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Publication Exploring the Impact of Primer–Template Mismatches on PCR Performance of DNA Polymerases Varying in Proofreading Activity(MDPI, 2024-02-07) Huang, Ke; Zhang, Jilei; Li, Jing; Qiu, Haixiang; Wei, Lanjing; Yang, Yi; Wang, ChengmingPolymerase chain reaction (PCR) is a widely used technique in gene expression analysis, diagnostics, and various molecular biology applications. However, the accuracy and sensitivity of PCR can be compromised by primer–template mismatches, potentially leading to erroneous results. In this study, we strategically designed 111 primer–template combinations with varying numbers, types, and locations of mismatches to meticulously assess their impact on qPCR performance while two distinctly different types of DNA polymerases were used. Notably, when a single-nucleotide mismatch occurred at the 3’ end of the primer, we observed significant decreases in the analytical sensitivity (0–4%) with Invitrogen™ Platinum™ Taq DNA Polymerase High Fidelity, while the analytical sensitivity remained unchanged with Takara Ex Taq Hot Start Version DNA Polymerase. Leveraging these findings, we designed a highly specific PCR to amplify Babesia while effectively avoiding the genetically close Theileria. Through elucidating the critical interplay between types of DNA polymerases and primer–template mismatches, this research provides valuable insights for improving PCR accuracy and performance. These findings have important implications for researchers aiming to achieve robust qPCR results in various molecular biology applications. Keywords: PCR, DNA Polymerases, nucleotide mismatch, ProofreadingPublication Antibody-directed evolution reveals a mechanism for enhanced neutralization at the HIV-1 fusion peptide site(Nature Research, 2023-11-21) Banach, Bailey B.; Pletnev, Sergei; Olia, Adam S.; Xu, Kai; Zhang, Baoshan; Rawi, Reda; Bylund, Tatsiana; Doria-Rose, Nicole A.; Nguyen, Thuy Duong; Fahad, Ahmed S.; Lee, Myungjin; Lin, Bob C; Liu, Tracy; Louder, Mark K.; Madan, Bharat; McKee, Krisha; O’Dell, Sijy; Sastry, Mallika; Schön, Arne; Bui, Natalie; Shen, Chen-Hsiang; Wolfe, Jacy R.; Chuang, Gwo-Yu; Mascola, John R; Kwong, Peter D.; DeKosky, Brandon J.The HIV-1 fusion peptide (FP) represents a promising vaccine target, but global FP sequence diversity among circulating strains has limited anti-FP antibodies to ~60% neutralization breadth. Here we evolve the FP-targeting antibody VRC34.01 in vitro to enhance FP-neutralization using site saturation mutagenesis and yeast display. Successive rounds of directed evolution by iterative selection of antibodies for binding to resistant HIV-1 strains establish a variant, VRC34.01_mm28, as a best-in-class antibody with 10-fold enhanced potency compared to the template antibody and ~80% breadth on a cross-clade 208-strain neutralization panel. Structural analyses demonstrate that the improved paratope expands the FP binding groove to accommodate diverse FP sequences of different lengths while also recognizing the HIV-1 Env backbone. These data reveal critical antibody features for enhanced neutralization breadth and potency against the FP site of vulnerability and accelerate clinical development of broad HIV-1 FP-targeting vaccines and therapeutics.Publication Controlled-Release Hydrogel Microspheres to Deliver Multipotent Stem Cells for Treatment of Knee Osteoarthritis(MDPI, 2023-11-15) Hamilton, Megan; Wang, Jinxi; Dhar, Prajnaparamita; Stehno-Bittel, LisaOsteoarthritis (OA) is the most common form of joint disease affecting articular cartilage and peri-articular tissues. Traditional treatments are insufficient, as they are aimed at mitigating symptoms. Multipotent Stromal Cell (MSC) therapy has been proposed as a treatment capable of both preventing cartilage destruction and treating symptoms. While many studies have investigated MSCs for treating OA, therapeutic success is often inconsistent due to low MSC viability and retention in the joint. To address this, biomaterial-assisted delivery is of interest, particularly hydrogel microspheres, which can be easily injected into the joint. Microspheres composed of hyaluronic acid (HA) were created as MSC delivery vehicles. Microrheology measurements indicated that the microspheres had structural integrity alongside sufficient permeability. Additionally, encapsulated MSC viability was found to be above 70% over one week in culture. Gene expression analysis of MSC-identifying markers showed no change in CD29 levels, increased expression of CD44, and decreased expression of CD90 after one week of encapsulation. Analysis of chondrogenic markers showed increased expressions of aggrecan (ACAN) and SRY-box transcription factor 9 (SOX9), and decreased expression of osteogenic markers, runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALPL). In