Mechanical property estimation of sarcoma-relevant extracellular vesicles using transmission electron microscopy.

Analysis of single extracellular vesicles (EVs) has the potential to yield valuable label-free information on their morphological structure, biomarkers and therapeutic targets, though such analysis is hindered by the lack of reliable and quantitative measurements of the mechanical properties of these compliant nanoscale particles. The technical challenge in mechanical property measurements arises from the existing tools and methods that offer limited throughput, and the reported elastic moduli range over several orders of magnitude. Here, we report on a flow-based method complemented by transmission electron microscopy (TEM) imaging to provide a high throughput, whole EV deformation analysis for estimating the mechanical properties of liposarcoma-derived EVs as a function of their size. Our study includes extracting morphological data of EVs from a large dataset of 432 TEM images, with images containing single to multiple EVs, and implementing the thin-shell deformation theory. We estimated the elastic modulus, E = 0.16 ± 0.02 MPa (mean±SE) for small EVs (sEVs; 30-150 nm) and E = 0.17 ± 0.03 MPa (mean±SE) for large EVs (lEVs; >150 nm). To our knowledge, this is the first report on the mechanical property estimation of LPS-derived EVs and has the potential to establish a relationship between EV size and EV mechanical properties.

Continuous flow microfluidics for colloidal particle assembly on porous substrates.

Self-assembly of colloidal particles for 'bottom-up' fabrication of various patterns and structures is critical for a range of applications including, but not limited to, energy generation and storage, nanomaterial structures, biomimetics, and biosensing. Multiple self-assembly techniques, such as substrate templating-via topological or chemical patterning-and solvent evaporation were discussed in our previous papers and have been developed for the deposition of patterned self-assembled structures, such as bands of colloidal particles, on various substrates. While the templating techniques are limited in applications due to the requirements for pattern-specific prior substrate engineering to fabricate the desired structure, solvent evaporation requires longer assembly times and precise control over environmental conditions. In this paper, a template-free, continuous flow process, which is facilitated by continuous solvent drainage through porous substrates, is demonstrated for the self-assembly of colloidal particles into high-aspect ratio (>103, length to width) structures, such as linear arrays or grid structures. Colloidal particles were assembled both on polymeric and metallic porous membranes, with rapid assembly times.

Mucormycosis threats: A systemic review.

During the catastrophic wave of Coronavirus disease 2019, health agencies started to report an infrequent but lethal mucormycosis or black fungal infection. Primarily, it causes sinusitis by affecting nasal, oral, lung, brain, ocular, and other body tissues. It becomes more fatal, especially in diabetic, cancer, and immune-compromised patients. Before 2020, the prevalence of mucormycosis was very rare but it has rapidly emerged globally from late 2020 to mid-2021. Recently, the mucormycosis got worse and epidemic with more than 30,000 cases reported across India. The etiology of infection can be diagnosed by molecular, serological, microscopic, and clinical methods. However, early diagnosis of this ailment is still a challenging task due to no standalone diagnostic tool available along with clinical manifestations of the ailment resembling other fungal diseases. The treatment of mucormycosis is also challenging and frequently requires long-term treatment. Amphotericin B was found to be an effective antifungal for preventing mucormycosis but it failed if infection disseminated to necrotizing tissues or adjacent organs. Removal of infected tissue/organ by surgery is an alternative treatment to control mucormycosis. In addition, reversal of underlying predisposing conditions based on therapy is also in practice for its prevention. This review highlights different aspects of mucormycosis such as pathogenesis, diagnosis, treatment, and their challenges and so on. We also emphasized the epidemiological shift during the recent outbreak and its influence on the different regions of India.

Application of Microfluidics for Bacterial Identification.

