Knockdown of YAP/TAZ sensitizes tamoxifen-resistant MCF7 breast cancer cells.

Although endocrine therapy with tamoxifen has improved survival in breast cancer patients, resistance to this therapy remains one of the major causes of breast cancer mortality. In the present study, we found that the expression level of YAP/TAZ in tamoxifen-resistant MCF7 (MCF7-TR) breast cancer cells was significantly increased compared with that in MCF7 cells. Knockdown of YAP/TAZ with siRNA sensitized MCF7-TR cells to tamoxifen. Furthermore, siRNA targeting PSAT1, a downstream effector of YAP/TAZ, enhanced sensitivity to tamoxifen in MCF7-TR cells. Additionally, mTORC1 activity and survivin expression were significantly decreased during cell death induced by combination treatment with YAP/TAZ or PSAT1 siRNA and tamoxifen. In conclusion, targeting the YAP/TAZ-PSAT1 axis could sensitize tamoxifen-resistant MCF7 breast cancer cells by modulating the mTORC1-survivin axis.

Oxidative Stress Amplifying Polyprodrug Micelles as Drug Carriers for Combination Anticancer Therapy.

Cancer cells are more vulnerable to reactive oxygen species (ROS)-mediated oxidative stress than normal cells due to disturbed redox balance. It can be postulated that ROS-generating drug carriers exert anticancer actions, leading to combination anticancer therapy with drug payloads. Here, we report a ROS-generating polyprodrug of cinnamaldehyde (CA) that not only serves as a drug carrier but also synergizes with drug payloads. The polyprodrug of CA (pCA) incorporates ROS-generating CA in the backbone of an amphiphilic polymer through an acid-cleavable acetal linkage. pCA could self-assemble with tumor-targeting lipopeptide (DSPE-PEG-RGD) and encapsulate doxorubicin (DOX) to form T-pCAD micelles. At acidic pH, T-pCAD micelles release both CA and DOX to exert synergistic anticancer actions. Animal studies using mouse xenograft models revealed that T-pCAD micelles accumulate in tumors preferentially and suppress the tumor growth significantly. Based on the oxidative stress amplification and acid-responsiveness, ROS-generating pCAD micelles hold tremendous potential as drug carriers for combination anticancer therapy.

Suture Fiber Reinforcement of a 3D Printed Gelatin Scaffold for Its Potential Application in Soft Tissue Engineering.

Gelatin has excellent biological properties, but its poor physical properties are a major obstacle to its use as a biomaterial ink. These disadvantages not only worsen the printability of gelatin biomaterial ink, but also reduce the dimensional stability of its 3D scaffolds and limit its application in the tissue engineering field. Herein, biodegradable suture fibers were added into a gelatin biomaterial ink to improve the printability, mechanical strength, and dimensional stability of the 3D printed scaffolds. The suture fiber reinforced gelatin 3D scaffolds were fabricated using the thermo-responsive properties of gelatin under optimized 3D printing conditions (-10 °C cryogenic plate, 40-80 kPa pneumatic pressure, and 9 mm/s printing speed), and were crosslinked using EDC/NHS to maintain their 3D structures. Scanning electron microscopy images revealed that the morphologies of the 3D printed scaffolds maintained their 3D structure after crosslinking. The addition of 0.5% (w/v) of suture fibers increased the printing accuracy of the 3D printed scaffolds to 97%. The suture fibers also increased the mechanical strength of the 3D printed scaffolds by up to 6-fold, and the degradation rate could be controlled by the suture fiber content. In in vitro cell studies, DNA assay results showed that human dermal fibroblasts' proliferation rate of a 3D printed scaffold containing 0.5% suture fiber was 10% higher than that of a 3D printed scaffold without suture fibers after 14 days of culture. Interestingly, the supplement of suture fibers into gelatin biomaterial ink was able to minimize the cell-mediated contraction of the cell cultured 3D scaffolds over the cell culture period. These results show that advanced biomaterial inks can be developed by supplementing biodegradable fibers to improve the poor physical properties of natural polymer-based biomaterial inks.

Ionizing radiation attracts tumor targeting and apoptosis by radiotropic lysyl oxidase traceable nanoparticles.

