Pyrroloquinoline quinone promotes human mesenchymal stem cell-derived mitochondria to improve premature ovarian insufficiency in mice through the SIRT1/ATM/p53 pathway.

BACKGROUND: DNA damage and oxidative stress induced by chemotherapy are important factors in the onset of premature ovarian insufficiency (POI). Studies have shown that mitochondria derived from mesenchymal stem cells (MSC-Mito) are beneficial for age-related diseases, but their efficacy alone is limited. Pyrroloquinoline quinone (PQQ) is a potent antioxidant with significant antiaging and fertility enhancement effects. This study aimed to investigate the therapeutic effect of MSC-Mito in combination with PQQ on POI and the underlying mechanisms involved. METHODS: A POI animal model was established in C57BL/6J mice by cyclophosphamide and busulfan. The effects of MSC-Mito and PQQ administration on the estrous cycle, ovarian pathological damage, sex hormone secretion, and oxidative stress in mice were evaluated using methods such as vaginal smears and ELISAs. Western blotting and immunohistochemistry were used to assess the expression of SIRT1, PGC-1α, and ATM/p53 pathway proteins in ovarian tissues. A cell model was constructed using KGN cells treated with phosphoramide mustard to investigate DNA damage and apoptosis through comet assays and flow cytometry. SIRT1 siRNA was transfected into KGN cells to further explore the role of the SIRT1/ATM/p53 pathway in combination therapy with MSC-Mito and PQQ for POI. RESULTS: The combined treatment of MSC-Mito and PQQ significantly restored ovarian function and antioxidant capacity in mice with POI. This treatment also reduced the loss of follicles at various stages, improving the disrupted estrous cycle. In vitro experiments demonstrated that PQQ facilitated the proliferation of MitoTracker-labelled MSC-Mito, synergistically restoring mitochondrial function and inhibiting oxidative stress in combination with MSC-Mito. Both in vivo and in vitro, the combination of MSC-Mito and PQQ increased mitochondrial biogenesis mediated by SIRT1 and PGC-1α while inhibiting the activation of ATM and p53, consequently reducing DNA damage-mediated cell apoptosis. Furthermore, pretreatment of KGN cells with SIRT1 siRNA reversed nearly all the aforementioned changes induced by the combined treatment. CONCLUSIONS: Our research findings indicate that PQQ facilitates MSC-Mito proliferation and, in combination with MSC-Mito, ameliorates chemotherapy-induced POI through the SIRT1/ATM/p53 signaling pathway.

Differential diagnosis of Crohn's disease and intestinal tuberculosis based on ATR-FTIR spectroscopy combined with machine learning.

BACKGROUND: Crohn's disease (CD) is often misdiagnosed as intestinal tuberculosis (ITB). However, the treatment and prognosis of these two diseases are dramatically different. Therefore, it is important to develop a method to identify CD and ITB with high accuracy, specificity, and speed. AIM: To develop a method to identify CD and ITB with high accuracy, specificity, and speed. METHODS: A total of 72 paraffin wax-embedded tissue sections were pathologically and clinically diagnosed as CD or ITB. Paraffin wax-embedded tissue sections were attached to a metal coating and measured using attenuated total reflectance fourier transform infrared spectroscopy at mid-infrared wavelengths combined with XGBoost for differential diagnosis. RESULTS: The results showed that the paraffin wax-embedded specimens of CD and ITB were significantly different in their spectral signals at 1074 cm-1 and 1234 cm-1 bands, and the differential diagnosis model based on spectral characteristics combined with machine learning showed accuracy, specificity, and sensitivity of 91.84%, 92.59%, and 90.90%, respectively, for the differential diagnosis of CD and ITB. CONCLUSION: Information on the mid-infrared region can reveal the different histological components of CD and ITB at the molecular level, and spectral analysis combined with machine learning to establish a diagnostic model is expected to become a new method for the differential diagnosis of CD and ITB.

Diverse pro-inflammatory ability of mutated spike protein derived from variant strains of SARS-CoV-2.

