Fusogenic Lipid Nanovesicle for Biomacromolecular Delivery.

Although biomacromolecules are promising cytosolic drugs which have attracted tremendous attention, the major obstacles were the cellular membrane hindering the entrance and the endosome entrapment inducing biomacromolecule degradation. How to avoid those limitations to realize directly cytosolic delivery was still a challenge. Here, we prepared oligoarginine modified lipid to assemble a nanovesicle for biomacromolecules delivery, including mRNA (mRNA) and proteins which could be directly delivered into the cytoplasm of dendritic cells through subendocytosis-mediated membrane fusion. We named this membrane fusion lipid nanovesicle as MF-LNV. The mRNA loaded MF-LNV as nanovaccines showed efficient antigen expression to elicit robust immuno responses for cancer therapy. What's more, the antigen protein loaded MF-LNV as nanovaccines elicits much stronger CD8+ T cell specific responses than lipid nanoparticles through normal uptake pathways. This MF-LNV represented a refreshing strategy for intracellular delivery of the biomacromolecule.

Nanomedicines for an Enhanced Immunogenic Cell Death-Based In Situ Cancer Vaccination Response.

Cancer vaccines have shown tremendous potential in preventing and treating cancer by providing immunogenic antigens to initiate specific tumor immune responses. An in situ vaccine prepared from an autologous tumor can mobilize a patient's own tumor cell lysate as a reservoir of specific antigens, thus triggering a broad immune response and diverse antitumor immunity in an individually tailored manner. Its efficacy is much better than that of conventional vaccines with a limited number of epitopes. Several conventional therapies, including radiotherapy (RT), chemotherapeutics, photodynamic therapy (PDT), and photothermal therapy (PTT) can activate an anticancer in situ vaccine response by inducing immunogenic cell death (ICD), triggering the exposure of tumor-associated antigens (TAAs), cancerous testis antigens, neoantigens, and danger-associated molecular patterns (DAMPs) with low cost. However, the immunogenicity of dying tumor cells is low, making released antigens and DAMPs insufficient to initiate a robust immune response against malignant cancer. Moreover, the immunosuppressive tumor microenvironment (TME) severely hinders the infiltration and sensitization of effector immune cells, causing tolerogenic immunological effects.Herein, we mainly focus on the research in developing nanoplatforms to surmount the major challenges met by ICD-based in situ vaccines. We first summarized a variety of nanotechnologies that enable enhanced immunogenicity of dying cancer cells by enhancing antigenicity and adjuvanticity. The robust antigenicity was obtained via regulating the tumor cells death mode or the dying state to amplify the recognition of tumor debris by professional antigen-presenting cells (APCs). The adjuvanticity was potentiated by raising the level or intensifying the activity of endogenous adjuvants or promoting the intelligent delivery of exogenous immunostimulants to activate immune cell recruitment and promote antigen presentation. Additionally, versatile approaches to reverse immunosuppressive TME to boost the in situ tumor vaccination response are also highlighted in detail. On one hand, by modulating the cell metabolism in TME, the expansion and activity of effector versus immunosuppressive cells can be optimized to improve the efficiency of in situ vaccines. On the other hand, regulating cellular components in TME, such as reversing adverse immune cell phenotypes or inhibiting the activity of interstitial cells, can also significantly enhance the ICD-based antitumor immunotherapy effect. Finally, our viewpoint on the future challenges and opportunities in this hopeful area is presented. We expect that this Account can offer much more insight into the design, planning, and development of cutting-edge in situ tumor vaccine platforms, promoting more attention and academic-industry collaborations, accelerating the advanced progress of in situ tumor vaccine-based immunotherapy in the clinic.

X-ray-Guided In Situ Genetic Engineering of Macrophages for Sustained Cancer Immunotherapy.

Effective repolarization of macrophages has emerged as a promising approach for anticancer therapy. However, there are very few studies on the effect of reprogramming macrophages from M2 phenotype to M1 phenotype without reconversion while maintaining an activated M1 phenotype. Moreover, these immunomodulatory methods have serious drawbacks due to the activation of normal monocytic cells. Therefore, it remains a challenge to selectively reprogram tumor-associated macrophages (TAMs) without systemic toxicities. Here, X-ray-guided and triggered remote control of a CRISPR/Cas9 genome editing system (X-CC9) that exclusively activates therapeutic agents at tumor sites is established. Under X-ray irradiation, X-CC9 selectively enhances M2-to-M1 repolarization within the tumor microenvironment, and significantly improves antitumor efficacy with robust immune responses in two animal models. This strategy provides an ideal method for improving the safety of macrophage polarization and may constitute a promising immunotherapy strategy.

Nuclear organization in crucifer genomes: nucleolus-associated telomere clustering is not a universal interphase configuration in Brassicaceae.

