Atovaquone enhances antitumor efficacy of TCR-T therapy by augmentation of ROS-induced ferroptosis in hepatocellular carcinoma.

T-cell receptor (TCR) engineered T-cell therapy has recently emerged as a promising adoptive immunotherapy approach for tumor treatment, yet hindered by tumor immune evasion resulting in poor therapeutic efficacy. The introduction of ferroptosis-targeted inducers offers a potential solution, as they empower T cells to induce ferroptosis and exert influence over the tumor microenvironment. Atovaquone (ATO) stands as a prospective pharmaceutical candidate with the potential to target ferroptosis, effectively provoking an excessive generation and accumulation of reactive oxygen species (ROS). In this study, we evaluated the effectiveness of a combination therapy comprising ATO and TCR-T cells against hepatocellular carcinoma (HCC), both in vitro and in vivo. The results of lactate dehydrogenase and cytokine assays demonstrated that ATO enhanced cytotoxicity mediated by AFP-specific TCR-T cells and promoted the release of IFN-γ in vitro. Additionally, in an established HCC xenograft mouse model, the combined therapy with low-dose ATO and TCR-T cells exhibited heightened efficacy in suppressing tumor growth, with no apparent adverse effects, comparable to the results achieved through monotherapy. The RNA-seq data unveiled a significant activation of the ferroptosis-related pathway in the combination therapy group in comparison to the TCR-T cells group. Mechanistically, the synergy between ATO and TCR-T cells augmented the release of IFN-γ by TCR-T cells, while concurrently elevating the intracellular and mitochondrial levels of ROS, expanding the labile iron pool, and impairing the integrity of the mitochondrial membrane in HepG2 cells. This multifaceted interaction culminated in the potentiation of ferroptosis within the tumor, primarily induced by an excess of ROS. In summary, the co-administration of ATO and TCR-T cells in HCC exhibited heightened vulnerability to ferroptosis. This heightened susceptibility led to the inhibition of tumor growth and the stimulation of an anti-tumor immune response. These findings suggest that repurposing atovaquone for adoptive cell therapy combination therapy holds the potential to enhance treatment outcomes in HCC.

Mild hyperphenylalaninemia (hpa) presenting as orthostatic tremor: a case report.

BACKGROUND: Orthostatic tremor (OT) is a type of postural tremor of the lower extremities that has not been described in either phenylketonuria (PKU) or hyperphenylalaninemia (HPA). Because little is known about the clinical features and therapeutic responses of OT in mild HPA, we describe a mild HPA patient who presented with OT as an initial symptom. CASE PRESENTATION: A 22-year-old male was admitted for bilateral leg tremor while standing, with symptom onset eight months prior. One month before admission, the tremor disappeared in the left leg but persisted in the right leg. Electromyography recorded from the right gastrocnemius revealed a 6-8 Hz tremor, which appeared when the patient was standing and disappeared when he was resting or walking. Blood screening showed a phenylalanine/tyrosine ratio of 2.06 and a phenylalanine level of 140 µmol/L. Urine metabolic screening was negative. Whole-exome sequencing confirmed the presence of a compound heterozygous mutation, c.158G > A and c.728G > A, in phenylalanine hydroxylase (PAH) gene. After three months of levodopa/benserazide tablets (250 mg, tid) and a low-phenylalanine diet treatment, the tremor disappeared. CONCLUSIONS: Young-onset mild HPA is a relatively rare autosomal recessive metabolic disease, and slow OT is a rare clinical feature. Metabolic screening and genetic testing are the keys to early diagnosis and treatment. For adolescents and young adults, appropriate medication and long-term dietary therapy remain important treatments. This case expanded the disease spectrum of slow OT.

The scaffold-protein IQGAP1 enhances and spatially restricts the actin-nucleating activity of Diaphanous-related formin 1 (DIAPH1).

