Fibroblast-to-myofibroblast transition (FMT) is cellular transformation process driving the pathogenesis of fibrotic disorders such as pulmonary fibrosis and scleroderma. Despite advances in anti-fibrotic therapies, existing treatments primarily slow disease progression rather than directly targeting extracellular matrix (ECM)-secreting myofibroblasts, and cannot reverse established fibrosis. In this work, a nanoparticle-enabled, anti-fibrotic approach is demonstrated to reverse the FMT in myofibroblasts via targeting cadherin-2 (CDH2), a cell-surface maker of myofibroblasts, using melanin nanoparticles with surface-engineered CDH2 antibody (CDH2-MelNPs). Treatment with CDH2-MelNPs promotes myofibroblast-to-fibroblast transition (MFT, reversed FMT), significantly suppressing ECM deposition, proliferation, migration, and invasive behavior of myofibroblasts, while concurrently mitigating tissue contractility and stiffness, the hallmarks of fibrosis. Mechanistic studies reveal that this nanoparticle-driven MFT is regulated by the inhibition of the Rho signaling, a critical regulator of FMT. Overall, these findings propose an alternative therapeutic avenue to potentially halt or reverse a broad spectrum of fibrotic diseases. The manipulation of fibroblast/myofibroblast phenotype using nanoparticles may also serve as a promising tool in tissue engineering, enabling precise control over tissue remodeling.

1.EMT in cancer metastasis and chemoresistance Cancer metastasis is contingent on the epithelial-mesenchymal transition(EMT)of cancer cells.During EMT,epithelial cancer cells transform into a mesenchymal phenotype,which endows cancer cells with enhanced migratory and invasive abilities,promoting their dissemination throughout the body.EMT also contributes signifi-cantly to chemoresistance,allowing cancer cells to survive and metastasize even after chemotherapy[1].

Type II myosin motors generate cytoskeletal forces that are central to cell division, embryogenesis, muscle contraction, and many other cellular functions. However, at present there is no method that can directly measure the forces generated by myosins in living cells. Here we describe a Förster resonance energy transfer (FRET)-based tension sensor that can measure forces generated by Nonmuscle Myosin IIB (NMIIB) in living cells with piconewton (pN) sensitivity. Fluorescence lifetime imaging microscopy (FLIM)-FRET measurements indicate that the forces generated by NMIIB exhibit significant spatial and temporal heterogeneity, with inferred tensions that vary widely in different regions of the cell. This initial report highlights the potential utility of myosin-based tension sensors in elucidating the roles of cytoskeletal contractility in a wide variety of contexts.

Endothelial-mesenchymal transition (EndoMT) of vascular endothelial cells has recently been considered as a key player in the early progression of a variety of vascular and nonvascular diseases, including atherosclerosis, cancer, and organ fibrosis. However, current strategies attempting to identify pharmacological inhibitors to block the regulatory pathways of EndoMT suffer from poor selectivity, unwanted side effects, and a heterogeneous response from endothelial cells with different origins. Furthermore, EndoMT inhibitors focus on preventing EndoMT, leaving the endothelial cells that have already undergone EndoMT unresolved. Here, we report the design of a simple but powerful nanoparticle system (i.e., N-cadherin targeted melanin nanoparticles) to convert cytokine-activated, mesenchymal-like endothelial cells back to their original endothelial phenotype. We term this process "Reversed EndoMT" (R-EndoMT). R-EndoMT allows the impaired endothelial barriers to recover their quiescence and intactness, with significantly reduced leukocyte and cancer cell adhesion and transmigration, which could potentially stop atheromatous plaque formation and cancer metastasis in the early stages. R-EndoMT is achieved on different endothelial cell types originating from arteries, veins, and capillaries, independent of activating cytokines. We reveal that N-cadherin targeted melanin nanoparticles reverse EndoMT by downregulating an N-cadherin dependent RhoA activation pathway. Overall, this approach offers a different prospect to treat multiple EndoMT-associated diseases by designing nanoparticles to reverse the phenotypical transition of endothelial cells.

