation. Ectopic miR-155-5p expression inhibited radiation-induced-EMT in vitro and in vivo. Knockdown of glycogen synthase kinase-3ß (GSK-3ß), the functional target of miR-155-5p, reversed the induction of EMT and enhanced the phosphorylation of p65, a subunit of NF-κB, which were mediated by the down-regulation of miR-155-5p. Moreover, our finding demonstrated that ectopic miR-155-5p expression alleviated RIPF in mice by the GSK-3ß/NF-κB pathway. Thus, radiation downregulates miR-155-5p in alveolar epithelial cells that induces EMT, which contributes to RIPF using GSK-3ß/NF-κB pathway. Our observation provides further understanding on the regulation of RIPF and identifies potential therapeutic targets.

Radiation-targeted immunotherapy: A new perspective in cancer radiotherapy.

In contemporary oncology, radiation therapy and immunotherapy stand as critical treatments, each with distinct mechanisms and outcomes. Radiation therapy, a key player in cancer management, targets cancer cells by damaging their DNA with ionizing radiation. Its effectiveness is heightened when used alongside other treatments like surgery and chemotherapy. Employing varied radiation types like X-rays, gamma rays, and proton beams, this approach aims to minimize damage to healthy tissue. However, it is not without risks, including potential damage to surrounding normal cells and side effects ranging from skin inflammation to serious long-term complications. Conversely, immunotherapy marks a revolutionary step in cancer treatment, leveraging the body's immune system to target and destroy cancer cells. It manipulates the immune system's specificity and memory, offering a versatile approach either alone or in combination with other treatments. Immunotherapy is known for its targeted action, long-lasting responses, and fewer side effects compared to traditional therapies. The interaction between radiation therapy and immunotherapy is intricate, with potential for both synergistic and antagonistic effects. Their combined use can be more effective than either treatment alone, but careful consideration of timing and sequence is essential. This review explores the impact of various radiation therapy regimens on immunotherapy, focusing on changes in the immune microenvironment, immune protein expression, and epigenetic factors, emphasizing the need for personalized treatment strategies and ongoing research to enhance the efficacy of these combined therapies in cancer care.

Diverse applications and development of aptamer detection technology.

Aptamers have received extensive attention in recent years because of their advantages of high specificity, high sensitivity and low immunogenicity. Aptamers can perform almost all functions of antibodies through the combination of spatial structure and target, which are called "chemical antibodies". At present, aptamers have been widely used in cell imaging, new drug development, disease treatment, microbial detection and other fields. Due to the diversity of modifications, aptamers can be combined with different detection technologies to construct aptasensors. This review focuses on the diversity of aptamers in the field of detection and the development of aptamer-based detection technology and proposes new challenges for aptamers in this field.

Low dose radiation therapy attenuates ACE2 depression and inflammatory cytokines induction by COVID-19 viral spike protein in human bronchial epithelial cells.

Purpose: Low-dose radiation therapy (LDRT) is an evidence-based anti-inflammatory treatment. In anti-COVID-19, our study suggests that low to moderate dose radiation of < 1.5 Gy can inhibit the induction of inflammatory cytokine and attenuate the ACE2 depression induced by spike protein in human bronchial epithelial cells in COVID-19 infection. Our study provided further mechanistic evidence to support LDRT as a cost-effective treatment for COVID-19 to relieve the severe inflammatory reaction and lung injury. Methods and materials: A cellular model was created by treating human bronchial epithelial cells (BEP2D) with SARS-CoV-2 spike protein. We used the qRT-PCR and ELISA analysis to identify the production of inflammatory cytokines. The BEP2D control cells and the spike-treated cells were irradiated using a single low to moderate dose radiation of 0.5 Gy, 1 Gy, and 1.5 Gy. The inflammatory cytokines and ACE2 expression were detected at different time points. Results: The soluble SARS-CoV-2 spike protein stimulated the formation of inflammatory cytokines IL-6 and TNF-α while reducing the ACE2 protein expression in human bronchial epithelial cells. A single low to moderate dose exposure of 0.5 Gy, 1 Gy, and 1.5 Gy could attenuate the IL-6 and TNF-α induction and rescue the depression of ACE2 by spike protein. Moreover, the spike protein increased the proteolytic degradation of ACE2 protein by promoting NEDD4-mediated ubiquitination of ACE2. Conclusions: The low-dose radiation can attenuate ACE2 depression and inflammatory response produced in the targeted human bronchial epithelial cells by spike protein. This coordinating effect of LDRT may relieve the severe inflammatory reaction and lung injury in COVID-19 patients.

The p53/RMRP/miR122 signaling loop promotes epithelial-mesenchymal transition during the development of silica-induced lung fibrosis by activating the notch pathway.

