Interaction of T-cell-specific adapter protein with Src- and Tec-family kinases.

Adapter proteins are flexible and dynamic modulators of cellular signalling that are important for immune cell function. One of these, the T-cell-specific adapter protein (TSAd), interacts with the non-receptor tyrosine kinases Src and Lck of the Src family kinases (SFKs) and Itk of the Tec family kinases (TFKs). Three tyrosine residues in the TSAd C-terminus are phosphorylated by Lck and serve as docking sites for the Src homology 2 (SH2) domains of Src and Lck. The TSAd proline-rich region (PRR) binds to the Src homology 3 (SH3) domains found in Lck, Src and Itk. Despite known interactors, the role TSAd plays in cellular signalling remains largely unknown. TSAd's ability to bind both SFKs and TFKs may point to its function as a general scaffold for both kinase families. Using GST-pulldown as well as peptide array experiments, we found that both the SH2 and SH3 domains of the SFKs Fyn and Hck, as well as the TFKs Tec and Txk, interact with TSAd. This contrasts with Itk, which interacts with TSAd only through its SH3 domain. Although our analysis showed that TSAd is both co-expressed and may interact with Fyn, we were unable to co-precipitate Fyn with TSAd from Jurkat cells, as detected by Western blotting and affinity purification mass spectrometry. This may suggest that TSAd-Fyn interaction in intact cells may be limited by other factors, such as the subcellular localization of the two molecules or the co-expression of competing binding partners.

Research progress on oncoprotein hepatitis B X­interacting protein (Review).

Hepatitis B X­interacting protein (HBXIP) is a membrane protein located on the lysosomal surface and encoded by the Lamtor gene. It is expressed by a wide range of tumor types, including breast cancer, esophageal squamous cell carcinoma and hepatocellular carcinoma, and its expression is associated with certain clinicopathological characteristics. In the past decade, research on the oncogenic mechanisms of HBXIP has increased and the function of HBXIP in normal cells has been gradually elucidated. In the present review, the following was discussed: The normal physiological role of the HBXIP carcinogenic mechanism; the clinical significance of high levels of HBXIP expression in different tumors; HBXIP regulation of transcription, post­transcription and post­translation processes in tumors; the role of HBXIP in improving the antioxidant capacity of tumor cells; the inhibition of ferroptosis of tumor cells and regulating the metabolic reprogramming of tumor cells; and the role of HBXIP in promoting the malignant progression of tumors. In conclusion, the present review summarized the existing knowledge of HBXIP, established its carcinogenic mechanism and discussed future related research on HBXIP.

BCAR4 facilitates trastuzumab resistance and EMT in breast cancer via sponging miR-665 and interacting with YAP1.

Breast cancer antiestrogen resistance 4 (BCAR4) has been suggested that can modulate cell behavior, resulting in tumorigenesis and chemoresistance. However, the underlying mechanisms of BCAR4 in trastuzumab resistance (TR) is still elusive. Here, we explored the function and the underlying mechanism of BCAR4 involving in TR. We found that BCAR4 is significantly upregulated in trastuzumab-resistant BC cells. Knockdown of BCAR4 could sensitize the BC cells to trastuzumab and suppress epithelial-mesenchymal transition (EMT). Mechanically, BCAR4 promotes yes-associated protein 1 (YAP1) expression by competitively sponging miR-665, to activated TGF-ß signaling. Reciprocally, YAP1 could occupy the BCAR4 promoter to enhance its transcription, suggesting that there exists a positive feedback regulation between YAP1 and BCAR4. Targeting the BCAR4/miR-665/YAP1 axis may provide a novel insight of therapeutic approaches for TR in BC.

Surviving without BRCA2: MLH1 gets R-looped in to curtail genomic instability.

While breast cancer 2 (BRCA2) loss of heterozygosity (LOH) promotes cancer initiation, it can also induce death in nontransformed cells. In contrast, mismatch repair gene mutL homolog 1 (MLH1) is a tumor-suppressor gene that protects cells from cancer development through repairing mismatched base pairs during DNA mismatch repair (MMR). Sengodan et al., in this issue of the JCI, reveal an interplay between the 2 genes: MLH1 promoted the survival of BRCA2-deficient cells independently of its MMR function. MLH1 protected replication forks from degradation, while also resolving R-loops, thereby reducing genomic instability. Moreover, MLH1 expression was regulated directly by estrogen, shedding light into the hormone-responsive nature of many BRCA2 mutant breast cancers. These results provide important insight into the genetics that drive the initiation of BRCA2-mutated breast cancers.

