Non-invasive Cell Research Articles

Non-Invasive Three-Dimensional Cell Analysis in Bioinks by Raman Imaging.

3D bioprinting is an emerging biofabrication strategy using bioinks, comprising cells and biocompatible materials, to produce functional tissue models. Despite progress in building increasingly complex objects, biological analyses in printed constructs remain challenging. Especially, methods that allow non-invasive and non-destructive evaluation of embedded cells are largely missing. Here, we implemented Raman imaging for molecular-sensitive investigations on bioprinted objects. Different aspects such as culture formats (2D, 3D-cast, and 3D-printed), cell types (endothelial cells and fibroblasts), and the selection of the biopolymer (alginate, alginate/nanofibrillated cellulose, alginate/gelatin) were considered and evaluated. Raman imaging allowed for marker-independent identification and localization of subcellular components against the surrounding biomaterial background. Furthermore, single-cell analysis of spectral signatures, performed by multivariate analysis, demonstrated discrimination between endothelial cells and fibroblasts and identified cellular features influenced by the bioprinting process. In summary, Raman imaging was successfully established to analyze cells in 3D culture in situ and evaluate them with regard to the localization of different cell types and their molecular phenotype as a valuable tool for quality control of bioprinted objects.

Intranasally applied human olfactory mucosa neural progenitor cells migrate to damaged brain regions.

Aim:To determine if intranasally administered olfactory mucosa progenitor cells (OMPCs) migrate to damaged areas of brain.Materials & methods:Rowett Nude (RNU) adult rats were injured using the Marmarou model then 2 weeks later received intranasally-delivered human OMPC. After 3 weeks, rats were sacrificed and brain sectioned. The mean distances from the human OMPCs to markers for degenerative neuronal cell bodies (p-c-Jun+), axonal swellings on damaged axons (β-APP+) and random points in immunostained sections were quantified. One-way ANOVA was used to analyze data.Results:The human OMPCs were seen in specific areas of the brain near degenerating cell bodies and damaged axons.Conclusion:Intranasally delivered human OMPC selectively migrate to brain injury sites suggesting a possible noninvasive stem cell delivery for brain injury.

P-237 Non-invasive metabolic live cell imaging of early embryo development using adapted confocal microscopy; a safety study