Bacterial infections continue to pose serious public health challenges. Though anti-bacterial therapeutics are effective remedies for treating these infections, the emergence of antibiotic resistance has imposed new challenges to treatment. Often, there is a delay in prescribing antibiotics at initial symptom presentation as it can be challenging to clinically differentiate bacterial infections from other organisms (e.g., viruses) causing infection. Moreover, bacterial infections can arise from food, water, or other sources. These challenges have demonstrated the need for rapid identification of bacteria in liquids, food, clinical spaces, and other environments. Conventional methods of bacterial identification rely on culture-based approaches which require long processing times and higher pathogen concentration thresholds. In the past few years, microfluidic devices paired with various bacterial identification methods have garnered attention for addressing the limitations of conventional methods and demonstrating feasibility for rapid bacterial identification with lower biomass thresholds. However, such culture-free methods often require integration of multiple steps from sample preparation to measurement. Research interest in using microfluidic methods for bacterial identification is growing; therefore, this review article is a summary of current advancements in this field with a focus on comparing the efficacy of polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), and emerging spectroscopic methods.

Voltage-gated nanofluidic devices for protein capture, concentration, and release.

A nanofluidic device with spatially, non-uniformly distributed gate electrodes is reported. In this nanofluidic architecture, multiple nanochannels connect microfluidic reservoirs for the formation of a planar, hybrid microfluidic-nanofluidic device. The gate electrodes are individually addressable, fluidically isolated, and enable a non-uniform electric field distribution within the nanochannels permitting the capture of proteins and a local increase in their concentration. The removal of the gate potential allows the model protein, bovine serum albumin, to move away from the electrodes after concentration at the electrodes for the release of the captured protein. A maximum increase in the protein concentration of nearly an order of magnitude was observed as evaluated by fluorescence intensity.

Microfluidic Approaches and Methods Enabling Extracellular Vesicle Isolation for Cancer Diagnostics.

Advances in cancer research over the past half-century have clearly determined the molecular origins of the disease. Central to the use of molecular signatures for continued progress, including rapid, reliable, and early diagnosis is the use of biomarkers. Specifically, extracellular vesicles as biomarker cargo holders have generated significant interest. However, the isolation, purification, and subsequent analysis of these extracellular vesicles remain a challenge. Technological advances driven by microfluidics-enabled devices have made the challenges for isolation of extracellular vesicles an emerging area of research with significant possibilities for use in clinical settings enabling point-of-care diagnostics for cancer. In this article, we present a tutorial review of the existing microfluidic technologies for cancer diagnostics with a focus on extracellular vesicle isolation methods.

Electroceutical treatment of infected chronic wounds in a dog and a cat.

OBJECTIVES: To describe the use of an innovative printed electroceutical dressing (PED) to treat non-healing, infected chronic wounds in one dog and one cat and report outcomes. ANIMALS: A 4-year-old female spayed Mastiff and a 1-year-old female spayed domestic shorthair cat. STUDY DESIGN: Short case series. METHODS: Both cases had chronic wounds (duration: approximately 1 year for the dog and 6 3/4 months for the cat) that remained open and infected despite various wound management strategies. Both animals were treated with the PED. Observations from the records regarding wound size, antimicrobial susceptibility, and the time to healing were recorded. RESULTS: After 10 days of PED treatment in the dog and 17 days of PED treatment in the cat, the wounds had decreased in size by approximately 4.2 times in the dog and 2.5 times in the cat. Culture of punch biopsies yielded negative results. Wounds were clinically healed at 67 days in the dog and 47 days in the cat. No further treatment of the wounds was required beyond that point. CONCLUSION: Application of a PED led to closure of two chronic wounds and resolution of their persistent infection. CLINICAL SIGNIFICANCE: PEDs may provide a new treatment modality to mitigate infection and promote healing of chronic wounds.

Endoscopic ultrasound-directed transgastric ERCP (EDGE): a systematic review describing the outcomes, adverse events, and knowledge gaps.