vivo analysis revealed that HA microspheres remained in the joint for up to 6 weeks. Rats that had undergone destabilization of the medial meniscus and had overt OA were treated with empty HA microspheres, MSC-laden microspheres, MSCs alone, or a control vehicle. Pain measurements taken before and after the treatment illustrated temporarily decreased pain in groups treated with encapsulated cells. Finally, the histopathological scoring of each group illustrated significantly less OA damage in those treated with encapsulated cells compared to controls. Overall, these studies demonstrate the potential of using HA-based hydrogel microspheres to enhance the therapeutic efficacy of MSCs in treating OA.Publication In-plane Extended Nano-coulter Counter (XnCC) for the Label-free Electrical Detection of Biological Particles(Wiley, 2022-06-14) Zhao, Zheng; Vaidyanathan, Swarnagowri; Bhanja, Payel; Gamage, Sachindra; Saha, Subhrajit; McKinney, Collin; Choi, Junseo; Park, Sunggook; Pahattuge, Thilanga; Wijerathne, Harshani; Jackson, Joshua M.; Huppert, Mateusz L.; Witek, Małgorzata A.; Soper, Steven A.We report an in-plane extended nanopore Coulter counter (XnCC) chip fabricated in a thermoplastic via imprinting. The fabrication of the sensor utilized both photolithography and focused ion beam milling to make the microfluidic network and the in-plane pore sensor, respectively, in Si from which UV resin stamps were generated followed by thermal imprinting to produce the final device in the appropriate plastic (cyclic olefin polymer, COP). As an example of the utility of this in-plane extended nanopore sensor, we enumerated SARS-CoV-2 viral particles (VPs) affinity-selected from saliva and extracellular vesicles (EVs) affinity-selected from plasma samples secured from mouse models exposed to different ionizing radiation doses.Publication Subthreshold white noise vibration alters trembling sway in older adults(Elsevier, 2023-06-28) Gerber, Eryn D.; Giraldo, Camilo; Whorley, Brett; Nichols, Paris; Ring, Scott; Luchies, Carl W.Background Somatosensory deficit is a significant contributor to falls in older adults. Stochastic resonance has shown promise in recent studies of somatosensation-based balance disorders, improving many measures of stability both inside and outside of the clinic. However, our understanding of this effect from a physiological perspective is poorly understood. Therefore, the primary goal of this study is to explore the influence of subthreshold vibratory stimulation on sway under the rambling-trembling framework. Methods 10 Healthy older adults (60–65 years) volunteered to participate in this study. Each participant underwent two randomized testing sessions on separate days, one experimental and one placebo. During each session, the participants' baseline sway was captured during one 90-s quiet standing trial. Their sensation threshold was then captured using a custom vibratory mat and 4–2-1 vibration perception threshold test. Finally, participants completed another 90-s quiet standing trial while the vibratory mat vibrated at 90% of their measured threshold (if experimental) or with the mat off (if placebo). While they completed these trials, an AMTI force plate collected force and moment data in the anteroposterior (AP) and mediolateral (ML), from which the center of pressure (COP), rambling (RM), and trembling (TR) time series were calculated. From each of these time series, range, variability (root-mean-square), and predictability (sample entropy) were extracted. One-tailed paired t-tests were used to compare baseline and during-vibration measures. Results No significant differences were found during the placebo session. For the experimental session, significant increases were found in AP TR range, ML TR RMS, AP COP predictability, and AP & ML TR predictability. The TR time series was particularly sensitive to vibration, suggesting a strong influence on peripheral/spinal mechanisms of postural control. Significance Though it is unclear whether observed effects are indicative of “improvements” or not, it does suggest that there was a measurable effect of subthreshold vibration on sway. This knowledge should be utilized in future studies of stochastic resonance, potentially acting as a mode of customization, tailoring vibration location, duration, magnitude, and frequency content to achieve the desired effect. One day, this work may aid in our ability to treat somatosensation-based balance deficits, ultimately reducing the incidence and severity of falls in older adults.Publication Design considerations for piezocomposite materials for electrical stimulation in medical implants(Taylor and Francis Group, 2022) Krech, Ember; Haas, Evan; Tideman, Grace; Reinsch, Bonnie; Friis, ElizabethIncidence of non-union following long bone fracture fixation and spinal fusion procedures is increasing, and