Multimodal Radiobioconjugates of Magnetic Nanoparticles Labeled with 44Sc and 47Sc for Theranostic Application.

This study was performed to synthesize multimodal radiopharmaceutical designed for the diagnosis and treatment of prostate cancer. To achieve this goal, superparamagnetic iron oxide (SPIO) nanoparticles were used as a platform for targeting molecule (PSMA-617) and for complexation of two scandium radionuclides, 44Sc for PET imaging and 47Sc for radionuclide therapy. TEM and XPS images showed that the Fe3O4 NPs have a uniform cubic shape and a size from 38 to 50 nm. The Fe3O4 core are surrounded by SiO2 and an organic layer. The saturation magnetization of the SPION core was 60 emu/g. However, coating the SPIONs with silica and polyglycerol reduces the magnetization significantly. The obtained bioconjugates were labeled with 44Sc and 47Sc, with a yield higher than 97%. The radiobioconjugate exhibited high affinity and cytotoxicity toward the human prostate cancer LNCaP (PSMA+) cell line, much higher than for PC-3 (PSMA-) cells. High cytotoxicity of the radiobioconjugate was confirmed by radiotoxicity studies on LNCaP 3D spheroids. In addition, the magnetic properties of the radiobioconjugate should allow for its use in guide drug delivery driven by magnetic field gradient.

Scandium-44 Radiolabeled Peptide and Peptidomimetic Conjugates Targeting Neuropilin-1 Co-Receptor as Potential Tools for Cancer Diagnosis and Anti-Angiogenic Therapy.

Pathological angiogenesis, resulting from an imbalance between anti- and pro-angiogenic factors, plays a pivotal role in tumor growth, development and metastasis. The inhibition of the angiogenesis process by the VEGF/VEGFR-2/NRP-1 pathway raises interest in the search for such interaction inhibitors for the purpose of the early diagnosis and treatment of angiogenesis-dependent diseases. In this work we designed and tested peptide-based radiocompounds that selectively bind to the neuropilin-1 co-receptor and prevent the formation of the pro-angiogenic VEGF-A165/NRP-1 complex. Three biomolecules, A7R and retro-inverso DR7A peptides, and the branched peptidomimetic Lys(hArg)-Dab-Pro-Arg (K4R), conjugated with macrocyclic chelator through two linkers' types, were labeled with theranostic scandium-44 radionuclide, and studied in vitro as potential targeted radiopharmaceuticals. ELISA (enzyme-linked immunosorbent assay) studies showed no negative effect of the introduced biomolecules' changes and high NRP-1 affinity in the case of A7R- and K4R-radiocompounds and a lack affinity for DR7A-radiocompounds. All radiopeptides showed a hydrophilic nature as well as high stability against ligand exchange reactions in cysteine/histidine solutions. Unfortunately, all radiocompounds showed unsatisfactory nano-scale stability in human serum, especially for use as therapeutic radioagents. Further work is ongoing and focused on the search for angiogenesis inhibitors that are more human serum stable.

Nanosatellite Payload for Research on Seed Germination in a 3D Printed Micropot.

In this paper, an autonomous payload proposal for a nanosatellite mission allowing for the cultivation of grains in space was presented. For the first time, a micropot made with 3D printing technology, enabling the parametric determination of plant growth, both on Earth and in the simulated microgravity condition, was presented. A completed system for dosing the nutrient solution and observing the growth of a single grain, where the whole size did not exceed 70 × 50 × 40 mm3, was shown. The cultivation of Lepidium sativum seeds was carried out in the developed system, in terrestrial conditions and simulated microgravity conditions, using the RPM (Random Position Machine) device. The differences in plant growth depending on the environment were observed. It could be seen that the grains grown in simulated microgravity took longer to reach the full development stage of the plant. At the same time, fewer grains reached this stage and only remained at the earlier stages of growth. The conducted research allowed for the presentation of the payload concept for a 3U CubeSat satellite for research into the development of plants in space.

MOF-Based Sorbents Used for the Removal of Hg2+ from Aqueous Solutions via a Sorption-Assisted Microfiltration.

