Acetyl-CoA metabolic accumulation promotes hepatocellular carcinoma metastasis via enhancing CXCL1-dependent infiltration of tumor-associated neutrophils.

High levels of acetyl-CoA are considered a key metabolic feature of metastatic cancers. However, the impacts of acetyl-CoA metabolic accumulation on cancer microenvironment remodeling are poorly understood. In this study, using human hepatocellular carcinoma (HCC) tissues and orthotopic xenograft models, we found a close association between high acetyl-CoA levels in HCCs, increased infiltration of tumor-associated neutrophils (TANs) in the cancer microenvironment and HCC metastasis. Cytokine microarray and enzyme-linked immunosorbent assays (ELISA) revealed the crucial role of the chemokine (C-X-C motif) ligand 1(CXCL1). Mechanistically, acetyl-CoA accumulation induces H3 acetylation-dependent upregulation of CXCL1 gene expression. CXCL1 recruits TANs, leads to neutrophil extracellular traps (NETs) formation and promotes HCC metastasis. Collectively, our work linked the accumulation of acetyl-CoA in HCC cells and TANs infiltration, and revealed that the CXCL1-CXC receptor 2 (CXCR2)-TANs-NETs axis is a potential target for HCCs with high acetyl-CoA levels.

Prognostic prediction using a gene signature developed based on exhausted T cells for liver cancer patients.

Background: Liver hepatocellular carcinoma (LIHC) is a solid primary malignancy with poor prognosis. This study discovered key prognostic genes based on T cell exhaustion and used them to develop a prognostic prediction model for LIHC. Methods: SingleR's annotations combined with Seurat was used to automatically annotate the single-cell clustering results of the LIHC dataset GSE166635 downloaded from the Gene Expression Omnibus (GEO) database and to identify clusters related to exhausted T cells. Patients were classified using ConsensusClusterPlus package. Next, weighted gene co-expression network analysis (WGCNA) package was employed to distinguish key gene module, based on which least absolute shrinkage and selection operator (Lasso) and multi/univariate cox analysis were performed to construct a RiskScore system. Kaplan-Meier (KM) analysis and receiver operating characteristic curve (ROC) were employed to evaluate the efficacy of the model. To further optimize the risk model, a nomogram capable of predicting immune infiltration and immunotherapy sensitivity in different risk groups was developed. Expressions of genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence and Cell Counting Kit-8 (CCK-8) were performed for analyzing cell functions. Results: We obtained 18,413 cells and clustered them into 7 immune and non-immune cell subpopulations. Based on highly variable genes among T cell exhaustion clusters, 3 molecular subtypes (C1, C2 and C3) of LIHC were defined, with C3 subtype showing the highest score of exhausted T cells and a poor prognosis. The Lasso and multivariate cox analysis selected 7 risk genes from the green module, which were closely associated with the C3 subtype. All the patients were divided into low- and high-risk groups based on the medium value of RiskScore, and we found that high-risk patients had higher immune infiltration and immune escape and poorer prognosis. The nomogram exhibited a strong performance for predicting long-term LIHC prognosis. In vitro experiments revealed that the 7 risk genes all had a higher expression in HCC cells, and that both liver HCC cell numbers and cell viability were reduced by knocking down MMP-9. Conclusion: We developed a RiskScore model for predicting LIHC prognosis based on the scRNA-seq and RNA-seq data. The RiskScore as an independent prognostic factor could improve the clinical treatment for LIHC patients.

IL-6 Promotes Hepatocellular Carcinoma Invasion by Releasing Exosomal miR-133a-3p.

Interleukin-6 (IL-6), an important inflammatory cytokine, is a key factor regulating cancer metastasis. Cancer cells can modulate their tumorigenic abilities by sorting specific microRNAs (miRNAs) as exosomes into the tumor microenvironment. The relationship between IL-6 and exosomal miRNAs related to hepatocellular carcinoma (HCC) metastasis remains to be elucidated. We examined the metastatic ability of HCC cells after IL-6 treatment and found that miR-133a-3p was sorted into exosomes after IL-6 stimulation and was subsequently released into the tumor microenvironment. In vitro analysis confirmed that exosomal miR-133a-3p acted as a tumor suppressor in HCC. Bioinformatic analysis revealed several signaling pathways and hub genes (CREB1, VCP, CALM1, and YES1) regulated by miR-133a-3p. Survival curves further verified the important roles of hub genes in the prognosis of patients with HCC. It is envisaged that the IL-6/miR-133a-3p axis may be related to the activation of CREB1, VCP, CALM1, and YES1. Our findings provide new insights into the role of exosomal miRNA-mediated tumor progression under inflammatory conditions.

Clinical and epidemiological features of 36 children with coronavirus disease 2019 (COVID-19) in Zhejiang, China: an observational cohort study.