KLF4 suppresses anticancer effects of brusatol via transcriptional upregulating NCK2 expression in melanoma.

Brusatol (Bru), a main extract from traditional Chinese medicine Brucea javanica, has been reported to exist antitumor effect in many tumors including melanoma. However, the underlying mechanism in its anti-melanoma effect still need further exploration. Here, we reported that the protein expression of KLF4 in melanoma cells were significantly downregulated in response to brusatol treatment. Overexpression of KLF4 suppressed brusatol-induced melanoma cell apoptosis; while knockdown of KLF4 enhanced antitumor effects of brusatol on melanoma cells not only in vitro but also in vivo. Further studies on the mechanism revealed that KLF4 bound to the promoter of NCK2 directly and facilitated NCK2 transcription, which suppressed the antitumor effect of brusatol on melanoma. Furthermore, our findings showed that miR-150-3p was dramatically upregulated under brusatol treatment which resulted in the downregulation of KLF4. Our results suggested that the miR-150-3p/KLF4/NCK2 axis might play an important role in the antitumour effects of brusatol in melanoma.

Babaodan overcomes cisplatin resistance in cholangiocarcinoma via inhibiting YAP1.

CONTEXT: Cholangiocarcinoma with highly heterogeneous, aggressive, and multidrug resistance has a poor prognosis. Although babaodan (BBD) combined with cisplatin improved non-small cell lung cancer efficacy, its impact on overcoming resistance in cholangiocarcinoma remains unexplored. OBJECTIVE: This study explored the role and mechanism of BBD on cisplatin resistance in cholangiocarcinoma cells (CCAs). MATERIALS AND METHODS: Cisplatin-resistant CCAs were exposed to varying concentrations of cisplatin (25-400 µg/mL) or BBD (0.25-1.00 mg/mL) for 48 h. IC50 values, inhibition ratios, apoptosis levels, DNA damage, glutathione (GSH) levels, oxidized forms of GSH, total GSH content, and glutaminase relative activity were evaluated using the cell counting kit 8, flow cytometry, comet assay, and relevant assay kits. RESULTS: BBD-reduced the cisplatin IC50 in CCAs from 118.8 to 61.83 µg/mL, leading to increased inhibition rate, apoptosis, and DNA damage, and decreased expression of B-cell lymphoma-2, p-Yes-associated protein 1/Yes-associated protein 1, solute carrier family 1 member 5, activating transcription factor 4, and ERCC excision repair 1 in a dose-dependent manner with maximum reductions of 78.97%, 51.98%, 54.03%, 56.59%, and 63.22%, respectively; bcl2-associated X and gamma histone levels were increased by 0.43-115.77% and 22.15-53.39%. The impact of YAP1 knockdown on cisplatin-resistant CCAs resembled BBD. GSH, oxidized GSH species, total GSH content, and glutaminase activity in cisplatin-resistant CCAs with BBD treatment also decreased, while YAP1 overexpression countered BBD's effects. DISCUSSION AND CONCLUSION: This study provides a scientific basis for BBD clinical application and provides a new direction for BBD biological mechanism research.

NFKBIZ regulates NFκB signaling pathway to mediate tumorigenesis and metastasis of hepatocellular carcinoma by direct interaction with TRIM16.

Hepatocellular carcinoma (HCC) is a malignant tumor with high incidence and mortality rates. NFKBIZ, a member of the nuclear factor kappa B inhibitory family, is closely related to tumor progression. However, the precise role of NFKBIZ in HCC remains unclear. To explore this, we conducted a series of experiments from clinic to cells. Western blot and qPCR revealed a significant downregulation of NFKBIZ in human HCC tissues. Clinical character analysis showed that the patients with lower NFKBIZ expression had poorer prognosis and higher clinical stage. By using CCK-8, wound healing, transwell invasion and migration assay, we discovered that NFKBIZ expression was reversely associated with the proliferation, invasion, and migration ability of HCC cells in vitro. Additionally, the results obtained from xenograft assay and lung metastasis models showed that NFKBIZ overexpression inhibited the growth and metastasis of HCC cells in vivo. Western blot and immunofluorescence assay further revealed that NFKBIZ mediated HCC cell growth and migration by regulating NFκB signaling transduction. Finally, flow cytometry, protein degradation assay and Co-immunoprecipitation indicated that TRIM16 can enhance NFKBIZ ubiquitination by direct interactions at its K48 site, which may thereby alleviate HCC cell apoptosis to induce the insensitivity to sorafenib. In conclusion, our study demonstrated that NFKBIZ regulated HCC tumorigenesis and metastasis by mediating NFκB signal transduction and TRIM16/NFKBIZ/NFκB axis may be the underlying mechanism of sorafenib insensitivity in HCC.

