ERK and c-Myc signaling in host-derived tumor endothelial cells is essential for solid tumor growth.

The limited efficacy of the current antitumor microenvironment strategies is due in part to the poor understanding of the roles and relative contributions of the various tumor stromal cells to tumor development. Here, we describe a versatile in vivo anthrax toxin protein delivery system allowing for the unambiguous genetic evaluation of individual tumor stromal elements in cancer. Our reengineered tumor-selective anthrax toxin exhibits potent antiproliferative activity by disrupting ERK signaling in sensitive cells. Since this activity requires the surface expression of the capillary morphogenesis protein-2 (CMG2) toxin receptor, genetic manipulation of CMG2 expression using our cell-type-specific CMG2 transgenic mice allows us to specifically define the role of individual tumor stromal cell types in tumor development. Here, we established mice with CMG2 only expressed in tumor endothelial cells (ECs) and determined the specific contribution of tumor stromal ECs to the toxin's antitumor activity. Our results demonstrate that disruption of ERK signaling only within tumor ECs is sufficient to halt tumor growth. We discovered that c-Myc is a downstream effector of ERK signaling and that the MEK-ERK-c-Myc central metabolic axis in tumor ECs is essential for tumor progression. As such, disruption of ERK-c-Myc signaling in host-derived tumor ECs by our tumor-selective anthrax toxins explains their high efficacy in solid tumor therapy.

Selective targeting of metastatic ovarian cancer using an engineered anthrax prodrug activated by membrane-anchored serine proteases.

Treatments for advanced and recurrent ovarian cancer remain a challenge due to a lack of potent, selective, and effective therapeutics. Here, we developed the basis for a transformative anticancer strategy based on anthrax toxin that has been engineered to be selectively activated by the catalytic power of zymogen-activating proteases on the surface of malignant tumor cells to induce cell death. Exposure to the engineered toxin is cytotoxic to ovarian tumor cell lines and ovarian tumor spheroids derived from patient ascites. Preclinical studies demonstrate that toxin treatment induces tumor regression in several in vivo ovarian cancer models, including patient-derived xenografts, without adverse side effects, supportive of progression toward clinical evaluation. These data lay the groundwork for developing therapeutics for treating women with late-stage and recurrent ovarian cancers, utilizing a mechanism distinct from current anticancer therapies.

ATM-CHK2-Beclin 1 axis promotes autophagy to maintain ROS homeostasis under oxidative stress.

The homeostatic link between oxidative stress and autophagy plays an important role in cellular responses to a wide variety of physiological and pathological conditions. However, the regulatory pathway and outcomes remain incompletely understood. Here, we show that reactive oxygen species (ROS) function as signaling molecules that regulate autophagy through ataxia-telangiectasia mutated (ATM) and cell cycle checkpoint kinase 2 (CHK2), a DNA damage response (DDR) pathway activated during metabolic and hypoxic stress. We report that CHK2 binds to and phosphorylates Beclin 1 at Ser90/Ser93, thereby impairing Beclin 1-Bcl-2 autophagy-regulatory complex formation in a ROS-dependent fashion. We further demonstrate that CHK2-mediated autophagy has an unexpected role in reducing ROS levels via the removal of damaged mitochondria, which is required for cell survival under stress conditions. Finally, CHK2-/- mice display aggravated infarct phenotypes and reduced Beclin 1 p-Ser90/Ser93 in a cerebral stroke model, suggesting an in vivo role of CHK2-induced autophagy in cell survival. Taken together, these results indicate that the ROS-ATM-CHK2-Beclin 1-autophagy axis serves as a physiological adaptation pathway that protects cells exposed to pathological conditions from stress-induced tissue damage.

Targeting TAOK1 with resveratrol inhibits esophageal squamous cell carcinoma growth in vitro and in vivo.