The actin cytoskeleton is a dynamic array of filaments that undergoes rapid remodeling to drive many cellular processes. An essential feature of filament remodeling is the spatio-temporal regulation of actin filament nucleation. One family of actin filament nucleators, the Diaphanous-related formins, is activated by the binding of small G-proteins such as RhoA. However, RhoA only partially activates formins, suggesting that additional factors are required to fully activate the formin. Here we identify one such factor, IQ motif containing GTPase activating protein-1 (IQGAP1), which enhances RhoA-mediated activation of the Diaphanous-related formin (DIAPH1) and targets DIAPH1 to the plasma membrane. We find that the inhibitory intramolecular interaction within DIAPH1 is disrupted by the sequential binding of RhoA and IQGAP1. Binding of RhoA and IQGAP1 robustly stimulates DIAPH1-mediated actin filament nucleation in vitro In contrast, the actin capping protein Flightless-I, in conjunction with RhoA, only weakly stimulates DIAPH1 activity. IQGAP1, but not Flightless-I, is required to recruit DIAPH1 to the plasma membrane where actin filaments are generated. These results indicate that IQGAP1 enhances RhoA-mediated activation of DIAPH1 in vivo Collectively these data support a model where the combined action of RhoA and an enhancer ensures the spatio-temporal regulation of actin nucleation to stimulate robust and localized actin filament production in vivo.

A humanized TCR retaining authentic specificity and affinity conferred potent anti-tumour cytotoxicity.

The affinity of T-cell receptor (TCR) determines the efficacy of TCR-based immunotherapy. By using human leucocyte antigen (HLA)-A*02 transgenic mice, a TCR was generated previously specific for human tumour testis antigen peptide MAGE-A3112-120 (KVAELVHFL) HLA-A*02 complex. We developed an approach to humanize the murine TCR by replacing the mouse framework with sequences of folding optimized human TCR variable domains for retaining binding affinity. The resultant humanized TCR exhibited higher affinity and conferred better anti-tumour activity than its parent murine MAGE-A3 TCR (SRm1). In addition, the affinity of humanized TCR was enhanced further to achieve improved T-cell activation. Our studies demonstrated that the human TCR variable domain frameworks could provide support for complementarity-determining regions from a murine TCR, and retain the original binding activity. It could be used as a generic approach of TCR humanization.

ImmTAC/Anti-PD-1 antibody combination to enhance killing of cancer cells by reversing regulatory T-cell-mediated immunosuppression.

Recently, bi-functional molecules that can redirect immune effectors to tumour cells have emerged as potentially robust mediators of tumour regression in clinical trials. Two modalities in particular, bi-specific antibodies for T-cell redirection and activation (BiTe) and immune-mobilizing monoclonal T-cell receptors against cancer (ImmTAC), are being evaluated in efficacy studies as 'off-the-shelf' reagents. Optimal therapy will require an understanding and means to address regulatory mechanisms of limiting efficacy. In light of this, we evaluated the impact of induced regulatory T (iTreg) cells on the efficacy of tumour cell killing redirected by ImmTAC and demonstrated down-regulation of T-cell proliferation and expression of CD25, CD107a, Granzyme B and Perforin by ImmTAC-redirected T cells. Significant recovery of ImmTAC potency, however, could be achieved when combined with an anti-programmed cell death protein 1 monoclonal antibody. Furthermore, we found that among lung cancer patients failing to respond to ImmTAC therapy, there was a significantly higher fraction of Treg cells in the peripheral blood mononuclear cells of lung cancer patients than in healthy donors. These results provide in vitro evidence for an iTreg cell-mediated immunosuppression of ImmTAC-redirected T-cell responses. Whilst immune checkpoint blockade can reverse the Treg cell suppression, it forms a rational basis for a combination of the blockade with ImmTAC in clinical trials.

Importin ß2 Mediates the Spatio-temporal Regulation of Anillin through a Noncanonical Nuclear Localization Signal.

The compartmentalization of cell cycle regulators is a common mechanism to ensure the precise temporal control of key cell cycle events. For instance, many mitotic spindle assembly factors are known to be sequestered in the nucleus prior to mitotic onset. Similarly, the essential cytokinetic factor anillin, which functions at the cell membrane to promote the physical separation of daughter cells at the end of mitosis, is sequestered in the nucleus during interphase. To address the mechanism and role of anillin targeting to the nucleus in interphase, we identified the nuclear targeting motif. Here, we show that anillin is targeted to the nucleus by importin ß2 in a Ran-dependent manner through an atypical basic patch PY nuclear localization signal motif. We show that although importin ß2 binding does not regulate anillin's function in mitosis, it is required to prevent the cytosolic accumulation of anillin, which disrupts cellular architecture during interphase. The nuclear sequestration of anillin during interphase serves to restrict anillin's function at the cell membrane to mitosis and allows anillin to be rapidly available when the nuclear envelope breaks down to remodel the cellular architecture necessary for successful cell division.