BACKGROUND: Since December, 2019, an outbreak of coronavirus disease 2019 (COVID-19) has spread globally. Little is known about the epidemiological and clinical features of paediatric patients with COVID-19. METHODS: We retrospectively retrieved data for paediatric patients (aged 0-16 years) with confirmed COVID-19 from electronic medical records in three hospitals in Zhejiang, China. We recorded patients' epidemiological and clinical features. FINDINGS: From Jan 17 to March 1, 2020, 36 children (mean age 8·3 [SD 3·5] years) were identified to be infected with severe acute respiratory syndrome coronavirus 2. The route of transmission was by close contact with family members (32 [89%]) or a history of exposure to the epidemic area (12 [33%]); eight (22%) patients had both exposures. 19 (53%) patients had moderate clinical type with pneumonia; 17 (47%) had mild clinical type and either were asymptomatic (ten [28%]) or had acute upper respiratory symptoms (seven [19%]). Common symptoms on admission were fever (13 [36%]) and dry cough (seven [19%]). Of those with fever, four (11%) had a body temperature of 38·5°C or higher, and nine (25%) had a body temperature of 37·5-38·5°C. Typical abnormal laboratory findings were elevated creatine kinase MB (11 [31%]), decreased lymphocytes (11 [31%]), leucopenia (seven [19%]), and elevated procalcitonin (six [17%]). Besides radiographic presentations, variables that were associated significantly with severity of COVID-19 were decreased lymphocytes, elevated body temperature, and high levels of procalcitonin, D-dimer, and creatine kinase MB. All children received interferon alfa by aerosolisation twice a day, 14 (39%) received lopinavir-ritonavir syrup twice a day, and six (17%) needed oxygen inhalation. Mean time in hospital was 14 (SD 3) days. By Feb 28, 2020, all patients were cured. INTERPRETATION: Although all paediatric patients in our cohort had mild or moderate type of COVID-19, the large proportion of asymptomatic children indicates the difficulty in identifying paediatric patients who do not have clear epidemiological information, leading to a dangerous situation in community-acquired infections. FUNDING: Ningbo Clinical Research Center for Children's Health and Diseases, Ningbo Reproductive Medicine Centre, and Key Scientific and Technological Innovation Projects of Wenzhou.

Longitudinal and quantitative assessment platform for concurrent analysis of anti-tumor efficacy and cardiotoxicity of nano-formulated medication in vivo.

Increasing nanomedicinal approaches have been developed to effectively inhibit tumor growth; however, critical questions such as whether a nanomedicinal approach can mitigate latent side effects are barely addressed. To this end, we established a zebrafish xenograft tumor model, combining pseudodynamic three-dimensional cardiac imaging and image analysis to enable simultaneous and quantitative determination of the change of tumor volume and cardiac function of zebrafish upon specific nanoformulation treatment. Doxorubicin (DOX), a well-known chemotherapeutic agent with cardiotoxicity, and a recently developed DOX-loaded nanocomposite were employed as two model drugs to demonstrate the effectiveness to utilize the proposed evaluation platform for rapid validation. The nanoformulation significantly mitigated DOX-associated cardiotoxicity, while retaining the efficacy of DOX in inhibiting tumor growth compared to administration of carrier-free DOX at the same dose. We anticipate that this platform possesses the potential as an efficient assessment system for nanoformulated cancer therapeutics with suspected toxicity and side effects to vital organs such as the heart.

Differential in situ sensing of extra- and intracellular glutathione by a novel redox-responsive silica matrix-Au nanoprobe.

We synthesized a biothiol-sensitive nanoprobe by assembling gold nanoparticles with a novel redox-responsive silica (ReSi) matrix using dithiobis (succinimidyl propionate) and (3-aminopropyl) trimethoxysilane. Thin layer disulfide-bonded networks of the ReSi could differentially respond to extra- and intracellular glutathione in cancer cells within 30 min; furthermore, targeted cellular uptake could be monitored in situ by fluorescence recovery. Sigmoidal dose-response pattern of the nanoprobes presented in this study were attributed to the buried disulfide-linked 3D nanostructure of the ReSi nanoshell, optimized at an appropriate thickness, enabling not only buffering of small redox disturbances in the extracellular milieu but also the satisfied sensitivity for rapid redox sensing. Such a ReSi-functionalized gold nanoparticle-based nanoconjugate possesses the potential to serve as an effective intracellular drug carrier for future cancer theranostics.

In vitro investigation of methylene blue-bearing, electrostatically assembled aptamer-silica nanocomposites as potential photodynamic therapeutics.

