The advancement of microRNA (miRNA) therapies has been hampered by difficulties in delivering miRNA to the injured kidney in a robust and sustainable manner. Using bioluminescence imaging in mice with unilateral ureteral obstruction (UUO), we report that mesenchymal stem cells (MSCs), engineered to overexpress miRNA-let7c (miR-let7c-MSCs), selectively homed to damaged kidneys and upregulated miR-let7c gene expression, compared with nontargeting control (NTC)-MSCs. miR-let7c-MSC therapy attenuated kidney injury and significantly downregulated collagen IVα1, metalloproteinase-9, transforming growth factor (TGF)-β1, and TGF-β type 1 receptor (TGF-βR1) in UUO kidneys, compared with controls. In vitro analysis confirmed that the transfer of miR-let7c from miR-let7c-MSCs occurred via secreted exosomal uptake, visualized in NRK52E cells using cyc3-labeled pre-miRNA-transfected MSCs with/without the exosomal inhibitor, GW4869. The upregulated expression of fibrotic genes in NRK52E cells induced by TGF-β1 was repressed following the addition of isolated exosomes or indirect coculture of miR-let7c-MSCs, compared with NTC-MSCs. Furthermore, the cotransfection of NRK52E cells using the 3'UTR of TGF-βR1 confirmed that miR-let7c attenuates TGF-β1-driven TGF-βR1 gene expression. Taken together, the effective antifibrotic function of engineered MSCs is able to selectively transfer miR-let7c to damaged kidney cells and will pave the way for the use of MSCs for therapeutic delivery of miRNA targeted at kidney disease.

The fungal pathogen Candida albicans causes macrophage death and escapes, but the molecular mechanisms remained unknown. Here we used live-cell imaging to monitor the interaction of C. albicans with macrophages and show that C. albicans kills macrophages in two temporally and mechanistically distinct phases. Early upon phagocytosis, C. albicans triggers pyroptosis, a proinflammatory macrophage death. Pyroptosis is controlled by the developmental yeast-to-hypha transition of Candida. When pyroptosis is inactivated, wild-type C. albicans hyphae cause significantly less macrophage killing for up to 8 h postphagocytosis. After the first 8 h, a second macrophage-killing phase is initiated. This second phase depends on robust hyphal formation but is mechanistically distinct from pyroptosis. The transcriptional regulator Mediator is necessary for morphogenesis of C. albicans in macrophages and the establishment of the wild-type surface architecture of hyphae that together mediate activation of macrophage cell death. Our data suggest that the defects of the Mediator mutants in causing macrophage death are caused, at least in part, by reduced activation of pyroptosis. A Mediator mutant that forms hyphae of apparently wild-type morphology but is defective in triggering early macrophage death shows a breakdown of cell surface architecture and reduced exposed 1,3 β-glucan in hyphae. Our report shows how Candida uses host and pathogen pathways for macrophage killing. The current model of mechanical piercing of macrophages by C. albicans hyphae should be revised to include activation of pyroptosis by hyphae as an important mechanism mediating macrophage cell death upon C. albicans infection.

Mitochondria fulfill central functions in cellular energetics, metabolism and signaling. The outer membrane TOM40 complex imports virtually all mitochondrial proteins, however, its architecture and the molecular mechanisms of preprotein translocation are unknown. We mapped the active translocator with resolution down to single amino acid residues, discovering distinct transport paths for hydrophilic and hydrophobic preproteins through the Tom40 channel. An N-terminal segment of Tom40 passes from the cytosol through the channel interior to recruit intermembrane space chaperones that guide the transfer of hydrophobic preproteins. The translocator possesses an intricate architecture with three Tom40 β-barrel channels sandwiched 2 between a central α-helical Tom22 receptor cluster and external regulatory Tom proteins. The preprotein-translocating trimeric complex is in exchange with a dimeric isoform that is crucial for assembly of new TOM40 complexes. The dynamic coupling of α-helical receptors, β-barrel channels and chaperones generates a versatile machinery that manages transport of ~1,000 different proteins into mitochondria.One Sentence Summary: Architecture of the mitochondrial TOM40 entry gate identifies preprotein paths and the blueprint for its assembly.Main Text: Mitochondria are essential organelles in eukaryotic cells. They are pivotal for cellular ATP production, numerous metabolic pathways and regulatory processes, and programmed cell death. During evolution of eukaryotes, most genes for mitochondrial proteins were transferred to the nucleus. The proteins are synthesized as preproteins in the cytosol and imported back into mitochondria. Different classes of preproteins have been identified that either contain N-terminal targeting sequences (presequences) or internal targeting information in the mature part (1-3). The protein translocator of the outer membrane (TOM40 complex) functions as the main entry gate of mitochondria (1-3). Most of the >1,000 different mitochondrial proteins are imported by the TOM40 complex, followed by transfer to distinct intramitochondrial machineries specialized for individual classes of preproteins. Whereas the structurally known membrane protein complexes consist of either α-helical or β-barrel proteins, the TOM40 complex is composed of both α-helical and β-barrel integral membrane proteins. The complex consists of the channel-forming β-barrel protein Tom40 and six other subunits each containing single α-helical transmembrane (TM) segments: the receptor proteins Tom20, Tom22 and Tom70, and the small regulatory subunits Tom5, Tom6 and Tom7 (1-3). Tom40, Tom22 and the small Tom proteins form the TOM40 core complex, whereas Tom20 and Tom70 are more loosely associated with the complex. The molecular architecture of the complex has not been elucidated. It is thus unknown how α-helical and β-barrel membrane proteins can be combined into a functional complex and how diverse classes of preproteins can be transported by the same transmembrane channel.To define the archite...

Neutron reflectometry has been used to study the structure of the biosurfactant, surfactin, at the air/water and at the hydrophobic solid/water interfaces. Three different deuterated surfactins were produced from the Bacillus subtilis strain: one perdeuterated, one with the four leucines perdeuterated, and one with everything except the four leucines perdeuterated. The neutron reflectivity profiles of these three samples in null reflecting water and in D20 with a seventh profile of the protonated surfactin in D2O were measured at pH 7.5. This combination of different isotopic compositions made it possible to deduce the distribution of each type of labeled fragment in the surfactin. Surfactin is found to adopt a ball-like structure with a thickness of 14 +/- A and an area per molecule of 147 +/- 5 A2. This makes it more like a hydrophobic nanoparticle, whose solubility in water is maintained only by its charge, than a conventional surfactant. This is probably what makes it surface-active at such low concentrations and what contributes to its forming very compact surface layers that are more dense than observed for most conventional amphiphiles. The reflectivity data were fitted by a model in which the structure of surfactin was divided into three fragments: the four leucines taken as a group, the hydrocarbon chain, and a hydrophilic group containing the two negative charges. An analysis of the reflectivity gave the following separations between fragments, where zero corresponds to the Gibbs plane for zero water adsorption: chain-water 7 A, hydrophile-water 1 A, and leucines-water 6.5 A, all +/- 1 A. The overall structure of the layer appears to be identical at a hydrophobic octadecyltrichlorosilane-coated silicon surface where the thickness of the surfactin layer is 15 +/- 1 A and the area per molecule is 145 +/- 5 A2. Finally, the structure of surfactin micelles has been examined by means of small-angle neutron scattering. The aggregation number was found to be unusually small at 20 +/- 5. The structure of the micelle is of the core-shell type with the hydrocarbon chain and the four hydrophobic leucines forming the core of the micelle.