The worldwide incidence and mortality rates of esophageal squamous cell carcinoma (ESCC) have increased over the last decade. Moreover, molecular targets that may benefit the therapeutics of patients with ESCC have not been fully characterized. Our study discovered that thousand and one amino-acid protein kinase 1 (TAOK1) is highly expressed in ESCC tumor tissues and cell lines. Knock-down of TAOK1 suppresses ESCC cell proliferation in vitro and patient-derived xenograft or cell-derived xenograft tumors growth in vivo. Moreover, TAOK1 overexpression promotes ESCC growth in vitro and in vivo. Additionally, we identified that the natural small molecular compound resveratrol binds to TAOK1 directly and diminishes the kinase activity of TAOK1. Targeting TAOK1 directly with resveratrol significantly inhibits cell proliferation, induces cell cycle arrest and apoptosis, and suppresses tumor growth in ESCC. Furthermore, the silencing of TAOK1 or the application of resveratrol attenuated the activation of TAOK1 downstream signaling effectors. Interestingly, combining resveratrol with paclitaxel, cisplatin, or 5-fluorouracil synergistically enhanced their therapeutic effects against ESCC. In conclusion, this work illustrates the underlying oncogenic function of TAOK1 and provides a theoretical basis for the application of targeting TAOK1 therapy to the clinical treatment of ESCC.

Uncertainty affects cancer-related fatigue among breast cancer women undergoing peripherally inserted central catheter chemotherapy: the chain mediating role of psychological resilience and self-care.

BACKGROUND: Breast cancer patients undergoing chemotherapy via peripherally inserted central catheter often experience serious behavioral and psychological challenges, with uncertainty and cancer-related fatigue being prevalent issues that profoundly impact prognosis. Therefore, this study aimed to investigate the relationship between uncertainty and cancer-related fatigue by employing a chain mediation model to examine the potential mediating roles of psychological resilience and self-care. METHODS: A cross-sectional study was conducted with 223 breast cancer patients receiving peripherally inserted central catheter chemotherapy at two tertiary affiliated hospitals of China Medical University in Liaoning, China, from February 2021 to December 2022. Participants completed self-reported questionnaires to assess uncertainty, psychological resilience, self-care, and cancer-related fatigue. The collected data were subsequently analyzed using Pearson's correlation analysis, hierarchical regression analysis, and mediation analysis. RESULTS: Uncertainty exhibited a significant positive correlation with cancer-related fatigue (p < 0.01) and a negative correlation with psychological resilience (p < 0.01) and self-care (p < 0.01). Uncertainty was found to impact cancer-related fatigue through three pathways: psychological resilience mediated the relationship between uncertainty and cancer-related fatigue (mediating effect = 0.240, 95% confidence interval: 0.188 to 0.298, effect ratio = 53.22%); self-care also mediated this relationship (mediating effect = 0.080, 95% confidence interval: 0.044 to 0.121, effect ratio = 17.74%); furthermore, there was a significant joint mediating effect of psychological resilience and self-care on the association between uncertainty and cancer-related fatigue (mediating effect = 0.042, 95% confidence interval: 0.021 to 0.068, effect ratio o = 9.31%). CONCLUSION: The findings of this study revealed that uncertainty not only directly influenced cancer-related fatigue, but also operated through the mediating effect of psychological resilience, self-care, and sequential mediation of psychological resilience and self-care. Interventions tailored for breast cancer patients receiving peripherally inserted central catheter chemotherapy should target these factors to help alleviate uncertainty, enhance psychological resilience, and improve self-care practices, thereby ameliorating cancer-related fatigue.

The secretome mouse provides a genetic platform to delineate tissue-specific in vivo secretion.

At present, it remains difficult to deconvolute serum in order to identify the cell or tissue origin of a given circulating protein. Here, by exploiting the properties of proximity biotinylation, we describe a mouse model that enables the elucidation of the in vivo tissue-specific secretome. As an example, we demonstrate how we can readily identify in vivo endothelial-specific secretion as well as how this model allows for the characterization of muscle-derived serum proteins that either increase or decrease with exercise. This genetic platform should, therefore, be of wide utility in understanding normal and disease physiology and for the rational design of tissue-specific disease biomarkers.

The Programmable Catalytic Core of 8-17 DNAzymes.

