In vivo biosynthesis of nutritional holoferritin nanoparticles: Preparation, characterization, iron content analysis, and synthetic pathway.

Natural holoferritin, containing average 2000 Fe3+/ferritin, has been considered as promising iron supplementary in food and medical science. However, the low extraction yields highly limited its practical application. Herein, we provided a facile strategy for holoferritin preparation through in vivo microorganism-directed biosynthesis, and the structure, iron content, and the composition of iron core have been investigated. The results revealed that in vivo biosynthesized holoferritin possesses great monodispersity and water-solubility. In addition, the in vivo biosynthesized holoferritin contains a comparative iron content as compared to natural holoferritin, giving the ratio of âˆ¼ 2500 iron/ferritin. Besides, the composition of iron core has been identified as ferrihydrite and FeOOH, and three steps might be involved in iron core formation. This work highlighted that the microorganism-directed biosynthesis could be an efficient strategy for preparation of holoferritin, which might be beneficial for its practical application for iron supplementation.

Genipin-mediated subunit-subunit crosslinking of ferritin nanocages: Structure, properties, and its application for food bioactive compound sealing.

Ferritin proteins are promising nano-carriers for bioactive compound delivery. However, the disassembly properties under acidic/alkaline conditions pose risks of cargo leakage. Herein, genipin-mediated chemical crosslinking method was provided as an alternative and effective strategy to construct robust ferritin nanocarrier through controlled-intramolecular conjugation. As indicated by SDS-/Native- PAGE, the crosslinking degree gradually increased with incubating time prolonging. CD results showed that the cross-linking would decrease α-helix content from 78.4 % to 52.7 % upon 6 h incubation. However, TEM images showed that the genipin-modification has subtle influence on its shell-like structure. Remarkably, the cross-linking can be well controlled by intramolecular subunit-subunit conjugation rather than intermolecular conjugation, giving an excellent monodispersity. Importantly, the covalent cross-linking can tight neighboring subunits and inhibit its disassociation, finally inhibiting the leakage of encapsulated-cargos from ferritin cavity under acidic environments. Such findings suggested that the genipin-mediated cross-linking strategy can fabricate robust nano-carriers for bioactive compound delivery.

Ferritin nanocage based delivery vehicles: From single-, co- to compartmentalized- encapsulation of bioactive or nutraceutical compounds.

Bioactive or nutraceutical ingredients have been widely used in pursuit of health and well-being. However, the environmental instability, poor solubility and bioavailability, and unspecific delivery highly limited their practical values. By virtue of the unique shell-like structure, definite disassembly/reassembly behavior, and excellent safety profile of ferritin protein, it stands out among of various nano-materials and is emerging as one of the most promising vehicles for the encapsulation and delivery of bioactive ingredients or drugs. In this review, we present a systematic overview of recent advances of ferritin-based delivery systems from single-encapsulation, co-encapsulation, to compartmentalized-encapsulation of bioactive ingredients or drugs. Different encapsulation strategies for cargo loading as well as their advantages and drawbacks have been critically reviewed. This study emphasized the importance of the construction of compartmentalized delivery systems through the usage of ferritin nanocages, which exhibit great potential for facilitating the synergistic functionality of different types of cargos. Lastly, the applications of ferritin nanocages for physicochemical improvements and functionality achievements of loaded cargos are summarized. In conclusion, ferritin protein nanocages not only are excellent nanocarriers, but also can act as"multi-seated" vehicles for co-encapsulation and compartmentalized encapsulation of different cargos simultaneously.

The construction of self-protective ferritin nanocage to cross dynamic gastrointestinal barriers with improved delivery efficiency.

Ferritin nanocages are promising nanocarriers for food bioactive compound delivery, but gastrointestinal barriers including disassociation by environmental acidity, degradation by protease, pose great challenges for cargo delivery. Herein, a self-protective ferritin that can cross gastrointestinal barriers is prepared through phosphorylation modification at 37 °C for 4 h. The results showed that the conjugation of phosphate group facilitates an acidic pI shift of ferritin from ∼5.0 to ∼4.0, allowing fast aggregation and precipitation in an intact spherical form rather than disassociation into subunits in acidic environments. Meanwhile, after incubation at simulated gastric juice for 30 min, almost 80 % STP-MjFer is retained, thus, the aggregation state and phosphate layers can improve its digestive stability. Besides, curcumin can be encapsulated within its cavity and the retention rate is âˆ¼ 9 times higher than that of MjFer nanocage in simulated gastrointestinal fluid. Overall, the self-protective ferritin nanocarrier displays great potential for cargo delivery in food science.

Compartmentalized chitooligosaccharide/ferritin particles for controlled co-encapsulation of curcumin and rutin.

Compartmentalized carriers, containing multiple compartments, are receiving growing attention and are envisioned to be efficient vehicles for simultaneous encapsulation of different bioactive compounds or drugs. Herein, a two-step fabrication of chitooligosaccharide/ferritin (COS/Fn) particles containing compartmentalized cavities was reported by self-assembly of ferritin nanocages and COS. The COS/Fn particles exhibit a sphere-like morphology with nano-scale diameters depending on the COS/Fn ratio. Electrostatic interaction and hydrogen bonds are responsible for the generation of COS/Fn particles. By taking advantage of the multiple compartments of COS/Fn particles, curcumin and rutin molecules can be simultaneously encapsulated within COS/Fn particles in a compartmentalized manner. More importantly, the loading ratio of curcumin and rutin within COS/Fn particles can be easily controlled. Besides, the COS/Fn particles exhibit great protective capability against thermal treatment for internalized cargos. Overall, this work may promote future efforts to apply ferritin nanocages for the compartmentalized encapsulation of different bioactive compounds or drugs.

