ssDNA Aptamer Specifically Targets and Selectively Delivers Cytotoxic Drug Doxorubicin to HepG2 Cells

Hepatocellular carcinoma (HCC) is the third leading cause of death due to cancer worldwide with over 500,000 people affected annually. Although chemotherapy has been widely used to treat patients with HCC, alternate modalities to specifically deliver therapeutic cargos to cancer cells have been sought in recent years due to the severe side effects of chemotherapy. In this respect, aptamer-based tumor targeted drug delivery has emerged as a promising approach to increase the efficacy of chemotherapy and reduce or eliminate drug toxicity. In this study, we developed a new HepG2-specific aptamer (HCA#3) by a procedure known as systematic evolution of ligands by exponential enrichment (SELEX) and exploited its role as a targeting ligand to deliver doxorubicin (Dox) to HepG2 cells in vitro. The selected 76-base nucleotide aptamer preferentially bound to HepG2 hepatocellular carcinoma cells but not to control cells. The aptamer HCA#3 was modified with paired CG repeats at the 5′-end to carry and deliver a high payload of intercalated Dox molecules at the CG sites. Four Dox molecules (mol/mol) were fully intercalated in each conjugate aptamer-Dox (ApDC) molecule. Biostability analysis showed that the ApDC molecules are stable in serum. Functional analysis showed that ApDC specifically targeted and released Dox within HepG2 cells but not in control cells, and treatment with HCA#3 ApDC induced HepG2 cell apoptosis but had minimal effect on control cells. Our study demonstrated that HCA#3 ApDC is a promising aptamer-targeted therapeutic that can specifically deliver and release a high doxorubicin payload in HCC cells.     

The Potential Biomarkers to Identify the Development of Steatosis in Hyperuricemia

Hyperuricemia (HU) often progresses to combine with non-alcoholic fatty liver disease (NAFLD) in the clinical scenario, which further exacerbates metabolic disorders; early detection of biomarkers, if obtained during the HU progression, may be beneficial for preventing its combination with NAFLD. This study aimed to decipher the biomarkers and mechanisms of the development of steatosis in HU. Four groups of subjects undergoing health screening, including healthy subjects, subjects with HU, subjects with HU combined with NAFLD (HU+NAFLD) and subjects with HU initially and then with HU+NAFLD one year later (HU→HU+NAFLD), were recruited in this study. The metabolic profiles of all subjects’ serum were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry. The metabolomic data from subjects with HU and HU+NAFLD were compared, and the biomarkers for the progression from HU to HU+NAFLD were predicted. The metabolomic data from HU→HU+NAFLD subjects were collected for further verification. The results showed that the progression was associated with disturbances of phospholipase metabolism, purine nucleotide degradation and Liver X receptor/retinoic X receptor activation as characterized by up-regulated phosphatidic acid, cholesterol ester (18:0) and down-regulated inosine. These metabolic alterations may be at least partially responsible for the development of steatosis in HU. This study provides a new paradigm for better understanding and further prevention of disease progression.     

MOLECULAR CHARACTERIZATION OF AUTOPHAGY‐RELATED GENE 5 FROM Spodoptera exigua AND EXPRESSION ANALYSIS UNDER VARIOUS STRESS CONDITIONS

Autophagy is not only involved in development, but also has been proved to attend immune response against invading pathogens. Autophagy protein 5 (ATG5) is an important autophagic protein, which plays a crucial role in autophagosome elongation. Although ATG5 has been well studied in mammal, yeast, and Drosophila, little is known about ATG5 in lepidopteran insects. We cloned putative SeAtg5 gene from Spodoptera exigua larvae by the rapid amplification of cDNA ends method, and its characteristics and the influences of multiple exogenous factors on its expression levels were then investigated. The results showed that the putative S. exigua SeATG5 protein is highly homologous to other insect ATG5 proteins, which has a conserved Pfm domain and multiple phosphorylation sites. Next, fluorescence microscope observation showed that mCherry‐SeATG5 was distributed in both nucleus and cytoplasm of Spodoptera litura Sl‐HP cells and partially co‐localized with BmATG6‐GFP, but it almost has no significant co‐localization with GFP‐HaATG8. Then, the Western blot analysis demonstrated that GFP‐SeATG5 conjugated with ATG12. Moreover, real‐time PCR revealed that its expression levels significantly increased at the initiation of pupation and the stage of adult. In addition, the expression levels of SeAtg5 can be enhanced by the starvation, UV radiation, and infection of baculovirus and bacterium. However, the expression levels of SeAtg5 decreased at 24 h post treatments in all these treatments except in starvation. These results suggested that SeATG5 might be involved in response of S. exigua under various stress conditions.     