Lysyl oxidase (LOX) is a cell-secreted amine oxidase that crosslinks collagen and elastin in extracellular microenvironment. LOX-traceable nanoparticles (LOXab-NPs) consisting of LOX antibodies (LOXab) and paclitaxel, can accumulate at high concentrations at radiation-treated target sites, as a tumor-targeting drug carrier for chemotherapy. Tumor-targeting and anticancer effects of PLGA based LOXab-NPs in vitro and in vivo were evaluated at radiation-targeted site. In the in vivo A549 lung carcinoma xenograft model, we showed highly specific tumor targeting (above 7.0 times higher) of LOXab-NPs on irradiated tumors. Notably, systemically administered NPs delayed tumor growth, reducing tumor volumes by more than 2 times compared with non-irradiated groups (222% vs. >500%) over 2 weeks. Radiotropic LOXab-NPs can serve as chemotherapeutic vehicles for combined targeted chemo-radiotherapy in clinical oncology.

Fabrication of Vertically Aligned Liquid Crystals Devices Using Nanoscale Self-Assembled Alignment Layers of Photoluminescent Pyrene Derivatives.

Nanoscale self-assembled molecular layers of 1-pyrene carboxylic acid, 1-pyrene butylic acid, and 1-amino pyrene were employed as photoluminescent alignment agents to form vertical arrays of liquid crystals (LCs). The LC device using alignment agent of 1-amino pyrene appeared to have a stable vertical alignment based on crossed polarization analysis. The electro-optical properties of the LCs fabricated using this self-assembled layer exhibited better performance than those of general LC cells containing a conventional polyimide layer. The proposed self-assembled alignment method is sufficient as a replacement for more commonly used polyimide layers, which require complex film forming processing and high production costs; conversely, the proposed technique allows for simple mixing using low concentrations of alignment agents (0.05 wt%). Moreover, as LCs were mixed with the pyrene derivatives, photoluminescence (PL) under ultraviolet (UV) light was exhibited. This proposed method has excellent LC alignment and PL property, both of which can be utilized in advanced display technologies for next-generation LC devices, such as polyimide-free alignment or color-filter-free color expression.

Chitosan for Tissue Engineering.

Chitosan, a deacetylated chitin, is one of the few natural polymers similar to glycosaminoglycans (GAGs) widely distributed throughout connective tissues. It has been believed that the excellent biocompatibility of chitosan is largely attributed to this structural similarity. Chitosan is also known to possess biodegradability, antimicrobial activity and low toxicity and immunogenicity which are essential for scaffolds. In addition, the existence of free amine groups in its backbone chain enables further chemical modifications to create the additional biomedical functionality. For these reasons, chitosan has found a tremendous variety of biomedical applications in recent years. This chapter introduces the basic contents of chitosan and discusses its applications to artificial skin, artificial bone, and artificial cartilage in tissue engineering purpose.

3D Bioprinting Technologies for Tissue Engineering Applications.

Three-dimensional (3D) printing (rapid prototyping or additive manufacturing) technologies have received significant attention in various fields over the past several decades. Tissue engineering applications of 3D bioprinting, in particular, have attracted the attention of many researchers. 3D scaffolds produced by the 3D bioprinting of biomaterials (bio-inks) enable the regeneration and restoration of various tissues and organs. These 3D bioprinting techniques are useful for fabricating scaffolds for biomedical and regenerative medicine and tissue engineering applications, permitting rapid manufacture with high-precision and control over size, porosity, and shape. In this review, we introduce a variety of tissue engineering applications to create bones, vascular, skin, cartilage, and neural structures using a variety of 3D bioprinting techniques.

Neuropeptide Substance-P-Conjugated Chitosan Nanofibers as an Active Modulator of Stem Cell Recruiting.

The goal to successful wound healing is essentially to immobilize and recruit appropriate numbers of host stem or progenitor cells to the wound area. In this study, we developed a chitosan nanofiber-immobilized neuropeptide substance-P (SP), which mediates stem cell mobilization and migration, onto the surfaces of nanofibers using a peptide-coupling agent, and evaluated its biological effects on stem cells. The amount of immobilized SP on chitosan nanofibers was modulated over the range of 5.89 ± 3.27 to 75.29 ± 24.31 ng when reacted with 10 to 500 ng SP. In vitro migration assays showed that SP-incorporated nanofibers induced more rapid migration of human mesenchymal stem cells on nanofibers compared to pristine samples. Finally, the conjugated SP evoked a minimal foreign body reaction and recruited a larger number of CD29- and CD44-positive stem cells into nanofibers in a mouse subcutaneous pocket model.