very costly for patients and the medical system. Direct current (DC) electrical stimulation has shown success as an adjunct therapy to stimulate bone healing and increase surgery success rates, though drawbacks of current devices and implantable battery packs have limited widespread use. Energy harvesting utilising piezoelectric materials has been widely studied for powering devices without a battery, and a preclinical animal study has shown efficacy of a piezocomposite spinal fusion implant resulting in faster, more robust fusion. Most piezoelectric energy harvesters operate most effectively at high frequencies, limiting power generation from loads experienced by orthopaedic implants during human motion. This work characterises the efficient power generation capability of a novel composite piezoelectric material under simulated walking loads. Building on compliant layer adaptive composite stacks (CLACS), the power generation of mixed-mode CLACS (MMCLACS) is defined. Utilising poling direction to capitalise on in-plane strain generation due to compliant layer expansion, MMCLACS significantly increased power output compared to a standard piezo stack. The combination of radial and through-thickness poled piezoelectric elements within a stack to create MMCLACS significantly increases power generation under low-frequency dynamic loads. This technology can be adapted to a variety of architectures and assembled as a load-bearing energy harvester within current implants. MMCLACS integrated with implants would provide enough power to deliver bone healing electrical stimulation directly to the fusion site, decreasing non-union rates, and also could provide quantitative assessment of healing progression through load sensing.Publication Post-ischemic reorganization of sensory responses in cerebral cortex(Frontiers Media, 2023-06-02) Hayley, P.; Tuchek, C.; Dalla, S.; Borrell, J.; Murphy, M. D.; Nudo, R. J.; Guggenmos, D. J.Introduction: Sensorimotor integration is critical for generating skilled, volitional movements. While stroke tends to impact motor function, there are also often associated sensory deficits that contribute to overall behavioral deficits. Because many of the cortico-cortical projections participating in the generation of volitional movement either target or pass-through primary motor cortex (in rats, caudal forelimb area; CFA), any damage to CFA can lead to a subsequent disruption in information flow. As a result, the loss of sensory feedback is thought to contribute to motor dysfunction even when sensory areas are spared from injury. Previous research has suggested that the restoration of sensorimotor integration through reorganization or de novo neuronal connections is important for restoring function. Our goal was to determine if there was crosstalk between sensorimotor cortical areas with recovery from a primary motor cortex injury. First, we investigated if peripheral sensory stimulation would evoke responses in the rostral forelimb area (RFA), a rodent homologue to premotor cortex. We then sought to identify whether intracortical microstimulation-evoked activity in RFA would reciprocally modify the sensory response. Methods: We used seven rats with an ischemic lesion of CFA. Four weeks after injury, the rats’ forepaw was mechanically stimulated under anesthesia and neural activity was recorded in the cortex. In a subset of trials, a small intracortical stimulation pulse was delivered in RFA either individually or paired with peripheral sensory stimulation. Results: Our results point to post-ischemic connectivity between premotor and sensory cortex that may be related to functional recovery. Premotor recruitment during the sensory response was seen with a peak in spiking within RFA after the peripheral solenoid stimulation despite the damage to CFA. Furthermore, stimulation in RFA modulated and disrupted the sensory response in sensory cortex. Discussion: The presence of a sensory response in RFA and the sensitivity of S1 to modulation by intracortical stimulation provides additional evidence for functional connectivity between premotor and somatosensory cortex. The strength of the modulatory effect may be related to the extent of the injury and the subsequent reshaping of cortical connections in response to network disruption.Publication Tailoring the secretome composition of mesenchymal stem cells to augment specific functions of epidermal regeneration: an in vitro diabetic model(Frontiers Media, 2023-06-12) Hodge, Jacob G.; Robinson, Jennifer L.; Mellott, Adam J.Introduction: Wound healing consists of a dynamic series of events that are highly dependent on paracrine factors for proper progression through the phases of wound healing. Inappropriate progression through the phases is associated with insufficient epidermal regeneration (i.e., re-epithelialization) of wounds