8-17 DNAzymes (8-17, 17E, Mg5, and 17EV1) are in vitro-selected catalytic DNA molecules that are capable of cleaving complementary RNAs. The conserved residues in their similar catalytic cores, together with the metal ions, were suggested to contribute to the catalytic reaction. Based on the contribution of the less conserved residues in the bulge loop residues (W12, A15, A15.0) and the internal stem, new catalytic cores of 8-17 DNAzymes were programmed. The internal stem CTC-GAG seems to be more favorable for the DNAzymes than CCG-GGC, while an extra W12.0 led to a significant loss of activity of DNAzymes, which is contrary to the positive effect of A15.0, by which a new active DNAzyme 17EM was derived. It conducts a faster reaction than 17E. It is most active in the presence of Pb2+, with the metal ion preference of Pb2+ >> Zn2+ > Mn2+ > Ca2+ ≈ Mg2+. In the Pb2+ and Zn2+-mediated reactions of 17EM and 17E, the same Na+- and pH dependence were also observed as what was observed for 17E and other 8-17 DNAzymes. Therefore, 17EM is another member of the 8-17 DNAzymes, and it could be applied as a potential biosensor for RNA and metal ions.

Discovery of (S)-N1-(thiazol-2-yl) pyrrolidine-1,2-dicarboxamide derivatives targeting PI3Ka/HDAC6 for the treatment of cancer.

Recently, PI3K and HDAC have been considered as promising targets for the cancer therapy. A couple of pan-PI3K/HDAC dual inhibitors have been developed as a new class of anticancer agents. Herein, we discovered a new series of (S)-N1-(thiazol-2-yl) pyrrolidine-1,2-dicarboxamide derivatives targeting PI3Kα/HDAC6. All the derivatives exerted dual-target inhibitory activities. Particularly, in the enzymatic selectivity assay, compound 21j was identified as a subtype-selective PI3Kα/HDAC6 dual inhibitor (IC50 = 2.9 and 26 nM against PI3Kα and HDAC6, respectively), which displayed high potency against L-363 cell line with IC50 value of 0.17 µM. In addition, 21j significantly inhibited phosphorylation of pAkt(Ser473) and induced accumulation of acetylated α-tubulin while having a negligible effect on the levels of acetylated Histone H3 and H4 at nanomolar level. Attributed to its favorable in vitro performance, 21j has the potential to alleviate the adverse effects resulted from pan-PI3K inhibition and pan-HDAC inhibition. It is valuable for further functional investigation as an anti-cancer agent.

Identification of the transcription factor Miz1 as an essential regulator of diphthamide biosynthesis using a CRISPR-mediated genome-wide screen.

Diphthamide is a unique post-translationally modified histidine residue (His715 in all mammals) found only in eukaryotic elongation factor-2 (eEF-2). The biosynthesis of diphthamide represents one of the most complex modifications, executed by protein factors conserved from yeast to humans. Diphthamide is not only essential for normal physiology (such as ensuring fidelity of mRNA translation), but is also exploited by bacterial ADP-ribosylating toxins (e.g., diphtheria toxin) as their molecular target in pathogenesis. Taking advantage of the observation that cells defective in diphthamide biosynthesis are resistant to ADP-ribosylating toxins, in the past four decades, seven essential genes (Dph1 to Dph7) have been identified for diphthamide biosynthesis. These technically unsaturated screens raise the question as to whether additional genes are required for diphthamide biosynthesis. In this study, we performed two independent, saturating, genome-wide CRISPR knockout screens in human cells. These screens identified all previously known Dph genes, as well as further identifying the BTB/POZ domain-containing transcription factor Miz1. We found that Miz1 is absolutely required for diphthamide biosynthesis via its role in the transcriptional regulation of Dph1 expression. Mechanistically, Miz1 binds to the Dph1 proximal promoter via an evolutionarily conserved consensus binding site to activate Dph1 transcription. Therefore, this work demonstrates that Dph1-7, along with the newly identified Miz1 transcription factor, are likely to represent the essential protein factors required for diphthamide modification on eEF2.

Revisiting Cryptocyanine Dye, NK-4, as an Old and New Drug: Review and Future Perspectives.