Inhibition of HAS2 induction enhances the radiosensitivity of cancer cells via persistent DNA damage.

Hyaluronan synthase 2 (HAS2), a synthetic enzyme for hyaluronan, regulates various aspects of cancer progression, including migration, invasion and angiogenesis. However, the possible association of HAS2 with the response of cancer cells to anticancer radiotherapy, has not yet been elucidated. Here, we show that HAS2 knockdown potentiates irradiation-induced DNA damage and apoptosis in cancer cells. Upon exposure to radiation, all of the tested human cancer cell lines exhibited marked (up to 10-fold) up-regulation of HAS2 within 24h. Inhibition of HAS2 induction significantly reduced the survival of irradiated radioresistant and -sensitive cells. Interestingly, HAS2 depletion rendered the cells to sustain irradiation-induced DNA damage, thereby leading to an increase of apoptotic death. These findings indicate that HAS2 knockdown sensitizes cancer cells to radiation via persistent DNA damage, further suggesting that the irradiation-induced up-regulation of HAS2 contributes to the radioresistance of cancer cells. Thus, HAS2 could potentially be targeted for therapeutic interventions aimed at radiosensitizing cancer cells.

Selectively micro-patterned fibronectin for regulating fate of human mesenchymal stem cell.

Microenvironment of the extracellular matrix can influence cellular responses through alternation of initial attachment and induce production of new tissue. To study the effect of such microenvironment on the relationship of cell cytoskeletal shape and its biological behaviors such as adhesion, proliferation and differentiation, we designed a patterned strip line of fibronectin on self assembled monolayers via microcontact printing. The physiological behavior of human mesenchymal stem cell (hMSC) on defined micro-patterns of fibronectin was evaluated after 4 h and 2 days of culture. Initial adhesion of hMSCs on a substrate with pattern spacing of 11 µm was stabilized faster than that on other substrates. Ratio of proliferating hMSC on 5 and 11 µm substrate constantly maintained a high rate. hMSCs on 5 and 11 µm substrate could adhere to substrate as spreading from fibronectin pattern line to several and lateral fibronectin pattern line. Their nucleus area could represent artificial increase by widely spreading on several fibronectin pattern lines. On the contrary to this, ratio of proliferating hMSC on 20 µm substrate constantly maintained a low rate less than even control and 0 µm substrate without fibronectin pattern. Tiny nucleus caused narrow and elongated hMSC morphology on 20 µm substrate gave the negative effect on the cell adhesion and proliferation. However, hMSCs on 20 µm substrate possessed not only slightly increased value of GO/G1 phase but also down regulation of CD marker expression compared with other groups. These results show initial adhesion and morphology of hMSC could regulate specific cellular behavior of hMSC.

Fabrication of conductive polymer-based nanofiber scaffolds for tissue engineering applications.

Natural and synthetic polymers, in particular those that are conductive, are of great interest in the field of tissue engineering and the pursuit of biomimetic extracellular matrix (ECM) structures for adhesion, proliferation, and differentiation of cells. In the present study, natural chitin and conductive polyaniline (PANi) blended solutions were electrospun to produce biodegradable and conductive biomimetic nanostructured scaffolds. The chitin/PANi (Chi-PANi) nanofibrous materials were characterized using field emission scanning electron microscopy, Fourier transform-infrared spectroscopy, wettability analysis, mechanical testing, and electrical conductivity measurements using a 4-point probe method. The calculated electrical conductivities of the PANi-containing nanofiber scaffolds significantly increased as the amount of PANi increased, reaching 5.21 ± 0.28 x 10(-3) S/cm for 0.3 wt% content of the conducting polymer. In addition, the viability of human mesenchymal stem cells (hMSCs) cultured on the Chi-PANi nanofiber scaffolds in vitro was found to be excellent. These results suggest that the Chi-PANi nanofiber scaffolds have great potential for use in tissue engineering applications that involve electrical stimulation.

Tumor-targeted and stimulus-responsive polymeric prodrug nanoparticles to enhance the anticancer therapeutic efficacy of doxorubicin.

