Multi-Targeted and On-Demand Non-Coding RNA Regulation Nanoplatform against Metastasis and Recurrence of Triple-Negative Breast Cancer.

Dysregulation of microRNAs (miRs) is the hallmark of triple-negative breast cancer (TNBC), which is closely involved with its growth, metastasis, and recurrence. Dysregulated miRs are promising targets for TNBC therapy, however, targeted and accurate regulation of multiple disordered miRs in tumors is still a great challenge. Here, a multi-targeting and on-demand non-coding RNA regulation nanoplatform (MTOR) is reported to precisely regulate disordered miRs, leading to dramatical suppression of TNBC growth, metastasis, and recurrence. With the assistance of long blood circulation, ligands of urokinase-type plasminogen activator peptide and hyaluronan located in multi-functional shells enable MTOR to actively target TNBC cells and breast cancer stem cell-like cells (BrCSCs). After entering TNBC cells and BrCSCs, MTOR is subjected to lysosomal hyaluronidase-induced shell detachment, leading to an explosion of the TAT-enriched core, thereby enhancing nuclear targeting. Subsequently, MTOR could precisely and simultaneously downregulate microRNA-21 expression and upregulate microRNA-205 expression in TNBC. In subcutaneous xenograft, orthotopic xenograft, pulmonary metastasis, and recurrence TNBC mouse models, MTOR shows remarkably synergetic effects on the inhibition of tumor growth, metastasis, and recurrence due to its on-demand regulation of disordered miRs. This MTOR system opens a new avenue for on-demand regulation of disordered miRs against growth, metastasis, and recurrence of TNBC.

An Acidity-Initiated Self-Assembly/Disassembly Nanoprobe to Switch on Fluorescence for Tumor-Targeted Near-Infrared Imaging.

The deep penetration, real-time monitoring ability, and high resolution of near-infrared (NIR) fluorescence imaging make it suitable for tumor diagnosis. However, the lack of specificity and selectivity restricts its further application. Here, for the first time, we applied a CBT-Cys click condensation reaction to synthesize an acidity-initiated molecular probe (AIM-Probe, Cys(StBu)-Lys(Cy 5.5)-EDA-PMA-CBT), which could self-assemble into nanoparticles (AIM-NP) with self-quenched fluorescence under glutathione (GSH) reduction. AIM-NP could accumulate in tumors after intravenous injection. Subsequently, the EDA-PMA part of AIM-Probe in AIM-NP is fractured by the unique subacid condition in the tumor microenvironment, and AIM-NP disassembles into a small AIM-cleaved molecule (PMA-CBT-Cys-Lys(Cy5.5)-EDA) along with fluorescence switching on. As a result, AIM-NP could switch on fluorescence at the tumor site, thereby achieving tumor-targeted imaging. To our knowledge, utilizing tumor acidity to initiate the disassembly of self-assembled nanoparticles through a CBT-Cys click condensation reaction has not been reported.

A programmable releasing versatile hydrogel platform boosts systemic immune responses via sculpting tumor immunogenicity and reversing tolerogenic dendritic cells.

Immunogenicity improvement is a valuable strategy for tumor immunotherapy. However, immunosuppressive factors bestow tolerogenic phenotype on tumor-infiltrating DCs, which exhibit weak antigen presentation and strong anti-inflammatory cytokines secretion abilities, limiting the effectiveness of tumor immunotherapy even if the tumor has adequate immunogenicity. Herein, we designed a programmable releasing versatile hydrogel platform (PIVOT) to sculpt tumor immunogenicity, increase intratumoral DCs and cDC1s abundance, and reverse the tolerogenic phenotype of DCs, thus promoting their maturation for boosting innate and adaptive immune responses. Responsive to tumoral reactive oxygen species (ROS), the hydrogel splits and promotes the activation of DCs and macrophages. Then, oxaliplatin is first released from PIVOT to sculpt tumor immunogenicity by inducing immunogenic cell death (ICD) and causing tumoral DNA fragments exposure simultaneously. Subsequently, the impaired DNA fragments bind to high mobility group protein 1 (HMGB1) forming the DNA-HMGB1 complex. Moreover, exogenous FMS-like tyrosine kinase 3 ligand (Flt-3L) recruits masses of DCs, especially cDC1s, which will endocytose the complex benefiting from TIM-3 blockade (αTIM3) that can reverse tolerogenic DCs. Finally, the endocytosis activates the cGAS-STING pathway of cDC1s, which promotes the secretion of type I IFN that triggers innate immune responses, and CXCL9 which recruits CD8+ effector T cells to initiate the following adaptive immune response against tumor progress. PIVOT achieves nearly 90 % tumor growth inhibition and induces systemic antitumor immune responses. In conclusion, this study focuses on ICD-mediated tumor immunogenicity sculpture and nucleic acid endocytosis-involved tolerogenic DCs reversal, providing a novel paradigm for enhancing DCs-based antitumor immune responses.