The Enhanced Permeability and Retention (EPR) effect, an elevated accumulation of drugs and nanoparticles in tumors versus in normal tissues, is a widely used concept in the field of cancer therapy. It assumes that the vasculature of solid tumors would possess abnormal, leaky endothelial cell barriers, allowing easy access of intravenous-delivered drugs and nanoparticles to tumor regions. However, the EPR effect is not always effective owing to the heterogeneity of tumor endothelium over time, location, and species. Herein, we introduce a unique nanoparticle-based approach, using MUC18-targeted gold nanorods coupled with mild hyperthermia, to specifically enhance tumor endothelial permeability. This improves the efficacy of traditional cancer therapy including photothermal therapy and anticancer drug delivery by increasing the transport of photo-absorbers and drugs across the tumor endothelium. Using single cell imaging tools and classic analytical approaches in molecular biology, we demonstrate that MUC18-targeted gold nanorods and mild hyperthermia enlarge the intercellular gaps of tumor endothelium by inducing circumferential actin remodeling, stress fiber formation, and cell contraction of adjacent endothelial cells. Considering MUC18 is overexpressed on a variety of tumor endothelium and cancer cells, this approach paves a new avenue to improve the efficacy of cancer therapy by actively enhancing the tumor endothelial permeability.

We present spectrally-resolved multi-photon induced fluorescence (TPIF) and second harmonic generation (SHG) imaging of porcine arterial wall tissue sections, both native tissues and damaged tissues subject to a proprietary therapy based on photo-activated crosslinking of collagen and other structural proteins in the arterial wall, a proposed treatment for peripheral artery disease termed non-invasive vascular scaffolding (NVS). We employ a spectrally-resolved multi-photon imaging system, based on a closed loop piezoelectric stage, a transmission grating and an EMCCD. From the spectrally-resolved multiphoton emission, we form ratiometric images of select spectral bands associated primarily with collagen (SHG) and elastin (TPIF). The ratiometric images aid in identifying representative regions for comparing the native and treated tissue sections, as well as reducing sensitivity to variations in intensity caused by scattering or attenuation of the excitation beam. Our aim is to use these ratios as a metric of the tissue structure and composition, indicating the relative contribution of collagen and elastin to the observed nonlinear signals. We note the photochemical modification results in a recovery of SHG intensity similar to the native artery, with the hypothesis that cross-linking of the compressed collagen fibrils in the arterial wall during the light activation step results in the formation of the NVS.

Cytotoxicity of nanoparticles is routinely characterized by biochemical assays such as cell viability and membrane integrity assays. However, these approaches overlook cellular biophysical properties including changes in the actin cytoskeleton, cell stiffness, and cell morphology, particularly when cells are exposed to "non-cytotoxic" doses of nanoparticles. Zeolitic imidazolate framework-8 nanoparticles (ZIF-8 NPs), a member of metal–organic framework family, has received increasing interest in various fields such as environmental and biomedical sciences. ZIF-8 NPs may enter the blood circulation system after unintended oral and inhalational exposure or intended intravenous injection for diagnostic and therapeutic applications, yet the effect of ZIF-8 NPs on vascular endothelial cells is not well understood. Here, the biophysical impact of "non-cytotoxic" dose ZIF-8 NPs on human aortic endothelial cells (HAECs) is investigated. We demonstrate that "non-cytotoxic" doses of ZIF-8 NPs, pre-defined by a series of biochemical assays, can increase the endothelial permeability of HAEC monolayers by causing cell junction disruption and intercellular gap formation, which can be attributed to actin reorganization within adjacent HAECs. Nanomechanical atomic force microscopy and super resolution fluorescence microscopy further confirm that "non-cytotoxic" doses of ZIF-8 NPs change the actin structure and cell morphology of HAECs at the single cell level. Finally, the underlying mechanism of actin reorganization induced by the "non-cytotoxic" dose ZIF-8 NPs is elucidated. Together, this study indicates that the "non-cytotoxic" doses of ZIF-8 NPs, intentionally or unintentionally introduced into blood circulation, may still pose a threat to human health, considering increased endothelial permeability is essential to the progression of a variety of diseases. From a broad view of cytotoxicity evaluation, it is important to consider the biophysical properties of cells, since they can serve as novel and more sensitive markers to assess nanomaterial's cytotoxicity.