Molecular dynamics simulations to explore the binding mode between the amyloid-ß protein precursor (APP) and adaptor protein Mint2.

Alzheimer's disease (AD) presents a significant challenge in neurodegenerative disease management, with limited therapeutic options available for its prevention and treatment. At the heart of AD pathogenesis is the amyloid-ß (Aß) protein precursor (APP), with the interaction between APP and the adaptor protein Mint2 being crucial. Despite previous explorations into the APP-Mint2 interaction, the dynamic regulatory mechanisms by which Mint2 modulates APP binding remain poorly understood. This study undertakes molecular dynamics simulations across four distinct systems-free Mint2, Mint2 bound to APP, a mutant form of Mint2, and the mutant form bound to APP-over an extensive 400 ns timeframe. Our findings reveal that the mutant Mint2 experiences significant secondary structural transformations, notably the formation of an α-helix in residues S55-K65 upon APP binding, within the 400 ns simulation period. Additionally, we observed a reduction in the active pocket size of the mutant Mint2 compared to its wild-type counterpart, enhancing its APP binding affinity. These insights hold promise for guiding the development of novel inhibitors targeting the Mints family, potentially paving the way for new therapeutic strategies in AD prevention and treatment.

FTO attenuates the cytotoxicity of cisplatin in KGN granulosa cell-like tumour cells by regulating the Hippo/YAP1 signalling pathway.

Premature ovarian failure (POF) is a devastating condition for women under 40 years old. Chemotherapy, especially the use of cisplatin, has been demonstrated to promote the apoptosis of granulosa cells in primary and secondary follicles, leading to POF. Our previous studies demonstrated that fat mass- and obesity-associated (FTO) plays an essential role in protecting granulosa cells from cisplatin-induced cytotoxicity. Various studies have suggested that the Hippo/YAP signalling pathway plays a significant role in regulating cell apoptosis and proliferation. Additionally, YAP1 is the main downstream target of the Hippo signalling pathway and is negatively regulated by the Hippo signalling pathway. However, whether the Hippo/YAP signalling pathway is involved in the protective effect of FTO on granulosa cells has not been determined. In this study, we found that after cisplatin treatment, the apoptosis of granulosa cells increased in a concentration-dependent manner, accompanied by the downregulation of FTO and YAP1. Furthermore, overexpression of FTO decreased cisplatin-induced granulosa cell apoptosis, inhibited the Hippo/YAP kinase cascade-induced phosphorylation of YAP1, and promoted the entry of YAP1 into the nucleus. The downstream targets of YAP1 (CTGF, CYR61, and ANKRD1) were also increased. Si-RNA-mediated downregulation of FTO promoted cisplatin-induced granulosa cell apoptosis, activated the Hippo/YAP kinase cascade, and inhibited the YAP1 entry into the nucleus. These effects were completely reversed by the small molecule inhibitor of YAP1-verteporfin (VP). Taken together, these data suggested that FTO-YAP1 plays a positive role in regulating the proliferation of injured granulosa cells induced by cisplatin.

BAG3 localizes to mitochondria in cardiac fibroblasts and regulates mitophagy.