Polymeric prodrug nanoparticles have gained increasing attention in the field of anticancer drug delivery because of their dual functions as a drug carrier and a therapeutic agent. Doxorubicin (DOX) is a highly effective chemotherapeutic agent for various cancers but causes cardiotoxicity. In this work, we developed polymeric prodrug (pHU) nanoparticles that serve as both a drug carrier of DOX and a therapeutic agent. The composition of pHU includes antiangiogenic hydroxybenzyl alcohol (HBA) and ursodeoxycholic acid (UDCA), covalently incorporated through hydrogen peroxide (H2O2)-responsive peroxalate. To enhance cancer cell specificity, pHU nanoparticles were surface decorated with taurodeoxycholic acid (TUDCA) to facilitate p-selectin-mediated cancer targeting. TUDCA-coated and DOX-loaded pHU nanoparticles (t-pHUDs) exhibited controlled release of DOX triggered by H2O2, characteristic of the tumor microenvironment. t-pHUDs also effectively suppressed cancer cell migration and vascular endothelial growth factor (VEGF) expression in response to H2O2. In animal studies, t-pHUDs exhibited highly potent anticancer activity. Notably, t-pHUDs, with their ability to accumulate preferentially in tumors due to the p-selectin targeting, surpassed the therapeutic efficacy of equivalent DOX and pHU nanoparticles alone. What is more, t-pHUDs significantly suppressed VEGF expression in tumors and mitigated hepato- and cardiotoxicity of DOX. Given their cancer targeting ability, enhanced therapeutic efficacy and minimized off-target toxicity, t-pHUDs present an innovative and targeted approach with great translational potential as an anticancer therapeutic agent.

Glycinamide Facilitates Nanocomplex Formation and Functions Synergistically with Bone Morphogenetic Protein 2 to Promote Osteoblast Differentiation In Vitro and Bone Regeneration in a Mouse Calvarial Defect Model.

BACKGROUND: This study aimed to identify glycine analogs conducive to the formation of cell-absorbable nanocomplexes, enhancing collagen synthesis and subsequent osteogenesis in combination with BMP2 for improved bone regeneration. METHODS: Glycine and its derivatives were assessed for their effects on osteogenic differentiation in MC3T3-E1 cells and human bone marrow mesenchymal stem cells (BMSCs) under osteogenic conditions or with BMP2. Osteogenic differentiation was assessed through alkaline phosphatase staining and real-time quantitative polymerase chain reaction (RT-qPCR). Nanocomplex formation was examined via scanning electron microscopy, circular dichroism, and ultraviolet-visible spectroscopy. In vivo osteogenic effects were validated using a mouse calvarial defect model, and bone regeneration was evaluated through micro-computed tomography and histomorphometric analysis. RESULTS: Glycine, glycine methyl ester, and glycinamide significantly enhanced collagen synthesis and ALP activity in conjunction with an osteogenic medium (OSM). GA emerged as the most effective inducer of osteoblast differentiation marker genes. Combining GA with BMP2 synergistically stimulated ALP activity and the expression of osteoblast markers in both cell lines. GA readily formed nanocomplexes, facilitating cellular uptake through strong electrostatic interactions. In an in vivo calvarial defect mouse model, the GA and BMP2 combination demonstrated enhanced bone volume, bone volume/tissue volume ratio, trabecular numbers, and mature bone formation compared to other combinations. CONCLUSION: GA and BMP2 synergistically promoted in vitro osteoblast differentiation and in vivo bone regeneration through nanocomplex formation. This combination holds therapeutic promise for individuals with bone defects, showcasing its potential for clinical intervention.

The Effect of the Mechanical Properties of the 3D Printed Gelatin/Hyaluronic Acid Scaffolds on hMSCs Differentiation Towards Chondrogenesis.

