Publications

SARS-CoV-2 can cause neurological issues, including cognitive dysfunction in COVID-19 survivors. Endothelial dysfunction, a key mechanism in COVID-19, is also a risk factor for vascular dementia (VaD). Reduced nitric oxide (NO) bioavailability is a pathogenic factor of endothelial dysfunction and platelet aggregation in COVID-19 patients, and endothelial NO synthase (eNOS) levels decline with advancing age, a risk factor for both COVID-19 morbidity and VaD. SARS-CoV-2 also induces cellular senescence and senescence-associated secretory phenotype (SASP). We hypothesized that eNOS deficiency would worsen neuroinflammation, senescence, blood-brain barrier (BBB) permeability, and hypercoagulability in eNOS-deficient mice. Six-month-old eNOS^+/- (pre-cognitively impaired experimental VaD) and wild-type (WT) male mice were infected with mouse-adapted (MA10) SARS-CoV-2. Mice were evaluated for weight loss, viral load, and markers of inflammation and senescence 3 days post-infection. eNOS^+/- mice showed more weight loss (~15%) compared to WT mice (~5%) and increased inflammatory markers (Ccl2, Cxcl9, Cxcl10, IL-1β, and IL-6) and senescence markers (p53 and p21). They also exhibited higher microglial activation (Iba1) and increased plasma coagulation and BBB permeability, despite comparable lung viral loads and absence of virus in the brain. This is the first experimental evidence demonstrating that eNOS deficiency exacerbates SARS-CoV-2-induced morbidity, neuroinflammation, and brain senescence, linking eNOS to COVID-19-related neuropathology.

Objective: Tamoxifen is the most used agent to treat estrogen receptor-positive (ER+) breast cancer (BC). While it decreases the risk of cancer recurrence by 50%, many patients develop resistance to this treatment, culminating in highly aggressive disease. Tamoxifen resistance comes from the repression of ER transcriptional activity that switches the cancer cells to proliferation via nonhormonal signaling pathways. Here, we evaluate a potential strategy to overcome tamoxifen resistance by focused ultrasound (FUS), a noninvasive approach for the mechanical excitation of cancer cells.

Methods: Resistant and nonresistant ER+ BC cells and xenografts from patients with ER+ BC were treated with tamoxifen, FUS or their combination. The apoptosis, proliferation rate, gene expression and activity of estrogen receptor, and morphological changes were measured in treated cells and tissues.

Results: FUS caused the mechanical disruption of tamoxifen-resistant BC cells that in turn led to the upregulation of ERα-encoding gene expression and long-term re-sensitization of the cells to tamoxifen. Patient-derived xenografts treated with Tamoxifen and FUS demonstrated a significant reduction in tumor viability and proliferation and a strong structural damage to tumor cells and extracellular matrix.

Conclusion: FUS can improve ER+ BC treatment by re-sensitizing the cancer cells to tamoxifen.

Most coagulation tests are photo-optical turbidimetric assays that require the removal of cellular components from whole blood for optical clearing. If the resulting blood plasma samples are hemolyzed, they may become unsuitable for turbidimetric analysis. To resolve this issue, whole-blood analogs to plasma turbidimetric assays need to be developed. Using samples collected from non-smokers (normal group), smokers (thrombotic group), and hemophilia A (bleeding group) patients, we demonstrate that the reaction time assessed from whole blood viscosity data of the drop-of-blood acoustic tweezing spectroscopy (ATS) technique strongly correlates (R_p ≥ 0.95) with PT/aPTT values obtained from plasma turbidimetric data. Linear correlation (R_p ≥ 0.88) was also obtained between the viscous and elastic outputs of the ATS technique and the fibrinogen concentration. The integration of ATS data enabled the assessment of the functional level of fibrin cross-linkers such as factor XIII. Overall, ATS allows comprehensive sample-sparing analysis of whole blood coagulation for reliable and safe diagnosis of bleeding/thrombosis risks.

Background: Atherosclerosis is a common co-morbidity of type 2 diabetes mellitus. Monocyte recruitment by an activated endothelium and the pro-inflammatory activity of the resulting macrophages are critical components of atherosclerosis. Exosomal transfer of microRNAs has emerged as a paracrine signaling mechanism regulating atherosclerotic plaque development. MicroRNAs-221 and -222 (miR-221/222) are elevated in vascular smooth muscle cells (VSMCs) of diabetic patients. We hypothesized that the transfer of miR-221/222 via VSMC-derived exosomes from diabetic sources (DVEs) promotes increased vascular inflammation and atherosclerotic plaque development.