Abstract Study question Is it safe to use metabolic imaging to measure nicotinamide adenine dinucleotide (NADH) associated auto-fluorescence during embryo development using adapted confocal microscopy? Summary answer Non-invasive metabolic imaging is safe as no differences were observed between controls and illuminated embryos in terms of embryo development, blastocyst formation and implantation potential. What is known already Developing non-invasive methods that are reliable to assess oocyte and embryo quality has been a significant aim for assisted reproductive technologies. Changes in metabolic activity could lead to cell death or abnormal embryo development and low implantation potential. This could potentially be predicted by incorporating non-invasive measurements of metabolism. Metabolic imaging in embryos has been investigated through complex methodologies, however, scientific evidence for its utility during embryo development using simple technology remains unexplored. Measurements of metabolic activity could be a useful tool as the auto-fluorescence of molecules such as NADH is a straightforward representation of mitochondrial function. Study design, size, duration Super-ovulated female mice (n = 30) were subjected to mating with 10 males. In-vivo produced embryos collected at the 2-cell stage were divided in control group (n = 151), sham control group (n = 151) and illuminated group (n = 152). Illuminated embryos were assessed for NADH levels during embryo development every 3 hours using arbitrary units of autofluorescence (AU). Produced blastocysts were assessed for total cell and inner-cell-mass (ICM) number (Oct4 immuno-staining) and implantation potential through outgrowth assays in separate experiments. Participants/materials, setting, methods F1 (CBA/C57Bl6) mouse strain was used. NADH auto-fluorescence levels were measured during embryo development using adapted confocal microcopy (Olympus FV1200). A confocal Z-stacking function was used to record 15 focal planes using a 20x/0.95NA air objective of entire embryos, opening the confocal pinhole system completely. Then, images were collected and analysed using FIJI software (version: 2.0.0-rc-69/1.52n;ImageJ). Blastocyst cell number, formation rates and outgrowth rates for 4 days post blastocyst formation were compared between study groups. Main results and the role of chance Embryo culture experiments showed no significant differences in blastocyst formation rates between study groups (Control: 71.7%; Sham: 64.9%; Illuminated 71.7%; p > 0.05). Similarly, the total number of cells (Control: 82.9±5.6; Sham: 76.5±3.3; Illuminated: 77.1±4.2; ± Standard error of mean [SEM]) and ICM cells (Control: 10.8±1.3; Sham: 9.4±0.7; Illuminated: 11.9±0.8; ± SEM) did not differ between groups (p > 0.05). Outgrowth assays presented similar outgrowth areas during day5 to day8 post-blastocyst development between study groups (p > 0.05). Illuminated embryos presented significantly different NADH activity levels during embryo development, particularly between the 2-cell stage (987.1±36.2AU), morulae stage (1226±31.5AU) and blastocyst stage (649±42.9AU; ± SEM; p < 0.05). Embryos that did not reach the blastocyst stage presented a significantly different NADH activity profile during embryo development compared to those that did(p < 0.05). Additionally, abnormal embryos also presented significantly decreased NADH activity levels at the 2-cell stage (Normal: 987.1±36.2; abnormal: 726.9±121.7AU; p < 0.05) to the morulae stage (Normal: 1226±31.5; Abnormal:893.3±189AU; p < 0.05). Our study indicates that measuring NADH activity levels during early embryo development present no negative effects in embryo developmental rates, blastocyst formation and implantation potential. Thus, non-invasive measurements of NADH could be applied to determine embryo metabolic activity during embryo development using simple technology and imaging techniques. Limitations, reasons for caution The study was conducted using a mouse model focused in early embryo development and implantation potential. Thus, studies on live birth are required to fully assess safety to further validate potential wider applications. Validation in ageing models is also required to assess potential applications for embryo selection. Wider implications of the findings Non-invasive measurements of metabolic activity could be applied to determine embryo metabolic activity using simple and safe technology. Further applications could link the use of simple non-invasive metabolic imaging with the latest time-lapse technology and artificial intelligence applications. Trial registration number N/A

Validation and scalability of homemade polycaprolactone macrobeads grafted with thermo-responsive poly(N-isopropylacrylamide) for mesenchymal stem cell expansion and harvesting.

In this study, polycaprolactone (PCL) macrobeads were prepared by an oil‐in‐water (o/w) emulsion solvent evaporation method with poly(vinyl alcohol) (PVA) as an emulsifier and conjugated to poly(N‐isopropylacrylamide) (PNIPAAm) to be used as cell carriers with noninvasive cell detachment properties (thermo‐response). Following previous studies with PCL‐PNIPAAm carriers, our objectives were to confirm the successful conjugation on homemade macrobeads and to show the advantages of homemade production over commercial beads to control morphological, biological, and fluidization properties. The effects of PCL concentration on the droplet formation and of flow rate and PVA concentration on the size of the beads were demonstrated. The size of the beads, all spherical, ranged from 0.5 to 3.7 mm with four bead categories based on production parameters. The morphology and size of the beads were observed by scanning electron microscopy to show surface roughness enhancing cell attachment and proliferation compared to commercial beads. The functionalization steps with PNIPAAm were then characterized and confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy, and energy dispersion spectroscopy. PNIPAAm‐grafted macrobeads allowed mesenchymal stem cells (MSCs) to spread and grow for up to 21 days. By reducing the temperature to 25°C, the MSCs were successfully detached from the PCL‐PNIPAAm beads as observed with fluorescence microscopy. Furthermore, we validated the scalability potential of both macrobeads production and conjugation with PCL, to produce easily kilograms of thermo‐responsive macrocarriers in a lab environment. This could help moving such approaches towards clinically and industrially relevant processes were cell expansion is needed at very large scale.