BACKGROUND : Endoscopic ultrasound-directed transgastric endoscopic retrograde cholangiopancreatography (EDGE) has emerged as a viable completely endoscopic method for performing pancreaticobiliary interventions in patients with Roux-en-Y gastric bypass anatomy. The aims of this systematic review were: (1) to describe the indications, outcomes, and complications of EDGE; and (2) to identify deficiencies in our knowledge of important technical approaches and clinical outcomes. METHODS : A systematic review was conducted via comprehensive searches of Medline, Scopus, CINAHL, and Cochrane to identify studies focusing on EDGE outcomes. Simple descriptive statistics were derived from case series only. Case reports were only included to qualitatively describe additional indications, techniques, and adverse events. RESULTS : The initial search identified 2143 abstracts. Nine case series and eight case reports were included. In the nine case series, 169 patients underwent EDGE. The technical success rate was 99 % (168 /169) for gastrogastrostomy/jejunogastrostomy creation and 98 % (166 /169) for subsequent ERCP. Minor adverse events specifically related to EDGE occurred in 18 % (31/169) and included intraprocedural stent migration/malposition (n = 27) and abdominal pain (n = 4). Moderate adverse events specific to EDGE occurred in 5 % (9/169): including bleeding (2 %), persistent fistula (1 %), and perforation (1 %). Severe adverse events occurred in one patient with a perforation requiring surgery. Deficiency in reporting on the clinical significance of adverse events was identified. CONCLUSION : Based on limited observational data, in expert hands, EDGE has a high rate of technical success and an acceptable rate of adverse events. As a novel procedure, many knowledge gaps need to be addressed to inform the design of meaningful comparative studies and guide informed consent.

Exploration of the Pharmacodynamics for Pseudomonas aeruginosa Biofilm Eradication by Tobramycin.

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen which is involved in numerous infections. It is of growing concern within the field of antibiotic resistance and tolerance and often exhibits multidrug resistance. Previous studies have shown the emergence of antibiotic-resistant and -tolerant variants within the zone of clearance of a biofilm lawn after exposure to aminoglycosides. As concerning as the tolerant variant emergence is, there was also a zone of killing (ZOK) immediately surrounding the antibiotic source from which no detectable bacteria emerged or were cultured. In this study, the ZOK was analyzed using both in vitro and in silico methods to determine if there was a consistent antibiotic concentration versus time constraint (area under the curve [AUC]) which is able to completely kill all bacteria in the lawn biofilms in our in vitro model. Our studies revealed that by achieving an average AUC of 4,372.5 µg·h/mL, complete eradication of biofilms grown on both agar and hydroxyapatite was possible. These findings show that appropriate antibiotic concentrations and treatment duration may be able to treat antibiotic-resistant and -tolerant biofilm infections.

Cross-flow microfiltration for isolation, selective capture and release of liposarcoma extracellular vesicles.

We present a resource-efficient approach to fabricate and operate a micro-nanofluidic device that uses cross-flow filtration to isolate and capture liposarcoma derived extracellular vesicles (EVs). The isolated extracellular vesicles were captured using EV-specific protein markers to obtain vesicle enriched media, which was then eluted for further analysis. Therefore, the micro-nanofluidic device integrates the unit operations of size-based separation with CD63 antibody immunoaffinity-based capture of extracellular vesicles in the same device to evaluate EV-cargo content for liposarcoma. The eluted media collected showed ∼76% extracellular vesicle recovery from the liposarcoma cell conditioned media and ∼32% extracellular vesicle recovery from dedifferentiated liposarcoma patient serum when compared against state-of-art extracellular vesicle isolation and subsequent quantification by ultracentrifugation. The results reported here also show a five-fold increase in amount of critical liposarcoma-relevant extracellular vesicle cargo obtained in 30 min presenting a significant advance over existing state-of-art.

Direct current electric field regulates endothelial permeability under physiologically relevant fluid forces in a microfluidic vessel bifurcation model.

Previous in vitro studies have reported on the use of direct current electric fields (DC-EFs) to regulate vascular endothelial permeability, which is important for tissue regeneration and wound healing. However, these studies have primarily used static 2D culture models that lack the fluid mechanical forces associated with blood flow experienced by endothelial cells (ECs) in vivo. Hence, the effect of DC-EF on ECs under physiologically relevant fluid forces is yet to be systematically evaluated. Using a 3D microfluidic model of a bifurcating vessel, we report the role of DC-EF on regulating endothelial permeability when co-applied with physiologically relevant fluid forces that arise at the vessel bifurcation. The application of a 70 V m-1 DC-EF simultaneously with 1 µL min-1 low perfusion rate (generating 3.8 dyn cm-2 stagnation pressure at the bifurcation point and 0.3 dyn cm-2 laminar shear stress in the branched vessel) increased the endothelial permeability 7-fold compared to the static control condition (i.e., without flow and DC-EF). When the perfusion rate was increased to 10 µL min-1 (generating 38 dyn cm-2 stagnation pressure at the bifurcation point and 3 dyn cm-2 laminar shear stress in the branched vessel) while maintaining the same electrical stimulation, a 4-fold increase in endothelial permeability compared to the static control was observed. The lower increase in endothelial permeability for the higher fluid forces but the same DC-EF suggests a competing role between fluid forces and the applied DC-EF. Moreover, the observed increase in endothelial permeability due to combined DC-EF and flow was transient and dependent on the Akt signalling pathway. Collectively, these findings provide significant new insights into how the endothelium serves as an electro-mechanical interface for regulating vessel permeability.