Methods: Exosomes were obtained from VSMCs, following exposure to non-targeting or miR-221/-222 siRNA (-KD), isolated from diabetic (DVEs) and non-diabetic (NVEs) sources and their miR-221/-222 content was measured using droplet digital PCR (ddPCR). Expression of adhesion molecules and the adhesion of monocytes was measured following exposure to DVEs and NVEs. Macrophage phenotype following exposure to DVEs was determined by measuring mRNA markers and secreted cytokines. Age-matched apolipoprotein-E-deficient mice null (ApoE^-/-) mice were maintained on Western diet for 6 weeks and received injections of saline, NVEs, NVE-KDs, DVEs or DVE-KDs every other day. Atherosclerotic plaque formation was measured using Oil Red Oil staining.

Results: Exposure of human umbilical vein and coronary artery endothelial cells to DVEs, but not NVEs, NVE-KDs, or DVE-KDs promoted increased intercellular adhesion molecule-1 expression and monocyte adhesion. DVEs but not NVEs, NVE-KDs, or DVE-KDs also promoted pro-inflammatory polarization of human monocytes in a miR-221/222 dependent manner. Finally, intravenous administration of DVEs, but not NVEs, resulted in a significant increase in atherosclerotic plaque development.

Conclusion: These data identify a novel paracrine signaling pathway that promotes the cardiovascular complications of diabetes mellitus.

Objective: Intravenous microbubble oscillation in the presence of ultrasound has the potential to yield a wide range of therapeutic benefits. However, the likelihood of vessel damage caused by mechanical effects has not been quantified as a function of the numerous important parameters in therapeutic ultrasound procedures. In this study, we examined the effects of microbubbles injected into the vasculature of the earthworm. It was found that the elastic properties of earthworm blood vessels are similar to those of arteries in older humans, and that earthworms are well suited to the large number of experiments necessary to investigate safety of procedures involving microbubble oscillation in sonicated vessels.

Methods: Microbubbles were infused into earthworm vessels, and the rupture time during sonication was recorded as a function of ultrasound frequency, pulse repetition frequency and acoustic pressure.

Discussion: A modified mechanical index (MMI) was defined that successfully captured the trends in rupture probability and rupture time for the different parameter values, creating a database of vessel rupture thresholds. In the absence of bubbles, the product of MMI squared and rupture time was approximately constant, indicating a possible radiation-force effect.

Conclusion: The MMI was an effective correlating parameter in the presence of bubbles, though the mathematical dependence is not yet apparent. The results of the study are expected to be valuable in designing more refined studies in vertebrate models, as well as informing computational models.

Knowledge of rheological properties, such as viscosity and elasticity, is necessary for efficient material processing and transportation as well as biological analysis. Existing rheometers operate with large sample volume and induce sample contact with container or device walls, which are inadequate for rheological analysis of sensitive fluids limited in availability. In this work, we introduce acoustic tweezing spectroscopy (ATS), a novel noncontact rheological technique that operates with a single 4–6 μl drop of fluid sample. In ATS, a sample drop is acoustically levitated and then exposed to a modulated acoustic signal to induce its forced oscillation. The time-dependent sample viscosity and elasticity are measured from the resulting drop response. The ATS measurements of polymeric solutions (dextran, xanthan gum, gelatin) agree well with previously reported data. The ATS predicts that the shear viscosity of blood plasma increases from 1.5 cP at 1.5 min of coagulation onset to 3.35 cP at 9 min, while its shear elastic modulus grows from a negligible value to 10.7 Pa between 3.5 min and 6.5 min. Coagulation increases whole blood viscosity from 5.4 cP to 20.7 cP and elasticity from 0.1 Pa to 19.2 Pa at 15 min. In summary, ATS provides the opportunity for sensitive small-volume rheological analysis in biomedical research and medical, pharmaceutical, and chemical industries.

Despite the initial success in treatment of localized prostate cancer (PCa) using surgery, radiation or hormonal therapy, recurrence of aggressive tumors dictates morbidity and mortality. Focused ultrasound (FUS) is being tested as a targeted, noninvasive approach to eliminate the localized PCa foci, and strategies to enhance the anticancer potential of FUS have a high translational value. Since aggressive cancer cells utilize oxidative stress (Ox-stress) and endoplasmic reticulum stress (ER-stress) pathways for their survival and recurrence, we hypothesized that pre-treatment with drugs that disrupt stress-signaling pathways in tumor cells may increase FUS efficacy. Using four different PCa cell lines, i.e., LNCaP, C4-2B, 22Rv1 and DU145, we tested the in vitro effects of FUS, alone and in combination with two clinically tested drugs that increase Ox-stress (i.e., CDDO-me) or ER-stress (i.e., nelfinavir). As compared to standalone FUS, significant (p < 0.05) suppressions in both survival and recurrence of PCa cells were observed following pre-sensitization with low-dose CDDO-me (100 nM) and/or nelfinavir (2 µM). In drug pre-sensitized cells, significant anticancer effects were evident at a FUS intensity of as low as 0.7 kW/cm². This combined mechanochemical disruption (MCD) approach decreased cell proliferation, migration and clonogenic ability and increased apoptosis/necrosis and reactive oxygen species (ROS) production. Furthermore, although activated in cells that survived standalone FUS, pre-sensitization with CDDO-me and/or nelfinavir suppressed both total and activated (phosphorylated) NF-κB and Akt protein levels. Thus, a combined MCD therapy may be a safe and effective approach towards the targeted elimination of aggressive PCa cells.