Assessing the growth kinetics and stoichiometry of Escherichia coli at the single-cell level.

Microfluidic cultivation and single-cell analysis are inherent parts of modern microbial biotechnology and microbiology. However, implementing biochemical engineering principles based on the kinetics and stoichiometry of growth in microscopic spaces remained unattained. We here present a novel integrated framework that utilizes distinct microfluidic cultivation technologies and single-cell analytics to make the fundamental math of process-oriented biochemical engineering applicable at the single-cell level. A combination of non-invasive optical cell mass determination with sub-pg sensitivity, microfluidic perfusion cultivations for establishing physiological steady-states, and picoliter batch reactors, enabled the quantification of all physiological parameters relevant to approximate a material balance in microfluidic reaction environments. We determined state variables (biomass concentration based on single-cell dry weight and mass density), biomass synthesis rates, and substrate affinities of cells grown in microfluidic environments. Based on this data, we mathematically derived the specific kinetics of substrate uptake and growth stoichiometry in glucose-grown Escherichia coli with single-cell resolution. This framework may initiate microscale material balancing beyond the averaged values obtained from populations as a basis for integrating heterogeneous kinetic and stoichiometric single-cell data into generalized bioprocess models and descriptions.

Label-free, non-invasive, and repeatable cell viability bioassay using dynamic full-field optical coherence microscopy and supervised machine learning.

We present a novel method that can assay cellular viability in real-time using supervised machine learning and intracellular dynamic activity data that is acquired in a label-free, non-invasive, and non-destructive manner. Cell viability can be an indicator for cytology, treatment, and diagnosis of diseases. We applied four supervised machine learning models on the observed data and compared the results with a trypan blue assay. The cell death assay performance by the four supervised models had a balanced accuracy of 93.92 ± 0.86%. Unlike staining techniques, where criteria for determining viability of cells is unclear, cell viability assessment using machine learning could be clearly quantified.

Efficiency assessment of irrigation as an alternative method for improving the regenerative potential of non-healing wounds.

The application of mesenchymal stem/stromal cells (MSC) in regenerative medicine offers hope for the effective treatment of incurable or difficult‐to‐heal diseases. However, it requires the development of unified protocols for both safe and efficient cell acquisition and clinical usage. The therapeutic effect of fat grafts (containing stem cells) in non‐healing wounds has been discussed in previous studies, although the application requires local or general anaesthesia. The treatment of MSC derived from adipose tissue (ASC) could be a less invasive method, and efficient delivery could lead to more favourable outcomes, which should encourage clinicians to use such therapeutic approaches more frequently. Therefore, the aim of this study was to optimise the methods of ASC isolation, culture and administration while maintaining their high survival, proliferation and colonisation potential. The ASC were isolated by an enzymatic method and were characterised according to International Society for Cellular Therapy and International Federation for Adipose Therapeutics and Science guidelines. To assess the opportunity to obtain a sufficient number of cells for transplantation, long‐term cell cultures in two oxygen concentrations (5% vs. 21%) were conducted. For these cultures, the population doubling time, the cumulative time for cell population doublings and the rate of cell senescence were estimated. In a developed and pre‐defined protocol, ASC can be efficiently cultured at physiological oxygen concentrations (5%), which leads to faster proliferation and slower cell senescence. Subsequently, to select the optimal and minimally invasive methods of ASC transplantation, direct cell application with an irrigator or with skin dressings was analysed. Our results confirmed that both the presented methods of cell application allow for the safe delivery of isolated ASC into wounds without losing their vitality. Cells propagated in the described conditions and applied in non‐invasive cell application (with an irrigation system and dressings) to treat chronic wounds can be a potential alternative or supplement to more invasive clinical approaches.