The severity of COVID-19 has been reported to differ among SARS-CoV-2 mutant variants. The overactivation of macrophages is involved in severe COVID-19, yet the effects of SARS-CoV-2 mutations on macrophages remain poorly understood. To clarify the effects, we examined whether mutations of spike proteins (S-proteins) affect macrophage activation. CD14+ monocyte-derived macrophages were stimulated with the recombinant S-protein of the wild-type, Delta, and Omicron strains or live viral particles of individual strains. Regarding IL-6 and TNF-α, Delta or Omicron S-protein had stronger or weaker pro­inflammatory ability, respectively, than the wild-type. Similar trends were observed between S-proteins and viral particles. S-protein mutations could be related to the diversity in macrophage activation and severity rates in COVID-19 caused by various SARS-CoV-2 strains.

DNA-PK and ATM drive phosphorylation signatures that antagonistically regulate cytokine responses to herpesvirus infection or DNA damage.

The DNA-dependent protein kinase, DNA-PK, is an essential regulator of DNA damage repair. DNA-PK-driven phosphorylation events and the activated DNA damage response (DDR) pathways are also components of antiviral intrinsic and innate immune responses. Yet, it is not clear whether and how the DNA-PK response differs between these two forms of nucleic acid stress-DNA damage and DNA virus infection. Here, we define DNA-PK substrates and the signature cellular phosphoproteome response to DNA damage or infection with the nuclear-replicating DNA herpesvirus, HSV-1. We establish that DNA-PK negatively regulates the ataxia-telangiectasia-mutated (ATM) DDR kinase during viral infection. In turn, ATM blocks the binding of DNA-PK and the nuclear DNA sensor IFI16 to viral DNA, thereby inhibiting cytokine responses. However, following DNA damage, DNA-PK enhances ATM activity, which is required for IFN-ß expression. These findings demonstrate that the DDR autoregulates cytokine expression through the opposing modulation of DDR kinases.

Randomized Phase II Study of Gemcitabine With or Without ATR Inhibitor Berzosertib in Platinum-Resistant Ovarian Cancer: Final Overall Survival and Biomarker Analyses.

PURPOSE: The multicenter, open-label, randomized phase 2 NCI-9944 study (NCT02595892) demonstrated that addition of ATR inhibitor (ATRi) berzosertib to gemcitabine increased progression-free survival (PFS) compared to gemcitabine alone (hazard ratio [HR]=0.57, one-sided log-rank P = .044, which met the one-sided significance level of 0.1 used for sample size calculation). METHODS: We report here the final overall survival (OS) analysis and biomarker correlations (ATM expression by immunohistochemistry, mutational signature 3 and a genomic biomarker of replication stress) along with post-hoc exploratory analyses to adjust for crossover from gemcitabine to gemcitabine/berzosertib. RESULTS: At the data cutoff of January 27, 2023 (>30 months of additional follow-up from the primary analysis), median OS was 59.4 weeks with gemcitabine/berzosertib versus 43.0 weeks with gemcitabine alone (HR 0.79, 90% CI 0.52 to 1.2, one-sided log-rank P = .18). An OS benefit with addition of berzosertib to gemcitabine was suggested in patients stratified into the platinum-free interval ≤3 months (N = 26) subgroup (HR, 0.48, 90% CI 0.22 to 1.01, one-sided log-rank P =.04) and in patients with ATM-negative/low (N = 24) tumors (HR, 0.50, 90% CI 0.23 to 1.08, one-sided log-rank P = .06). CONCLUSION: The results of this follow-up analysis continue to support the promise of combined gemcitabine/ATRi therapy in platinum resistant ovarian cancer, an active area of investigation with several ongoing clinical trials.

Context matters: DNA virus infection reshapes DNA damage response pathways.

The cellular DNA damage response pathway can have vastly different outcomes depending on the source of its activation. Justice and colleagues apply phosphoproteomics to uncover a divergence in DNA-PK and ATM kinase activities in the contexts of DNA damage and DNA virus infection.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

Regulation of transcription patterns, poly(ADP-ribose), and RNA-DNA hybrids by the ATM protein kinase.

The ataxia telangiectasia mutated (ATM) protein kinase is a master regulator of the DNA damage response and also an important sensor of oxidative stress. Analysis of gene expression in ataxia-telangiectasia (A-T) patient brain tissue shows that large-scale transcriptional changes occur in patient cerebellum that correlate with the expression level and guanine-cytosine (GC) content of transcribed genes. In human neuron-like cells in culture, we map locations of poly(ADP-ribose) and RNA-DNA hybrid accumulation genome-wide with ATM inhibition and find that these marks also coincide with high transcription levels, active transcription histone marks, and high GC content. Antioxidant treatment reverses the accumulation of R-loops in transcribed regions, consistent with the central role of reactive oxygen species in promoting these lesions. Based on these results, we postulate that transcription-associated lesions accumulate in ATM-deficient cells and that the single-strand breaks and PARylation at these sites ultimately generate changes in transcription that compromise cerebellum function and lead to neurodegeneration over time in A-T patients.

