A Biomimetic Drug Delivery System by Integrating Grapefruit Extracellular Vesicles and Doxorubicin-Loaded Heparin-Based Nanoparticles for Glioma Therapy.

Existing nanoparticle-mediated drug delivery systems for glioma systemic chemotherapy remain a great challenge due to poor delivery efficiency resulting from the blood brain barrier/blood-(brain tumor) barrier (BBB/BBTB) and insufficient tumor penetration. Here, we demonstrate a distinct design by patching doxorubicin-loaded heparin-based nanoparticles (DNs) onto the surface of natural grapefruit extracellular vesicles (EVs), to fabricate biomimetic EV-DNs, achieving efficient drug delivery and thus significantly enhancing antiglioma efficacy. The patching strategy allows the unprecedented 4-fold drug loading capacity compared to traditional encapsulation for EVs. The biomimetic EV-DNs are enabled to bypass BBB/BBTB and penetrate into glioma tissues by receptor-mediated transcytosis and membrane fusion, greatly promoting cellular internalization and antiproliferation ability as well as extending circulation time. We demonstrate that a high-abundance accumulation of EV-DNs can be detected at glioma tissues, enabling the maximal brain tumor uptake of EV-DNs and great antiglioma efficacy in vivo.

Conserved Trigger Loop Histidine of RNA Polymerase II Functions as a Positional Catalyst Primarily through Steric Effects.

In all domains of life, multisubunit RNA polymerases (RNAPs) catalyze both the extension of mRNA transcripts by nucleotide addition and the hydrolysis of RNA, which enables proofreading by removal of misincorporated nucleotides. A highly conserved catalytic module within RNAPs called the trigger loop (TL) functions as the key controller of these activities. The TL is proposed to act as a positional catalyst of phosphoryl transfer and transcript cleavage via electrostatic and steric contacts with substrates in its folded helical form. The function of a near-universally conserved TL histidine that contacts NTP phosphates is of particular interest. Despite its exceptional conservation, substitutions of the TL His with Gln support efficient catalysis in bacterial and yeast RNAPs. Unlike bacterial TLs, which contain a nearby Arg, the TL His is the only acid-base catalyst candidate in the eukaryotic RNAPII TL. Nonetheless, replacement of the TL His with Leu is reported to support cell growth in yeast, suggesting that even hydrogen bonding and polarity at this position may be dispensable for efficient catalysis by RNAPII. To test how a TL His-to-Leu substitution affects the enzymatic functions of RNAPII, we compared its rates of nucleotide addition, pyrophosphorolysis, and RNA hydrolysis to those of the wild-type RNAPII enzyme. The His-to-Leu substitution slightly reduced rates of phosphoryl transfer with little if any effect on intrinsic transcript cleavage. These findings indicate that the highly conserved TL His is neither an obligate acid-base catalyst nor a polar contact for NTP phosphates but instead functions as a positional catalyst mainly through steric effects.

Phospholipid scramblase 1 functionally interacts with angiogenin and regulates angiogenin-enhanced rRNA transcription.

BACKGROUND: Angiogenin (ANG) can translocate to the target cell nucleus and accumulate in the nucleolus to enhance rRNA transcription, thus promoting cell proliferation. However, the regulation of ANG-enhanced rRNA transcription remains unknown. Previously we identified phospholipid scramblase 1 (PLSCR1) as a potential ANG-interacting protein in yeast two-hybrid screening. METHODS: The interaction was re-confirmed in yeast cells and further verified by in vitro pull down, in vivo co-immunoprecipitation (Co-IP), fluorescent resonance energy transfer (FRET) and immunofluorescence analyses. The rRNA transcription level was determined by real-time quantitative PCR and Northern blot. RESULTS: PLSCR1 was identified as a novel ANG-interacting protein. Notably, PLSCR1 interacted with ANG in the cell nucleus and regulated rRNA transcription. Furthermore, depletion of cellular ANG expression abolished PLSCR1-enhanced rRNA transcription, which could be rescued by exogenous ANG. CONCLUSION: Our data suggest that PLSCR1 positively regulates rRNA transcription through interacting with ANG, thus deepening our understanding on rRNA transcription regulation.

A new species of Andricus Hartig, 1840 (Hymenoptera, Cynipidae) from China, with references to DNA taxonomy and Wolbachia infection.

In the present paper, a new species of cynipid gall wasp, Andricuselodeoides Liu & Pang, is described from several provinces in southern China. The new species is closely related to the recently redescribed A.mairei (Kieffer, 1906). In addition to differences in adult and gall morphology, the new species is also readily separated by COI sequences, with a 6.2-8.9% genetic distance between populations of the new species and those of A.mairei. A contrasting difference in sex ratios was also observed between the two species, with A.elodeoides extremely female-biased (95.5-97.8% female) while A.mairei male-biased to more balanced (5.4-43.5% female). PCR screening for Wolbachia infection further revealed contrasting infection rates between populations of A.elodeoides and A.mairei: the Wolbachia infection rate was 0% in A.elodeoides and 100% in A.mairei. Cytoplasmic incompatibility induced by Wolbachia is proposed as a potential mechanism of speciation of the sympatric A.elodeoides and A.mairei.

Nucleolar follistatin promotes cancer cell survival under glucose-deprived conditions through inhibiting cellular rRNA synthesis.