Mercury is considered to be one of the most important chemicals of public health concern. Therefore, it is necessary to develop an effective method of removing mercury ions from aqueous solutions to protect people from exposure to this element. This paper presents research on the application of a sorption-assisted microfiltration (SAMF) hybrid process for the removal of Hg2+ from aqueous solutions. As adsorbents used in the process, the metal-organic-framework-UiO-66-type materials have been considered. The methods of synthesis of two types of metal-organic-framework (MOF) sorbents were developed: UiO-66_MAA modified with mercaptoacetic acid (MAA) and a composite of UiO-66 with cellulose. The results of the experiments performed proved that the separation of Hg2+ from water solutions conducted in such a system was effective; however, a relatively long initial contact time of reagents before filtration was required. The experimental results can be used to optimize the parameters of the SAMF process in order to obtain an effective method of Hg2+ removal from aqueous solutions.

Microfluidic-Assisted Human Cancer Cells Culturing Platform for Space Biology Applications.

In the paper, the lab-on-chip platform applicable for the long-term cultivation of human cancer cells, as a solution meeting the demands of the CubeSat biological missions, is presented. For the first time, the selected cancer cell lines-UM-UC-3 and RT 112 were cultured on-chip for up to 50 days. The investigation was carried out in stationary conditions (without medium microflow) in ambient temperature and utilizing the microflow perfusion system in the incubation chamber assuring typical cultivation atmosphere (37 °C). All the experiments were performed to imitate the conditions that are provided before the biological mission starts (waiting for the rocket launch) and when the actual experiment is initialized on a CubeSat board in space microgravity. The results of the tests showed appropriate performance of the lab-on-chip platform, especially in the context of material and technological biocompatibility. Cultured cells were characterized by adequate morphology-high attachment rate and visible signs of proliferation in each of the experimental stage. These results are a good basis for further tests of the lab-on-chip platform in both terrestrial and space conditions. At the end of the manuscript, the authors provide some considerations regarding a potential 3-Unit CubeSat biological mission launched with Virgin Orbit company. The lab-on-chip platform was modelled to fit a 2-Unit autonomous laboratory payload.

Cubosomal Lipid Formulation for Combination Cancer Treatment: Delivery of a Chemotherapeutic Agent and Complexed α-Particle Emitter 213Bi.

Here, we propose tailored lipid liquid-crystalline carriers (cubosomes), which incorporate an anticancer drug (doxorubicin) and complexed short-lived α-emitter (bismuth-213), as a strategy to obtain more effective action toward the cancer cells. Cubosomes were formulated with doxorubicin (DOX) and an amphiphilic ligand (DOTAGA-OA), which forms stable complexes with 213Bi radionuclide. The behavior of DOX incorporated into the carrier together with the chelating agent was investigated, and the drug liberation profile was determined. The experiments revealed that the presence of the DOTAGA-OA ligand affects the activity of DOX when they are incorporated into the same carrier. This unexpected influence was explained based on the results of release studies, which proved the contribution of electrostatics in molecular interactions between the positively charged DOX and negatively charged DOTAGA-OA in acidic and neutral solutions. A significant decrease in the viability of HeLa cancer cells was achieved using sequential cell exposure: first to the radiolabeled cubosomes containing 213Bi complex and next to DOX-doped cubosomes. Therefore, the sequential procedure for the delivery of both drugs encapsulated in cubosomes is suggested for further biological and in vivo studies.

Effect of Carbon Black and Hybrid Steel-Polypropylene Fiber on the Mechanical and Self-Sensing Characteristics of Concrete Considering Different Coarse Aggregates' Sizes.

The effect of combining filler (carbon black) and fibrous materials (steel fiber and polypropylene fiber) with various sizes of coarse particles on the post-cracking behavior of conductive concrete was investigated in this study. Steel fibers (SF) and carbon black (CB) were added as monophasic, diphasic, and triphasic materials in the concrete to enhance the conductive properties of reinforced concrete. Polypropylene fiber (PP) was also added to steel fiber and carbon to improve the post-cracking behavior of concrete beams. This research mainly focused on the effects of macro fibers on toughness parameters and energy absorption capacity, as well as enhancing the self-sensing of multiple cracks and post-cracking behavior. Fractional changes in resistance and crack opening displacement (COD-FCR) and the relationship of load-deflection-FCR with different coarse aggregates of (5-10 mm and 15-20 mm) sizes were investigated, and the law of resistance signal changes with single and multiple cracking through load-time-FCR curves was explored. Results indicated that the smaller size coarse aggregates (5-10 mm) showed higher compressive strength: up to 8.3% and 14.83% with diphasic (SF + CB), respectively. The flexural strength of PC-10 increased 22.60 and 51.2%, respectively, with and without fibers, compared to PC-20. The diphasic and triphasic conductive material with the smaller size of aggregates (5-10 mm) increased the FCR values up to 38.95% and 42.21%, respectively, as compared to those of greater size coarse aggregates (15-20 mm). The hybrid uses of fibrous and filler materials improved post-cracking behavior as well as the self-sensing ability of reinforced concrete.