Discovery of Selective and Potent ATR Degrader for Exploration its Kinase-Independent Functions in Acute Myeloid Leukemia Cells.

ATR has emerged as a promising target for anti-cancer drug development. Several potent ATR inhibitors are currently undergoing various stages of clinical trials, but none have yet received FDA approval due to unclear regulatory mechanisms. In this study, we discovered a potent and selective ATR degrader. Its kinase-independent regulatory functions in acute myeloid leukemia (AML) cells were elucidated using this proteolysis-targeting chimera (PROTAC) molecule as a probe. The ATR degrader, 8 i, exhibited significantly different cellular phenotypes compared to the ATR kinase inhibitor 1. Mechanistic studies revealed that ATR deletion led to breakdown in the nuclear envelope, causing genome instability and extensive DNA damage. This would increase the expression of p53 and triggered immediately p53-mediated apoptosis signaling pathway, which was earlier and more effective than ATR kinase inhibition. Based on these findings, the in vivo anti-proliferative effects of ATR degrader 8 i were assessed using xenograft models. The degrader significantly inhibited the growth of AML cells in vivo, unlike the ATR inhibitor. These results suggest that the marked anti-AML activity is regulated by the kinase-independent functions of the ATR protein. Consequently, developing potent and selective ATR degraders could be a promising strategy for treating AML.

Depletion of CNOT4 modulates the DNA damage responses following ionizing radiation (IR).

BACKGROUND: The Ccr4-Not complex (CNOT complex in mammals) is a unique and highly conserved complex with numerous cellular functions. Until now, there has been relatively little known about the importance of the CNOT complex subunits in the DNA damage response (DDR) in mammalian cells. CNOT4 is a subunit of the complex with E3 ubiquitin ligase activity that interacts transiently with the CNOT1 subunit. Here, we attempt to investigate the role of human CNOT4 subunit in the DDR in human cells. MATERIAL AND METHODS: In this study, cell viability in the absence of CNOT4 was assessed using a Cell Titer-Glo Luminescence assay up to 4 days post siRNA transfection. In a further experiment, CNOT4-depleted HeLa cells were exposed to 3Gy ionizing radiation (IR). Ataxia telangiectasia-mutated (ATM) and ATM Rad3-related (ATR) signaling pathways were then investigated by western blotting for phosphorylated substrates. In addition, foci formation of histone 2A family member X (γH2AX), replication protein A (RPA), TP53 binding protein 1 (53BP1), and DNA repair protein RAD51 homolog 1 was also determined by immunofluorescence microscopy comparing control and CNOT4-depleted HeLa cells 0, 8, and 24 h post IR treatment. RESULTS: Our results from cell viability assays showed a significant reduction of cell growth activity at 24 (P value 0.02) and 48 h (P value 0.002) post siRNA. Western blot analysis showed slightly reduced or slightly delayed DDR signaling in CNOT4-depleted HeLa cells after IR. More significantly, we observed increased formation of γH2AX, RPA, 53BP1, and RAD51 foci after IR in CNOT4-depleted cells compared with the control cells. CONCLUSION: We conclude that depletion of CNOT4 affects various aspects of the cellular response to DNA damage.

Ataxia telangiectasia and Rad3-related (ATR) inhibition by VE-822 potently reversed 5-flourouracil resistance in colorectal cancer cells through targeting DNA damage response.