Endothelial barrier function is co-regulated at vessel bifurcations by fluid forces and sphingosine-1-phosphate.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid mediator of endothelial barrier function. Prior studies have implicated mechanical stimulation due to intravascular laminar shear stress in co-regulating S1P signaling in endothelial cells (ECs). Yet, vascular networks in vivo consist of vessel bifurcations, and this geometry generates hemodynamic forces at the bifurcation point distinct from laminar shear stress. However, the role of these forces at vessel bifurcations in regulating S1P-dependent endothelial barrier function is not known. In this study, we implemented a microfluidic platform that recapitulates the flow dynamics of vessel bifurcations with in situ quantification of the permeability of microvessel analogues. Co-application of S1P with impinging bifurcated fluid flow, which is characterized by approximately zero shear stress and 38 dyn•cm-2 stagnation pressure at the vessel bifurcation point, promotes vessel stabilization. Similarly, co-treatment of S1P with 3 dyn•cm-2 laminar shear stress is also protective of endothelial barrier function. Moreover, it is shown that vessel stabilization due to bifurcated fluid flow and laminar shear stress is dependent on S1P receptor 1 or 2 signaling. Collectively, these findings demonstrate the endothelium-protective function of fluid forces at vessel bifurcations and their involvement in coordinating S1P-dependent regulation of vessel permeability.

Combined electrokinetic and shear flows control colloidal particle distribution across microchannel cross-sections.

Recent experimental observations on combined electrokinetic and shear flows of colloidal suspensions in rectangular cross-section microfluidic channels have shown unusual cross-stream colloidal particle migration and dynamic assembly. Although a new electrophoresis-induced lift force has been postulated to cause the lateral migration of colloidal particles, little is known about how fluid properties and flow conditions impact this force and therefore subsequent colloidal particle migration. Furthermore, no experimental quantification of this electrophoresis-induced lift force is available. We report several key advances by demonstrating that the kinematic viscosity of the fluid can be used to modulate the spatial distribution of particles over the entire microchannel cross-section, with suppression of the colloidal particle migration observed with increase in fluid kinematic viscosity. Colloidal particle migration of ∼10 µm from not only the top and bottom microchannel walls but also from the side walls is shown with the corresponding electrophoresis-induced lift force of up to ∼30 fN. The breadth of flow conditions tested capture the channel Reynolds number in the 0.1-1.1 range, with inertial migration of colloidal particles shown in flow regimes where the migration was previously thought to be ineffective, if not for the electrophoresis-induced lift force. The ability of the electrophoresis-induced lift force to migrate colloidal particles across the entire microchannel cross-section establishes a new paradigm for three-dimensional control of colloidal particles within confined microchannels.

Evaluation of electrical properties of ex vivo human hepatic tissue with metastatic colorectal cancer.

OBJECTIVE: To probe the distribution of electrical properties in tumor-bearing human hepatic tissues with metastatic colorectal cancer. APPROACH: Electrochemical impedance spectroscopy (EIS) and a non-contact electromagnetic probe were used for distinguishing spatial heterogeneities in fresh, unfixed human hepatic tissues ex vivo from patients with metastatic colorectal cancer (CRC). MAIN RESULTS: Point-wise EIS measurements reported over a frequency range of 100 Hz-1 MHz showed that the interface tissue between visible tumor and normal tissue exhibits an electrically different domain (p < 0.05) from both normal tissue (over 100 Hz-100 kHz) and tumor tissue (over 100 Hz-1 MHz). Observations of the microstructure on tumor-bearing hepatic tissue from hematoxylin and eosin stained images and the equivalent circuit modelling were used to validate the impedance measurements and characterize previously unidentified interfacial domain between normal and tumor tissue. Lastly, in a proof of concept study, a new in-house designed non-contact electromagnetic probe, as opposed to the invasive EIS measurements, was demonstrated for distinguishing tumor tissue from the normal tissue in a hepatic tissue specimen from a patient with metastatic CRC. SIGNIFICANCE: EIS measurements, correlated with histological observations, show potential for mapping electrical properties in tumor-bearing human hepatic tissue.