Design and Evaluation of 223Ra-Labeled and Anti-PSMA Targeted NaA Nanozeolites for Prostate Cancer Therapy-Part II. Toxicity, Pharmacokinetics and Biodistribution.

Metastatic castration-resistant prostate cancer (mCRPC) is a progressive and incurable disease with poor prognosis for patients. Despite introduction of novel therapies, the mortality rate remains high. An attractive alternative for extension of the life of mCRPC patients is PSMA-based targeted radioimmunotherapy. In this paper, we extended our in vitro study of 223Ra-labeled and PSMA-targeted NaA nanozeolites [223RaA-silane-PEG-D2B] by undertaking comprehensive preclinical in vitro and in vivo research. The toxicity of the new compound was evaluated in LNCaP C4-2, DU-145, RWPE-1 and HPrEC prostate cells and in BALB/c mice. The tissue distribution of 133Ba- and 223Ra-labeled conjugates was studied at different time points after injection in BALB/c and LNCaP C4-2 tumor-bearing BALB/c Nude mice. No obvious symptoms of antibody-free and antibody-functionalized nanocarriers cytotoxicity and immunotoxicity was found, while exposure to 223Ra-labeled conjugates resulted in bone marrow fibrosis, decreased the number of WBC and platelets and elevated serum concentrations of ALT and AST enzymes. Biodistribution studies revealed high accumulation of 223Ra-labeled conjugates in the liver, lungs, spleen and bone tissue. Nontargeted and PSMA-targeted radioconjugates exhibited a similar, marginal uptake in tumour lesions. In conclusion, despite the fact that NaA nanozeolites are safe carriers, the intravenous administration of NaA nanozeolite-based radioconjugates is dubious due to its high accumulation in the lungs, liver, spleen and bones.

Possibilities of Increasing Effectiveness of RC Structure Strengthening with FRP Materials.

Modern composite materials based on non-metallic continuous fibres are increasingly used in civil engineering to strengthen building structures. In the strengthening of reinforced concrete (RC) structures, the utilisation of externally bonded fibre-reinforced polymer (FRP) composites is only up to 35% because of the pilling-off failure mechanism. This problem can be solved using pre-tensioned composite laminates. Due to more complex behaviour, the strengthening of structures by means of prestressing technology needs a careful design approach and a full understanding of the behaviour of both the materials and elements. The advantages and risks of the presented technology, which may determine the success of the entire project, will be highlighted in the paper. The possibility of using a flexible adhesive layer in carbon fibre reinforced polymer (CFRP) strengthening applications for flexural strengthening of RC elements, as an innovative solution in civil engineering, will also be presented. Parallel introduction of the flexible adhesive layer (made of polyurethane masses) and a traditional epoxy adhesive layer in one strengthening system was investigated in the laboratory tests. This solution was used for the repair and protection of a previously damaged RC beam against brittle failure.

Radiochemical separation of 224Ra from 232U and 228Th sources for 224Ra/212Pb/212Bi generator.

The application of diagnostic and therapeutic radionuclides in nuclear medicine has grown significantly and has translated into the increased interest in radionuclide generators and their development. 224Ra and its shorter-lived daughters, 212Pb and 212Bi, are very interesting radionuclides from Targeted Alpha Therapy point of view for treatment of small cancers or metastatic forms. The purpose of the present work was to develop a simple generator for rapid elution of carrier-free 224Ra from 232U or 228Th sources by radiochemical separation based on extraction chromatography with the utilization of a home-made material. The bis(2-ethylhexyl) hydrogen phosphate (HDEHP) extractant was immobilized on polytetrafluroethylene (PTFE) grains and its ability to selectively adsorb 232U and 228Th, with simultaneous high elution recovery of 224Ra, was checked over few years. The 224Ra was quantitatively eluted with small volume (3-5 mL) of 0.1 M HNO3 with low breakthrough (<0.005%) and was used for further milking of 212Bi and 212Pb from DOWEX 50WX12 by 0.75 M and 2.0 M HCl, respectively. The elaborated here methods allowed high recovery of 224Ra, 212Pb and 212Bi radionuclides and their application in radiolabeling of various biomolecules.