Blood-brain barrier-penetrating single CRISPR-Cas9 nanocapsules for effective and safe glioblastoma gene therapy.

We designed a unique nanocapsule for efficient single CRISPR-Cas9 capsuling, noninvasive brain delivery and tumor cell targeting, demonstrating an effective and safe strategy for glioblastoma gene therapy. Our CRISPR-Cas9 nanocapsules can be simply fabricated by encapsulating the single Cas9/sgRNA complex within a glutathione-sensitive polymer shell incorporating a dual-action ligand that facilitates BBB penetration, tumor cell targeting, and Cas9/sgRNA selective release. Our encapsulating nanocapsules evidenced promising glioblastoma tissue targeting that led to high PLK1 gene editing efficiency in a brain tumor (up to 38.1%) with negligible (less than 0.5%) off-target gene editing in high-risk tissues. Treatment with nanocapsules extended median survival time (68 days versus 24 days in nonfunctional sgRNA-treated mice). Our new CRISPR-Cas9 delivery system thus addresses various delivery challenges to demonstrate safe and tumor-specific delivery of gene editing Cas9 ribonucleoprotein for improved glioblastoma treatment that may potentially be therapeutically useful in other brain diseases.

DNA Engineered Lymphocyte-Based Homologous Targeting Artificial Antigen-Presenting Cells for Personalized Cancer Immunotherapy.

Artificial antigen-presenting cells (aAPCs) constructed by integrating T cell activation ligands on biocompatible materials hold great potential in tumor immunotherapy. However, it remains challenging to develop aAPCs, which could mimic the characteristics of natural APCs, thereby realizing antigen-specific T cells activation in vivo. Here, we report the first effort to construct natural lymphocyte-based homologous targeting aAPCs (LC-aAPCs) with lipid-DNA-mediated noninvasive live cell surface engineering. Through a predesigned bottom-up self-assembly path, we achieved natural-APC-mimicking distribution of T cell activation ligands on LC-aAPCs, which would enable the optimized T cell activation. Moreover, the lipid-DNA-mediated self-assembly occurring on lipid bilayers would not affect the functions of homing receptors expressed on lymphocyte. Therefore, such LC-aAPCs could actively migrate to peripheral lymphatic organs and then effectively activate antigen-specific T cells. Combined with an immune checkpoint inhibitor, such LC-aAPCs could effectively inhibit the growth of different tumor models. Thus, our work provides a new design of aAPCs for in vivo applications in tumor immunotherapy, and the lipid-DNA-mediated noninvasive live cell surface engineering would be a powerful tool for designing cell-based therapeutics.

Development of self-powered multifunctional piezomagnetic nanoparticles for non-invasive post-surgical osteosarcoma theranogeneration

Non-invasive treatment approaches are gaining increased attention in recent times due to the potential improvement in treatment efficiency. Here we report a brand-new ultrasound (US), magnetic field (MF), and light responsive nanoparticle system based on piezoelectric barium titanate and superparamagnetic iron oxide nanoparticles termed as PiezoMagnetic nanoparticles (PMNPs) for the multimodal anticancer treatment, complimentary imaging, and non-invasive cell stimulation. Systematic electrical studies reveal that PMNPs exhibit superior ferroelectric and conducting properties and produce electrical signals analogous to native tissues when mechanically stimulated and using Food and Drug Administration (FDA) approved Low intensity Pulsed Ultrasound (LIPUS) non-invasively for cell stimulation. The green energy generation from PMNPs are further proved by the real-time demonstration of commercial LED lit-up. Thus, as a proof of concept, we have demonstrated a stepwise treatment efficacy of PMNPs for the post-surgical osteosarcoma therapy, diagnosis, and regeneration (termed as Theranogeneration). The PMNPs exhibited excellent anticancer properties to eradicate the residual osteosarcoma cells by magnetic hyperthermia, photothermal and photodynamic therapy along with complimentary imaging capabilities as X-ray computed tomography contrast agent and label free fluorescent probe. The conjugation of risedronate R (RIS)- a new generation bisphosphonates to PMNPs imparted bone seeking property to PMNPs. Therefore, as a result of noninvasive cell stimulation properties of PMNPs under LIPUS and LIPUS mediated RIS release from PMNPs resulted in enhanced bone regeneration and bone turnover which helps to repair the large bone defects occurs in osteosarcoma resection. Thus, the study corroborates the development of a potential bioactive piezoelectric nanoplatform that can be used to control the cell behaviour and fate in order to develop new types of therapies for multiple diseases including various types of cancers in remotely controlled approaches.