BACKGROUND: VE-822 is a novel inhibitor of ATR, a key kinase involved in the DNA damage response pathway. The role of ATR inhibition in reversing drug resistance in various cancer types has been investigated. Therefore, this study investigated the effects of ATR inhibition by VE-822 on reversing 5-fluorouracil (5-FU) resistance in colorectal cancer cell line (Caco-2). METHODS: Caco-2 and 5-FU resistance Caco-2 (Caco-2/5-FU) cells were treated with 5-FU and VE-822, alone and in combination. Cell proliferation and viability were assessed by MTT assay and Trypan Blue staining. P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP1) activities were measured by Rhodamine123 accumulation and uptake assay. The mRNA levels of P-gp, MRP-1, ataxia telangiectasia and Rad3-related (ATR) and checkpoint kinase 1 (CHK1) were measured by qRT-PCR. Western blot was used to measure the protein levels of P-gp, MRP-1, γ-H2AX, ATR and CHK1 in cells. 8-Oxo-2'-deoxyguanosine (8-oxo-dG) levels were determined via ELISA. Apoptosis was evaluated by ELISA death assay, DAPI staining and lactate dehydrogenase (LDH) assay. RESULTS: The Caco-2/5-FU cells showed lower levels of 5-FU mediated proliferation inhibition in comparison to Caco-2 cells. VE-822 decreased the IC50 value of 5-FU on resistant cells. In addition, the expression levels and activity of P-gp and MRP-1 were significantly decreased in resistant cells treated with VE-822 (P < 0.05). The combination of 5-FU and VE-822 increased apoptosis in Caco-2/5-FU cells by downregulating CHK1 and ATR and upregulating γ-H2AX and 8-oxo-dG. CONCLUSION: The simultaneous treatment of resistant colorectal cancer cells with 5-FU and ATR inhibitor, VE-822, was demonstrated to be effective in reversing drug resistance and potentiating 5-FU mediated anticancer effects via targeting DNA damage.

Therapeutic Options Targeting the Ataxia-Telangiectasia Mutated (ATM)-mediated DNA Damage Response, Macropinocytosis, and Adaptive Immunity in Ovarian Cancer.

Ataxia-telangiectasia mutated (ATM) is a pivotal protein with versatile kinase activity that responds to DNA damage. While its well-established role as a DNA repair protein is widely recognized, the understanding of its noncanonical functions in ovarian cancer remains limited. Numerous studies have investigated the potential of targeting ATM for ovarian cancer treatment. In addition to its involvement in homologous recombination repair (HRR), an increasing body of research suggests that ATM plays a role in cellular metabolism and adaptive immunity. This review focuses on the current evidence and provides a perspective on how targeting ATM in ovarian cancer can address HRR-deficient genotypes, influence macropinocytosis, and enhance immune checkpoint blockade (ICB) therapy. It underscores the diverse avenues through which targeting ATM is a potential tailored treatment for ovarian cancer.

ATR limits Rad18-mediated PCNA monoubiquitination to preserve replication fork and telomerase-independent telomere stability.

Upon replication fork stalling, the RPA-coated single-stranded DNA (ssDNA) formed behind the fork activates the ataxia telangiectasia-mutated and Rad3-related (ATR) kinase, concomitantly initiating Rad18-dependent monoubiquitination of PCNA. However, whether crosstalk exists between these two events and the underlying physiological implications of this interplay remain elusive. In this study, we demonstrate that during replication stress, ATR phosphorylates human Rad18 at Ser403, an adjacent residue to a previously unidentified PIP motif (PCNA-interacting peptide) within Rad18. This phosphorylation event disrupts the interaction between Rad18 and PCNA, thereby restricting the extent of Rad18-mediated PCNA monoubiquitination. Consequently, excessive accumulation of the tumor suppressor protein SLX4, now characterized as a novel reader of ubiquitinated PCNA, at stalled forks is prevented, contributing to the prevention of stalled fork collapse. We further establish that ATR preserves telomere stability in alternative lengthening of telomere (ALT) cells by restricting Rad18-mediated PCNA monoubiquitination and excessive SLX4 accumulation at telomeres. These findings shed light on the complex interplay between ATR activation, Rad18-dependent PCNA monoubiquitination, and SLX4-associated stalled fork processing, emphasizing the critical role of ATR in preserving replication fork stability and facilitating telomerase-independent telomere maintenance.

ZNF827 is a single-stranded DNA binding protein that regulates the ATR-CHK1 DNA damage response pathway.

The ATR-CHK1 DNA damage response pathway becomes activated by the exposure of RPA-coated single-stranded DNA (ssDNA) that forms as an intermediate during DNA damage and repair, and as a part of the replication stress response. Here, we identify ZNF827 as a component of the ATR-CHK1 kinase pathway. We demonstrate that ZNF827 is a ssDNA binding protein that associates with RPA through concurrent binding to ssDNA intermediates. These interactions are dependent on two clusters of C2H2 zinc finger motifs within ZNF827. We find that ZNF827 accumulates at stalled forks and DNA damage sites, where it activates ATR and promotes the engagement of homologous recombination-mediated DNA repair. Additionally, we demonstrate that ZNF827 depletion inhibits replication initiation and sensitizes cancer cells to the topoisomerase inhibitor topotecan, revealing ZNF827 as a therapeutic target within the DNA damage response pathway.