Genome sequences of Colletotrichum eleusines and C. echinochloae: two pathogens from Poaceae weeds.

We present two whole-genome sequences of Colletotrichum strains which were isolated from Eleusine indica and Echinochloa crus-galli using Nanopore and Illumina technologies, as part of screening for potential mycoherbicide. The genome sequences will provide important genetic information and will be useful for further research into secondary metabolites of Colletotrichum.

Colletotrichum echinochloae: A Potential Bioherbicide Agent for Control of Barnyardgrass (Echinochloa crus-galli (L.) Beauv.).

Barnyardgrass (Echinochloa crus-galli (L.) Beauv.) is one of the most troublesome weeds in transplanted and direct-seeded rice worldwide. To develop a strategy for the biocontrol of barnyardgrass, fungal isolates were recovered from barnyardgrass plants that exhibited signs of necrosis and wilt. An isolate B-48 with a high level of pathogenicity to barnyardgrass was identified after pathogenicity tests. From cultural and DNA sequence data, this strain was identified as Colletotrichum echinochloae. The inoculation of the barnyardgrass plant with C. echinochloae caused a significant reduction in fresh weight. The isolate B-48 was highly pathogenic to barnyardgrass at the three- to four-leaf stages. When inoculated at a concentration of 1 × 107 spores/mL, barnyardgrass could achieve a reduction in fresh weight of more than 50%. This strain was safe for rice and most plant species. The results of this study indicated that this strain could be a potential mycoherbicide for barnyardgrass control in paddy fields in the future.

Mortality of Alzheimer's Disease and Other Dementias in China: Past and Future Decades.

Objectives: This study aimed to explore the distribution features and trends of dementia mortality in China from 2011 to 2020 and make a prediction for the next decade. Methods: Mortality-relevant data were gathered from the Chinese Center for Disease Control and Prevention's Disease Surveillance Points system. Joinpoint regression was applied to evaluate the trends. Results: Crude Mortality Rate (CMR) of AD and other dementias increased from 3.7 per 100,000 to 6.2 per 100,000 in 2011-2020, with an Average Annual Percent Change (AAPC) of 5.3% (95% CI 4.4%-6.3%). Age-Standardized Mortality Rate (ASMR) slightly decreased from 5.0 per 100,000 to 4.1 per 100,000 in 2011-2020, with AAPC of -0.4% (95% CI -2.5%-1.8%). CMR will increase to 9.66 per 100,000 while ASMR will decline to 3.42 per 100,000 in the following decade. Conclusion: The upward trend in CMR and downward trend in ASMR suggested the further development of population aging and dementia mortality in the past and future decades. In China, there were gender, urban-rural, regional and age differences.

Mutant soluble ectodomain of fibroblast growth factor receptor-2 IIIc attenuates bleomycin-induced pulmonary fibrosis in mice.

We have developed a strong inhibitor (S252W mutant soluble ectodomain of fibroblast growth factor recptor-2 IIIc, msFGFR2) that binds FGFs strongly and blocks the activation of FGFRs. In vitro, msFGFR2 could inhibit the promoting effect of transforming growth factor (TGF)-ß1 on the proliferation of primary lung fibroblasts. In vivo, msFGFR2 alleviated lung fibrosis through inhibiting the expression of α-smooth muscle actin (SMA) and collagen deposit. In Western blotting of the right lung tissues and immunohistochemical assay, we found the level of p-FGFRs, p-mitogen activated protein kinase (MAPK) and p-Smad3 in the mice of bleomycin (BLM) group treated with msFGFR2 was down dramatically compared with the mice of BLM group, which suggested the activations of FGF and TGF-ß signals were blocked meanwhile. In summary, msFGFR2 attenuated BLM-induced fibrosis and is an attractive therapeutic candidate for human pulmonary fibrosis.

An anillin-CIN85-SEPT9 complex promotes intercellular bridge maturation required for successful cytokinesis.