Photodynamic therapy, that is, excitation of a photosensitizer with light to generate reactive oxygen species such as singlet oxygen, has emerged as a noninvasive technique for cancer theranostics. However, the clinical use of many photosensitizers is impeded by their hydrophobicity, the nonspecific damage they cause to normal tissues, and their susceptibility to environmental degradation. In this study, we developed a simple electrostatic adsorption strategy to fabricate aptamer-silica nanocomposites by sequentially functionalizing nanocomposites with the cell surface-associated mucin 1 aptamer for tumor targeting and a hydrophilic photosensitizer, methylene blue, for photodynamic therapy applications. We investigated the relationship between the biophysical properties and cellular uptake of such nanocomposites to improve their formulation. Effective generation of singlet oxygen was achieved with a low photosensitizer dosage (0.5 µM) and a short, low-power irradiation (1 min, 10 mW/cm(2)). With the current strategy, the efficiency of photodynamic therapy was determined by the cellular uptake of nanocomposites and the targeting molecules used.

Down-regulation of PERK-ATF4-CHOP pathway by Astragaloside IV is associated with the inhibition of endoplasmic reticulum stress-induced podocyte apoptosis in diabetic rats.

BACKGROUND: Endoplasmic reticulum (ER) stress-induced podocyte apoptosis plays a critical role in the development of diabetic nephropathy (DN). Here, we tested the hypothesis that suppression of PERK-ATF4-CHOP pathway by Astragaloside IV (AS-IV) is associated with inhibition of ER stress-induced podocyte apoptosis in streptozotocin (STZ)-induced diabetic rats. METHODS: Diabetic rats were treated with AS-IV at 5 and 10 mg·kg-1·d-1, p.o., for 12 weeks. Albuminuria examination, hematoxylin & eosin staining and TUNEL analysis were performed. Immunohistochemistry, western blot, and real-time PCR were used to detect renal expression of ER chaperone GRP78 and ER-associated apoptosis proteins. RESULTS: Treatment with AS-IV ameliorated albuminuria and renal histopathology in diabetic rats. Diabetic rats had significant increment in podocyte apoptosis as well as phosphorylated PERK and eIF2α in the kidneys, which were attenuated by AS-IV treatment. Furthermore, diabetic rats were found to have increased protein and mRNA expressions of GRP78 and ER-associated apoptosis proteins, such as ATF4, CHOP and TRB3, which were also attenuated by AS-IV treatment. Increased Bax expression and decreased Bcl-2 expression were detected in diabetic rats, and these changes were partially restored by AS-IV treatment. CONCLUSION: The protective effect of AS-IV on ER stress-induced podocyte apoptosis is associated with inhibition of PERK-ATF4-CHOP pathway. Down-regulation of PERK- ATF4-CHOP pathway by AS-IV may be a novel strategy for the treatment of DN.

Efficient soybean regeneration and Agrobacterium-mediated transformation using a whole cotyledonary node as an explant.

An optimized regeneration and Agrobacterium-mediated transformation protocol based on whole cotyledonary node explants was developed in soybean (Glycine max) cultivar Zhong Huang 13. Adding 6-benzylaminopurine (BAP) in a germinating medium could significantly increase regeneration efficiency; the optimal BAP concentration for shoot formation was 0.5 mg/L. The concentrations of plant growth regulators in a shoot induction medium were optimized by the orthogonal test [L9 (3(3))]. The best combination for shoot regeneration was a medium of Murashige & Skoog salts with B5 vitamins (MSB) supplemented with 3.5 mg/L BAP, 0.2 mg/L indole-3-butyric acid (IBA), and 0.2 mg/L kinetin (KT). Under this favorable condition, one node could regenerate 28-30 shoots. Soybean whole cotyledonary nodes were transformed by inoculation with A. tumefaciens strain EHA105 harboring a vector pBI121 containing a ß-glucuronidase gene (gus). GUS assay, polymerase chain reaction, and Southern blot analysis indicated that the gus gene was transformed into soybean plants with 23.1% transformation efficiency. Transgenic plants could be obtained within 5-6 weeks, which was about 4 weeks less than that of a traditional single cotyledonary node method.

Astragaloside IV ameliorates diabetic nephropathy involving protection of podocytes in streptozotocin induced diabetic rats.

Podocyte loss and dysfunction play key role during the development of diabetic nephropathy (DN). The aim of this study was to observe the protective effects of astragaloside IV on podocyte in diabetic rats and explore its mechanisms preliminary. Healthy male Sprague-Dawley (SD) rats were randomized into normal control group, diabetic nephropathy group and diabetic nephropathy with AS-IV treatment group. DN was induced by intraperitoneal injection of streptozotocin (STZ). AS-IV treatment started 2 weeks before STZ injection and lasted 14 weeks. 24h Urinary proteins were measured 4, 8 and 12 weeks after STZ injection. Body weight, blood glucose, blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured 12 weeks after STZ injection. Renal pathology, podocyte morphological changes, podocyte density, protein and mRNA expression of integrin α3, integrin ß1 and integrin-linked kinase (ILK) were detected by histopathology, electron microscopy, immunohistochemistry, western blot and real-time PCR, respectively. Hyperglycemia, proteinuria, mesangial expansion and podocyte loss, increased protein expression of ILK and decreased protein expression of integrin α3 and integrin ß1 were detected in diabetic rats. AS-IV treatment ameliorated podocyte loss, renal histopathology and podocyte foot process effacement, decreased proteinuria, partially restored protein expression of integrin α3, integrin ß1 and ILK. These findings suggested that AS-IV may protect podocyte and ameliorate diabetic nephropathy by inhibiting the expression of ILK and restoring the expression of integrin α3ß1 in diabetic rats.