Functionally-instructed modifiers of response to ATR inhibition in experimental glioma.

BACKGROUND: The DNA damage response (DDR) is a physiological network preventing malignant transformation, e.g. by halting cell cycle progression upon DNA damage detection and promoting DNA repair. Glioblastoma are incurable primary tumors of the nervous system and DDR dysregulation contributes to acquired treatment resistance. Therefore, DDR targeting is a promising therapeutic anti-glioma strategy. Here, we investigated Ataxia telangiectasia and Rad3 related (ATR) inhibition (ATRi) and functionally-instructed combination therapies involving ATRi in experimental glioma. METHODS: We used acute cytotoxicity to identify treatment efficacy as well as RNAseq and DigiWest protein profiling to characterize ATRi-induced modulations within the molecular network in glioma cells. Genome-wide CRISPR/Cas9 functional genomic screens and subsequent validation with functionally-instructed compounds and selected shRNA-based silencing were employed to discover and investigate molecular targets modifying response to ATRi in glioma cell lines in vitro, in primary cultures ex vivo and in zebrafish and murine models in vivo. RESULTS: ATRi monotherapy displays anti-glioma efficacy in vitro and ex vivo and modulates the molecular network. We discovered molecular targets by genome-wide CRISPR/Cas9 loss-of-function and activation screens that enhance therapeutic ATRi effects. We validated selected druggable targets by a customized drug library and functional assays in vitro, ex vivo and in vivo. CONCLUSION: In conclusion, our study leads to the identification of novel combination therapies involving ATRi that could inform future preclinical studies and early phase clinical trials.

Targeting ATR Pathway in Solid Tumors: Evidence of Improving Therapeutic Outcomes.

The DNA damage response (DDR) system is a complicated network of signaling pathways that detects and repairs DNA damage or induces apoptosis. Critical regulators of the DDR network include the DNA damage kinases ataxia telangiectasia mutated Rad3-related kinase (ATR) and ataxia-telangiectasia mutated (ATM). The ATR pathway coordinates processes such as replication stress response, stabilization of replication forks, cell cycle arrest, and DNA repair. ATR inhibition disrupts these functions, causing a reduction of DNA repair, accumulation of DNA damage, replication fork collapse, inappropriate mitotic entry, and mitotic catastrophe. Recent data have shown that the inhibition of ATR can lead to synthetic lethality in ATM-deficient malignancies. In addition, ATR inhibition plays a significant role in the activation of the immune system by increasing the tumor mutational burden and neoantigen load as well as by triggering the accumulation of cytosolic DNA and subsequently inducing the cGAS-STING pathway and the type I IFN response. Taken together, we review stimulating data showing that ATR kinase inhibition can alter the DDR network, the immune system, and their interplay and, therefore, potentially provide a novel strategy to improve the efficacy of antitumor therapy, using ATR inhibitors as monotherapy or in combination with genotoxic drugs and/or immunomodulators.

Promising Response of Olaparib in Patient With Germline ATM-Mutated Metastatic Gastric Cancer.

Gastric cancer ranks as the fifth leading cause of global cancer incidences, exhibiting varied prevalence influenced by geographical, ethnic, and lifestyle factors, as well as Helicobacter pylori infection. The ATM gene on chromosome 11q22 is vital for genomic stability as an initiator of the DNA damage response, and mutations in this gene have been associated with various cancers. Poly ADP-ribose polymerase (PARP) inhibitors, such as olaparib, have shown efficacy in cancers with homologous recombination repair deficiencies, notably in those with ATM mutations. Here, we present a case of a 66-year-old patient with germline ATM-mutated metastatic gastric cancer with very high CA 19-9 (48 000 units/mL) who demonstrated an exceptional response to the addition of olaparib to chemo-immunotherapy and subsequent olaparib maintenance monotherapy for 12 months. CA 19-9 was maintained at low level for 18 months. Despite the failure of a phase II clinical trial on olaparib in gastric cancer (NCT01063517) to meet its primary endpoint, intriguing findings emerged in the subset of ATM-mutated patients, who exhibited notable improvements in overall survival. Our case underscores the potential clinical utility of olaparib in germline ATM-mutated gastric cancer and emphasizes the need for further exploration through larger clinical trials. Ongoing research and clinical trials are essential for optimizing the use of PARP inhibitors, identifying biomarkers, and advancing personalized treatment strategies for gastric cancer.