Classification of life cycle form spectrum of artificial community of Pinus svlvestris var. mongolica

The plant life cycle form and its spectrum features of different plants in the artificial community of Pinus svlvestris var. mongolica at the botanical garden of Northeast Forestry University were classified by PCA method. For canopy layer plants, the vegetative growth (V) accounted for 46%, the sexual growth (S) accounted for 35%, and the clonal growth (C) accounted for the rest 19%. For succession layer plants, V accounted for more than 50%, and C accounted for a little higher than S did. For herbaceous layer plants, V accounted for nearly 47%, which was only 4% higher than that of S, and C only accounted for 11%. These findings indicate that the plant life cycle forms of canopy layer plants and herbaceous layer plants were VS transition form with V form as the main form, while the succession layer plants were V form. At the viewpoint of artificial community, the life cycle form can be described as V_0.49S_0.33C_0.18, a typical VS transition form, indicating that most of the species studied in this community are in a healthy status with the V form. Moreover, the relatively high (33%) percentage in the S form indicates that the community also tends to sexual growth for offspring, although it will be steady within a period of time for the vegetative growth of bodies.     

太子参花药发育及精细胞分离

太子参花药壁发育为基本型,腺质绒毡层。小孢子母细胞减数分裂为同时型,小孢子四分体为四面体型,成熟花粉具两个精细胞,为3胞花粉。在花粉表面具散孔,孔数22—30个,均匀分布于花粉粒表面上。花粉在10％甘露醇或15％蔗糖溶液中可直接爆破,精细胞易被释放并散开,通过显微操作仪可收集到一定数目的精细胞。FDA染色荧光显示释放出来的精细胞活力可维持25—50min。花粉在舍O．03％CaCl2、0．01％H3803、0．01％KH2P04和20％PEG、pH5．8的培养液中2—5min即萌发花粉管．花粉管生长2h可达815μm。一般花粉管伸长500—600μm时,一对精细胞才进入花粉管。DAPI染色后荧光观察．可观察到精细胞和营养细胞核在花粉管中的移动状况。爆破花粉管后可释放出一对精细胞。     

Oncogenic GBX2 promotes the malignant behaviors of bladder cancer cells by binding to the ITGA5 promoter and activating its transcription

Functional & Integrative Genomics - In bladder cancer patients, metastasis after surgical resection and serious adverse reactions brought by cisplatin-based systemic chemotherapy make it urgent...     

Hypoxic conditioned medium derived from bone marrow mesenchymal stromal cells protects against ischemic stroke in rats