BACKGROUND: Tissue engineering, including 3D bioprinting, holds great promise as a therapeutic tool for repairing cartilage defects. Mesenchymal stem cells have the potential to treat various fields due to their ability to differentiate into different cell types. The biomimetic substrate, such as scaffolds and hydrogels, is a crucial factor that affects cell behavior, and the mechanical properties of the substrate have been shown to impact differentiation during incubation. In this study, we examine the effect of the mechanical properties of the 3D printed scaffolds, made using different concentrations of cross-linker, on hMSCs differentiation towards chondrogenesis. METHODS: The 3D scaffold was fabricated using 3D bioprinting technology with gelatin/hyaluronic acid (HyA) biomaterial ink. Crosslinking was achieved by using different concentrations of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methlymorpholinium chloride n-hydrate (DMTMM), allowing for control of the scaffold's mechanical properties. The printability and stability were also evaluated based on the concentration of DMTMM used. The effects of the gelatin/HyA scaffold on chondrogenic differentiation was analyzed by utilizing various concentrations of DMTMM. RESULTS: The addition of HyA was found to improve the printability and stability of 3D printed gelatin/HyA scaffolds. The mechanical properties of the 3D gelatin/HyA scaffold could be regulated through the use of different concentrations of DMTMM cross-linker. In particular, the use of 0.25 mM DMTMM for crosslinking the 3D gelatin/HyA scaffold resulted in enhanced chondrocyte differentiation. CONCLUSION: The mechanical properties of 3D printed gelatin/HyA scaffolds cross-linked using various concentrations of DMTMM can influence the differentiation of hMSCs into chondrocytes.

Radiation induces acute and subacute vascular regression in a three-dimensional microvasculature model.

Radiation treatment is one of the most frequently used therapies in patients with cancer, employed in approximately half of all patients. However, the use of radiation therapy is limited by acute or chronic adverse effects and the failure to consider the tumor microenvironment. Blood vessels substantially contribute to radiation responses in both normal and tumor tissues. The present study employed a three-dimensional (3D) microvasculature-on-a-chip that mimics physiological blood vessels to determine the effect of radiation on blood vessels. This model represents radiation-induced pathophysiological effects on blood vessels in terms of cellular damage and structural and functional changes. DNA double-strand breaks (DSBs), apoptosis, and cell viability indicate cellular damage. Radiation-induced damage leads to a reduction in vascular structures, such as vascular area, branch length, branch number, junction number, and branch diameter; this phenomenon occurs in the mature vascular network and during neovascularization. Additionally, vasculature regression was demonstrated by staining the basement membrane and microfilaments. Radiation exposure could increase the blockage and permeability of the vascular network, indicating that radiation alters the function of blood vessels. Radiation suppressed blood vessel recovery and induced a loss of angiogenic ability, resulting in a network of irradiated vessels that failed to recover, deteriorating gradually. These findings demonstrate that this model is valuable for assessing radiation-induced vascular dysfunction and acute and chronic effects and can potentially improve radiotherapy efficiency.

Tumor-targeted redox-regulating and antiangiogenic phototherapeutics nanoassemblies for self-boosting phototherapy.

Cancer cells are equipped with abundant antioxidants such as glutathione (GSH) that eliminate reactive oxygen species (ROS) to deteriorate the therapeutic efficacy of photodynamic therapy (PDT). Another challenge in PDT is circumventing PDT-induced hypoxic condition that provokes upregulation of pro-angiogenic factor such as vascular endothelial growth factor (VEGF). It is therefore reasonable to expect that therapeutic outcomes of PDT could be maximized by concurrent delivery of photosensitizers with GSH depleting agents and VEGF suppressors. To achieve cooperative therapeutic actions of PDT with in situ GSH depletion and VEGF suppression, we developed tumor targeted redox-regulating and antiangiogenic phototherapeutic nanoassemblies (tRAPs) composed of self-assembling disulfide-bridged borylbenzyl carbonate (ssBR), photosensitizer (IR780) and tumor targeting gelatin. As a framework of tRAPs, ssBR was rationally designed to form nanoconstructs that serve as photosensitizer carriers with intrinsic GSH depleting- and VEGF suppressing ability. tRAPs effectively depleted intracellular GSH to render cancer cells more vulnerable to ROS and also provoked immunogenic cell death (ICD) of cancer cells upon near infrared (NIR) laser irradiation. In mouse xenograft models, tRAPs preferentially accumulated in tumors and dramatically eradicated tumors with laser irradiation. The design rationale of tRAPs provides a simple and versatile strategy to develop self-boosting phototherapeutic agents with great potential in targeted cancer therapy.

Self-assembling biomolecules for biosensor applications.