BACKGROUND: Understanding the roles of long noncoding RNAs (lncRNAs) in EMT would help with establishing novel avenues for further uncovering the mechanisms of lung fibrosis and identifying preventative and therapeutic targets. This study aimed to identify silica-induced specific lncRNAs and investigate the feedback loop regulation among their upstream and downstream genes. METHODS AND MATERIALS: A microarray assay, quantitative real-time polymerase chain reaction and Western blot analysis dual-luciferase reporter gene activity and chromatin immunoprecipitation assays were used. Moreover, a silica-induced lung fibrosis mouse model was used to verify the roles of the lncRNAs. RESULTS: Following silica exposure, both RNA component of mitochondrial RNA processing endoribonuclease (RMRP) and p53 were significantly upregulated during the EMT. The upregulation of p53 upon silica exposure activated RMRP expression, which promoted the EMT. When RMRP is overexpressed, additional RMRP acts as a sponge to bind to miR122, thus decreasing miR122 levels. Using microarrays, miR122 was identified as a potential upstream regulator of p53. This relationship was also verified using the dual-luciferase reporter gene. Hence, decreased miR122 levels result in an increase in p53 activity. More importantly, RMRP promotes the transcription of Notch 1, which, in turn, results in Notch pathway activation. We show that the p53/RMRP/miR122 pathway creates a positive feedback loop that promotes EMT progress by activating the Notch signaling pathway. CONCLUSION: Our data indicated that p53/RMRP/miR122 feedback loop might contribute to the EMT development by activating Notch pathway, which provides new sight into understanding of the complex network regulating silica-induced lung fibrosis.

ZGRF1 promotes end resection of DNA homologous recombination via forming complex with BRCA1/EXO1.

To maintain genomic stability, the mammalian cells has evolved a coordinated response to DNA damage, including activation of DNA repair and cell cycle checkpoint processes. Exonuclease 1 (EXO1)-dependent excision of DNA ends is important for the initiation of homologous recombination (HR) repair of DNA breaks, which is thought to play a key role in activating the ATR-CHK1 pathway to induce G2/M cell cycle arrest. But the mechanism is still not fully understood. Here, we report that ZGRF1 forms complexes with EXO1 as well as other repair proteins and promotes DNA repair through HR. ZGRF1 is recruited to DNA damage sites in a MDC1-RNF8-BRCA1 dependent manner. Furthermore, ZGRF1 is important for the recruitment of RPA2 to DNA damage sites and the following ATR-CHK1 mediated G2/M checkpoint in response to irradiation. ZGRF1 null cells show increased sensitivity to many DNA-damaging agents, especially PARPi and irradiation. Collectively,our findings identify ZGRF1 as a novel regulator of DNA end resection and G2/M checkpoint. ZGRF1 is a potential target of radiation and PARPi cancer therapy.

DNA-PKcs: A Multi-Faceted Player in DNA Damage Response.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is a member of the phosphatidylinositol 3-kinase related kinase family, which can phosphorylate more than 700 substrates. As the core enzyme, DNA-PKcs forms the active DNA-PK holoenzyme with the Ku80/Ku70 heterodimer to play crucial roles in cellular DNA damage response (DDR). Once DNA double strand breaks (DSBs) occur in the cells, DNA-PKcs is promptly recruited into damage sites and activated. DNA-PKcs is auto-phosphorylated and phosphorylated by Ataxia-Telangiectasia Mutated at multiple sites, and phosphorylates other targets, participating in a series of DDR and repair processes, which determine the cells' fates: DSBs NHEJ repair and pathway choice, replication stress response, cell cycle checkpoints, telomeres length maintenance, senescence, autophagy, etc. Due to the special and multi-faceted roles of DNA-PKcs in the cellular responses to DNA damage, it is important to precisely regulate the formation and dynamic of its functional complex and activities for guarding genomic stability. On the other hand, targeting DNA-PKcs has been considered as a promising strategy of exploring novel radiosensitizers and killing agents of cancer cells. Combining DNA-PKcs inhibitors with radiotherapy can effectively enhance the efficacy of radiotherapy, offering more possibilities for cancer therapy.

A Biomarker Panel of Radiation-Upregulated miRNA as Signature for Ionizing Radiation Exposure.

Ionizing radiation causes serious injury to the human body and has long-time impacts on health. It is important to find optimal biomarkers for the early quick screening of exposed individuals. A series of miRNAs signatures have been developed as the new biomarkers for diagnosis, survival, and prognostic prediction of cancers. Here, we have identified the ionizing radiation-inducible miRNAs profile through microarray analysis. The biological functions were predicted for the top six upregulated miRNAs by 4 Gy γ-rays: miR-1246, miR-1307-3p, miR-3197, miR-4267, miR-5096 and miR-7641. The miRNA-gene network and target gene-pathway network analyses revealed that DNAH3 is the target gene associated with all the six miRNAs. GOLGB1 is related to 4 miRNAs and other 26 genes targeted by 3 miRNAs. The upregulation of fifteen miRNAs were further verified at 4 h and 24 h after 0 to 10 Gy irradiation in the human lymphoblastoid AHH-1 cells, and some demonstrated a dose-dependent increased. Six miRNAs, including miR-145, miR-663, miR-1273g-3p, miR-6090, miR-6727-5p and miR-7641, were validated to be dose-dependently upregulated at 4 h or 24 h post-irradiation in both AHH-1 and human peripheral blood lymphocytes irradiated ex vivo. This six-miRNA signature displays the superiority as a radiation biomarker for the translational application of screening and assessment of radiation exposed individuals.

