Intracellular delivery of virus-like particles using a sheddable linker.

Intracellular targeting is essential for the efficient delivery of drugs and nanotherapeutics. Transporting nanomaterials into cells' cytoplasm for therapeutic purposes can be challenging due to the endosomal trap and lysosomal degradation of cargo. To overcome this issue, we utilized chemical synthesis to design a functional carrier that can escape the endosome and deliver biological materials into the cytoplasm. We synthesized a thiol-sensitive maleimide linker that connects the well-known mitochondria targeting lipophilic triphenylphosphonium cation (TPP) to the surface of a proteinaceous nanoparticle based on the engineered virus-like particle (VLP) Qß. TPP facilitates endosomal escape by its lipophilic and cationic nature, which disrupts the endosomal membrane. Once in the cytosol, glutathione reacts with the thiol-sensitive maleimide linkers, severs the TPP from the nanoparticle, halting its trafficking to the mitochondria, and marooning it in the cytosol. We successfully demonstrated cytosolic delivery of a VLP loaded with Green Fluorescent Protein (GFP) in vitro and small-ultrared fluorescent protein (smURFP) in vivo, where evenly distributed fluorescence is observed in A549 human lung adenocarcinoma cells and the epithelial cells of BALB/c mice lungs. As a proof of concept, we encapsulated luciferase-targeted siRNA (siLuc) inside the VLP decorated with the maleimide-TPP (M-TPP) linker. We observed enhanced luminescence silencing in luciferase-expressing HeLa cells using our sheddable TPP linker compared to control VLPs.

HSC-independent definitive hematopoiesis persists into adult life.

It is widely believed that hematopoiesis after birth is established by hematopoietic stem cells (HSCs) in the bone marrow and that HSC-independent hematopoiesis is limited only to primitive erythro-myeloid cells and tissue-resident innate immune cells arising in the embryo. Here, surprisingly, we find that significant percentages of lymphocytes are not derived from HSCs, even in 1-year-old mice. Instead, multiple waves of hematopoiesis occur from embryonic day 7.5 (E7.5) to E11.5 endothelial cells, which simultaneously produce HSCs and lymphoid progenitors that constitute many layers of adaptive T and B lymphocytes in adult mice. Additionally, HSC lineage tracing reveals that the contribution of fetal liver HSCs to peritoneal B-1a cells is minimal and that the majority of B-1a cells are HSC independent. Our discovery of extensive HSC-independent lymphocytes in adult mice attests to the complex blood developmental dynamics spanning the embryo-to-adult transition and challenges the paradigm of HSCs exclusively underpinning the postnatal immune system.

Carrier gas triggered controlled biolistic delivery of DNA and protein therapeutics from metal-organic frameworks.

The efficacy and specificity of protein, DNA, and RNA-based drugs make them popular in the clinic; however, these drugs are often delivered via injection, requiring skilled medical personnel, and producing biohazardous waste. Here, we report an approach that allows for their controlled delivery, affording either a burst or slow release without altering the formulation. We show that when encapsulated within zeolitic-imidazolate framework eight (ZIF-8), the biomolecules are stable in powder formulations and can be inoculated with a low-cost, gas-powered "MOF-Jet" into living animal and plant tissues. Additionally, their release profiles can be modulated through judicious selection of the carrier gas used in the MOF-Jet. Our in vitro and in vivo studies reveal that when CO2 is used, it creates a transient and weakly acidic local environment that causes a near-instantaneous release of the biomolecules through an immediate dissolution of ZIF-8. Conversely, when air is used, ZIF-8 biodegrades slowly, releasing the biomolecules over a week. This is the first example of controlled-biolistic delivery of biomolecules using ZIF-8, which provides a powerful tool for fundamental and applied science research.

Conserved FimK Truncation Coincides with Increased Expression of Type 3 Fimbriae and Cultured Bladder Epithelial Cell Association in Klebsiella quasipneumoniae.