and subsequent propagation of chronic wounds, such as diabetic ulcers, which are associated with increased patient morbidity. Recently, investigation into the dynamic secretome of Adipose-derived Mesenchymal Stem Cells (ASCs), have shown promise in augmenting the wound healing response of chronic diabetic wounds. However, currently utilized 2D culture techniques are known to drastically alter the regenerative phenotype of ASCs. In this study a novel tissue-mimetic 3D system was utilized as a means to culture ASCs. Methods: The capacity for the ASC secretome to augment epidermal regeneration activity was then evaluated after exposure of ASCs to “wound priming stimuli” in 2D and 3D. The priming stimuli consisted of coating the 2D and 3D systems with the wound matrix proteins, collagen type I, fibronectin, and fibrin. To understand the potential benefit of the ASC secretome in the context of diabetic wounds, keratinocytes (KCs) were exposed to super-physiological glucose levels to induce a diabetic-like phenotype (idKCs). Results: Relative to KCs, idKC exhibited a 52% and 23% decline in proliferation and migration, respectively. Subsequently, analyses of the ASC secretome were performed. ASC conditioned media (ASC-CM) from tissue-mimetic culture demonstrated a > 50% increase secretion of proteins and a 2-fold increase in secreted EVs, relative to 2D culture. Interestingly, the different priming stimuli did not alter the total amount of protein or EVs secreted within the tissue-mimetic system. However, evaluation of specific soluble proteins via ELISA revealed significant differences in key epidermal regeneration factors, such as EGF, IGF-1, FGF-2, MMP-1, TIMP-1, and TGFβ-1. Additionally, the relative effect of ASC-EVs from the 2D and 3D system on idKCs epidermal regeneration functionality varied significantly, with EVs from 3D-Collagen culture providing the most significant benefit on idKC activity. Discussion: Together, these data support the utilization of tissue-mimetic culture system to enhance the adaptability and secretory activity of MSC-like populations in order to generate tailored biologics, via priming stimuli, for specific wound healing applications.Publication Quality control in resting-state fMRI: the benefits of visual inspection(Frontiers Media, 2023-05-04) Lepping, Rebecca J.; Yeh, Hung-Wen; McPherson, Brent C.; Brucks, Morgan G.; Sabati, Mohammad; Karcher, Rainer T.; Brooks, William M.; Habiger, Joshua D.; Papa, Vlad B.; Martin, Laura E.Background: A variety of quality control (QC) approaches are employed in resting-state functional magnetic resonance imaging (rs-fMRI) to determine data quality and ultimately inclusion or exclusion of a fMRI data set in group analysis. Reliability of rs-fMRI data can be improved by censoring or “scrubbing” volumes affected by motion. While censoring preserves the integrity of participant-level data, including excessively censored data sets in group analyses may add noise. Quantitative motion-related metrics are frequently reported in the literature; however, qualitative visual inspection can sometimes catch errors or other issues that may be missed by quantitative metrics alone. In this paper, we describe our methods for performing QC of rs-fMRI data using software-generated quantitative and qualitative output and trained visual inspection. Results: The data provided for this QC paper had relatively low motion-censoring, thus quantitative QC resulted in no exclusions. Qualitative checks of the data resulted in limited exclusions due to potential incidental findings and failed pre-processing scripts. Conclusion: Visual inspection in addition to the review of quantitative QC metrics is an important component to ensure high quality and accuracy in rs-fMRI data analysis.Publication Engineering peptide-polymer hybrids for targeted repair and protection of cervical lesions(Frontiers Media, 2022-11-25) Spencer, Paulette; Ye, Qiang; Misra, Anil; Chandler, Josephine R.; Cobb, Charles M.; Tamerler, CandanBy 2060, nearly 100 million people in the U.S. will be over age 65 years. One-third of these older adults will have root caries, and nearly 80% will have dental erosion. These conditions can cause pain and loss of tooth structure that interfere with eating, speaking, sleeping, and quality of life. Current treatments for root caries and dental erosion have produced unreliable results. For example, the glass-ionomer-cement or composite-resin restorations used to treat these lesions have annual failure rates of 44% and 17%, respectively. These limitations and the pressing need to treat these conditions in the aging population are driving a focus on microinvasive strategies, such as sealants and varnishes. Sealants can inhibit caries on coronal surfaces, but they are ineffective for root caries. For healthy, functionally independent elders, chlorhexidine