ROS-sensitive PD-L1 siRNA cationic selenide nanogels for self-inhibition of autophagy and prevention of immune escape.

In the field of cancer therapy, inhibiting autophagy has emerged as a promising strategy. However, pharmacological disruption of autophagy can lead to the upregulation of programmed death-ligand 1 (PD-L1), enabling tumor immune evasion. To address this issue, we developed innovative ROS-responsive cationic poly(ethylene imine) (PEI) nanogels using selenol chemistry-mediated multicomponent reaction (MCR) technology. This procedure involved simple mixing of low-molecular-weight PEI (LMW PEI), γ-selenobutylacetone (γ-SBL), and poly(ethylene glycol) methacrylate (PEGMA). Through high-throughput screening, we constructed a library of AxSeyOz nanogels and identified the optimized A1.8Se3O0.5/siPD-L1 nanogels, which exhibited a size of approximately 200 nm, excellent colloidal stability, and the most effective PD-L1 silencing efficacy. These nanogels demonstrated enhanced uptake by tumor cells, excellent oxidative degradation ability, and inhibited autophagy by alkalinizing lysosomes. The A1.8Se3O0.5/siPD-L1 nanogels significantly downregulated PD-L1 expression and increased the expression of major histocompatibility complex class I (MHC-I), resulting in robust proliferation of specific CD8+ T cells and a decrease in MC38 tumor growth. As a result, the A1.8Se3O0.5/siPD-L1 nanogels inhibited tumor growth through self-inhibition of autophagy, upregulation of MHC-I, and downregulation of PD-L1. Designed with dynamic diselenide bonds, the A1.8Se3O0.5/siPD-L1 nanogels showed synergistic antitumor efficacy through self-inhibition of autophagy and prevention of immune escape.

Hierarchically tumor-activated nanoCRISPR-Cas13a facilitates efficient microRNA disruption for multi-pathway-mediated tumor suppression.

Rationale: CRISPR-Cas13a is an efficient tool for robust RNA knockdown with lower off-target effect, which may be a potentially powerful and safe tool for cancer gene therapy. However, therapeutic effect of current cancer gene therapy that targeting monogene was compromised by the multi-mutational signal pathway alterations of tumorigenesis. Methods: Here, hierarchically tumor-activated nanoCRISPR-Cas13a (CHAIN) is fabricated for multi-pathway-mediated tumor suppression by efficient microRNA disruption in vivo. A fluorinated polyetherimide (PEI; Mw=1.8KD) with graft rate of 33% (PF33) was utilized to compact the CRISPR-Cas13a megaplasmid targeting microRNA-21 (miR-21) (pCas13a-crRNA) via self-assemble to constitute a nanoscale 'core' (PF33/pCas13a-crRNA), which was further wrapped by modified hyaluronan (HA) derivatives (galactopyranoside-PEG2000-HA, GPH) to form CHAIN. Results: The dual-tumor-targeting and tumor-activated CHAIN not only manifested long-term circulation, but augmented tumor cellular uptake and endo/lysosomal escape, thus achieving efficient transfection of CRISPR-Cas13a megaplasmid (~ 13 kb) in tumor cells with minimal toxity. Efficient knockdown of miR-21 by CHAIN restored programmed cell death protein 4 (PDCD4) and reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) and further crippled downstream matrix metalloproteinases-2 (MMP-2), which undermined cancer proliferation, migration and invasion. Meanwhile, the miR-21-PDCD4-AP-1 positive feedback loop further functioned as an enhanced force for anti-tumor activity. Conclusion: Treatment with CHAIN in hepatocellular carcinoma mouse model achieved significant inhibition of miR-21 expression and rescued multi-pathway, which triggered substantial tumor growth suppression. By efficient CRISPR-Cas13a induced interference of one oncogenic microRNA, the CHAIN platform exerted promising capabilities in cancer treatment.

