Bourns College of Engineering

African Swine Fever Virus (ASFV) is a highly contagious pathogen with nearly 100% mortality in swine, causing severe global economic loss. Current detection methods rely on nucleic acid amplification, which requires specialized equipment and skilled operators, limiting accessibility in resource-constrained settings. To address these challenges, we developed the Covalently Immobilized Magnetic Nanoparticles Enhanced CRISPR (CIMNE-CRISPR) system. This amplification-free diagnostic system seamlessly combines target recognition, sequence-specific enrichment, and signal generation. This approach uses covalent immobilization of CRISPR-LbCas12a-crRNA complexes on Fe₃O₄@SiO₂ core-shell magnetic nanoparticles, which improves enzyme specificity and robustness over traditional adsorption. The CIMNE-CRISPR assay reached a limit of detection (LOD) of 8.1 × 10⁴ copies/μL and a limit of quantification (LOQ) of 4.2 × 10⁵ copies/μL, with a dynamic range spanning 10⁵ to 10¹⁰ copies/μL and a matrix factor of 100.29% in porcine plasma. It maintained great specificity and accurately detecting 10⁵ copies/μL of ASFV DNA even with high mutant concentrations (10¹³ copies/μL). The method demonstrated decent reproducibility across different nanoparticle synthesis batches, with an RSD of 9.63% and recovery rates between 97% and 103%, and features rapid processing well-suited for field diagnostics. Overall, this system's cost-effectiveness, simplicity, and reliability highlight its potential to pave the way for advanced CRISPR-based diagnostics, particularly for diverse viral and bacterial targets in agricultural, environmental, and zoonotic disease contexts.

The intricate interplay between biochemical and physical cues dictates pluripotent stem cell (PSC) differentiation to form various tissues. While biochemical modulation has been extensively studied, the role of biophysical microenvironments in early lineage commitment remains elusive. Here, we introduce a novel 3D cell culture system combining electrospun nanofibers with microfabricated polydimethylsiloxane (PDMS) patterns. This system enables the controlled formation of semispherical human induced pluripotent stem cell (hiPSC) colonies, facilitating the investigation of local mechanical stem cell niches on mechano-responsive signaling and lineage specification. Our system unveiled spatially organized RhoA activity coupled with actin-myosin cable formation, suggesting mechano-dependent hiPSC behaviors. Nodal network analysis of RNA-seq data revealed RhoA downstream regulation of YAP signaling, DNA histone modifications, and patterned germ layer specification. Notably, altering colony morphology through controlled PDMS microwell shaping effectively modulated the spatial distribution of mechano-sensitive mediators and subsequent differentiation. This study provides a cell culture platform to decipher the role of biophysical cues in early embryogenesis, offering valuable insights for material design in tissue engineering and regenerative medicine applications.

Erythrocyte-derived optical microparticles containing near infrared (NIR) dyes such as indocyanine green (ICG) present a promising platform for fluorescence imaging and laser treatment of abnormal vasculature, including port wine birthmarks. Herein, we have investigated the effects of blood type utilized in fabricating these microparticles, and the number density of the particles on their circulation time in mice by real-time NIR fluorescence imaging of the dermal vasculature. We find that the emission half-life of microparticles engineered from human O+ blood type increases by approximately two-fold as compared to those engineered from B+ blood type. Increasing the number density of the microparticles fabricated from O+ blood type from ~0.5 millions/µl to 1.6 millions/µl is associated with nearly a fourfold increase in the emission half-life of the particles. These findings emphasize the importance of blood type and number density in engineering erythrocyte-derived particles for clinical applications as treatment of PWBs.