Klebsiella spp. commonly cause both uncomplicated urinary tract infection (UTI) and recurrent UTI (rUTI). Klebsiella quasipneumoniae, a relatively newly defined species of Klebsiella, has been shown to be metabolically distinct from Klebsiella pneumoniae, but its type 1 and type 3 fimbriae have not been studied. K. pneumoniae uses both type 1 and type 3 fimbriae to attach to host epithelial cells. The type 1 fimbrial operon is well conserved between Escherichia coli and K. pneumoniae apart from fimK, which is unique to Klebsiella spp. FimK contains an N-terminal DNA binding domain and a C-terminal phosphodiesterase (PDE) domain that has been hypothesized to cross-regulate type 3 fimbriae expression via modulation of cellular levels of cyclic di-GMP. Here, we find that a conserved premature stop codon in K. quasipneumoniae fimK results in truncation of the C-terminal PDE domain and that K quasipneumoniae strain KqPF9 cultured bladder epithelial cell association and invasion are dependent on type 3 but not type 1 fimbriae. Further, we show that basal expression of both type 1 and type 3 fimbrial operons as well as cultured bladder epithelial cell association is elevated in KqPF9 relative to uropathogenic K. pneumoniae TOP52. Finally, we show that complementation of KqPF9ΔfimK with the TOP52 fimK allele reduced type 3 fimbrial expression and cultured bladder epithelial cell attachment. Taken together these data suggest that the C-terminal PDE of FimK can modulate type 3 fimbrial expression in K. pneumoniae and its absence in K. quasipneumoniae may lead to a loss of type 3 fimbrial cross-regulation. IMPORTANCE K. quasipneumoniae is often indicated as the cause of opportunistic infections, including urinary tract infection, which affects >50% of women worldwide. However, the virulence factors of K. quasipneumoniae remain uninvestigated. Prior to this work, K. quasipneumoniae and K. pneumoniae had only been distinguished phenotypically by metabolic differences. This work contributes to the understanding of K. quasipneumoniae by evaluating the contribution of type 1 and type 3 fimbriae, which are critical colonization factors encoded by all Klebsiella spp., to K. quasipneumoniae bladder epithelial cell attachment in vitro. We observe clear differences in bladder epithelial cell attachment and regulation of type 3 fimbriae between uropathogenic K. pneumoniae and K. quasipneumoniae that coincide with a structural difference in the fimbrial regulatory gene fimK.

Mast Cell Repopulating Ability Is Lost During the Transition From Pre-HSC to FL HSC.

Recent advances in developmental immunology have revealed a hematopoietic stem cell (HSC)-independent origin for various innate immune lineages, including mast cells (MCs). It is now established that adult bone marrow (BM) long-term HSCs do not regenerate MCs but, instead, the physiological production of MCs starts before the emergence of HSCs in the aorta-gonad-mesonephros (AGM) region and is mostly completed before birth. However, while the AGM region represents a major site of MC generation during ontogeny, whether the first emerging HSCs in the AGM or fetal liver (FL) possess the potential to regenerate MCs is unknown. Here, we combined three fate-mapping mouse models with detailed HSC transplantation assays to determine the potential of AGM and FL HSCs to produce MCs. We show that HSCs from E11.5 AGM and E12.5 FL efficiently repopulated MCs in recipients. In stark contrast, HSCs from ≥E14.5 FL failed to reconstitute MCs. An Endothelial (EC) fate-mapping study confirmed the EC origin of the majority of MCs. Additionally, our HSC-labeling showed that HSCs do not produce MCs in a physiological setting. Hence, although most MCs are generated and maintained via an HSC-independent pathway, the earliest HSCs to emerge in the AGM and seed the early FL can produce MCs, but only during a minimal time window. Our results challenge the stem cell theory in hematology and EC-derived mast cells may contribute to the pathogenesis of postnatal mast cell disorders.