Complex Age- and Cancer-Related Changes in Human Blood Transcriptome-Implications for Pan-Cancer Diagnostics.

Early cancer detection is the key to a positive clinical outcome. While a number of early diagnostics methods exist in clinics today, they tend to be invasive and limited to a few cancer types. Thus, a clear need exists for non-invasive diagnostics methods that can be used to detect the presence of cancer of any type. Liquid biopsy based on analysis of molecular components of peripheral blood has shown significant promise in such pan-cancer diagnostics; however, existing methods based on this approach require improvements, especially in sensitivity of early-stage cancer detection. The improvement would likely require diagnostics assays based on multiple different types of biomarkers and, thus, calls for identification of novel types of cancer-related biomarkers that can be used in liquid biopsy. Whole-blood transcriptome, especially its non-coding component, represents an obvious yet under-explored biomarker for pan-cancer detection. In this study, we show that whole transcriptome analysis using RNA-seq could indeed serve as a viable biomarker for pan-cancer detection. Furthermore, a class of long non-coding (lnc) RNAs, very long intergenic non-coding (vlinc) RNAs, demonstrated superior performance compared with protein-coding mRNAs. Finally, we show that age and presence of non-blood cancers change transcriptome in similar, yet not identical, directions and explore implications of this observation for pan-cancer diagnostics.

Very long intergenic non-coding (vlinc) RNAs directly regulate multiple genes in cis and trans.

BACKGROUND: The majority of the human genome is transcribed in the form of long non-coding (lnc) RNAs. While these transcripts have attracted considerable interest, their molecular mechanisms of function and biological significance remain controversial. One of the main reasons behind this lies in the significant challenges posed by lncRNAs requiring the development of novel methods and concepts to unravel their functionality. Existing methods often lack cross-validation and independent confirmation by different methodologies and therefore leave significant ambiguity as to the authenticity of the outcomes. Nonetheless, despite all the caveats, it appears that lncRNAs may function, at least in part, by regulating other genes via chromatin interactions. Therefore, the function of a lncRNA could be inferred from the function of genes it regulates. In this work, we present a genome-wide functional annotation strategy for lncRNAs based on identification of their regulatory networks via the integration of three distinct types of approaches: co-expression analysis, mapping of lncRNA-chromatin interactions, and assaying molecular effects of lncRNA knockdowns obtained using an inducible and highly specific CRISPR/Cas13 system. RESULTS: We applied the strategy to annotate 407 very long intergenic non-coding (vlinc) RNAs belonging to a novel widespread subclass of lncRNAs. We show that vlincRNAs indeed appear to regulate multiple genes encoding proteins predominantly involved in RNA- and development-related functions, cell cycle, and cellular adhesion via a mechanism involving proximity between vlincRNAs and their targets in the nucleus. A typical vlincRNAs can be both a positive and negative regulator and regulate multiple genes both in trans and cis. Finally, we show vlincRNAs and their regulatory networks potentially represent novel components of DNA damage response and are functionally important for the ability of cancer cells to survive genotoxic stress. CONCLUSIONS: This study provides strong evidence for the regulatory role of the vlincRNA class of lncRNAs and a potentially important role played by these transcripts in the hidden layer of RNA-based regulation in complex biological systems.

A CRISPR/Cas13-based approach demonstrates biological relevance of vlinc class of long non-coding RNAs in anticancer drug response.

Long non-coding (lnc) RNAs represent a fascinating class of transcripts that remains highly controversial mainly due to ambiguity surrounding overall biological relevance of these RNAs. Multitude of reverse genetics studies showing functionality of lncRNAs are unfortunately based on assays that are either plagued by non-specific effects and/or cannot unambiguously assign observed phenotypes to the transcript per se. Here, we show application of the novel CRISPR/Cas13 RNA knockdown system that has superior specificity compared to other transcript-targeting knockdown methods like RNAi. We applied this method to a novel widespread subclass of nuclear lncRNAs - very long intergenic non-coding (vlinc) RNAs - in a high-throughput phenotypic assay based on survival challenge in response to anticancer drug treatments. We used multiple layers of controls including mismatch control for each targeting gRNA to ensure uncovering true phenotype-transcript relationships. We found evidence supporting importance for cellular survival for up to 60% of the tested protein-coding mRNAs and, importantly, 64% of vlincRNAs. Overall, this study demonstrates utility of CRISPR/Cas13 as a highly sensitive and specific tool for reverse genetics study of both protein-coding genes and lncRNAs. Furthermore, importantly, this approach provides evidence supporting biological significance of the latter transcripts in anticancer drug response.

Novel approach reveals genomic landscapes of single-strand DNA breaks with nucleotide resolution in human cells.

Single-strand breaks (SSBs) represent the major form of DNA damage, yet techniques to map these lesions genome-wide with nucleotide-level precision are limited. Here, we present a method, termed SSiNGLe, and demonstrate its utility to explore the distribution and dynamic changes in genome-wide SSBs in response to different biological and environmental stimuli. We validate SSiNGLe using two very distinct sequencing techniques and apply it to derive global profiles of SSBs in different biological states. Strikingly, we show that patterns of SSBs in the genome are non-random, specific to different biological states, enriched in regulatory elements, exons, introns, specific types of repeats and exhibit differential preference for the template strand between exons and introns. Furthermore, we show that breaks likely contribute to naturally occurring sequence variants. Finally, we demonstrate strong links between SSB patterns and age. Overall, SSiNGLe provides access to unexplored realms of cellular biology, not obtainable with current approaches.