Cleavage of one cell into two is the most dramatic event in the life of a cell. Plasma membrane fission occurs within a narrow intercellular bridge (ICB) between the daughter cells, but the mechanisms underlying ICB formation and maturation are poorly understood. Here we identify CIN85 as an ICB assembly factor and demonstrate its requirement for robust and timely cytokinesis. CIN85 interacts directly with the N-terminal region of anillin and SEPT9 and thereby facilitates SEPT9-containing filament localization to the plasma membrane of the ICB. In contrast, the C-terminal pleckstrin homology (PH) domain of anillin binds to septin units lacking SEPT9 but enriched in SEPT11. Anillin's interactions with distinct septin units are required to promote ICB elongation and maturation that, we propose, generate the physical space into which the abscission machinery is recruited to drive the final membrane scission event releasing two independent daughter cells.

SAGE1: a Potential Target Antigen for Lung Cancer T-Cell Immunotherapy.

A fundamental understanding of cancer-specific antigens is crucial for successful T-cell immunotherapy. Sarcoma antigen 1 (SAGE1) is a cancer/testis antigen that has not yet been verified for T-cell immunotherapy applications. Here, we examined SAGE1 RNA expression and carried out IHC analyses, revealing that SAGE1 is expressed in 50% of non-small cell lung-cancer samples (n = 40). To verify the immunogenicity of SAGE1, we discovered a novel HLA-A*24:02 (HLA-A24)-restricted SAGE1 epitope (SAGE1597-606, VFSTAPPAFI) using mass spectrometry and identified SAGE1597-606-specific T-cell clones and T-cell receptors (TCR) from peripheral bloods of HLA-A24+ donors. The highest affinity TCR VF3 (KD = 4.3 µM) demonstrated the highest antitumor potency. Moreover, VF3-transduced T cells mediated the efficient killing of HLA-A24+/SAGE1+ tumor cells in vitro and effectively inhibited the growth of lung cancer xenografts in mice. Together, our data suggest that SAGE1 could be a target for T-cell immunotherapies against lung cancer, while its specific TCRs could be candidates for developing reagents to treat SAGE1+ tumors.

Inhibition of polar actin assembly by astral microtubules is required for cytokinesis.

During cytokinesis, the actin cytoskeleton is partitioned into two spatially distinct actin isoform specific networks: a ß-actin network that generates the equatorial contractile ring, and a γ-actin network that localizes to the cell cortex. Here we demonstrate that the opposing regulation of the ß- and γ-actin networks is required for successful cytokinesis. While activation of the formin DIAPH3 at the cytokinetic furrow underlies ß-actin filament production, we show that the γ-actin network is specifically depleted at the cell poles through the localized deactivation of the formin DIAPH1. During anaphase, CLIP170 is delivered by astral microtubules and displaces IQGAP1 from DIAPH1, leading to formin autoinhibition, a decrease in cortical stiffness and localized membrane blebbing. The contemporaneous production of a ß-actin contractile ring at the cell equator and loss of γ-actin from the poles is required to generate a stable cytokinetic furrow and for the completion of cell division.

High-affinity human programmed death-1 ligand-1 variant promotes redirected T cells to kill tumor cells.

Tumor cells can escape immune surveillance through the programmed cell death protein 1 (PD-1) axis suppressing T cells. However, we recently demonstrated that high-affinity variants of soluble human programmed death-ligand 1 (shPD-L1) could diminish the suppression. We propose that in comparison to the wild-type shPD-L1, the further affinity enhancement will confer the molecule with opposite characteristics that augment T-cell activation and immunotherapeutic drug potential. In this study, a new shPD-L1 variant, L3C7c, has been generated to demonstrate ∼167 fold greater affinity than wild-type hPD-L1. The L3C7c-Fc fusion protein demonstrated completely opposite effects of conventional PD-1 axis by promoting redirected T-cell proliferation, activation and cytotoxicity in vitro, as being slightly better than that of anti-PD1-Ab (Pembrolizumab). Moreover, L3C7c-Fc was more effective than Pembrolizumab in enhancing redirected T cells' ability to suppress Mel624 melanoma growth in vivo. As a downsized L3C7c-Fc variant, L3C7v-Fc improved the anti-tumor efficacy in vivo when combined with dendritic cell vaccines. In conclusion, our studies demonstrate that high-affinity hPD-L1 variants could be developed as the next generation reagents for tumor immunotherapy based on the blockade of the PD-1 axis.