Ultrastructure imaging of Pseudomonas aeruginosa lawn biofilms and eradication of the tobramycin-resistant variants under in vitro electroceutical treatment.

Electrochemically generated bactericidal compounds have been shown to eradicate bacterial lawn biofilms through electroceutical treatment. However, the ultrastructure of biofilms exposed to these species has not been studied. Moreover, it is unknown if the efficacy of electroceutical treatment extends to antibiotic-resistant variants that emerge in lawn biofilms after antibiotic treatment. In this report, the efficacy of the in vitro electroceutical treatment of Pseudomonas aeruginosa biofilms is demonstrated both at room temperature and in an incubator, with a ~4 log decrease (p < 0.01) in the biofilm viability observed over the anode at both conditions. The ultrastructure changes in the lawn biofilms imaged using transmission electron microscopy demonstrate significant bacterial cell damage over the anode after 24 h of electroceutical treatment. A mix of both damaged and undamaged cells was observed over the cathode. Finally, both eradication and prevention of the emergence of tobramycin-resistant variants were demonstrated by combining antibiotic treatment with electroceutical treatment on the lawn biofilms.

Multi-Layer Micro-Nanofluidic Device for Isolation and Capture of Extracellular Vesicles Derived from Liposarcoma Cell Conditioned Media.

We report on isolation, capture, and subsequent elution for analysis of extracellular vesicles derived from human liposarcoma cell conditioned media, using a multi-layer micro-nanofluidic device operated with tangential flow separation. Our device integrates size-based separation followed by immunoaffinity-based capture of extracellular vesicles in the same device. For liposarcomas, this is the first report on isolating, capturing, and then eluting the extracellular vesicles using a micro-nanofluidic device. The results show a significantly higher yield of the eluted extracellular vesicles (~84%) compared to the current methods of ultracentrifugation (~6%) and ExoQuick-based separations (~16%).

Printed Electroceutical Dressings for the Inhibition of Biofilms and Treatment of Chronic Wounds.

We report on an innovative, fabric-based conformable, and easily fabricated electroceutical wound dressing that inhibits bacterial biofilm infections and shows significant promise for healing chronic wounds. Cyclic voltammetry demonstrates the ability of the electroceutical to produce reactive oxygen species, primarily HOCl that is responsible for bacterial inhibition. In vitro investigation with the lawn biofilm grown on a soft tissue mimic assay shows the efficacy of the dressing against both gram-positive and gram-negative bacteria in the biofilm form. In vivo, the printed electroceutical dressing was utilized as an intervention treatment for a canine subject with a non-healing wound due to a year-long persistent polymicrobial infection. The clinical case study with the canine subject exhibited the applicability in a clinical setting with the results showing infection inhibition within 11 days of initial treatment. This printed electroceutical dressing was integrated with a Bluetooth® enabled circuit allowing remote monitoring of the current flow within the wound bed. The potential to monitor wounds remotely in real-time with a Bluetooth® enabled circuit proposes a new physical biomarker for management of infected, chronic wounds.

Competing Fluid Forces Control Endothelial Sprouting in a 3-D Microfluidic Vessel Bifurcation Model.