Integrated microfluidic device to monitor unseen Escherichia coli contamination in mammalian cell culture

Microbial contamination in in vitro cell culture technologies is a common problem endangering the reliability of the collected data and biosafety of the biological products. Conventionally, microbial contamination can be detected by monitoring the pH and turbidity of cell culture media when using light microscope and/or molecular probes. These methods have limitations such as incompatibility with some cell lines, requirement for human intervention, and long experimental times. Here we present a new integrated microfluidic device (IMD) for the detection of the presence of Escherichia coli (E. coli) in benign prostatic hyperplasia (BPH-1) cell culture. Cell culture media acidity was monitored using an optofluidic sensor, and the cellular glucose metabolism using an electrochemical sensor having in-line continuous monitoring capability. Using this IMD, detection of a bacterial contamination at stages in which it is undetectable using a light microscope and plate counting methods is achieved. Additionally, since even the short-term (less than 8 h) presence of E. coli can change the cellular metabolism in the long term (at least up to 24 h), the use of non-invasive continuous cell culture monitoring platforms significantly reduce the risk of an undetected contamination, increasing the reliability of the conducted experiments with no interference to the cell culture process.

Urine-Derived Stem Cells for Regenerative Medicine: Basic Biology, Applications, and Challenges.

Regenerative medicine based on stem cell research has the potential to provide advanced health care for human beings. Recent studies demonstrate that stem cells in human urine can serve as an excellent source of graft cells for regenerative therapy, mainly due to simple, low-cost, and noninvasive cell isolation. These cells, termed human urine-derived stem cells (USCs), are highly expandable and can differentiate into various cell lineages. They share many biological properties with mesenchymal stem cells, such as potent paracrine effects and immunomodulation ability. The advantage of USCs has motivated researchers to explore their applications in regenerative medicine, including genitourinary regeneration, musculoskeletal repair, skin wound healing, and disease treatment. Although USCs have showed many positive outcomes in preclinical studies, and although the possible applications of USCs for animal therapy have been reported, many issues need to be addressed before clinical translation. This article provides a comprehensive review of USC biology and recent advances in their application for tissue regeneration. Challenges in the clinical translation of USC-based therapy are also discussed. Impact statement Recently, stem cells isolated from urine, referred to as urine-derived stem cells (USCs), have gained much interest in the field of regenerative medicine. Many advantages of human USCs have been found for cell-based therapy: (i) the cell isolation procedure is simple and low cost; (ii) they have remarkable proliferation ability, multidifferentiation potential, and paracrine effects; and (iii) they facilitate tissue regeneration in many animal models. With the hope to facilitate the development of USC-based therapy, we describe the current understanding of USC biology, summarize recent advances in their applications, and discuss future challenges in clinical translation.