Molecular self-assembly has received considerable attention in biomedical fields as a simple and effective method for developing biomolecular nanostructures. Self-assembled nanostructures can exhibit high binding affinity and selectivity by displaying multiple ligands/receptors on their surface. In addition, the use of supramolecular structure change upon binding is an intriguing approach to generate binding signal. Therefore, many self-assembled nanostructure-based biosensors have been developed over the past decades, using various biomolecules (e.g., peptides, DNA, RNA, lipids) and their combinations with non-biological substances. In this review, we provide an overview of recent developments in the design and fabrication of self-assembling biomolecules for biosensing. Furthermore, we discuss representative electrochemical biosensing platforms which convert the biochemical reactions of those biomolecules into electrical signals (e.g., voltage, ampere, potential difference, impedance) to contribute to detect targets. This paper also highlights the successful outcomes of self-assembling biomolecules in biosensor applications and discusses the challenges that this promising technology needs to overcome for more widespread use.

Nebivolol Sensitizes BT-474 Breast Cancer Cells to FGFR Inhibitors.

BACKGROUND/AIM: The fibroblast growth factor receptor (FGFR) signaling pathway is abnormally activated in human cancers, including breast cancer. Therefore, targeting the FGFR signaling pathway is a potent strategy to treat breast cancer. The purpose of this study was to find drugs that could increase sensitivity to FGFR inhibitor effects in BT-474 breast cancer cells, and to investigate the combined effects and underlying mechanisms of these combinations for BT-474 breast cancer cell survival. MATERIALS AND METHODS: Cell viability was measured by MTT assay. Protein expression was determined by western blot analysis. mRNA expression was detected by Real-time PCR. Drug synergy effect was determined by isobologram analysis. RESULTS: Nebivolol, a third generation ß1-blocker, synergistically increased the sensitivity of BT-474 breast cancer cells to the potent and selective FGFR inhibitors erdafitinib (JNJ-42756493) and AZD4547. A combination of nebivolol and erdafitinib markedly reduced AKT activation. Suppression of AKT activation using specific siRNA and a selective inhibitor further enhanced cell sensitivity to combined treatment with nebivolol and erdafitinib, whereas SC79, a potent activator of AKT, reduced cell sensitivity to nebivolol and erdafitinib. CONCLUSION: Enhanced sensitivity of BT-474 breast cancer cells to nebivolol and erdafitinib was probably associated with down-regulation of AKT activation. Combined treatment with nebivolol and erdafitinib is a promising strategy for breast cancer treatment.

Characterization of surface properties and cytocompatibility of ion-etched chitosan films.

Surface modification of biomaterials has been highlighted by biomedical engineers as a facile method for improving cell-biomaterial interactions without the expense and time required to develop new materials. In the present study, we investigated the influence of ion-etching on the surface characteristics of chitosan films using XPS and ATR FT-IR. The physiological behavior of human dermal fibroblasts (hDFs) grown on such surfaces was studied by evaluating adhesive and proliferative properties, and by examining surface morphologies of hDFs using AFM. hDFs displayed different shapes depending on the ion-etching time. hDFs grown on chitosan films ion-etched for 5 min displayed better development of lamellipodia and filopodia around the hDF periphery than did cells grown on nonmodified chitosan film, whereas hDFs did not spread well on films ion-etched for 20 min. Films ion-etched for 5 min or less had higher NH(2) and COOH contents, leading to enhanced hDF adhesion and proliferation.

Detection and quantification of a radiation-associated mitochondrial DNA deletion by a nested real-time PCR in human peripheral lymphocytes.

In this study we implemented a new assay using a nested real-time polymerase chain reaction (PCR) to detect radiation-induced common deletion (CD) in mitochondrial DNA (mtDNA) of human peripheral lymphocytes. A standard curve for real-time PCR was established by applying a plasmid DNA containing human normal mtDNA or mutated mtDNA. Human peripheral lymphocyte DNA was amplified and quantified by real-time PCR using primer sets for total damaged or mutated mtDNA, plus probes labeled with the fluorescent dyes. The first-round PCR generated multiple products were used as the template for a second-round PCR. We herein describe a nested real-time PCR assay capable of quantifying mtDNA bearing the CD in human peripheral lymphocytes following exposure (in vitro) to (137)Cs γ-rays in a dose range of 0.5 up to 5Gy. The reproducibility of this assay was evident for both unirradiated and irradiated samples by examining human blood lymphocytes from 14 donors. This technique was sensitive enough to detect deletions in mtDNA at low dose levels, as low as 0.5Gy, and higher levels of CD mtDNA were evident at higher doses (≥1Gy), however, there was no consistent dose-response relationship.