In recent years, studies have shown that the secretome of bone marrow mesenchymal stromal cells (BMSCs) contains many growth factors, cytokines, and antioxidants, which may provide novel approaches to treat ischemic diseases. Furthermore, the secretome may be modulated by hypoxic preconditioning. We hypothesized that conditioned medium (CM) derived from BMSCs plays a crucial role in reducing tissue damage and improving neurological recovery after ischemic stroke and that hypoxic preconditioning of BMSCs robustly improves these activities. Rats were subjected to ischemic stroke by middle cerebral artery occlusion and then intravenously administered hypoxic CM, normoxic CM, or Dulbecco modified Eagle medium (DMEM, control). Cytokine antibody arrays and label-free quantitative proteomics analysis were used to compare the differences between hypoxic CM and normoxic CM. Injection of normoxic CM significantly reduced the infarct area and improved neurological recovery after stroke compared with administering DMEM. These outcomes may be associated with the attenuation of apoptosis and promotion of angiogenesis. Hypoxic preconditioning significantly enhanced these therapeutic effects. Fourteen proteins were significantly increased in hypoxic CM compared with normoxic CM as measured by cytokine arrays. The label-free quantitative proteomics analysis revealed 163 proteins that were differentially expressed between the two groups, including 107 upregulated proteins and 56 downregulated proteins. Collectively, our results demonstrate that hypoxic CM protected brain tissue from ischemic injury and promoted functional recovery after stroke in rats and that hypoxic CM may be the basis of a potential therapy for stroke patients.     

Thymoquinone ameliorates pressure overload‐induced cardiac hypertrophy by activating the AMPK signalling pathway

Prolonged pathological myocardial hypertrophy leads to end‐stage heart failure. Thymoquinone (TQ), a bioactive component extracted from Nigella sativa seeds, is extensively used in ethnomedicine to treat a broad spectrum of disorders. However, it remains unclear whether TQ protects the heart from pathological hypertrophy. This study was conducted to examine the potential utility of TQ for treatment of pathological cardiac hypertrophy and if so, to elucidate the underlying mechanisms. Male C57BL/6J mice underwent either transverse aortic constriction (TAC) or sham operation, followed by TQ treatment for six consecutive weeks. In vitro experiments consisted of neonatal rat cardiomyocytes (NRCMs) that were exposed to phenylephrine (PE) stimulation to induce cardiomyocyte hypertrophy. In this study, we observed that systemic administration of TQ preserved cardiac contractile function, and alleviated cardiac hypertrophy, fibrosis and oxidative stress in TAC‐challenged mice. The in vitro experiments showed that TQ treatment attenuated the PE‐induced hypertrophic response in NRCMs. Mechanistical experiments showed that supplementation of TQ induced reactivation of the AMP‐activated protein kinase (AMPK) with concomitant inhibition of ERK 1/2, p38 and JNK1/2 MAPK cascades. Furthermore, we demonstrated that compound C, an AMPK inhibitor, abolished the protective effects of TQ in in vivo and in vitro experiments. Altogether, our study disclosed that TQ provides protection against myocardial hypertrophy in an AMPK‐dependent manner and identified it as a promising agent for the treatment of myocardial hypertrophy.     

Lipid nanoparticle-encapsulated mRNA antibody provides long-term protection against SARS-CoV-2 in mice and hamsters

Monoclonal antibodies represent important weapons in our arsenal to against the COVID-19 pandemic. However, this potential is severely limited by the time-consuming process of developing effective antibodies and the relative high cost of manufacturing. Herein, we present a rapid and cost-effective lipid nanoparticle (LNP) encapsulated-mRNA platform for in vivo delivery of SARS-CoV-2 neutralization antibodies. Two mRNAs encoding the light and heavy chains of a potent SARS-CoV-2 neutralizing antibody HB27, which is currently being evaluated in clinical trials, were encapsulated into clinical grade LNP formulations (named as mRNA-HB27-LNP). In vivo characterization demonstrated that intravenous administration of mRNA-HB27-LNP in mice resulted in a longer circulating half-life compared with the original HB27 antibody in protein format. More importantly, a single prophylactic administration of mRNA-HB27-LNP provided protection against SARS-CoV-2 challenge in mice at 1, 7 and even 63 days post administration. In a close contact transmission model, prophylactic administration of mRNA-HB27-LNP prevented SARS-CoV-2 infection between hamsters in a dose-dependent manner. Overall, our results demonstrate a superior long-term protection against SARS-CoV-2 conferred by a single administration of this unique mRNA antibody, highlighting the potential of this universal platform for antibody-based disease prevention and therapy against COVID-19 as well as a variety of other infectious diseases.Subject terms: Biological techniques, Immunology