varnish applied every 3 months inhibits root caries, but this bitter-tasting varnish stains the teeth. Fluoride gel inhibits root caries, but requires prescriptions and daily use, which may not be feasible for some older patients. Silver diamine fluoride can both arrest and inhibit root caries but stains the treated tooth surface black. The limitations of current approaches and high prevalence of root caries and dental erosion in the aging population create an urgent need for microinvasive therapies that can: (a) remineralize damaged dentin; (b) inhibit bacterial activity; and (c) provide durable protection for the root surface. Since cavitated and non-cavitated root lesions are difficult to distinguish, optimal approaches will treat both. This review will explore the multi-factorial elements that contribute to root surface lesions and discuss a multi-pronged strategy to both repair and protect root surfaces. The strategy integrates engineered peptides, novel polymer chemistry, multi-scale structure/property characterization and predictive modeling to develop a durable, microinvasive treatment for root surface lesions.Publication Engineered Peptides Enable Biomimetic Route for Collagen Intrafibrillar Mineralization(MDPI, 2023-03-28) Cloyd, Aya K.; Boone, Kyle; Ye, Qiang; Snead, Malcolm L.; Spencer, Paulette; Tamerler, CandanOvercoming the short lifespan of current dental adhesives remains a significant clinical need. Adhesives rely on formation of the hybrid layer to adhere to dentin and penetrate within collagen fibrils. However, the ability of adhesives to achieve complete enclosure of demineralized collagen fibrils is recognized as currently unattainable. We developed a peptide-based approach enabling collagen intrafibrillar mineralization and tested our hypothesis on a type-I collagen-based platform. Peptide design incorporated collagen-binding and remineralization-mediating properties using the domain structure conservation approach. The structural changes from representative members of different peptide clusters were generated for each functional domain. Common signatures associated with secondary structure features and the related changes in the functional domain were investigated by attenuated total reflectance Fourier-transform infrared (ATR-FTIR) and circular dichroism (CD) spectroscopy, respectively. Assembly and remineralization properties of the peptides on the collagen platforms were studied using atomic force microscopy (AFM). Mechanical properties of the collagen fibrils remineralized by the peptide assemblies was studied using PeakForce-Quantitative Nanomechanics (PF-QNM)-AFM. The engineered peptide was demonstrated to offer a promising route for collagen intrafibrillar remineralization. This approach offers a collagen platform to develop multifunctional strategies that combine different bioactive peptides, polymerizable peptide monomers, and adhesive formulations as steps towards improving the long-term prospects of composite resins.Publication Autonomous-Strengthening Adhesive Provides Hydrolysis-Resistance and Enhanced Mechanical Properties in Wet Conditions(MDPI, 2022-08-27) Ezazi, Mohammadamin; Ye, Qiang; Misra, Anil; Tamerler, Candan; Spencer, PauletteThe low-viscosity adhesive that is used to bond composite restorative materials to the tooth is readily damaged by acids, enzymes, and oral fluids. Bacteria infiltrate the resulting gaps at the composite/tooth interface, demineralize the tooth, and further erode the adhesive. This paper presents the preparation and characterization of a low-crosslink-density hydrophilic adhesive that capitalizes on sol-gel reactions and free-radical polymerization to resist hydrolysis and provide enhanced mechanical properties in wet environments. Polymerization behavior, water sorption, and leachates were investigated. Dynamic mechanical analyses (DMA) were conducted using water-saturated adhesives to mimic load transfer in wet conditions. Data from all tests were analyzed using appropriate statistical tests (α = 0.05). The degree of conversion was comparable for experimental and control adhesives at 88.3 and 84.3%, respectively. HEMA leachate was significantly lower for the experimental (2.9 wt%) compared to control (7.2 wt%). After 3 days of aqueous aging, the storage and rubbery moduli and the glass transition temperature of the experimental adhesive (57.5MPa, 12.8MPa, and 38.7 °C, respectively) were significantly higher than control (7.4MPa, 4.3 MPa, and 25.9 °C, respectively). The results indicated that the autonomic sol-gel reaction continues in the wet environment, leading to intrinsic reinforcement of the polymer network, improved hydrolytic stability, and enhanced mechanical properties.Publication Silica-Coated Magnetic Nanoparticles for Vancomycin Conjugation(American Chemical Society, 2022-08-16) Abdelaziz, Moustafa