Arabidopsis thaliana has become a major plant research model, where interphase nuclear organization exhibits unique features, including nucleolus-associated telomere clustering. The chromocenter (CC)-loop model, or rosette-like configuration, describes intranuclear chromatin organization in Arabidopsis as megabase-long loops anchored in, and emanating from, peripherally positioned CCs, with those containing telomeres associating with the nucleolus. To investigate whether the CC-loop organization is universal across the mustard family (crucifers), the nuclear distributions of centromeres, telomeres and nucleoli were analyzed by fluorescence in situ hybridization in seven diploid species (2n = 10-16) representing major crucifer clades with an up to 26-fold variation in genome size (160-4260 Mb). Nucleolus-associated telomere clustering was confirmed in Arabidopsis (157 Mb) and was newly identified as the major nuclear phenotype in other species with a small genome (215-381 Mb). In large-genome species (2611-4264 Mb), centromeres and telomeres adopted a Rabl-like configuration or dispersed distribution in the nuclear interior; telomeres only rarely associated with the nucleolus. In Arabis cypria (381 Mb) and Bunias orientalis (2611 Mb), tissue-specific patterns deviating from the major nuclear phenotypes were observed in anther and stem tissues, respectively. The rosette-like configuration, including nucleolus-associated telomere clustering in small-genome species from different infrafamiliar clades, suggests that genomic properties rather than phylogenetic position determine the interphase nuclear organization. Our data suggest that nuclear genome size, average chromosome size and degree of longitudinal chromosome compartmentalization affect interphase chromosome organization in crucifer genomes.

Transcriptome analysis reveals metabolic alteration due to consecutive monoculture and abiotic stress stimuli in Rehamannia glutinosa Libosch.

KEY MESSAGE: We deeply investigated the mechanism underlying metabolic regulation in response to consecutive monoculture (replanting disease) and different abiotic stresses that unfolded the response mechanism to consecutive monoculture problem through RNA-seq analysis. The consecutive monoculture problem (CMP) resulted of complex environmental stresses mediated by multiple factors. Previous studies have noted that multiple stress factors in consecutive monoculture soils or plants severely limited the interpretation of the critical molecular mechanism, and made a predict that the specifically responding factor was autotoxic allelochemicals. To identify the specifically responding genes, we compared transcriptome changes in roots of Rehamannia glutinosa Libosch using consecutive monoculture, salt, drought, and ferulic acid as stress factors. Comparing with normal growth, 2502, 2672, 2485, and 1956 genes were differentially expressed in R. glutinosa under consecutive monoculture practice, salt, drought, and ferulic acid stress, respectively. In addition, 510 genes were specifically expressed under consecutive monoculture, which were not present under the other stress conditions. Integrating the biological and enrichment analyses of the differentially expressed genes, the result demonstrated that the plants could alter enzyme genes expression to reconstruct the complicated metabolic pathways, which used to tolerate the CMP and abiotic stresses. Furthermore, most of the affected pathway genes were closely related to secondary metabolic processes, and the influence of consecutive monoculture practice on the transcriptome genes expression profile was very similar to the profile under salt stress and then to the profile under drought stress. The outlined schematic diagram unfolded the putative signal regulation mechanism in response to the CMP. Genes that differentially up- or down-regulated under consecutive monoculture practice may play important roles in the CMP or replanting disease in R. glutinosa.

Rapid proliferation and nucleolar organizer targeting centromeric retrotransposons in cotton.

Centromeric chromatin in most eukaryotes is composed of highly repetitive centromeric retrotransposons and satellite repeats that are highly variable even among closely related species. The evolutionary mechanisms that underlie the rapid evolution of centromeric repeats remain unknown. To obtain insight into the evolution of centromeric repeats following polyploidy, we studied a model diploid progenitor (Gossypium raimondii, D-genome) of the allopolyploid (AD-genome) cottons, G. hirsutum and G. barbadense. Sequence analysis of chromatin-immunoprecipitated DNA showed that the G. raimondii centromeric repeats originated from retrotransposon-related sequences. Comparative analysis showed that nine of the 10 analyzed centromeric repeats were absent from the centromeres in the A-genome and related diploid species (B-, F- and G-genomes), indicating that they colonized the centromeres of D-genome lineage after the divergence of the A- and D- ancestral species or that they were ancestrally retained prior to the origin of Gossypium. Notably, six of the nine repeats were present in both the A- and D-subgenomes in tetraploid G. hirsutum, and increased in abundance in both subgenomes. This finding suggests that centromeric repeats may spread and proliferate between genomes subsequent to polyploidization. Two repeats, Gr334 and Gr359 occurred in both the centromeres and nucleolar organizer regions (NORs) in D- and AD-genome species, yet localized to just the NORs in A-, B-, F-, and G-genome species. Contained within is a story of an established centromeric repeat that is eliminated and allopolyploidization provides an opportunity for reinvasion and reestablishment, which broadens our evolutionary understanding behind the cycles of centromeric repeat establishment and targeting.

A and D genomes spatial separation at somatic metaphase in tetraploid cotton: evidence for genomic disposition in a polyploid plant.

Chromosomal dispositions were analyzed on the metaphase plate of tetraploid cotton (AADD). At metaphase, the two subgenomes, A and D, were separated in a radial pattern in which the small D subgenome chromosomes tended to concentrate at the center and the large A subgenome chromosomes were scattered about the periphery on the metaphase plate. Although the ordered chromosome arrangement was disturbed in an artificial hexaploid (AADDGG), the separation pattern could be recovered after the majority of the additional genome (GG) chromosomes were removed by backcrossing the artificial hexaploid with the tetraploid cotton (AADD). A similar genome separation phenomenon was also found in synthesized tetraploid cotton (AAGG). These results indicate that the genome separation pattern could be established immediately after tetraploid cotton formation and could be stably inherited in tetraploid cotton. Given the evidence of parental genome separation in other plants and animals, we speculated that genome separation might be a normal phenomenon in diploid and polyploid species. These finding will shed light on the chromosome conformation in plant cells.