Non-metallic nanomaterials in cancer theranostics: a review of silica- and carbon-based drug delivery systems.

The rapid development in nanomaterials has brought great opportunities to cancer theranostics, which aims to combine diagnostics and therapy for cancer treatment and thereby improve the healthcare of patients. In this review we focus on the recent progress of several cancer theranostic strategies using mesoporous silica nanoparticles and carbon-based nanomaterials. Silicon and carbon are both group IV elements; they have been the most abundant and significant non-metallic substances in human life. Their intrinsic physical/chemical properties are of critical importance in the fabrication of multifunctional drug delivery systems. Responsive nanocarriers constructed using these nanomaterials have been promising in cancer-specific theranostics during the past decade. In all cases, either a controlled texture or the chemical functionalization is coupled with adaptive properties, such as pH-, light-, redox- and magnetic field- triggered responses. Several studies in cells and mice models have implied their underlying therapeutic efficacy; however, detailed and long-term in vivo clinical evaluations are certainly required to make these bench-made materials compatible in real bedside circumstances.

Aptamer-conjugated and drug-loaded acoustic droplets for ultrasound theranosis.

Tumor therapy requires multi-functional treatment strategies with specific targeting of therapeutics to reduce general toxicity and increase efficacy. In this study we fabricated and functionally tested aptamer-conjugated and doxorubicin (DOX)-loaded acoustic droplets comprising cores of liquid perfluoropentane compound and lipid-based shell materials. Conjugation of sgc8c aptamers provided the ability to specifically target CCRF-CEM cells for both imaging and therapy. High-intensity focused ultrasound (HIFU) was introduced to trigger targeted acoustic droplet vaporization (ADV) which resulted in both mechanical cancer cell destruction by inertial cavitation and chemical treatment through localized drug release. HIFU insonation showed a 56.8% decrease in cell viability with aptamer-conjugated droplets, representing a 4.5-fold increase in comparison to non-conjugated droplets. In addition, the fully-vaporized droplets resulted in the highest DOX uptake by cancer cells, compared to non-vaporized or partially vaporized droplets. Optical studies clearly illustrated the transient changes that occurred upon ADV of droplet-targeted CEM cells, and B-mode ultrasound imaging revealed contrast enhancement by ADV in ultrasound images. In conclusion, our fabricated droplets functioned as a hybrid chemical and mechanical strategy for the specific destruction of cancer cells upon ultrasound-mediated ADV, while simultaneously providing ultrasound imaging capability.

Release of photoactivatable drugs from plasmonic nanoparticles for targeted cancer therapy.

Chemotherapy is an important modality in cancer treatment. The major challenges of recent works are to improve drug loading, increase selectivity to target cells, and control the precise release of drugs. In the present study, we devised a smart drug carrier, an aptamer/hairpin DNA-gold nanoparticle (apt/hp-Au NP) conjugate for targeted delivery of drugs. The DNA aptamer sgc8c, which possesses strong affinity for protein tyrosine kinase 7 (PTK7), abundantly expressed on the surface of CCRF-CEM (T-cell acute lymphoblastic leukemia) cells, was assembled onto the surface of Au NPs. The repeated d(CGATCG) sequence within the hpDNA on the Au NP surface was used for the loading of the anticancer drug doxorubicin (Dox). After optimization, 25 (±3) sgc8c and 305 (±9) Dox molecules were successfully loaded onto the AuNP (13 nm) surface. The binding capability of apt/hp-Au NP conjugates toward targeted cells was investigated by flow cytometry and atomic absorption spectroscopy, which showed that the aptamer-functionalized nanoconjugates were selective for targeting of cancer cells. A cell toxicity (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, MTT) assay also demonstrated that these drug-loaded nanoconjugates could kill targeted cancer cells more effectively than nontargeted (control) cells. Most importantly, when illuminated with plasmon-resonant light (532 nm), Dox:nanoconjugates displayed enhanced antitumor efficacy with few side effects. The marked release of Dox from these nanoconjugates in living cells was monitored by increasing fluorescence signals upon light exposure. In vitro studies confirmed that aptamer-functionalized hp-Au NPs can be used as carriers for targeted delivery of drugs with remote control capability by laser irradiation with high spatial/temporal resolution.