NK-4 plays a key role in the treatment of various diseases, such as in hay fever to expect anti-allergic effects, in bacterial infections and gum abscesses to expect anti-inflammatory effects, in scratches, cuts, and mouth sores from bites inside the mouth for enhanced wound healing, in herpes simplex virus (HSV)-1 infections for antiviral effects, and in peripheral nerve disease that causes tingling pain and numbness in hands and feet, while NK-4 is used also to expect antioxidative and neuroprotective effects. We review all therapeutic directions for the cyanine dye NK-4, as well as the pharmacological mechanism of NK-4 in animal models of related diseases. Currently, NK-4, which is sold as an over-the-counter drug in drugstores, is approved for treating allergic diseases, loss of appetite, sleepiness, anemia, peripheral neuropathy, acute suppurative diseases, wounds, heat injuries, frostbite, and tinea pedis in Japan. The therapeutic effects of NK-4's antioxidative and neuroprotective properties in animal models are now under development, and we hope to apply these pharmacological effects of NK-4 to the treatment of more diseases. All experimental data suggest that different kinds of utility of NK-4 in the treatment of diseases can be developed based on the various pharmacological properties of NK-4. It is expected that NK-4 could be developed in more therapeutic strategies to treat many types of diseases, such as neurodegenerative and retinal degenerative diseases.

Studies on the Effect of Lipofectamine and Cell-Penetrating Peptide on the Properties of 10-23 DNAzyme.

Cationic polymeric materials and cell-penetrating peptides (CPPs) were often used as the delivery vectors in the evaluation of nucleic acid therapeutics. 10-23 DNAzyme is a kind of potential antisense therapeutics by catalytic cleavage of the disease-related RNAs. Here, lipofectamine 2000 and Tat peptide were evaluated for their effect on the catalytic activity of 10-23 DNAzyme, with the observed rate constant, thermal stability, CD spectra, and PAGE analysis, with a duplex DNA mimicking DNAzyme-substrate as a control. It was shown that the cationic carriers had a negative effect on the catalytic performance of the 10-23 DNAzyme. Significantly, the destabilizing effect of the cationic carriers on the duplex formation was noteworthy, as a duplex formation is an essential prerequisite in the silencing mechanisms of antisense and RNAi.

Guidelines for the prevention and management of children and adolescents with COVID-19.

Children are the future of the world, but their health and future are facing great uncertainty because of the coronavirus disease 2019 (COVID-19) pandemic. In order to improve the management of children with COVID-19, an international, multidisciplinary panel of experts developed a rapid advice guideline at the beginning of the outbreak of COVID-19 in 2020. After publishing the first version of the rapid advice guideline, the panel has updated the guideline by including additional stakeholders in the panel and a comprehensive search of the latest evidence. All recommendations were supported by systematic reviews and graded using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) system. Expert judgment was used to develop good practice statements supplementary to the graded evidence-based recommendations. The updated guideline comprises nine recommendations and one good practice statement. It focuses on the key recommendations pertinent to the following issues: identification of prognostic factors for death or pediatric intensive care unit admission; the use of remdesivir, systemic glucocorticoids and antipyretics, intravenous immunoglobulin (IVIG) for multisystem inflammatory syndrome in children, and high-flow oxygen by nasal cannula or non-invasive ventilation for acute hypoxemic respiratory failure; breastfeeding; vaccination; and the management of pediatric mental health. CONCLUSION: This updated evidence-based guideline intends to provide clinicians, pediatricians, patients and other stakeholders with evidence-based recommendations for the prevention and management of COVID-19 in children and adolescents. Larger studies with longer follow-up to determine the effectiveness and safety of systemic glucocorticoids, IVIG, noninvasive ventilation, and the vaccines for COVID-19 in children and adolescents are encouraged. WHAT IS KNOWN: • Several clinical practice guidelines for children with COVID-19 have been developed, but only few of them have been recently updated. • We developed an evidence-based guideline at the beginning of the COVID-19 outbreak and have now updated it based on the results of a comprehensive search of the latest evidence. WHAT IS NEW: • The updated guideline provides key recommendations pertinent to the following issues: identification of prognostic factors for death or pediatric intensive care unit admission; the use of remdesivir, systemic glucocorticoids and antipyretics, intravenous immunoglobulin for multisystem inflammatory syndrome in children, and high-flow oxygen by nasal cannula or non-invasive ventilation for acute hypoxemic respiratory failure; breastfeeding; vaccination; and the management of pediatric mental health.

Retracted: Aggravation of Cerebral Ischemia/Reperfusion Injury by Peroxisome Proliferator-Activated Receptor-Gamma Deficiency via Endoplasmic Reticulum Stress.