M.; Hefnawy, Amr; Anter, Asem; Abdellatif, Menna M.; Khalil, Mahmoud A. F.; Khalil, Islam A.Drug resistance is a global health challenge with thousands of deaths annually caused by bacterial multidrug resistance (MDR). Efforts to develop new antibacterial molecules do not meet the mounting needs imposed by the evolution of MDR. An alternative approach to overcome this challenge is developing targeted formulations that can enhance the therapeutic efficiency and limit side effects. In this aspect, vancomycin is a potent antibacterial agent that has inherent bacterial targeting properties by binding to the D-Ala-D-Ala moiety of the bacterial peptidoglycan. However, the use of vancomycin is associated with serious side effects that limit its clinical use. Herein, we report the development of vancomycin-conjugated magnetic nanoparticles using a simple conjugation method for targeted antibacterial activity. The nanoparticles were synthesized using a multistep process that starts by coating the nanoparticles with a silica layer, followed by binding an amide linker and then binding the vancomycin glycopeptide. The developed vancomycin-conjugated magnetic nanoparticles were observed to exhibit a spherical morphology and a particle size of 16.3 ± 2.6 nm, with a silica coating thickness of 5 nm and a total coating thickness of 8 nm. The vancomycin conjugation efficiency on the nanoparticles was measured spectrophotometrically to be 25.1%. Additionally, the developed formulation retained the magnetic activity of the nanoparticles, where it showed a saturation magnetization value of 51 emu/g, compared to 60 emu/g for bare magnetic nanoparticles. The in vitro cell biocompatibility demonstrated improved safety where vancomycin-conjugated nanoparticles showed IC50 of 183.43 μg/mL, compared to a much lower value of 54.11 μg/mL for free vancomycin. While the antibacterial studies showed a comparable activity of the developed formulation, the minimum inhibitory concentration was 25 μg/mL, compared to 20 μg/mL for free vancomycin. Accordingly, the reported formulation can be used as a platform for the targeted and efficient delivery of other drugs.Publication CT-based lung motion differences in patients with usual interstitial pneumonia and nonspecific interstitial pneumonia(Frontiers Media, 2022-10-04) Choi, Jiwoong; Chae, Kum Ju; Jin, Gong Yong; Lin, Ching-Long; Laroia, Archana T.; Hoffman, Eric A.; Lee, Chang HyunWe applied quantitative CT image matching to assess the degree of motion in the idiopathic ILD such as usual interstitial pneumonia (UIP) and nonspecific interstitial pneumonia (NSIP). Twenty-one normal subjects and 42 idiopathic ILD (31 UIP and 11 NSIP) patients were retrospectively included. Inspiratory and expiratory CT images, reviewed by two experienced radiologists, were used to compute displacement vectors at local lung regions matched by image registration. Normalized three-dimensional and two-dimensional (dorsal-basal) displacements were computed at a sub-acinar scale. Displacements, volume changes, and tissue fractions in the whole lung and the lobes were compared between normal, UIP, and NSIP subjects. The dorsal-basal displacement in lower lobes was smaller in UIP patients than in NSIP or normal subjects (p = 0.03, p = 0.04). UIP and NSIP were not differentiated by volume changes in the whole lung or upper and lower lobes (p = 0.53, p = 0.12, p = 0.97), whereas the lower lobe air volume change was smaller in both UIP and NSIP than normal subjects (p = 0.02, p = 0.001). Regional expiratory tissue fractions and displacements showed positive correlations in normal and UIP subjects but not in NSIP subjects. In summary, lung motionography quantified by image registration-based lower lobe dorsal-basal displacement may be used to assess the degree of motion, reflecting limited motion due to fibrosis in the ILD such as UIP and NSIP.Publication Novel hydrogel system eliminates subculturing and improves retention of nonsenescent mesenchymal stem cell populations(Future Science Group, 2022-10-12) Hodge, Jacob G.; Robinson, Jennifer L.; Mellott, Adam J.Aim: To compare the physiological behavior of mesenchymal stem/stromal cells (MSCs) within an expandable tissue-mimetic 3D system relative to in vitro expansion in a traditional 2D system. Methods: Adipose-derived MSCs (ASCs) were continuously cultured for 6 weeks on either 2D culture plastic or in a 3D hydrogel system that eliminated subculturing. ASCs were assessed for senescence, ‘stem-like’ MSC markers, and ability for their secretome to augment a secondary cell population. Results: The 3D hydrogel system resulted in an enhanced retention of more regenerative, nonsenescent ASC populations that exhibited increased expression of ‘stem-like’ MSC surface markers. Conclusion: This study introduces a proof-of-concept design for a novel modular 3D system that