Self-Assembled Multivalent Aptamer Nanoparticles with Potential CAR-like Characteristics Could Activate T Cells and Inhibit Melanoma Growth.

In this study, the CAR-like multivalent aptamer nanoparticles (X-polymers) were assembled with the dimer of murine CD28 RNA aptamer (CD28Apt7), the tetramer of CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) RNA aptamer (Del60), and a folic acid labeled ssDNA fragment in a stable nucleic acid three-way junction scaffold (3WJ). Results showed that the X-polymers could recognize both the mCD28 and mCTLA-4 molecules. Confocal imaging and flow cytometry assays showed that the X-polymers could target both T cells and B16 cells in vitro. With the first costimulatory signals provided by the CD3 antibodies, the X-polymers could increase T cell proliferation and reverse the inhibitory effect of interleukin-2 (IL-2) secreting caused by exogenous B7.1 molecules on T cells in vitro. Results of our study also showed that X-polymers could inhibit mouse melanoma B16 cell growth both in vitro and in vivo. Our study demonstrated for the first time that the multivalent aptamer nanoparticle-activated T cells could fulfill the function of CAR-T, which promised a novel approach to developing a multi-functional design of aptamer drugs with potential CAR-like characteristics to enhance the safety of CAR-T cell immunotherapy.

HUWE1-dependent DNA-PKcs neddylation modulates its autophosphorylation in DNA damage response.

DNA-dependent protein kinase catalytic subunit (DNA-PKcs) is the core component of DNA-PK complex in the non-homologous end-joining (NHEJ) repair of DNA double-strand breaks, and its activity is strictly controlled by DNA-PKcs phosphorylation. The ubiquitin-like protein, NEDD8 is involved in regulation of DNA damage response, but it remains mysterious whether and how NEDD8-related neddylation affects DNA-PKcs and the NHEJ process. Here, we show that DNA-PKcs is poly-neddylated at its kinase domain. The neddylation E2-conjugating enzyme UBE2M and E3 ligase HUWE1 (HECT, UBA, and WWE domain containing E3 ubiquitin protein ligase 1) are responsible for the DNA-PKcs neddylation. Moreover, inhibition of HUWE1-dependent DNA-PKcs neddylation impairs DNA-PKcs autophosphorylation at Ser2056. Finally, depletion of HUWE1-dependent DNA-PKcs neddylation reduces the efficiency of NHEJ. These studies provide insights how neddylation modulates the activity of NHEJ core complex.

Aptamer selection and application in multivalent binding-based electrical impedance detection of inactivated H1N1 virus.

The type A influenza viruses are the most virulent and variable human pathogens with epidemic or even pandemic threat. The development of sensitive, specific and safe field testing methods is in particular need and quite challenging. We report here the selection and practical utilization of the inactivated influenza virus-specific aptamers. The DNA aptamers against inactivated intact H1N1 virus particles were identified through the systematic evolution of ligands by exponential enrichment (SELEX) procedure. The discriminative aptamers and their truncated sequences showed selectively high affinity to inactive H1N1 virus and H3N2 virus with the Kd in the low nanomolar range and collective binding properties. The truncated sequences were first applied in a sandwich enzyme-linked oligonucleotide assay (ELONA) with a H1N1 detection limit (LOD, S/N = 3) of 0.3 ng/µL and then in an electrochemical impedance (EIS) aptasensor with more than 300 times improved LOD (0.9 pg/µL) and the excellent selectivity over other viruses (> 100 times). Therefore the developed aptasensors represent the safer, simpler, and possibly better virus-variation adaptable means of virus diagnostics.

Identification of Functional mimotopes of human Vasorin Ectodomain by Biopanning.

Human vasorin (VASN) as a type I transmembrane protein, is a potential biomarker of hepatocellular carcinoma, which could expedite HepG2 cell proliferation and migration significantly in vitro. The ectodomain of VASN was proteolytically released to generate soluble VASN (sVASN), which was validated to be the active form. Among several monoclonal antibodies produced against sVASN, the clone V21 was found to bind with the recombinant human sVASN (rhsVASN) with the highest affinity and specificity, and also have inhibitory effects on proliferation and migration of HepG2 cells. Hence the phage-displayed peptide library was screened against the antibody V21. The positive phage clones were isolated and sequenced, and one unique consensus motifs was obtained. The result of sequence alignment showed that the conserved motif had similarity to VASN(Cys432-Cys441), embedded in the epidermal growth factor (EGF)-like domain. The synthetic mimotope peptide V21P1 and V21P2 were confirmed to bind with V21 and could compete with rhsVASN in ELISA assay. And they could also almost completely reverse the inhibitory effect of V21 on HepG2 migration and proliferation. Furthermore, the antibodies produced against V21P1 were able to bind not only with the peptide V21P1, but also with rhsVASN and the natural VASN from HepG2 cell. Our results showed that V21 seemed to be a functional antibody. The mimotopes toward V21 might mimic the functional domain of VASN, which would be helpful to exploit VASN functions and act as a candidate target for developing therapeutic antibodies against VASN.