The increased use of antimicrobial compounds such as copper into nanoparticles changes how living cells interact with these novel materials. The increased use of antimicrobial nanomaterials combats infectious disease and food spoilage. Fungal infections are particularly difficult to treat because of the few druggable targets, and Saccharomyces cerevisiae provides an insightful model organism to test these new materials. However, because of the novel characteristics of these materials, it is unclear how these materials interact with living cells and if resistance to copper-based nanomaterials could occur. Copper nanoparticles built on carboxymethylcellulose microfibril strands with copper (CMC-Cu) are a promising nanomaterial when imported into yeast cells and induce cell death. The α-arrestins are cargo adaptors that select which molecules are imported into eukaryotic cells. We screened α-arrestins mutants and identified Aly2, Rim8, and Rog3 α-arrestins, which are necessary for the internalization of CMC-Cu nanoparticles. Internal reactive oxygen species in these mutants were lower and corresponded to the increased viability in the presence of CMC-Cu. Using lattice light-sheet microscopy on live cells, we determined that CMC-Cu were imported into yeast within 30 min of exposure. Initially, the cytoplasmic pH decreased but returned to basal level 90 min later. However, there was heterogeneity in response to CMC-Cu exposure, which could be due to the heterogeneity of the particles or differences in the metabolic states within the population. When yeast were exposed to sublethal concentrations of CMC-Cu no resistance occurred. Internalization of CMC-Cu increases the potency of these antimicrobial nanomaterials and is likely key to preventing fungi from evolving resistance.

Cancer stem-like cells (CSCs) play key roles in chemoresistance, tumor metastasis, and clinical relapse. However, current CSC inhibitors lack specificity, efficacy, and applicability to different cancers. Herein, we introduce a nanomaterial-based approach to photothermally induce the differentiation of CSCs, termed "photothermal differentiation", leading to the attenuation of cancer cell stemness, chemoresistance, and metastasis. MoS2 nanosheets and a moderate photothermal treatment were applied to target a CSC surface receptor (i.e., CD44) and modulate its downstream signaling pathway. This treatment forces the more stem-like cancer cells to lose the mesenchymal phenotype and adopt an epithelial, less stem-like state, which shows attenuated self-renewal capacity, more response to anticancer drugs, and less invasiveness. This approach could be applicable to various cancers due to the broad availability of the CD44 biomarker. The concept of using photothermal nanomaterials to regulate specific cellular activities driving the differentiation of CSCs offers a new avenue for treating refractory cancers.

Understanding the specific mechanisms responsible for anabolic and catabolic responses to static or dynamic force are largely poorly understood. Because of this, most research groups studying mechanotransduction due to dynamic forces employ an empirical approach in deciding what frequencies to apply during experiments. While this has been shown to elucidate valuable information regarding how cells respond under controlled provocation, it is often difficult or impossible to determine a true optimal frequency for force application, as many intracellular complexes are involved in receiving, propagating, and responding to a given stimulus. Here we present a novel adaptation of an analytical technique from the fields of civil and mechanical engineering that may open the door to direct measurement of mechanobiological cellular frequencies which could be used to target specific cell signaling pathways leveraging synergy between outside-in and inside-out mechanotransduction approaches. This information could be useful in identifying how specific proteins are involved in the homeostatic balance, or disruption thereof, of cells and tissue, furthering the understanding of the pathogenesis and progression of many diseases across a wide variety of cell types, which may one day lead to the development of novel mechanobiological therapies for clinical use.

Cancer metastasis leads to most deaths in cancer patients, and the epithelial-mesenchymal transition (EMT) is the key mechanism that endows the cancer cells with strong migratory and invasive abilities. Here, we present a nanomaterial-based approach to reverse the EMT in cancer cells by targeting an EMT inducer, CD146, using engineered black phosphorus nanosheets (BPNSs) and a mild photothermal treatment. We demonstrate this approach can convert highly metastatic, mesenchymal-type breast cancer cells to an epithelial phenotype (i.e., reversing EMT), leading to a complete stoppage of cancer cell migration. By using advanced nanomechanical and super-resolution imaging, complemented by immunoblotting, we validate the phenotypic switch in the cancer cells, as evidenced by the altered actin organization and cell morphology, downregulation of mesenchymal protein markers, and upregulation of epithelial protein markers. We also elucidate the molecular mechanism behind the reversal of EMT. Our results reveal that CD146-targeted BPNSs and a mild photothermal treatment synergistically contribute to EMT reversal by downregulating membrane CD146 and perturbing its downstream EMT-related signaling pathways. Considering CD146 overexpression has been confirmed on the surface of a variety of metastatic, mesenchymal-like cancer cells, this approach could be applicable for treating various cancer metastasis via modulating the phenotype switch in cancer cells.

Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3’-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3’-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

Zeolitic imidazolate framework-8 (ZIF-8) nanoparticles have emerged as a promising platform for drug delivery and controlled release. Considering most ZIF-8 nanoparticle drug carriers are designed to be administered intravenously, and thus would directly contact vascular smooth muscle cells (VSMCs) in many circumstances, the potential interactions of ZIF-8 nanoparticles with VSMCs require investigation. Here, the effects of low doses of ZIF-8 nanoparticles on VSMC morphology, actin organization, and contractility are investigated. Two nanoscale imaging tools, atomic force microscopy, and direct stochastic optical reconstruction microscopy, show that even at the concentrations (12.5 and 25 µg/ml) that were deemed “safe” by conventional biochemical cell assays (MTT and LDH assays), ZIF-8 nanoparticles can still cause changes in cell morphology and actin cytoskeleton organization at the cell apical and basal surfaces. These cytoskeletal structural changes impair the contractility function of VSMCs in response to Angiotensin II, a classic vasoconstrictor. Based on intracellular zinc and actin polymerization assays, we conclude that the increased intracellular Zn2+ concentration due to the uptake and dissociation of ZIF-8 nanoparticles could cause the actin cytoskeleton dis-organization, as the elevated Zn2+ directly disrupts the actin assembly process, leading to altered actin organization such as branches and networks. Since the VSMC phenotype change and loss of contractility are fundamental to the development of atherosclerosis and related cardiovascular diseases, it is worth noting that these low doses of ZIF-8 nanoparticles administered intravenously could still be a safety concern in terms of cardiovascular risks. Moving forward, it is imperative to re-consider the “safe” nanoparticle dosages determined by biochemical cell assays alone, and take into account the impact of these nanoparticles on the biophysical characteristics of VSMCs, including changes in the actin cytoskeleton and cell morphology.

Cluster of differentiation 146 (CD146), a cancer cell adhesion molecule, is over-expressed on the surfaces of melanoma, breast, ovarian, and prostate cancer cells, and its high expression indicates the migration tendency of these cancer cells and poor patient prognosis. Here, we hypothesize that targeting the CD146 with low-dose gold nanorods combined with mild hyperthermia can stop the migration of these cancer cells. Two metastatic cancer cells including a melanoma and a breast cancer cell line are selected as the model systems. Cell migration assays show that the migration of both cell lines can be completely stopped by the treatment. Atomic force microscopy and super resolution fluorescence microscopy reveal the alterations of actin cytoskeleton and cell morphology correspond to the inhibited cell migration. Further mechanistic analysis indicates the treatment disrupts the actin cytoskeleton by a synergistic mechanism including depleting membrane CD146 and interfering ezrin-radixin-moesin phosphorylation. As a result, we believe targeting CD146 with low-dose gold nanorods and mild hyperthermia could be a versatile, effective, and safe approach for stopping cancer metastasis. More broadly, the concept of targeting cancer cell surface markers that connect the underlying actin cytoskeleton, offers enormous potential in treating cancer metastasis, which accounts for more than 90% of cancer-associated mortality.

Immunoassays typically must be stored under refrigerated conditions because antibodies, after being immobilized to solid surfaces, tend to lose their recognition capabilities to target antigens under non-refrigerated conditions. This requirement hinders application of immunoassays in resource-limited settings including rural clinics in tropical regions, disaster struck areas, and low-income countries, where refrigeration may not be feasible. In this work, a facile approach based on a reversable zeolitic imidazolate framework-8 (ZIF-8) coating is introduced to stabilize surface-bound antibodies on enzyme-linked immunosorbent assay (ELISA) plates under non-refrigerated conditions. Using a sandwich ELISA for the detection of neutrophil gelatinase-associated lipocalin (NGAL), a urine biomarker for acute kidney injury, as a model system, ZIF-8 is demonstrated to be able to uniformly coat the surface-bound anti-NGAL IgG, and stabilize the dynamic range and detection sensitivity of the assay after storage at an elevated temperature (50 °C) for at least 4 weeks. The stabilization efficacy of the ZIF-8 coating is comparable to the current "gold standard" refrigeration approach, and superior to the commonly used sucrose coating method. This approach will greatly improve the shelf-life and stability of antibody-coated ELISAs and other types of assays which utilize surface-bound antibodies, thus extending biomedical research and medical diagnostics to resource-limited settings.

