School of Medicine

To understand the mechanisms driving fluid flow behavior in nanofluidics so that they may be used for on-chip biomedical and chemical applications, the fluids 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.

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.

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.

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.

BACKGROUND: Obesity is an independent risk factor for complications after abdominal hernia repair. Glucagon-like-peptide-1 (GLP-1) receptor agonists are gaining popularity as pharmacologic weight loss adjuncts and may help patients reach weight loss goals for surgery. We examine our early experience utilizing GLP-1 agonists versus lifestyle modifications alone to achieve weight loss in patients before elective hernia repair. METHODS: This single-center, retrospective review identified obese patients who underwent elective hernia repair from 2014 to 2023. Patients were asked to achieve a BMI ≤ 33 kg/m2 before surgery. Patients who lost weight with GLP-1 therapy in addition to lifestyle changes were compared to a control cohort that achieved similar preoperative weight loss without GLP-1 therapy. Primary outcome was mean time from GLP-1 agonist initiation and initial surgery clinic visit to surgery. Secondary outcomes were 30-day morbidity, mortality, and reoperation rates, and hernia recurrence. RESULTS: Forty-six patients with ventral/incisional, flank, umbilical, parastomal, inguinal, and hiatal hernias were identified (GLP-1 N = 24, control N = 22). 81.8% (N = 18) of controls had a ventral/incisional hernia, compared to 45.8% (N = 11) of GLP-1 patients (p = 0.03). Mean BMI at GLP-1 agonist initiation was similar to mean BMI at initial clinic visit for controls (38.1 ± 4.9 vs 38.2 ± 2.7 kg/m2, p = 0.66). Preoperative mean percentage total weight loss (14.9 ± 7.5 vs 12.4 ± 6.9 kg, p = 0.39) and mean BMI reduction (6.0 ± 3.8 vs 4.9 ± 2.3 kg/m2, p = 0.43) were similar between groups. The mean time from GLP-1 agonist initiation to surgery was significantly shorter than initial clinic visit to surgery for controls (6.3 ± 4.0 vs 14.7 ± 17.6 months, p = 0.03). There was no statistically significant difference in time from initial clinic visit to surgery between groups (7.6 ± 4.4 vs 14.7 ± 17.6 months, p = 0.06). There was no significant difference in 30-day morbidity between groups (8.3 vs 27.3%, p = 0.13). CONCLUSION: GLP-1 agonists accelerate preoperative weight loss for obese hernia patients without negatively impacting postoperative outcomes.

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BACKGROUND: The use of standing electric motorized scooters (eScooters) has skyrocketed since its first release in 2016. This quickly popularized form of transportation has been associated with significant injury and even death. These eScooter-related traumatic injuries led to local advocacy efforts, resulting in safety restrictions including speed limit geofencing, sidewalk restrictions, and limiting the number of eScooter providers in high-density population areas. We hypothesized that these local safety restrictions decreased the number of eScooter-related injuries presenting to our trauma center. . METHODS: This is a retrospective cohort study of eScooter-crash patients presenting to our Level 1 trauma center from July 2018 to June 2023. Variables included patient demographics, injury severity score (ISS), and mortality. The primary outcome was the rate of eScooter patients presenting over time in relation to the implementation of local-regional safety regulations. RESULTS: A total of 381 patients presented after eScooter crashes. Males were 73.8% of patients. The average age was 38.6 years; 45+ years was the most common age group at 33%, followed by ages 25-34 (31%). The mean ISS was 9±6, with ISS 0-9 (65.1%), 10-15 (24.4%), 16-24 (8.4%), and >25 (20.1%). There were three (0.8%) deaths. The median number of eScooter patients per month with prespeed limits was nine and post five (p=0.005), showing a 44.4% decrease in injured patients. After February 2022 restrictions, the rate precipitously declined with a median of two (p=0.033), reflecting an additional 60% decrease in injured patients. CONCLUSION: Local advocacy resulting in increased safety regulations was associated with a significant reduction in injured patients secondary to eScooter use. This demonstrates the importance of advocacy efforts in response to changes in injury patterns and mechanisms of injury. We believe that our work can serve as a model for other urban centers seeking to reduce eScooter-related injuries and implement effective safety measures. LEVEL OF EVIDENCE: IV, prognostic/epidemiologic.

BACKGROUND: Traumatic rib fractures are associated with pain lasting weeks to months and a decreased ability to inspire deeply or cough to clear secretions. Ultrasound-guided percutaneous cryoneurolysis involves reversibly ablating peripheral nerve(s) using exceptionally low temperature with a transdermal probe, resulting in a prolonged nerve block with a duration measured in months. We hypothesized that cryoneurolysis would improve analgesia and inspired volume following rib fracture. METHODS: Adults with 1-6 traumatic rib fractures were randomized to either active cryoneurolysis and a sham peripheral nerve block (PNB), or sham cryoneurolysis and active PNB in a participant/observer-masked fashion. The primary endpoint was the maximum inspired volume the day after the procedure as measured with an incentive spirometer. RESULTS: The day after the procedure, the unadjusted median [IQR] maximum inspired volume for participants who received cryoneurolysis (n=11) was 2,250 mL [1,500; 2,500] versus 1,300 mL [750; 2,500] for PNB (n=9, mean difference 496; 95%CI -428 to 1420; t-test P=0.269). When adjusted for covariates (e.g., baseline lung volume), the cryoneurolysis group had an estimated 793 mL greater mean volume than PNB (95%CI 273 to 1,312; ANCOVA P=0.005). Improvement from baseline in maximum inspired volume for cryoneurolysis was 1,000 mL [1,000; 1,375] versus 300 mL [0; 1,000] for PNB (t-test P=0.002). This was equivalent to an improvement over baseline of 100% [90%, 188%] for cryoneurolysis versus 30% [0%, 50%] for PNB (t-test P=0.003). Average daily pain scores were generally lower for the cryoneurolysis group throughout the first month. Total cumulative oxycodone equivalents were 5 mg [0, 13] for cryoneurolysis vs 45 mg [43, 135] for PNB (t-test P=0.013). CONCLUSIONS: Ultrasound-guided percutaneous cryoneurolysis improves maximum inspired lung volume while concurrently decreasing pain and opioid consumption after traumatic rib fracture. These results should be considered preliminary, requiring confirmation with a trial including a larger sample size.