Robotic Patch Clamp Based on Noninvasive 3-D Cell Morphology Measurement for Higher Success Rate

The formation of the gigaseal, a giga Omega-scale seal between the micropipette electrode and cell surface, is the keystone to measuring the extremely weak electric signals of cell ion channels in patch clamp technique. To determine a point on the cell surface facilitating gigaseal formation, 3D cell morphology information is required. However, the current 3D cell morphology measurement methods relying on special devices or easily causing clogging issues of the micropipette electrode due to contact on cells, are usually not applicable in the traditional patch clamp system. Addressing this, a noninvasive 3D cell morphology measurement method was developed in this paper for robotic patch clamp with a higher success rate of gigaseal formation. First, the measured bath impedance of the micropipette electrode was modeled and then utilized to measure the cell surface height noninvasively. Using the measured cell surface heights at key positions, the 3D cell morphology was fitted to determine a contact position on the cell surface to facilitate gigaseal formation. Finally, a robotic whole-patch clamp process was conducted at the determined contact position. Experimental results demonstrated that 100% and 90% success rates of noninvasive 3D cell morphology measurements were achieved on the cultured HEK-293 cells and pyramidal neurons in mouse brain slices, respectively, with an average measurement error of cell surface height less than 0.15 μm. At the determined contact position, significant improvements in success rates of gigaseal formation and whole-cell patch clamp operation were achieved in comparison to the results at the positions recommended in related work. Our research may spike inspiration to improve the success rates of gigaseal formation and patch clamp operation based on 3-D cell morphologies.

Hyperspectral CARS microscopy and quantitative unsupervised analysis of deuterated and non-deuterated fatty acid storage in human cells.

Coherent anti-Stokes Raman scattering (CARS) implemented as a vibrational micro-spectroscopy modality eradicates the need for potentially perturbative fluorescent labeling while still providing high-resolution, chemically specific images of biological samples. Isotopic substitution of hydrogen atoms with deuterium introduces minimal change to molecular structures and can be coupled with CARS microscopy to increase chemical contrast. Here, we investigate HeLa cells incubated with non-deuterated or deuterium-labeled fatty acids, using an in-house-developed hyperspectral CARS microscope coupled with an unsupervised quantitative data analysis algorithm, to retrieve Raman susceptibility spectra and concentration maps of chemical components in physically meaningful units. We demonstrate that our unsupervised analysis retrieves the susceptibility spectra of the specific fatty acids, both deuterated and non-deuterated, in good agreement with reference Raman spectra measured in pure lipids. Our analysis, using the cell-silent spectral region, achieved excellent chemical specificity despite having no prior knowledge and considering the complex intracellular environment inside cells. The quantitative capabilities of the analysis allowed us to measure the concentration of deuterated and non-deuterated fatty acids stored within cytosolic lipid droplets over a 24 h period. Finally, we explored the potential use of deuterium-labeled lipid droplets for non-invasive cell tracking, demonstrating an effective application of the technique for distinguishing between cells in a mixed population over a 16 h period. These results further demonstrate the chemically specific capabilities of hyperspectral CARS microscopy to characterize and distinguish specific lipid types inside cells using an unbiased quantitative data analysis methodology.

Noninvasive manipulation of cell adhesion for cell harvesting with piezoelectric composite film

Cell sheet harvesting has recently emerged as a promising strategy for scaffold-free cell transplantation and tissue engineering. It is critical to achieve the disconnection between cell and its adherent support while maintaining the availability of cell sheet. Hence, the regulation of cell adhesion behavior often becomes the primary consideration for designing biomaterials used for cell sheet harvesting. The stimuli-responsive biomaterials can rationally modulate cell behaviors, especially adhesion performance. However, modulation of cell adhesion through noninvasive and highly efficient method is still greatly needed. Herein, a noninvasive low-intensity ultrasound-mediated cell harvesting is demonstrated based on the piezoelectric effect of polyvinylidene fluoride (PVDF)/barium titanate (BaTiO3, BTO) composite film with superior electromechanical conversion ability. We found that the piezoelectric effect triggered by ultrasound (US) can modulate the absorption and secondary structure of adhesive protein fibronectin (FN) on the composite film, which further influenced cell adhesion force to finally obtain free-standing cell sheets. The detached cell sheets with accurate comtrol retained their viability and excellent proliferative capability. These findings highlight the ability of the piezoelectric biomaterials for cell sheet engineering to serve as a noninvasive and accurately controllable platform via regulating surface protein adsorption and conformation, which indicates great application prospect of this piezoelectricity-based cell harvesting method in regenerative medicine.