The co-chaperone Bcl2-associated athanogene 3 (BAG3) is a central node in protein quality control in the heart. In humans and animal models, decreased BAG3 expression is associated with cardiac dysfunction and dilated cardiomyopathy. Although previous studies focused on BAG3 in cardiomyocytes, cardiac fibroblasts are also critical drivers of pathologic remodeling. Yet, the role of BAG3 in cardiac fibroblasts is almost completely unexplored. Here, we show that BAG3 is expressed in primary rat neonatal cardiac fibroblasts and preferentially localizes to mitochondria. Knockdown of BAG3 reduces mitophagy and enhances fibroblast activation, which is associated with fibrotic remodeling. Heat shock protein 70 (Hsp70) is a critical binding partner for BAG3 and inhibiting this interaction in fibroblasts using the drug JG-98 decreased autophagy, decreased mitofusin-2 expression, and disrupted mitochondrial morphology. Together, these data indicate that BAG3 is expressed in cardiac fibroblasts, where it facilitates mitophagy and promotes fibroblast quiescence. This suggests that depressed BAG3 levels in heart failure may exacerbate fibrotic pathology, thus contributing to myocardial dysfunction through sarcomere-independent pathways.NEW & NOTEWORTHY We report BAG3's localization to mitochondria and its role in mitophagy for the first time in primary ventricular cardiac fibroblasts. We have also collected the first evidence showing that loss of BAG3 increases cardiac fibroblast activation into myofibroblasts, which are major drivers of cardiac fibrosis and pathological remodeling during heart disease.

The CB1 receptor interacts with cereblon and drives cereblon deficiency-associated memory shortfalls.

Cereblon/CRBN is a substrate-recognition component of the Cullin4A-DDB1-Roc1 E3 ubiquitin ligase complex. Destabilizing mutations in the human CRBN gene cause a form of autosomal recessive non-syndromic intellectual disability (ARNSID) that is modelled by knocking-out the mouse Crbn gene. A reduction in excitatory neurotransmission has been proposed as an underlying mechanism of the disease. However, the precise factors eliciting this impairment remain mostly unknown. Here we report that CRBN molecules selectively located on glutamatergic neurons are necessary for proper memory function. Combining various in vivo approaches, we show that the cannabinoid CB1 receptor (CB1R), a key suppressor of synaptic transmission, is overactivated in CRBN deficiency-linked ARNSID mouse models, and that the memory deficits observed in these animals can be rescued by acute CB1R-selective pharmacological antagonism. Molecular studies demonstrated that CRBN interacts physically with CB1R and impairs the CB1R-Gi/o-cAMP-PKA pathway in a ubiquitin ligase-independent manner. Taken together, these findings unveil that CB1R overactivation is a driving mechanism of CRBN deficiency-linked ARNSID and anticipate that the antagonism of CB1R could constitute a new therapy for this orphan disease.

Lian-Mei-Yin formula alleviates diet-induced hepatic steatosis by suppressing Yap1/FOXM1 pathway-dependent lipid synthesis.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, with a global prevalence of 25%. Patients with NAFLD are more likely to suffer from advanced liver disease, cardiovascular disease, or type II diabetes. However, unfortunately, there is still a shortage of FDA-approved therapeutic agents for NAFLD. Lian-Mei-Yin (LMY) is a traditional Chinese medicine formula used for decades to treat liver disorders. It has recently been applied to type II diabetes which is closely related to insulin resistance. Given that NAFLD is another disease involved in insulin resistance, we hypothesize that LMY might be a promising formula for NAFLD therapy. Herein, we verify that the LMY formula effectively reduces hepatic steatosis in diet-induced zebrafish and NAFLD model mice in a time- and dose-dependent manner. Mechanistically, LMY suppresses Yap1-mediated Foxm1 activation, which is crucial for the occurrence and development of NAFLD. Consequently, lipogenesis is ameliorated by LMY administration. In summary, the LMY formula alleviates diet-induced NAFLD in zebrafish and mice by inhibiting Yap1/Foxm1 signaling-mediated NAFLD pathology.

XAF1 overexpression inhibits the malignant progression and cisplatin resistance of NSCLC by activating endoplasmic reticulum stress.