Solid tumor development is frequently accompanied by energy-deficient conditions such as glucose deprivation and hypoxia. Follistatin (FST), a secretory protein originally identified from ovarian follicular fluid, has been suggested to be involved in tumor development. However, whether it plays a role in cancer cell survival under energy-deprived conditions remains elusive. In this study, we demonstrated that glucose deprivation markedly enhanced the expression and nucleolar localization of FST in HeLa cells. The nucleolar localization of FST relied on its nuclear localization signal (NLS) comprising the residues 64-87. Localization of FST to the nucleolus attenuated rRNA synthesis, a key process for cellular energy homeostasis and cell survival. Overexpression of FST delayed glucose deprivation-induced apoptosis, whereas down-regulation of FST exerted the opposite effect. These functions depended on the presence of an intact NLS because the NLS-deleted mutant of FST lost the rRNA inhibition effect and the cell protective effect. Altogether, we identified a novel nucleolar function of FST, which is of importance in the modulation of cancer cell survival in response to glucose deprivation.

An acoustic/thermo-responsive hybrid system for advanced doxorubicin delivery in tumor treatment.

The efficiency of drug delivery and bioavailability to tumor cells are crucial for effective cancer chemotherapy. Herein, a doxorubicin (DOX) encapsulated lysolipid-based thermosensitive liposome decorated with cRGD peptide (RTSL) is conjugated on the surface of an IR780-loaded microbubble (IMB) to synthesize RTSL-IMBs. Sequentially taking advantage of acoustic-assisted early extravasation and thermo-triggered interstitium ultrafast drug release, RTSL-IMBs combine with ultrasound (US) and laser irradiation can advance drug delivery and bioavailability. In vitro experiments demonstrate that RTSL-IMBs associated with a two-step protocol (subsequently US irradiation for 1 min and laser irradiation for 5 min) can dramatically enhance the cellular uptake and bioavailability of DOX. In vivo fluorescence imaging studies reveal that the combination of RTSL-IMBs and US shows a 2.8-fold intratumoral drug accumulation increase at 0.5 h post-injection, while it will take 48 h to reach the same level of intratumoral drug accumulation for the RTSL-IMB group alone. Interestingly, the following localized application of a laser can further increase drug accumulation and slow tumor clearance. Histological analysis demonstrates that the combinational RTSL-IMBs, US and laser significantly improve the drug penetration distance and delivery efficiency in the tumor core. In this study, the acoustic/thermo-responsive hybrid system shows potential for advancing DOX chemotherapy in breast cancer cell MCF-7 xenograft nude mice.

cRGD-functionalized nanoparticles for combination therapy of anti-endothelium dependent vessels and anti-vasculogenic mimicry to inhibit the proliferation of ovarian cancer.

Accumulating evidence has disclosed effective anti-angiogenic strategies should simultaneously inhibit endothelium-dependent vessels (EDV) and tumor cell-mediated vasculogenic mimicry (VM). The αvß3 integrin-targeting peptide cRGD has the ability to inhibit EDV and we have found cRGD can also suppress the formation of VM in ovarian cancer cells. Herein, a cRGD-based combination strategy was developed to suppress the proliferation of tumor cells by anti-EDV and anti-VM. We firstly engineered two cRGD functionalized nanoparticles (cRGD-NPs1 and cRGD-NPs2) by self-assembly using heparin conjugated with cRGD and folate. In vitro experiments demonstrated cRGD-NPs2 exhibited more significant cytotoxicity and higher intracellular uptake ability than cRGD-NPs1. Also, cRGD-NPs2 could efficiently discourage EDV, VM and proliferation in HUVECs and SKOV3 (VM+) cells. In vivo studies showed cRGD-NPs2 could specifically accumulate in ovarian cancer tissues and exerted a superior anti-tumor effect in SKOV3 xenografts. The mechanisms responsible for creating anti-EDV and anti-VM action of cRGD-NPs2 related to combined effects of cRGD, heparin and folate. The results demonstrated cRGD-NPs2 represented a versatile anti-angiogenic medicine via their combined inhibitory effect. STATEMENT OF SIGNIFICANCE: Accumulating documents indicate tumor cell-mediated vasculogenic mimicry (VM) is positively correlated with poor prognosis, occurrence of distant metastasis and low survival rate in cancer patients, suggesting VM is a potential therapeutic target for cancer treatment. Thus, effective anti-angiogenic strategies should simultaneously inhibit VM as well as endothelium-dependent vessels (EDV). Integrin αvß3 is a crucial inducer involved in the formation of both EDV and VM. In this study, we engineered αvß3 integrin-targeting peptide cRGD functionalized nanoparticles (cRGD-NPs) by self-assembly using heparin conjugated with cRGD and folate. The prepared cRGD-NPs represent a promising anti-angiogenic medicine in that they are able to inhibit endothelial sprouting angiogenesis and tumor cell-mediated VM. This work may provide useful information with which to construct effective anti-angiogenic nanomedicines.

Identification and characterization of follistatin as a novel angiogenin-binding protein.

Angiogenin enhances tumorigenesis. However, the mechanisms of angiogenin-induced angiogenesis and cancer cell proliferation remain elusive. In this study, follistatin was identified as a binding partner of angiogenin by a yeast two-hybrid screen and confirmed by a pull-down experiment. The interaction of fluorescently tagged angiogenin and follistatin was monitored in real time by a laser confocal microscope and shown to localize at the sub-nuclear region of HeLa cells. Additional yeast two-hybrid analysis revealed that domains 2 and 3 of follistatin were the minimal structure requirement for angiogenin binding. These findings provide new clues for further studies on the mechanisms of angiogenin-induced angiogenesis or cancer cell growth.