Volumetric 23Na Single and Triple-Quantum Imaging at 7T: 3D-CRISTINA

PurposeTo measure multi-quantum coherence (MQC) 23Na signals for noninvasive cell physiological information in the whole-brain, the 2D-CRISTINA method was extended to 3D. This experimental study investigated the use and results of a new sequence, 3D-CRISTINA, on a phantom and healthy volunteers. MethodsThe 3D Cartesian single and triple-quantum imaging of 23Na (3D-CRISTINA) was developed and implemented at 7T, favoring a non-selective volume excitation for increased signal-to-noise ratio (SNR) and lower energy deployment than its 2D counterpart. Two independent phase cycles were used in analogy to the 2D method. A comparison of 6-steps cycles and 12-steps cycles was performed.We used a phantom composed of different sodium and agarose concentrations, 50mM to 150mM Na+, and 0–5% agarose for sequence validation. Four healthy volunteers were scanned at 7T for whole brain MQC imaging. The sequence 3D-CRISTINA was developed and tested at 7T. ResultsAt 7T, the 3D-CRISTINA acquisition allowed to reduce the TR to 230ms from the previous 390ms for 2D, resulting in a total acquisition time of 53min for a 3D volume of 4×4×8mm resolution. The phase cycle evaluation showed that the 7T acquisition time could be reduced by 4-fold with moderate single and triple-quantum signals SNR loss. The healthy volunteers demonstrated clinical feasibility at 7T and showed a difference in the MQC signals ratio of White Matter (WM) and Grey Matter (GM). ConclusionVolumetric CRISTINA multi-quantum imaging allowed whole-brain coverage. The non-selective excitation enabled a faster scan due to a decrease in energy deposition which enabled a lower repetition time. Thus, it should be the preferred choice for future in vivo multi-quantum applications compared to the 2D method. A more extensive study is warranted to explore WM and GM MQC differences.

Repeated exposure of the oral mucosa over 12\xa0months with cold plasma is not carcinogenic in mice

Peri-implantitis may result in the loss of dental implants. Cold atmospheric pressure plasma (CAP) was suggested to promote re-osseointegration, decrease antimicrobial burden, and support wound healing. However, the long-term risk assessment of CAP treatment in the oral cavity has not been addressed. Treatment with two different CAP devices was compared against UV radiation, carcinogen administration, and untreated conditions over 12 months. Histological analysis of 406 animals revealed that repeated CAP exposure did not foster non-invasive lesions or squamous cell carcinoma (SCCs). Carcinogen administration promoted non-invasive lesions and SCCs. Molecular analysis by a qPCR screening of 144 transcripts revealed distinct inflammatory profiles associated with each treatment regimen. Interestingly, CAP treatment of carcinogen-challenged mucosa did not promote but instead left unchanged or reduced the proportion of non-invasive lesions and SCC formation. In conclusion, repeated CAP exposure of murine oral mucosa was well tolerated, and carcinogenic effects did not occur, motivating CAP applications in patients for dental and implant treatments in the future.

Cell proliferation detected using [18F]FLT PET/CT as an early marker of abdominal aortic aneurysm