BACKGROUND: XIAP-associated factor 1 (XAF1) has been found to participate in the progression of multiple human cancers. Nevertheless, its role as well as the reaction mechanism in non-small cell lung cancer (NSCLC) still remains obscure. METHODS: In this study, the protein expression of XAF1 in NSCLC cell lines was evaluated using western blot. With the employment of CCK-8 assay, EdU staining, wound healing and transwell, capabilities of NSCLC cells to proliferate, migrate and invade were assessed. Cell apoptotic level and cell cycle were resolved utilizing flow cytometry. Western blot was applied for the estimation of apoptosis- and endoplasmic reticulum (ER) stress-related proteins. RESULTS: It was discovered that XAF1 expression was conspicuously reduced in NSCLC cell lines. XAF1 overexpression suppressed H1299 cell proliferative, invasive and migrative capabilities, but exhibited promotive effects on cell cycle arrest. Meanwhile, XAF1 overexpression inhibited cisplatin resistance in H1299 and H1299/DDP cells by promoting cell apoptosis and enhanced the expression levels of ER stress-related proteins CHOP, GRP78 and ATF4. What's more, 4-PBA treatment reversed the impacts of XAF1 overexpression on the proliferative, invasive, migrative and apoptotic capabilities of H1299 cells, as well as cell cycle and cisplatin resistance. CONCLUSION: In conclusion, XAF1 overexpression impeded the advancement of NSCLC and repressed cisplatin resistance of NSCLC cells through inducing ER stress, which indicated that XAF1 might be a novel targeted-therapy for NSCLC.

T cell specific deletion of Casitas B lineage lymphoma-b reduces atherosclerosis, but increases plaque T cell infiltration and systemic T cell activation.

Introduction: Atherosclerosis is a lipid-driven inflammatory disease of the arterial wall, and the underlying cause of the majority of cardiovascular diseases. Recent advances in high-parametric immunophenotyping of immune cells indicate that T cells constitute the major leukocyte population in the atherosclerotic plaque. The E3 ubiquitin ligase Casitas B-lymphoma proto-oncogene-B (CBL-B) is a critical intracellular regulator that sets the threshold for T cell activation, making CBL-B a potential therapeutic target to modulate inflammation in atherosclerosis. We previously demonstrated that complete knock-out of CBL-B aggravated atherosclerosis in Apoe-/- mice, which was attributed to increased macrophage recruitment and increased CD8+ T cell activation in the plaque. Methods: To further study the T cell specific role of CBL-B in atherosclerosis, Apoe-/- CD4cre Cblb fl/fl (Cbl-bcKO) mice and Apoe-/-CD4WTCblbfl/fl littermates (Cbl-bfl/fl) were fed a high cholesterol diet for ten weeks. Results: Cbl-bcKO mice had smaller atherosclerotic lesions in the aortic arch and root compared to Cbl-bfl/fl, and a substantial increase in CD3+ T cells in the plaque. Collagen content in the plaque was decreased, while other plaque characteristics including plaque necrotic core, macrophage content, and smooth muscle cell content, remained unchanged. Mice lacking T cell CBL-B had a 1.4-fold increase in CD8+ T cells and a 1.8-fold increase in regulatory T cells in the spleen. Splenic CD4+ and CD8+ T cells had increased expression of C-X-C Motif Chemokine Receptor 3 (CXCR3) and interferon-γ (IFN-γ), indicating a T helper 1 (Th1)-like/effector CD8+ T cell-like phenotype. Conclusion: In conclusion, Cbl-bcKO mice have reduced atherosclerosis but show increased T cell accumulation in the plaque accompanied by systemic T cell activation.

CDCA5 promoted cell invasion and migration by activating TGF-ß1 pathway in human ovarian cancer cells.

BACKGROUND: The gene cell division cycle associated 5 (CDCA5), also called sororin, has oncogenic characteristics and is upregulated in various carcinomas. Nevertheless, the involvement of CDCA5 in ovarian cancer (OC), a highly aggressive form of cancer, and the underlying mechanism of metastasis remain inadequately investigated. RESULTS: The bioinformatics data revealed a negative correlation between the patient's survival and CDCA5 expression, which was overexpressed in OC. Functional assays also confirmed high expression levels of CDCA5 in OC tissues and cells. This suggests that CDCA5 may potentially enhance the motility, migration, and proliferation of OC cells invitro. It impedes DNA damage and apoptosis in OC cells, inhibiting xenograft development in nude mice. The RNA sequencing results suggest CDCA5 is majorly associated with biological functions related to the extracellular matrix (ECM) and influences the transforming growth factor (TGF) signaling pathway. Moreover, subsequent functional investigations elucidated that CDCA5 facilitated the migration and invasion of OC cells viathe TGF-ß1/Smad2/3 signaling pathway activation. CONCLUSIONS: CDCA5 may be a strong potential therapeutic target for the treatment and management of OC.