This paper is retracted at the author's request. Reference: Yueping Chen, Shihui Liu, Guangyong Chen: Aggravation of Cerebral Ischemia/Reperfusion Injury by Peroxisome Proliferator-Activated Receptor-Gamma Deficiency via Endoplasmic Reticulum Stress. Med Sci Monit, 2019; 25:7518-7526. DOI: 10.12659/MSM.915914.

Effect of NK-5962 on Gene Expression Profiling of Retina in a Rat Model of Retinitis Pigmentosa.

PURPOSE: NK-5962 is a key component of photoelectric dye-coupled polyethylene film, designated Okayama University type-retinal prosthesis (OUReP™). Previously, we found that NK-5962 solution could reduce the number of apoptotic photoreceptors in the eyes of the Royal College of Surgeons (RCS) rats by intravitreal injection under a 12 h light/dark cycle. This study aimed to explore possible molecular mechanisms underlying the anti-apoptotic effect of NK-5962 in the retina of RCS rats. METHODS: RCS rats received intravitreal injections of NK-5962 solution in the left eye at the age of 3 and 4 weeks, before the age of 5 weeks when the speed in the apoptotic degeneration of photoreceptors reaches its peak. The vehicle-treated right eyes served as controls. All rats were housed under a 12 h light/dark cycle, and the retinas were dissected out at the age of 5 weeks for RNA sequence (RNA-seq) analysis. For the functional annotation of differentially expressed genes (DEGs), the Metascape and DAVID databases were used. RESULTS: In total, 55 up-regulated DEGs, and one down-regulated gene (LYVE1) were found to be common among samples treated with NK-5962. These DEGs were analyzed using Gene Ontology (GO) term enrichment, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Reactome pathway analyses. We focused on the up-regulated DEGs that were enriched in extracellular matrix organization, extracellular exosome, and PI3K-Akt signaling pathways. These terms and pathways may relate to mechanisms to protect photoreceptor cells. Moreover, our analyses suggest that SERPINF1, which encodes pigment epithelium-derived factor (PEDF), is one of the key regulatory genes involved in the anti-apoptotic effect of NK-5962 in RCS rat retinas. CONCLUSIONS: Our findings suggest that photoelectric dye NK-5962 may delay apoptotic death of photoreceptor cells in RCS rats by up-regulating genes related to extracellular matrix organization, extracellular exosome, and PI3K-Akt signaling pathways. Overall, our RNA-seq and bioinformatics analyses provide insights in the transcriptome responses in the dystrophic RCS rat retinas that were induced by NK-5962 intravitreal injection and offer potential target genes for developing new therapeutic strategies for patients with retinitis pigmentosa.

A Titanium Nitride Nanozyme for pH-Responsive and Irradiation-Enhanced Cascade-Catalytic Tumor Therapy.

Nanozyme-based catalytic tumor therapy is an emerging therapeutic method with high reactivity in response to tumor microenvironments (TMEs). To overcome the current limitations of deficient catalytic activity of nanozymes, we studied the contributing factors of enzymatic activity based on non-metallic-atom doping and irradiation. Nitrogen doping significantly enhanced the peroxidase activity of Ti-based nanozymes, which was shown experimentally and theoretically. Based on the excellent NIR-adsorption-induced surface plasmon resonance and photothermal effect, the enzymatic activity of TiN nanoparticles (NPs) was further improved under NIR laser irradiation. Hence, an acidic TME-responsive and irradiation-mediated cascade nanocatalyst (TLGp) is presented by using TiN-NP-encapsulated liposomes linked with pH-responsive PEG-modified glucose oxidase (GOx). The integration of pH-responsive GOx-mediated H2 O2 self-supply, nitrogen-doping, and irradiation-enhanced enzymatic activity of TiN NPs and mild-photothermal therapy enables an effective tumor inhibition by TLGp with minimal side effects in vivo.

Anthrax Pathogenesis.

Anthrax is caused by the spore-forming, gram-positive bacterium Bacillus anthracis. The bacterium's major virulence factors are (a) the anthrax toxins and (b) an antiphagocytic polyglutamic capsule. These are encoded by two large plasmids, the former by pXO1 and the latter by pXO2. The expression of both is controlled by the bicarbonate-responsive transcriptional regulator, AtxA. The anthrax toxins are three polypeptides-protective antigen (PA), lethal factor (LF), and edema factor (EF)-that come together in binary combinations to form lethal toxin and edema toxin. PA binds to cellular receptors to translocate LF (a protease) and EF (an adenylate cyclase) into cells. The toxins alter cell signaling pathways in the host to interfere with innate immune responses in early stages of infection and to induce vascular collapse at late stages. This review focuses on the role of anthrax toxins in pathogenesis. Other virulence determinants, as well as vaccines and therapeutics, are briefly discussed.