Tumor Environment Regression Therapy Implemented by Switchable Prune-to-Essence Nanoplatform Unleashed Systemic Immune Responses.

Coevolution of tumor cells and surrounding stroma results in protective protumoral environment, in which abundant vessel, stiff structure and immunosuppression promote each other, cooperatively incurring deterioration and treatment compromise. Reversing suchenvironment may transform tumors from treatment-resistant to treatment-vulnerable. However, effective reversion requires synergistic comprehensive regression of such environment under precise control. Here, the first attempt to collaboratively retrograde coevolutionary tumor environment to pre-oncogenesis status, defined as tumor environment regression therapy, is made for vigorous immune response eruption by a switchable prune-to-essence nanoplatform (Pres) with simplified composition and fabrication process. Through magnetic targeting and multimodal imaging of Pres, tumor environment regression therapy is guided, optimized and accomplished in a trinity way: Antiangiogenesis is executed to rarefy vessels to impede tumor progression. By seizing the time, cancer associated fibroblasts are eliminated to diminish collagen and loosen the stiff structure for deep penetration of Pres, which alternately functioned in deeper tumors, forming a positive feedback loop. Through this loop, immune cell infiltration, immunosuppression mitigation and immunogenic cells death induction are all fulfilled and further escalated in the regressed environment. These transformations consequently unleashed systemic immune responses and generated immune memory against carcinoma. This study provides new insights intotreatment of solid tumors.

A self-sustained nanoplatform reverses TRAIL-resistance of pancreatic cancer through coactivating of exogenous and endogenous apoptotic pathway.

Since the 5-year survival rate of pancreatic cancer is only 10.0%, new therapies are urgently needed. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis specifically on tumor cells, nevertheless its clinical application was seriously restricted by resistance and short in vivo half-life. Herein, a novel multifunctional R6ST protein equipped with cell penetrating peptides R6, intrinsic apoptosis inducing tetrapeptide AVPI and soluble TRAIL was designed and constructed. Then, it was recruited to prepare self-sustained nanoplatform (SSN) to reverse TRAIL-resistance of pancreatic cancer through simultaneously promoting extrinsic and intrinsic apoptotic pathway, as well to elongate circulation time. Once administrated, high tumor accumulation and cellular uptake of SSN were achieved through prolonged circulation time, targeting ability of soluble TRAIL to death receptors and positive-charged R6, and further enhanced through reversed upregulation of death receptors on TRAIL-resistant tumor cells by the cumulated artesunate released in cytoplasm as a positive feedback loop. Furthermore, this loop simultaneously promoted extrinsic apoptosis of TRAIL fragment via the upregulated death receptors on TRAIL-resistant pancreatic cancer cells and intrinsic apoptosis of AVPI tetrapeptide via the efficient accumulation and uptake of R6ST on SSN. Hence, SSN exhibited synergistic antitumor effect and provided a new strategy for TRAIL-resistant pancreatic cancer therapy.

Virus-esque nucleus-targeting nanoparticles deliver trojan plasmid for release of anti-tumor shuttle protein.