Impaired oxysterol-liver X receptor signaling underlies aberrant cortical neurogenesis in a stem cell model of neurodevelopmental disorder.

The mechanisms by which genomic risks contribute to the onset of neuropsychiatric conditions remain a key challenge and a prerequisite for successful development of effective therapies. 15q11.2 copy number variation (CNV) containing the CYFIP1 gene is associated with autism and schizophrenia. Using stem cell models, we show that 15q11.2 deletion (15q11.2del) and CYFIP1 loss of function (CYFIP1-LoF) lead to premature neuronal differentiation, while CYFIP1 gain of function (CYFIP1-GoF) favors neural progenitor maintenance. CYFIP1 dosage changes led to dysregulated cholesterol metabolism and altered levels of 24S,25-epoxycholesterol, which can mimic the 15q11.2del and CYFIP1-LoF phenotypes by promoting cortical neuronal differentiation and can restore the impaired neuronal differentiation of CYFIP1-GoF neural progenitors. Moreover, the neurogenic activity of 24S,25-epoxycholesterol is lost following genetic deletion of liver X receptor (LXRß), while compound deletion of LXRß in CYFIP1-/- background rescued their premature neurogenesis. This work delineates LXR-mediated oxysterol regulation of neurogenesis as a pathological mechanism in neural cells carrying 15q11.2 CNV and provides a potential target for therapeutic strategies for associated disorders.

The activation of the adaptor protein STING depends on its interactions with the phospholipid PI4P.

Activation of the endoplasmic reticulum (ER)-resident adaptor protein STING, a component of a cytosolic DNA-sensing pathway, induces the transcription of genes encoding type I interferons (IFNs) and other proinflammatory factors. Because STING is activated at the Golgi apparatus, control of the localization and activation of STING is important in stimulating antiviral and antitumor immune responses. Through a genome-wide CRISPR interference screen, we found that STING activation required the Golgi-resident protein ACBD3, which promotes the generation of phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network, as well as other PI4P-associated proteins. Appropriate localization and activation of STING at the Golgi apparatus required ACBD3 and the PI4P-generating kinase PI4KB. In contrast, STING activation was enhanced when the lipid-shuttling protein OSBP, which removes PI4P from the Golgi apparatus, was inhibited by the US Food and Drug Administration-approved antifungal itraconazole. The increase in the abundance of STING-activating phospholipids at the trans-Golgi network resulted in the increased production of IFN-ß and other cytokines in THP-1 cells. Furthermore, a mutant STING that could not bind to PI4P failed to traffic from the ER to the Golgi apparatus in response to a STING agonist, whereas forced relocalization of STING to PI4P-enriched areas elicited STING activation in the absence of stimulation with a STING agonist. Thus, PI4P is critical for STING activation, and manipulating PI4P abundance may therapeutically modulate STING-dependent immune responses.

BAG5 regulates HSPA8-mediated protein folding required for sperm head-tail coupling apparatus assembly.

Teratozoospermia is a significant cause of male infertility, but the pathogenic mechanism of acephalic spermatozoa syndrome (ASS), one of the most severe teratozoospermia, remains elusive. We previously reported Spermatogenesis Associated 6 (SPATA6) as the component of the sperm head-tail coupling apparatus (HTCA) required for normal assembly of the sperm head-tail conjunction, but the underlying molecular mechanism has not been explored. Here, we find that the co-chaperone protein BAG5, expressed in step 9-16 spermatids, is essential for sperm HTCA assembly. BAG5-deficient male mice show abnormal assembly of HTCA, leading to ASS and male infertility, phenocopying SPATA6-deficient mice. In vivo and in vitro experiments demonstrate that SPATA6, cargo transport-related myosin proteins (MYO5A and MYL6) and dynein proteins (DYNLT1, DCTN1, and DNAL1) are misfolded upon BAG5 depletion. Mechanistically, we find that BAG5 forms a complex with HSPA8 and promotes the folding of SPATA6 by enhancing HSPA8's affinity for substrate proteins. Collectively, our findings reveal a novel protein-regulated network in sperm formation in which BAG5 governs the assembly of the HTCA by activating the protein-folding function of HSPA8.