Key tissue targets responsible for anthrax-toxin-induced lethality.

Bacillus anthracis, the causative agent of anthrax disease, is lethal owing to the actions of two exotoxins: anthrax lethal toxin (LT) and oedema toxin (ET). The key tissue targets responsible for the lethal effects of these toxins are unknown. Here we generated cell-type-specific anthrax toxin receptor capillary morphogenesis protein-2 (CMG2)-null mice and cell-type-specific CMG2-expressing mice and challenged them with the toxins. Our results show that lethality induced by LT and ET occurs through damage to distinct cell types; whereas targeting cardiomyocytes and vascular smooth muscle cells is required for LT-induced mortality, ET-induced lethality occurs mainly through its action in hepatocytes. Notably, and in contradiction to what has been previously postulated, targeting of endothelial cells by either toxin does not seem to contribute significantly to lethality. Our findings demonstrate that B. anthracis has evolved to use LT and ET to induce host lethality by coordinately damaging two distinct vital systems.

Aggravation of Cerebral Ischemia/Reperfusion Injury by Peroxisome Proliferator-Activated Receptor-Gamma Deficiency via Endoplasmic Reticulum Stress.

BACKGROUND Ischemic stroke is a dominant contributor to disability and mortality worldwide and is recognized as an important health concern. As a transcription factor triggered via stress, peroxisome proliferator-activated receptor-gamma (PPAR-γ) has a crucial impact on differentiation, cell death, and cell growth. However, the role of PPAR-γ and its precise mechanism in cerebral ischemia injury (CII) remain unclear. MATERIAL AND METHODS The male C57Bl/6 mice (12 weeks old, n=52) were subjected to middle cerebral artery occlusion (MCAO). Infarct volume was evaluated by 2, 3, 5-Triphenyltetrazolium chloride staining. Cell apoptosis was measured by terminal dUTP nick-end labeling (TUNEL) staining. The expression of apoptotic-related protein was examined by Western blotting. Neuron2A cells were transfected with PPAR-γ-specific siRNA and then were subjected to oxygen-glucose exhaustion and reoxygenation. RESULTS It was observed that PPAR-γ-deficient mice displayed extended infarct trigon in the MCAO stroke model. Neuronal deficiency was more severe in PPAR-γ-deficient models. Additionally, expression of cell death-promoting Bcl-2 associated X and active caspase-3 was reinforced, while that of cell death-counteracting Bcl-2 was repressed in PPAR-γ-deficient mice. This was characterized by reinforced endoplasmic reticulum (ER) stress reactions in in vivo brain specimens as well as in vitro neurons in ischemia/reperfusion (I/R) injury. CONCLUSIONS This research proved that PPAR-γ protected the brain from cerebral I/R injury by repressing ER stress and indicated that PPAR-γ is a potential target in the treatment of ischemia.

Solid tumor therapy by selectively targeting stromal endothelial cells.

Engineered tumor-targeted anthrax lethal toxin proteins have been shown to strongly suppress growth of solid tumors in mice. These toxins work through the native toxin receptors tumor endothelium marker-8 and capillary morphogenesis protein-2 (CMG2), which, in other contexts, have been described as markers of tumor endothelium. We found that neither receptor is required for tumor growth. We further demonstrate that tumor cells, which are resistant to the toxin when grown in vitro, become highly sensitive when implanted in mice. Using a range of tissue-specific loss-of-function and gain-of-function genetic models, we determined that this in vivo toxin sensitivity requires CMG2 expression on host-derived tumor endothelial cells. Notably, engineered toxins were shown to suppress the proliferation of isolated tumor endothelial cells. Finally, we demonstrate that administering an immunosuppressive regimen allows animals to receive multiple toxin dosages and thereby produces a strong and durable antitumor effect. The ability to give repeated doses of toxins, coupled with the specific targeting of tumor endothelial cells, suggests that our strategy should be efficacious for a wide range of solid tumors.