Embedded pillar microstructures are an efficient approach for controlling and sculpting shear flow in a microchannel but have not yet demonstrated to be effective for deformability-based cell separation and sorting. Although simple pillar configurations (lattice, line sequence) work well for size-based separation of rigid particles, these have a low separation efficiency for circulating cells. The objective of this study is to optimize sequenced microstructures for separation of deformable cells. This is achieved by numerical analysis of pairwise cell migration in a microchannel with multiple pillars, where size, longitudinal spacing, and lateral location as well as the cell elasticity and size vary. This study reveals two basic pillar configurations optimized for deformability-based separation: “duplet” that consists of two closely spaced pillars positioned far below the centerline and above the centerline halfway to the wall; and “triplet” composed of three widely spaced pillars located below, above and at the centerline, respectively. The duplet configuration is well suited for deformable cell separation in short channels, whereas the triplet or a combination of duplets and triplets provides even better separation in long channels. These optimized pillar microstructures can dramatically improve microfluidic methods for sorting and isolation of blood and rare circulating tumor cells.

Many patients develop coagulation abnormalities due to chronic and hereditary disorders, infectious disease, blood loss, extracorporeal circulation, and oral anticoagulant misuse. These abnormalities lead to bleeding or thrombotic complications, the risk of which is assessed by coagulation analysis. Current coagulation tests pose safety concerns for neonates and small children due to large sample volume requirement and may be unreliable for patients with coagulopathy. This study introduces a containerless drop-of-blood method for coagulation analysis, termed "integrated quasi-static acoustic tweezing thromboelastometry" (i-QATT™), that addresses these needs. In i-QATT™, a single drop of blood is forced to levitate and deform by the acoustic radiation force. Coagulation-induced changes in drop turbidity and firmness are measured simultaneously at different instants. The parameters describing early, intermediate, and late stages of the coagulation process are evaluated from the resulting graphical outputs. i-QATT™ rapidly (<10 min) detected hyper- and hypo-coagulable states and identified single deficiency in coagulation factors VII, VIII, IX, X, and XIII. The linear relationship (r² > 0.9) was established between fibrinogen concentration and two i-QATT™ parameters: maximum clot firmness and maximum fibrin level. Factor XIII activity was uniquely measured by the fibrin network formation time (r² = 0.9). Reaction time, fibrin formation rate, and time to firm clot formation were linearly correlated with heparin concentration (r² > 0.7). tPA-induced hyperfibrinolysis was detected in the clot firmness output at 10 min. i-QATT™ provides comprehensive coagulation analysis in point-of-care or laboratory settings, well suited to the needs of neonatal and pediatric patients and adult patients with anemia or blood collection issues.

Although vertebrates are indispensable to biomedical research, studies are often limited by factors such as cost, lengthy internal review, and ethical considerations. We present the earthworm as an alternative, low-cost, invertebrate applicable to certain preliminary vasculature studies. Due to the surgical availability of the earthworm's dorsal vessels, ventral vessels, and five pairs of pseudo hearts, earthworms are readily accessible, offer low-cost maintenance, and require administration of only small doses of a given compound. The earthworm model provides a simple closed vascular circulatory system with a hemoglobin structure similar to human blood. A protocol is provided for anaesthetizing the earthworms and performing surgical incisions to expose relevant blood vessels. Micropipettes for compound administration are formed by heating and pulling glass with a pipette puller and using a beveling system to create a micron-scale fine needle tip. The tips are then used with a micropositioner and microinjector to inject arbitrary compounds into the vascular system of an earthworm, repeatably, with the availability of large sample sizes and small compound volumes. Details on the intricacies of injection procedure are provided. The small vessel size of the earthworm is challenging, particularly in the case of the ventral vessel; however, mastery of the techniques presented offers high repeatability as a low-cost solution, making studies of very large sample size practical.