Gene therapy has gathered vast interest and been proved promising and prospective. While gene therapy evolves fast, demands of high transfecting efficiency and less toxic gene vectors are not sufficiently fulfilled. The progression of materials is doing the favor from which therapeutic application benefited is helping reshape treatments of cancer. In this work, we synthesized fluorinated branched polyethylenimine (PF33) and RGD-R8-PEG-HA (RRPH). When mixed with plasmids, the PF33 could form a compact nanoparticle PFC (Fluorinated PEI/plasmid Complex) and showed high transfection efficiency (>70% in A549 cells). Peptide modification and PEGylation on HA constituted the RRPH, and coating on the PFC would enable the ultimate nanoparticle RRPHC (RRPH coating PFC Complex) achieve long-term circulation and tumor tissue-penetration while maintaining the high transfection efficiency of PFC. Observations about the behavior in cellular organisms of RRPHC revealed its nucleus-targeting tendency. The in vivo distribution images revealed the RRPHC nanoparticles, compared to HAC (HA coated PFC, used as control) could achieve extended accumulation specifically on tumor regions rather than stay in other organs. While loaded with plasmids encoding our rationally designed trojan Apoptin (pSTA), RRPHC could establish compounds for the massive production of membrane-penetrating protein. Hence these cancer-killing proteins would charge at nucleus once phosphorylated and finish the task of destruction. Both in vitro and in vivo treatment using RRPHC/pSTA nanoparticles resulted in remarkable tumor suppression and the cytotoxicity tests demonstrated its low toxicity. In summary, pSTA encapsulating RRPHC nanoparticles may have potential applications in cancer gene therapy.

A 14 nucleotide deletion mutation in the coding region of the PpBBX24 gene is associated with the red skin of "Zaosu Red" pear (Pyrus pyrifolia White Pear Group): a deletion in the PpBBX24 gene is associated with the red skin of pear.

Red skin is an important quality trait for pear fruits and is determined by the concentration and composition of anthocyanins. The regulatory mechanism underlying anthocyanin accumulation is a popular topic in fruit research. Red mutants are ideal materials for studying the molecular mechanism of color diversity in pear. Although several red pear mutants have been cultivated and are in production, no exact locus containing the responsible genetic mutation has been identified. In this study, by combining the bulked segregant analysis with whole-genome sequencing, we identified a 14 nucleotide deletion mutation in the coding region of the PpBBX24 gene from the red pear mutant "Zaosu Red". We further verified that the deletion was present only in the red mutant of "Zaosu" and in its red offspring, which was different from that which occurred in other red pear fruits. This deletion results in a coding frame shift such that there is an early termination of the PpBBX24 gene and loss of key NLS and VP domains from PpBBX24. The lost domains may reduce or alter the normal function of PpBBX24. In addition, we found that the transcript levels of the PpMYB10 and PpHY5 genes in red samples were significantly higher than those in green samples, whereas the results for the normal-type PpBBX24 gene were the opposite. We ultimately revealed that the 14 nucleotide deletion mutation in the coding region of the PpBBX24 gene is associated with the red skin of the "Zaosu Red" pear. This finding of somatic mutational events will be helpful for breeding new red pear cultivars and for understanding the regulatory mechanisms involved in pear skin pigmentation.

A de novo genome assembly of the dwarfing pear rootstock Zhongai 1.

'Zhongai 1' [(Pyrus ussuriensis × communis) × spp.] is an excellent pear dwarfing rootstock common in China. It is dwarf itself and has high dwarfing efficiency on most of main Pyrus cultivated species when used as inter-stock. Here we describe the draft genome sequences of 'Zhongai 1' which was assembled using PacBio long reads, Illumina short reads and Hi-C technology. We estimated the genome size is approximately 511.33 Mb by K-mer analysis and obtained a final genome of 510.59 Mb with a contig N50 size of 1.28 Mb. Next, 506.31 Mb (99.16%) of contigs were clustered into 17 chromosomes with a scaffold N50 size of 23.45 Mb. We further predicted 309.86 Mb (60.68%) of repetitive sequences and 43,120 protein-coding genes. The assembled genome will be a valuable resource and reference for future pear breeding, genetic improvement, and comparative genomics among related species. Moreover, it will help identify genes involved in dwarfism, early flowering, stress tolerance, and commercially desirable fruit characteristics.