Abstract: The biophysical microenvironment of cells dynamically evolves during embryonic development, leading to defined tissue specification. A versatile and highly adaptive magneto‐responsive hydrogel system composed of magnetic nanorods (MNRs) and a stress‐responsive polymeric matrix is developed to dynamically regulate the physical stem cell niche. The anisotropic magnetic/shape factor of nanorods is utilized to maximize the strains on the polymeric network, thus regulating the hydrogel modulus in a physiologically relevant range under a minimal magnitude of the applied magnetic fields below 4.5 mT. More significantly, the pre‐alignment of MNRs induces greater collective strains on the polymeric network, resulting in a superior stiffening range, over a 500% increase as compared to that with randomly oriented nanorods. The pre‐alignment of nanorods also enables a fast and reversible response under a magnetic field of the opposite polarity as well as spatially controlled heterogeneity of modulus within the hydrogel by applying anisotropic magnetic fields. The mechano‐modulative capability of this system is validated by a mechanotransduction model with human‐induced pluripotent stem cells where the locally controlled hydrogel modulus regulates the activation of mechano‐sensitive signaling mediators and subsequent stem cell differentiation. Therefore, this magneto‐responsive hydrogel system provides a platform to investigate various cellular behaviors under dynamic mechanical microenvironments.

To address limitations in current approaches for treating large peripheral nerve defects, the presented study evaluated the feasibility of functional material-mediated physical stimuli on peripheral nerve regeneration. Electrospun piezoelectric poly(vinylidene fluoride-trifluoroethylene) nanofibers were utilized to deliver mechanical actuation-activated electrical stimulation to nerve cells/tissues in a non-invasive manner. Using morphologically and piezoelectrically optimized nanofibers for neurite extension and Schwann cell maturation based on in vitro experiments, piezoelectric nerve conduits were synthesized and implanted in a rat sciatic nerve transection model to bridge a critical-sized sciatic nerve defect (15 mm). A therapeutic shockwave system was utilized to periodically activate the piezoelectric effect of the implanted nerve conduit on demand. The piezoelectric nerve conduit-mediated mechano-electrical stimulation (MES) induced enhanced peripheral nerve regeneration, resulting in full axon reconnection with myelin regeneration from the proximal to the distal ends over the critical-sized nerve gap. In comparison, a control group, in which the implanted piezoelectric conduits were not activated in vivo, failed to exhibit such nerve regeneration. In addition, at both proximal and distal ends of the implanted conduits, a decreased number of damaged myelination (ovoids), an increased number of myelinated nerves, and a larger axonal diameter were observed under the MES condition as compared to the control condition. Furthermore, unlike the control group, the MES condition exhibited a superior functional nerve recovery, assessed by walking track analysis and polarization-sensitive optical coherence tomography, demonstrating the significant potential of the piezoelectric conduit-based physical stimulation approach for the treatment of peripheral nerve injury.

Gastric cancer (GC) is one of the most common and lethal types of cancer affecting over one million people, leading to 768,793 deaths globally in 2020 alone. The key for improving the survival rate lies in reliable screening and early diagnosis. Existing techniques including barium-meal gastric photofluorography and upper endoscopy can be costly and time-consuming and are thus impractical for population screening. We look instead for small extracellular vesicles (sEVs, currently also referred as exosomes) sized ⌀ 30-150 nm as a candidate. sEVs have attracted a significantly higher level of attention during the past decade or two because of their potentials in disease diagnoses and therapeutics. Here, we report that the composition information of the collective Raman-active bonds inside sEVs of human donors obtained by surface-enhanced Raman spectroscopy (SERS) holds the potential for non-invasive GC detection. SERS was triggered by the substrate of gold nanopyramid arrays we developed previously. A machine learning-based spectral feature analysis algorithm was developed for objectively distinguishing the cancer-derived sEVs from those of the non-cancer sub-population. sEVs from the tissue, blood, and saliva of GC patients and non-GC participants were collected (n = 15 each) and analyzed. The algorithm prediction accuracies were reportedly 90, 85, and 72%. "Leave-a-pair-of-samples out" validation was further performed to test the clinical potential. The area under the curve of each receiver operating characteristic curve was 0.96, 0.91, and 0.65 in tissue, blood, and saliva, respectively. In addition, by comparing the SERS fingerprints of individual vesicles, we provided a possible way of tracing the biogenesis pathways of patient-specific sEVs from tissue to blood to saliva. The methodology involved in this study is expected to be amenable for non-invasive detection of diseases other than GC.