Sprouting angiogenesis-the infiltration and extension of endothelial cells from pre-existing blood vessels-helps orchestrate vascular growth and remodeling. It is now agreed that fluid forces, such as laminar shear stress due to unidirectional flow in straight vessel segments, are important regulators of angiogenesis. However, regulation of angiogenesis by the different flow dynamics that arise due to vessel branching, such as impinging flow stagnation at the base of a bifurcating vessel, are not well understood. Here we used a recently developed 3-D microfluidic model to investigate the role of the flow conditions that occur due to vessel bifurcations on endothelial sprouting. We observed that bifurcating fluid flow located at the vessel bifurcation point suppresses the formation of angiogenic sprouts. Similarly, laminar shear stress at a magnitude of ~3 dyn/cm2 applied in the branched vessels downstream of the bifurcation point, inhibited the formation of angiogenic sprouts. In contrast, co-application of ~1 µm/s average transvascular flow across the endothelial monolayer with laminar shear stress induced the formation of angiogenic sprouts. These results suggest that transvascular flow imparts a competing effect against bifurcating fluid flow and laminar shear stress in regulating endothelial sprouting. To our knowledge, these findings are the first report on the stabilizing role of bifurcating fluid flow on endothelial sprouting. These results also demonstrate the importance of local flow dynamics due to branched vessel geometry in determining the location of sprouting angiogenesis.

Disposable Patterned Electroceutical Dressing (PED-10) Is Safe for Treatment of Open Clinical Chronic Wounds.

Objective: To evaluate if patterned electroceutical dressing (PED) is safe for human chronic wounds treatment as reported by wound care providers. Approach: This work reports a pilot feasibility study with the primary objective to determine physically observable effects of PED application on host tissue response from a safety evaluation point of view. For this pilot study, patients receiving a lower extremity amputation with at least one open wound on the part to be amputated were enrolled. Patients were identified through the Ohio State University Wexner Medical Center (OSUWMC) based on inclusion and exclusion criteria through prescreening through the Comprehensive Wound Center's (CWC) Limb Preservation Program and wound physicians and/or providers at OSUWMC. Wounds were treated with the PED before amputation surgery. Results: The intent of the study was to identify if PED was safe for clinical application based on visual observations of adverse or lack of adverse events on skin and wound tissue. The pilot testing performed on a small cohort (N = 8) of patients showed that with engineered voltage regulation of current flow to the open wound, the PED can be used with little to no visually observable adverse effects on chronic human skin wounds. Innovation: The PED was developed as a second-generation tunable electroceutical wound care dressing, which could potentially be used to treat wounds with deeper infections compared with current state of the art that treats wounds with treatment zone limited to the surface near topical application. Conclusion: Technology advances in design and fabrication of electroceutical dressings were leveraged to develop a tunable laboratory prototype that could be used as a disposable low-cost electroceutical wound care dressing on chronic wounds. Design revisions of PED-1 (1 kΩ ballast resistor) circumvented previously observed adverse effects on the skin in the vicinity of an open wound. PED-10 (including a 10 kΩ ballast resistor) was well tolerated in the small cohort of patients (N = 8) on whom it was tested, and the observations reported here warrant a larger study to determine the clinical impact on human wound healing and infection control.

Electroceutical Treatment of Pseudomonas aeruginosa Biofilms.

Electroceutical wound dressings, especially those involving current flow with silver based electrodes, show promise for treating biofilm infections. However, their mechanism of action is poorly understood. We have developed an in vitro agar based model using a bioluminescent strain of Pseudomonas aeruginosa to measure loss of activity and killing when direct current was applied. Silver electrodes were overlaid with agar and lawn biofilms grown for 24 h. A 6 V battery with 1 kΩ ballast resistor was used to treat the biofilms for 1 h or 24 h. Loss of bioluminescence and a 4-log reduction in viable cells was achieved over the anode. Scanning electron microscopy showed damaged cells and disrupted biofilm architecture. The antimicrobial activity continued to spread from the anode for at least 2 days, even after turning off the current. Based on possible electrochemical ractions of silver electrodes in chlorine containing medium; pH measurements of the medium post treatment; the time delay between initiation of treatment and observed bactericidal effects; and the presence of chlorotyrosine in the cell lysates, hypochlorous acid is hypothesized to be the chemical agent responsible for the observed (destruction/killing/eradication) of these biofilm forming bacteria. Similar killing was obtained with gels containing only bovine synovial fluid or human serum. These results suggest that our in vitro model could serve as a platform for fundamental studies to explore the effects of electrochemical treatment on biofilms, complementing clinical studies with electroceutical dressings.