Genome-wide characterization of long terminal repeat -retrotransposons in apple reveals the differences in heterogeneity and copy number between Ty1-copia and Ty3-gypsy retrotransposons.

The conserved domains of reverse transcriptase (RT) genes of Ty1-copia and Ty3-gypsy groups of long terminal repeat (LTR) retrotransposons were isolated from the Malus domestica genome using degenerate oligonucleotide primers. Sequence analysis showed that 45% of Ty1-copia and 63% of Ty3-gypsy RT sequences contained premature stop codons and/or indels disrupting the reading frame. High heterogeneity among RT sequences of both Ty1-copia and Ty3-gypsy group retrotransposons was observed, but Ty3-gypsy group retrotransposons in the apple genome are less heterogeneous than Ty1-copia elements. Retrotransposon copy number was estimated by dot blot hybridizations for Ty1-copia (approximately 5,000) and Ty3-gypsy (approximately 26,000). All elements of the two types of LTR retrotransposons comprise approximately 38% of the M. domestica genome, with the Ty3-gypsy group contribution being higher (33.5%) than the Ty1-copia one (4.6%). Transcription was not detected by reverse transcription-polymerase chain reaction for either Ty1-copia or Ty3-gypsy retrotransposons in the leaves of plants in vitro or in leaf explants cultured on medium supplemented with high concentration benzylaminopurine. This research reveals the differences in heterogeneity and copy number between Ty1-copia and Ty3-gypsy retrotransposons in the apple genome. Ty1-copia retrotransposon has higher heterogeneity than Ty3-gypsy retrotransposon, but the latter has a higher copy number, which implies that Ty3-gypsy retrotransposons may play a more important role in the apple genome evolution.

Global Proteomics Deciphered Novel-Function of Osthole Against Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive cardiovascular-disease with high mortality lacking high-efficiency drug. Our efforts attempted to delineate therapeutic action of osthole produced by Angelica Pubescens Maxim, which has the capacity to treat PAH by exploiting an iTRAQ-based proteomic method. Excitingly, osthole was observed to significantly restore 98 of 315 differential proteins significantly modified by PAH progression. They were primarily annotated into 24 signaling pathways. Four mostly affected proteins (RPL15, Cathepsin S, Histone H3.3 and HMGB1) were experimentially validated which belonged to ribosome pathway, oxidative phosphorylation pathway, systemic lupus erythematosus pathway, complement and coagulation cascades pathway, whose modifications and modulations mostly accounted for therapeutic capacity of this compound against PAH. Altogether, our findings demonstrated that global proteomics is a promising systems-biology approach for deciphering therapeutic actions and associated mechanisms of natural products derived from traditional Chinese medicine. Importantly, osthole is supposed to be a candidate compound for new drug development to treat PAH.

Involvement of Auxin and Brassinosteroid in Dwarfism of Autotetraploid Apple (Malus × domestica).

The plant height is an important trait in fruit tree. However, the molecular mechanism on dwarfism is still poorly understood. We found that colchicine-induced autotetraploid apple plants (Malus × domestica) exhibited a dwarf phenotype. The vertical length of cortical parenchyma cells was shorter in autotetraploids than in diploids, by observing paraffin sections. Hormone levels of indoleacetic acid (IAA) and brassinosteroid (BR) were significantly decreased in 3- and 5-year-old autotetraploid plants. Digital gene expression (DGE) analysis showed that the differentially expressed genes were mainly involved in IAA and BR pathways. microRNA390 was significantly upregulated according to microarray analysis. Exogenous application of IAA and BR promoted stem elongation of both apple plants grown in medium. The results show that dwarfing in autotetraploid apple plants is most likely regulated by IAA and BR. The dwarf phenotype of autotetraploid apple plants could be due to accumulation of miR390 after genome doubling, leading to upregulation of apple trans-acting short-interfering RNA 3 (MdTAS3) expression, which in turn downregulates the expression of MdARF3. Overall, this leads to partial interruption of the IAA and BR signal transduction pathway. Our study provides important insights into the molecular mechanisms underlying dwarfism in autopolyploid apple plants.