Variant ataxia telangiectasia identified during evaluation for short stature.

Ataxia telangiectasia (A-T) (OMIM 208900) is an autosomal recessive multisystem disorder characterised by progressive cerebellar ataxia, telangiectasias, immunodeficiency and a predisposition to malignancy. 'Variant' A-T has later onset of neurological symptoms and slower progression compared with the 'classic' form. A woman presented with short stature in late childhood. Karyotype revealed rearrangements involving chromosomes 7 and 14. A chromosomal breakage disorder gene panel demonstrated compound heterozygote mutations in her ATM gene including one mutation c.7271T>G with residual ATM function, confirming the diagnosis of variant A-T. Since diagnosis, she has developed progressive cerebellar ataxia and telangiectasias. Long-standing restrictive and aversive feeding behaviours presented challenges for her management and necessitated gastrostomy.

Study of Monoclonal Antibody Aggregation at the Air-Liquid Interface under Flow by ATR-FTIR Spectroscopic Imaging.

Throughout bioprocessing, transportation, and storage, therapeutic monoclonal antibodies (mAbs) experience stress conditions that may cause protein unfolding and/or chemical modifications. Such structural changes may lead to the formation of aggregates, which reduce mAb potency and may cause harmful immunogenic responses in patients. Therefore, aggregates need to be detected and removed or ideally prevented from forming. Air-liquid interfaces, which arise during various stages of bioprocessing, are one of the stress factors causing mAb aggregation. In this study, the behavior of an immunoglobulin G (IgG) at the air-liquid interface was investigated under flow using macro attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging. This chemically specific imaging technique allows observation of adsorption of IgG to the air-liquid interface and detection of associated secondary structural changes. Chemical images revealed that IgG rapidly accumulated around an injected air bubble under flow at 45 °C; however, no such increase was observed at 25 °C. Analysis of the second derivative spectra of IgG at the air-liquid interface revealed changes in the protein secondary structure associated with increased intermolecular ß-sheet content, indicative of aggregated IgG. The addition of 0.01% w/v polysorbate 80 (PS80) reduced the amount of IgG at the air-liquid interface in a static setup at 30 °C; however, this protective effect was lost at 45 °C. These results suggest that the presence of air-liquid interfaces under flow may be detrimental to mAb stability at elevated temperatures and demonstrate the power of ATR-FTIR spectroscopic imaging for studying the structural integrity of mAbs under bioprocessing conditions.

Disassembly of the TRIM56-ATR complex promotes cytoDNA/cGAS/STING axis-dependent intervertebral disc inflammatory degeneration.

As the leading cause of disability worldwide, low back pain (LBP) is recognized as a pivotal socioeconomic challenge to the aging population and is largely attributed to intervertebral disc degeneration (IVDD). Elastic nucleus pulposus (NP) tissue is essential for the maintenance of IVD structural and functional integrity. The accumulation of senescent NP cells with an inflammatory hypersecretory phenotype due to aging and other damaging factors is a distinctive hallmark of IVDD initiation and progression. In this study, we reveal a mechanism of IVDD progression in which aberrant genomic DNA damage promoted NP cell inflammatory senescence via activation of the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) axis but not of absent in melanoma 2 (AIM2) inflammasome assembly. Ataxia-telangiectasia-mutated and Rad3-related protein (ATR) deficiency destroyed genomic integrity and led to cytosolic mislocalization of genomic DNA, which acted as a powerful driver of cGAS/STING axis-dependent inflammatory phenotype acquisition during NP cell senescence. Mechanistically, disassembly of the ATR-tripartite motif-containing 56 (ATR-TRIM56) complex with the enzymatic liberation of ubiquitin-specific peptidase 5 (USP5) and TRIM25 drove changes in ATR ubiquitination, with ATR switching from K63- to K48-linked modification, c thereby promoting ubiquitin-proteasome-dependent dynamic instability of ATR protein during NP cell senescence progression. Importantly, an engineered extracellular vesicle-based strategy for delivering ATR-overexpressing plasmid cargo efficiently diminished DNA damage-associated NP cell senescence and substantially mitigated IVDD progression, indicating promising targets and effective approaches to ameliorate the chronic pain and disabling effects of IVDD.