GCC2-ALK as a targetable fusion in lung adenocarcinoma and its enduring clinical responses to ALK inhibitors.

OBJECTIVES: ALK, RET and ROS1 fusions have been identified as treatable targets in 5%-15% of non-small-cell lung cancers, and thanks to the advanced sequencing technologies, their new partner genes have been steadily detected. Here we identified a rare fusion of ALK (GCC2-ALK) in a patient with advanced lung adenocarcinoma and monitored the treatment efficacy of ALK inhibitors on this patient. We further performed in vitro functional studies of this fusion protein for evaluating its oncogenic potential. MATERIALS AND METHODS: The GCC2-ALK fusion gene was identified by targeted next generation sequencing (NGS) from the tumor DNA samples, and its fusion product was confirmed by Sanger sequencing the cDNA product. The functional study of GCC2-ALK was performed in Ba/F3 cells with cell proliferation and viability assays. The activation of downstream signaling pathways of ALK and their responses to crizotinib inhibition were studied in HEK-293 and 293T cells with ectopic expression of GCC2-ALK. In parallel, disease progression in the patient was monitored by computed tomography scanning and targeted NGS of either liquid or tissue biopsy samples throughout and after crizotinib treatment. RESULTS: Similarly to EML4-ALK, the GCC2-ALK fusion protein promotes IL-3-independent growth of Ba/F3 cells. Ectopic expression of GCC2-ALK leads to hyper-activation of ALK downstream signaling that can be inhibited by crizotinib. Crizotinib treatment of the patient resulted in 18 months of progression free survival without any trace of GCC2-ALK fusion in the liquid biopsies. Re-biopsy of a lung lesion at progression identified the re-occurrence of GCC2-ALK. The patient was then administrated with a second-generation ALK inhibitor, ceritinib, and received partial response until the last follow-up. CONCLUSION: We identified and functionally validated GCC2-ALK as a constitutively activated fusion in NSCLC. The patient was benefited from crizotinib treatment initially and then ceritinib after progression, suggesting GCC2-ALK as a novel therapeutic target for ALK inhibitors.

BMP-2 induces EMT and breast cancer stemness through Rb and CD44.

Bone morphogenetic protein 2 (BMP-2) has been reported to facilitate epithelial-to-mesenchymal transition (EMT) and bone metastasis in breast cancer xenograft models. To investigate the role of BMP-2 in the development of breast cancer stem cells (BCSCs), and to further elucidate the mechanisms underlying its influence on breast cancer metastasis, we conducted a comprehensive molecular study using breast cancer cell lines and clinical samples. Our results showed that downregulation of Rb by BMP-2 was associated with ubiquitin-mediated degradation activated by phosphorylation of Rb via the PI3K/AKT signal pathway. In addition, the Smad signaling pathways are implicated in upregulation of CD44 protein expression by BMP-2. It was suggested that cross-talk exists between Rb and CD44 signaling pathways, as recombinant human BMP-2 (rhBMP-2) was found to regulate CD44 expression partly through Rb signals. In clinical tissues, BMP-2 was positively and negatively correlated with CD44 and Rb expression, respectively. Based on the in vitro and in vivo results, we have established an integrated mechanism by which rhBMP-2 induces EMT and stemness of breast cancer cells via the Rb and CD44 signaling pathways, which then contribute to breast cancer metastasis. These findings may be helpful for developing new strategies for the treatment and prognosis of advanced breast cancer.

[Leaching kinetics of josephinite tailings with sulfuric acid].

Leaching is the most important step of josephinite tailing recycle technology. This step can separate the valuable metal Mg from Si and other impure metal. Effects of sulfuric acid on leaching Mg efficiency from josephinite tailings were investigated. To obtain the leaching behavior, a modified unreacted shrinking core model that based on the experimental data was used to determine the dissolution kinetic parameters. The model was significant and showed that the dissolution of Mg2+ in josephinite tailing was controlled by the produce layer diffusion, apparent activation reaction energy E = 34.04 kJ x mol(-1). The produce layers obstruct the forward reaction of the dissolution of Mg2+.