can improve in vitro expansion of stem-like cell populations for future regenerative therapies.Publication The feasibility of ultrasound-assisted endovascular laser thrombolysis in an acute rabbit thrombosis model(Wiley, 2021-07-02) Singh, Rohit; Jo, Janggun; Riegel, Matthew; Forrest, M. Laird; Yang, XinmaiPurpose This study aimed to test the feasibility of combined ultrasound and laser technique, namely, ultrasound-assisted endovascular laser thrombolysis (USELT), for thrombolysis by conducting in vivo tests in a rabbit thrombosis model. Materials and methods An acute thrombus was created in the right jugular vein of rabbit and then was treated with ultrasound only, laser only, and USELT to dissolve the blood clot. A total of 20 rabbits were used. Out of which, the first three rabbits were used to titrate the laser and ultrasound parameters. Then, five rabbits were treated with ultrasound only, five rabbits were treated with laser only, and seven rabbits were treated with USELT. During USELT, 532-nm laser pulses were delivered endovascularly directly to the clot through a fiber optic, and 0.5 MHz ultrasound pulses were applied noninvasively to the same region. A laser fluence of 4 to 12 mJ/cm2 and ultrasound amplitude of 1 to 2 MPa were used. Recanalization of the jugular vein was assessed by performing ultrasound Doppler imaging immediately after the treatment. The maximum blood flow speed after the treatment as compared to its value before the treatment was used to calculate the blood flow recovery in vessel. Results The blood flow was fully recovered (100%) in three rabbits, partially recovered in two rabbits (more than 50% and less than 100%) with mean percentage recovery of 69.73% and poorly recovered in two rabbits (<50%) with mean percentage recovery of 6.2% in the USELT group. In contrast, the treatment group with ultrasound or laser alone did not show recanalization of vein in any case, all the five rabbits were poorly/not recovered with a mean percentage recovery of 0%. Conclusions The USELT technology was shown to effectively dissolve the blood clots in an acute rabbit jugular vein thrombosis model.Publication Fluidic operation of a polymer-based nanosensor chip for analysing single molecules(Cambridge University Press, 2022-06-27) Vaidyanathan, Swarnagowri; Gamage, Sachindra; Dathathreya, Kavya; Kryk, Renee; Manoharan, Anishkumar; Zhao, Zheng; Zhang, Lulu; Choi, Junseo; Park, Daniel; Park, Sunggook; Soper, Steven A.Most medical diagnostic tests are expensive, involve slow turnaround times from centralized laboratories and require highly specialized equipment with seasoned technicians to carry out the assay. To facilitate realization of precision medicine at the point of care, we have developed a mixed-scale nanosensor chip featuring high surface area pillar arrays where solid-phase reactions can be performed to detect and identify nucleic acid targets found in diseased patients. Products formed can be identified and detected using a polymer nanofluidic channel. To guide delivery of this platform, we discuss the operation of various components of the device and simulations (COMSOL) used to guide the design by investigating parameters such as pillar array loading, and hydrodynamic and electrokinetic flows. The fabrication of the nanosensor is discussed, which was performed using a silicon (Si) master patterned with a combination of focused ion beam milling and photolithography with deep reactive ion etching. The mixed-scale patterns were transferred into a thermoplastic via thermal nanoimprint lithography, which facilitated fabrication of the nanosensor chip making it appropriate for in vitro diagnostics. The results from COMSOL were experimentally verified for hydrodynamic flow using Rhodamine B as a fluorescent tracer and electrokinetic flow using single fluorescently labelled oligonucleotides (single-stranded DNAs, ssDNAs).Publication Effect of Photo-Mediated Ultrasound Therapy on Nitric Oxide and Prostacyclin from Endothelial Cells(National Academy of Sciences, 2022-03-03) Karthikesh, Madhumithra Subramanian; Wu, Sa; Paulus, Yannis; Wang, Xueding; Yang, XinmaiSeveral studies have investigated the effect of photo-mediated ultrasound therapy (PUT) on the treatment of neovascularization. This study explores the impact of PUT on the release of the vasoactive agents nitric oxide (NO) and prostacyclin (PGI2) from the endothelial cells in an in vitro blood vessel model. In this study, an in vitro vessel model containing RF/6A chorioretinal endothelial cells was used. The vessels were treated with ultrasound-only (0.5, 1.0, 1.5 and 2.0 MPa peak negative pressure at 0.5 MHz with 10% duty cycle), laser-only (5, 10, 15 and 20 mJ/cm2 at 532 nm with a pulse width of 5 ns), and synchronized laser and ultrasound (PUT) treatments. Passive cavitation detection was used to determine the cavitation