School of Medicine

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To understand the mechanisms driving fluid flow behavior in nanofluidics so that they may be used for on-chip biomedical and chemical applications, the fluid's motion itself needs to be observable and measurable, a difficult challenge at these small scales. We present a new method for measuring both slow and fast flows in nanofluidics using high-speed digital holographic microscopy. We measure the evaporation-driven flow in 25 and 7 nm tall nanoslit channels, showing that the consequent flow speed is about 15 times slower than open atmospheric evaporation due to the confinement of the nanoslit channel. We also measured the surface acoustic wave-driven flow in the 25 nm channel, showing flow at a speed of 0.12 m/s from acoustic wave propagation at 39.7 MHz interacting with the fluid in the channel. A process to eliminate the many sources of noise to produce these results is provided, showing that─in particular─spatial averaging is useful to determine the fluid flow and the dewetting of the fluid in the nanoslit channel over time.

The maturation and stabilization of appropriate synaptic connections is a vital step in neural circuit development; however, the molecular signals underlying these processes are not fully understood. We show that astrocytes, through production of glypican 5 (GPC5), are required for maturation and refinement of synapses in the mouse cortex during the critical period. In the absence of astrocyte GPC5, thalamocortical synapses show structural immaturity, including smaller presynaptic terminals, decreased postsynaptic density area, and presence of more postsynaptic partners at multisynaptic connections. This structural immaturity is accompanied by a delay in developmental incorporation of GLUA2-containing AMPARs at intracortical synapses. The functional impact of this is that mice lacking astrocyte GPC5 exhibit increased levels of ocular dominance plasticity in adulthood. This demonstrates that astrocyte GPC5 is necessary for maturation and stabilization of synaptic connections, which has implications for disorders with altered synaptic function where GPC5 levels are altered, including Alzheimer's disease and frontotemporal dementia.

Although inflammation has been widely associated with cancer development, how it affects the outcomes of immunotherapy and chemotherapy remains incompletely understood. Here, we show that CKLF-like MARVEL transmembrane domain-containing member 4 (CMTM4) is highly expressed in multiple human and murine cancer types including Lewis lung carcinoma, triple-negative mammary cancer and melanoma. In lung carcinoma, loss of CMTM4 significantly reduces tumor growth and impairs NF-κB, mTOR, and PI3K/Akt pathway activation. Furthermore, we demonstrate that CMTM4 can regulate epidermal growth factor (EGF) signaling post-translationally by promoting EGFR recycling and preventing its Rab-dependent degradation. Consequently, CMTM4 knockout sensitizes human lung tumor cells to EGFR inhibitors. In addition, CMTM4 knockout tumors stimulated with EGF show a decreased ability to produce inflammatory cytokines including granulocyte colony-stimulating factor (G-CSF), leading to decreased recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and therefore establishing a less suppressive tumor immune environment in both lung and mammary cancers. We also present evidence indicating that CMTM4-targeting siRNA-loaded liposomes reduce lung tumor growth in vivo and prolong animal survival. Knockout of CMTM4 enhances immune checkpoint blockade or chemotherapy to further reduce lung tumor growth. These data suggest that CMTM4 represents a novel target for the inhibition of tumor inflammation, and improvement of the immune response and tumor drug sensitivity.

BACKGROUND: The increasing demand for organs has pushed transplant providers to expand kidney acceptance criteria. The use of kidneys from donors with AKI has been shown to provide good long-term graft survival. We aim to evaluate and compare the outcomes of deceased donor kidney transplantation from donors with acute kidney injury (AKI), either with or without renal replacement therapy (AKI-RRT) before donation. METHODS: A single-center retrospective review of all patients who underwent deceased donor kidney transplantation from AKI donors between 2009 and 2020 was performed. AKI donors were defined on the basis of donor terminal creatinine ≥2.0 mg/dL or use of RRT before donation. We compared the outcomes of recipients receiving a kidney from a donor with AKI versus AKI-RRT. Data are presented as medians (interquartile ranges) and numbers (percentages). RESULTS: Four hundred ninety-six patients were identified, of whom 300 (60.4%) were men with a median age of 57 y at transplantation. Thirty-nine patients received an AKI-RRT, whereas 457 received an AKI kidney. Donors in the AKI-RRT group were younger (28 versus 40), had less incidence of hypertension (15.3% versus 31.9%), and were more likely to be imported (94.9% versus 76.8%). There was a higher incidence of delayed graft function (72% versus 44%, P < 0.001) in the AKI-RRT group. Recipients in both groups had similar 90-d (100% versus 95.2%) and 1-y (100% versus 91.9%) graft survival. With a median follow-up of 5 y, there was no difference in death-censored graft survival in both groups (P = 0.83). CONCLUSIONS: Careful selection of kidneys from donors with AKI on RRT can be safely used for kidney transplantation with favorable clinical outcomes.

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Small extracellular vesicles (sEVs) mediate intercellular signaling to coordinate the proliferation of cell types that promote re-epithelialization of skin following injury. Cyclin-dependent kinase 1 (CDK1) drives cell division and is a key regulator of entry to the cell cycle. To understand the potential of sEV-mediated delivery of CDK1 to reverse impaired wound healing, we generated CDK1-loaded sEVs (CDK1-sEVs) and evaluated their ability to mediate cell proliferation, re-epithelialization, and downstream signaling responses in the wound bed. We found that treatment of human keratinocytes with CDK1-sEVs increased phosphorylation of the CDK1 target, eukaryotic translation inhibition factor 4E-binding protein 1 (4E-BP1), and histone H3 within 24 h via AKT and ERK phosphorylation, driving increased proliferation and cell migration. Treatment of the wound bed of diabetic obese mice, a model of delayed wound healing, with a single dose of CDK1-sEVs accelerated wound closure, increased re-epithelialization, and promoted the proliferation of keratinocytes. These studies show that delivery of CDK1 by sEVs can stimulate selective and transient proliferation of cell types that increase re-epithelialization and promote proliferation of keratinocytes to accelerate wound healing.

Mitochondria are crucial for cellular metabolism and signalling. Mitochondrial activity is modulated by mitochondrial fission and fusion, which are required to properly balance metabolic functions, transfer material between mitochondria, and remove defective mitochondria. Mitochondrial fission occurs at mitochondria-endoplasmic reticulum (ER) contact sites, and requires the formation of actin filaments that drive mitochondrial constriction and the recruitment of the fission protein DRP1. The role of actin in mitochondrial fusion remains entirely unexplored. Here we show that preventing actin polymerisation on either mitochondria or the ER disrupts both fission and fusion. We show that fusion but not fission is dependent on Arp2/3, whereas both fission and fusion require INF2 formin-dependent actin polymerization. We also show that mitochondria-associated actin marks fusion sites prior to the fusion protein MFN2. Together, our work introduces a method for perturbing organelle-associated actin and demonstrates a previously unknown role for actin in mitochondrial fusion.