Transmembrane MUC18 is highly expressed on most metastatic cancers. Herein, we demonstrate that targeting MUC18 with polydopamine nanoparticles (PDA NPs) and a mild photothermal effect can completely cease the migration of melanoma and breast cancer cells without killing the cells. The inhibited cell migration can be attributed to the altered actin cytoskeleton, cell stiffness, and cell morphology, as revealed by nanomechanical and super resolution fluorescence imaging techniques. Further mechanistic studies at the molecular level show that MUC18 targeted PDA NPs and a mild photothermal treatment produce a synergistic effect on the actin cytoskeleton by downregulating the transmembrane MUC18 and interrupting ezrin-radixin-moesin phosphorylation, thereby releasing the actin cytoskeleton from the cell membrane and compromising force transduction through the actin cytoskeleton to the transmembrane MUC18. Overall, the concept of targeting transmembrane metastatic markers and disrupting their downstream effectors (i.e., actin and actin-binding proteins) opens up a new avenue to cancer therapy.

Background:Animal models of muscle injury have primarily relied on methods which do not mimic the chronic scarring that typically occurs adjacent to the myotendinous junction (MTJ). The goal of this study was three-fold: (i) to create a strain-induced in vivo model of rectus femoris MTJ injury in rats; (ii) to document clinical manifestations of injury using longitudinal tracking of individual animals via voluntary and compulsory (treadmill) mobility analyses and (iii) to validate and assess the model for persistent scarring through serial histologic assessment and development of a semi-quantitative grading scheme to characterize injury response over time.Methods:Strain-induced MTJ injury was generated in male Sprague Dawley rats via needle tension directed along the transverse axis between the rectus femoris muscle and distal tendon that attaches to the patella. Animals received mobility assessments (gait analysis using a DigiGait Treadmill System and weight bearing using a Tekscan Rodent Walkway System) at days 0, 1, 3, 6, 13, 20, and 27 of the experimental protocol. Rats were euthanized at 1, 3, 7, 14, and 28 days post-injury (n = 6 rats per time-point) and hindlimbs were processed for histology.Results:Significant changes in locomotor parameters included injured and contralateral limb paw area, max dA/dt (limb deceleration/breaking time), stride time, stance time, force time impulse, and fore/hind symmetry, and injured limb maximum force. The most significant and consistent histologic finding was a pathologic fibrotic adhesive lesion at the muscle and tendon interface along the proximal aspect of the patella just distal to the injury site. This lesion was composed of reactive fibroblasts, disorganized collagen fibers, vascular profiles, and a myxomatous ground substance stroma.Conclusions:This work is the first to characterize the clinical and pathologic development of a chronic model of rectus femoris MTJ injury, which resulted in altered mobility likely caused by a strain-induced fibrotic scar along the anterior patella. Notably, both the functional and pathologic changes recapitulated the course of injury progression similar to what is described in humans. This work provides a unique model to study MTJ injury mechanisms for the identification of enhanced treatment options for patients who suffer from activity-related muscle conditions.

Upon completion, participants will be able to understand the relationship between parental nutrition and the development of central line-associated blood stream infections.

Solar pyrolysis of renewable biomass has huge potential for sustainable production of fuel or chemical feedstock for the activated carbon materials. Here, we report a method for producing high-quality carbon nanofiber (CNF) precursor and subsequently CNFs as a low-cost and eco-friendly energy storage material. Specifically, pine-wood is pyrolyzed with solar energy to generate a phenol-rich bioliquid precursor that is found to be a strong candidate for synthesizing binder-free flexible electrode materials via electrospinning. The CNFs prepared with 30% solar-driven bioliquids and 70% polyacrylonitrile have a high specific surface area and rich microstructure, which are the key to its electrochemical performance in terms of specific capacitance (349 F g(-1) at the current density of 0.5 A g(-1)) with notable rate performance, reversibility, and cycling stability in 6 M KOH aqueous electrolyte. Thus, solar bioliquids are feasible CNF precursors, and such derived CNFs have potential to be applied in energy storage devices.