To improve the quality of MRI-based cerebral blood flow (CBF) measurements, a deep convolutional neural network (dCNN) was trained to combine single- and multi-delay arterial spin labeling (ASL) and structural images to predict gold-standard ¹⁵O-water PET CBF images obtained on a simultaneous PET/MRI scanner. The dCNN was trained and tested on 64 scans in 16 healthy controls (HC) and 16 cerebrovascular disease patients (PT) with 4-fold cross-validation. Fidelity to the PET CBF images and the effects of bias due to training on different cohorts were examined. The dCNN significantly improved CBF image quality compared with ASL alone (mean ± standard deviation): structural similarity index (0.854 ± 0.036 vs. 0.743 ± 0.045 [single-delay] and 0.732 ± 0.041 [multi-delay], P < 0.0001); normalized root mean squared error (0.209 ± 0.039 vs. 0.326 ± 0.050 [single-delay] and 0.344 ± 0.055 [multi-delay], P < 0.0001). The dCNN also yielded mean CBF with reduced estimation error in both HC and PT (P < 0.001), and demonstrated better correlation with PET. The dCNN trained with the mixed HC and PT cohort performed the best. The results also suggested that models should be trained on cases representative of the target population.

Erythrocyte-based carriers can serve as theranostic platforms for delivery of imaging and therapeutic payloads. Engineering these carriers at micro- or nanoscales makes them potentially useful for broad clinical applications ranging from vascular diseases to tumor theranostics. Longevity of these carriers in circulation is important in delivering a sufficient amount of their payloads to the target. We have investigated the circulation dynamics of micro (∼4.95 μm diameter) and nano (∼91 nm diameter) erythrocyte-derived carriers in real time using near-infrared fluorescence imaging, and evaluated the effectiveness of such carrier systems in mediating photothermolysis of cutaneous vasculature in mice. Fluorescence emission half-lives of micro- and nanosized carriers in response to a single intravenous injection were ∼49 and ∼15 min, respectively. A single injection of microsized carriers resulted in a 3-fold increase in signal-to-noise ratio that remained nearly persistent over 1 h of imaging time. Our results also suggest that a second injection of the carriers 7 days later can induce a transient inflammatory response, as manifested by the apparent leakage of the carriers into the perivascular tissue. The administration of the carriers into the mice vasculature reduced the threshold laser fluence to induce photothermolysis of blood vessels from >65 to 20 J/cm². We discuss the importance of membrane physicochemical and mechanical characteristics in engineering erythrocyte-derived carriers and considerations for their clinical translation.

Noncoding RNA (ncRNA) has a key role in regulating gene expression, mediating fundamental processes and diseases via a variety of yet unknown mechanisms. Here, we review recent applications of conventional and enhanced Molecular Dynamics (MD) simulations methods to address the mechanistic function of large biomolecular systems that are tightly involved in the ncRNA function and that are of key importance in life sciences. This compendium focuses of three biomolecular systems, namely the CRISPR-Cas9 genome editing machinery, group II intron ribozyme and the ribonucleoprotein complex of the spliceosome, which edit and process ncRNA. We show how the application of a novel accelerated MD simulations method has been key in disclosing the conformational transitions underlying RNA binding in the CRISPR-Cas9 complex, suggesting a mechanism for RNA recruitment and clarifying the conformational changes required for attaining genome editing. As well, we discuss the use of mixed quantum-classical MD simulations in deciphering the catalytic mechanism of RNA splicing as operated by group II intron ribozyme, one of the largest ncRNA structures crystallized so far. Finally, we debate the future challenges and opportunities in the field, discussing the recent application of MD simulations for unraveling the functional biophysics of the spliceosome, a multi-mega Dalton complex of proteins and small nuclear RNAs that performs RNA splicing in humans. This showcase of applications highlights the current talent of MD simulations to dissect atomic-level details of complex biomolecular systems instrumental for the design of finely engineered genome editing machines. As well, this review aims at inspiring future investigations of several other ncRNA regulatory systems, such as micro and small interfering RNAs, which achieve their function and specificity using RNA-based recognition and targeting strategies.