activities during treatment. The levels of NO and PGI2 generally increased when the applied ultrasound pressure and laser fluence were low. The increases in NO and PGI2 levels were significantly reduced by 37.2% and 42.7%, respectively, from 0.5 to 1.5 MPa when only ultrasound was applied. The increase in NO was significantly reduced by 89.5% from 5 to 20 mJ/cm2, when only the laser was used. In the PUT group, for 10 mJ/cm2 laser fluence, the release of NO decreased by 76.8% from 0.1 to 1 MPa ultrasound pressure. For 0.5 MPa ultrasound pressure in the PUT group, the release of PGI2 started to decrease by 144% from 15 to 20 mJ/cm2 laser fluence. The decreases in NO and PGI2 levels coincided with the increased cavitation activities in each group. In conclusion, PUT can induce a significant reduction in the release of NO and PGI2 in comparison with ultrasound-only and laser-only treatments.Publication Estrogen Signaling Dictates Musculoskeletal Stem Cell Behavior: Sex Differences in Tissue Repair(Mary Ann Liebert, 2022-08-08) Knewtson, Kelsey E.; Ohl, Nathan R.; Robinson, Jennifer L.Sexual dimorphisms in humans and other species exist in visually evident features such as body size and less apparent characteristics, including disease prevalence. Current research is adding to a growing understanding of sex differences in stem cell function and response to external stimuli, including sex hormones such as estrogens. These differences are proving significant and directly impact both the understanding of stem cell processes in tissue repair and the clinical implementation of stem cell therapies. Adult stem cells of the musculoskeletal system, including those used for development and repair of muscle, bone, cartilage, fibrocartilage, ligaments, and tendons, are no exception. Both in vitro and in vivo studies have found differences in stem cell number, proliferative and differentiation capabilities, and response to estrogen treatment between males and females of many species. Maintaining the stemness and reducing senescence of adult stem cells is an important topic with implications in regenerative therapy and aging. As such, this review discusses the effect of estrogens on musculoskeletal system stem cell response in multiple species and highlights the research gaps that still need to be addressed. The following evidence from investigations of sex-related phenotypes in adult progenitor and stem cells are pieces to the big puzzle of sex-related effects on aging and disease and critical information for both fundamental tissue repair and regeneration studies and safe and effective clinical use of stem cells.Publication Activity dependent stimulation increases synaptic efficacy in spared pathways in an anesthetized rat model of spinal cord contusion injury(IOS Press, 2022-04-08) Borrell, Jordan A.; Krizsan-Agbas, Dora; Nudo, Randolph J.; Frost, Shawn B.Background: Closed-loop neuromodulation systems have received increased attention in recent years as potential therapeutic approaches for treating neurological injury and disease. Objective: The purpose of this study was to assess the ability of intraspinal microstimulation (ISMS), triggered by action potentials (spikes) recorded in motor cortex, to alter synaptic efficacy in descending motor pathways in an anesthetized rat model of spinal cord injury (SCI). Methods: Experiments were carried out in adult, male, Sprague Dawley rats with a moderate contusion injury at T8. For activity-dependent stimulation (ADS) sessions, a recording microelectrode was used to detect neuronal spikes in motor cortex that triggered ISMS in the spinal cord grey matter. SCI rats were randomly assigned to one of four experimental groups differing by: a) cortical spike-ISMS stimulus delay (10 or 25 ms) and b) number of ISMS pulses (1 or 3). Four weeks after SCI, ADS sessions were conducted in three consecutive 1-hour conditioning bouts for a total of 3 hours. At the end of each conditioning bout, changes in synaptic efficacy were assessed using intracortical microstimulation (ICMS) to examine the number of spikes evoked in spinal cord neurons during 5-minute test bouts. A multichannel microelectrode recording array was used to record cortically-evoked spike activity from multiple layers of the spinal cord. Results: The results showed that ADS resulted in an increase in cortically-evoked spikes in spinal cord neurons at specific combinations of spike-ISMS delays and numbers of pulses. Efficacy in descending motor pathways was increased throughout all dorsoventral depths of the hindlimb spinal cord. Conclusions: These results show that after an SCI, ADS can increase synaptic efficacy in spared pathways between motor cortex and spinal cord. This study provides further support for the potential of ADS therapy as an effective method for enhancing descending motor control after SCI.