BackgroundAbdominal aortic aneurysm (AAA) is a focal aortic dilatation progressing towards rupture. Non-invasive AAA-associated cell proliferation biomarkers are not yet established. We investigated the feasibility of the cell proliferation radiotracer, fluorine-18-fluorothymidine ([18F]FLT) with positron emission tomography/computed tomography (PET/CT) in a progressive pre-clinical AAA model (angiotensin II, AngII infusion). Methods and ResultsFourteen-week-old apolipoprotein E-knockout (ApoE−/−) mice received saline or AngII via osmotic mini-pumps for 14 (n = 7 and 5, respectively) or 28 (n = 3 and 4, respectively) days and underwent 90-minute dynamic [18F]FLT PET/CT. Organs were harvested from independent cohorts for gamma counting, ultrasound scanning, and western blotting. [18F]FLT uptake was significantly greater in 14- (n = 5) and 28-day (n = 3) AAA than in saline control aortae (n = 5) (P < 0.001), which reduced between days 14 and 28. Whole-organ gamma counting confirmed greater [18F]FLT uptake in 14-day AAA (n = 9) compared to saline-infused aortae (n = 4) (P < 0.05), correlating positively with aortic volume (r = 0.71, P < 0.01). Fourteen-day AAA tissue showed increased expression of thymidine kinase-1, equilibrative nucleoside transporter (ENT)-1, ENT-2, concentrative nucleoside transporter (CNT)-1, and CNT-3 than 28-day AAA and saline control tissues (n = 3 each) (all P < 0.001). Conclusions[18F]FLT uptake is increased during the active growth phase of the AAA model compared to saline control mice and late-stage AAA.

Features in the Resonance Behavior of Magnetization in Arrays of Triangular and Square Nanodots

Collective modes of the gyrotropic motion of a magnetic vortex core in ordered arrays of triangular and square ferromagnetic film nanodots have been theoretically investigated. The dispersion relations have been derived. The dipole–dipole interaction of the magnetic moments of the magnetic vortex cores of elements has been taken into account in the approximation of a small shift from the equilibrium position. It is shown that the effective rigidity of the magnetic subsystem of triangular elements is noticeably higher than that of the subsystem of square elements of the same linear sizes. The potential application of the polygonal film nanodisks as nanoscalpels for noninvasive tumor cell surgery is discussed

CNS Hypomyelination Disrupts Axonal Conduction and Behavior in Larval Zebrafish.

Myelination is essential for central nervous system (CNS) formation, health and function. As a model organism, larval zebrafish have been extensively employed to investigate the molecular and cellular basis of CNS myelination, because of their genetic tractability and suitability for non-invasive live cell imaging. However, it has not been assessed to what extent CNS myelination affects neural circuit function in zebrafish larvae, prohibiting the integration of molecular and cellular analyses of myelination with concomitant network maturation. To test whether larval zebrafish might serve as a suitable platform with which to study the effects of CNS myelination and its dysregulation on circuit function, we generated zebrafish myelin regulatory factor (myrf) mutants with CNS-specific hypomyelination and investigated how this affected their axonal conduction properties and behavior. We found that myrf mutant larvae exhibited increased latency to perform startle responses following defined acoustic stimuli. Furthermore, we found that hypomyelinated animals often selected an impaired response to acoustic stimuli, exhibiting a bias toward reorientation behavior instead of the stimulus-appropriate startle response. To begin to study how myelination affected the underlying circuitry, we established electrophysiological protocols to assess various conduction properties along single axons. We found that the hypomyelinated myrf mutants exhibited reduced action potential conduction velocity and an impaired ability to sustain high-frequency action potential firing. This study indicates that larval zebrafish can be used to bridge molecular and cellular investigation of CNS myelination with multiscale assessment of neural circuit function.SIGNIFICANCE STATEMENT Myelination of CNS axons is essential for their health and function, and it is now clear that myelination is a dynamic life-long process subject to modulation by neuronal activity. However, it remains unclear precisely how changes to myelination affects animal behavior and underlying action potential conduction along axons in intact neural circuits. In recent years, zebrafish have been employed to study cellular and molecular mechanisms of myelination, because of their relatively simple, optically transparent, experimentally tractable vertebrate nervous system. Here we find that changes to myelination alter the behavior of young zebrafish and action potential conduction along individual axons, providing a platform to integrate molecular, cellular, and circuit level analyses of myelination using this model.