Volatile organic compounds sensing based on Bennet doubler-inspired triboelectric nanogenerator and machine learning-assisted ion mobility analysis.

Ion mobility analysis is a well-known analytical technique for identifying gas-phase compounds in fast-response gas-monitoring systems. However, the conventional plasma discharge system is bulky, operates at a high temperature, and inappropriate for volatile organic compounds (VOCs) concentration detection. Therefore, we report a machine learning (ML)-enhanced ion mobility analyzer with a triboelectric-based ionizer, which offers good ion mobility selectivity and VOC recognition ability with a small-sized device and non-strict operating environment. Based on the charge accumulation mechanism, a multi-switched manipulation triboelectric nanogenerator (SM-TENG) can provide a direct current (DC) bias at the order of a few hundred, which can be further leveraged as the power source to obtain a unique and repeatable discharge characteristic of different VOCs, and their mixtures, with a special tip-plate electrode configuration. Aiming to tackle the grand challenge in the detection of multiple VOCs, the ML-enhanced ion mobility analysis method was successfully demonstrated by extracting specific features automatically from ion mobility spectrometry data with ML algorithms, which significantly enhance the detection ability of the SM-TENG based VOC analyzer, showing a portable real-time VOC monitoring solution with rapid response and low power consumption for future internet of things based environmental monitoring applications.

Lipidic Cubic-Phase Nanoparticles (Cubosomes) Loaded with Doxorubicin and Labeled with 177Lu as a Potential Tool for Combined Chemo and Internal Radiotherapy for Cancers.

Lipid liquid-crystalline nanoparticles (cubosomes) were used for the first time as a dual-modality drug delivery system for internal radiotherapy combined with chemotherapy. Monoolein (GMO)-based cubosomes were prepared by loading the anticancer drug, doxorubicin and a commonly used radionuclide, low-energy beta (ß-)-emitter, 177Lu. The radionuclide was complexed with a long chain derivative of DOTAGA (DOTAGA-OA). The DOTAGA headgroup of the chelator was exposed to the aqueous channels of the cubosomes, while, concerning OA, the hydrophobic tail was embedded in the nonpolar region of the lipid bilayer matrix, placing the radioactive dopant in a stable manner inside the cubosome. The cubosomes containing doxorubicin and the radionuclide complex increased the cytotoxicity measured by the viability of the treated HeLa cells compared with the effect of single-drug cubosomes containing either the DOX DOTAGA-OA or DOTAGA-OA-177Lu complex. Multifunctional lipidic nanoparticles encapsulating the chemotherapeutic agent together with appropriately complexed (ß-) radionuclide are proposed as a potential strategy for effective local therapy of various cancers.

Lab-on-Chip Platform for Culturing and Dynamic Evaluation of Cells Development.

This paper presents a full-featured microfluidic platform ensuring long-term culturing and behavioral analysis of the radically different biological micro-objects. The platform uses all-glass lab-chips and MEMS-based components providing dedicated micro-aquatic habitats for the cells, as well as their intentional disturbances on-chip. Specially developed software was implemented to characterize the micro-objects metrologically in terms of population growth and cells' size, shape, or migration activity. To date, the platform has been successfully applied for the culturing of freshwater microorganisms, fungi, cancer cells, and animal oocytes, showing their notable population growth, high mobility, and taxis mechanisms. For instance, circa 100% expansion of porcine oocytes cells, as well as nearly five-fold increase in E. gracilis population, has been achieved. These results are a good base to conduct further research on the platform versatile applications.

Production of Sc medical radioisotopes with proton and deuteron beams.

Proton and deuteron beams (15.3 and 6.8 MeV, respectively) extracted from the PETtrace medical cyclotron at the Radiopharmaceuticals Production and Research Centre in the University of Warsaw, Heavy Ion Laboratory, 28 MeV protons from the C30 cyclotron at the National Centre for Nuclear Research, Swierk, near Warsaw and 33 MeV protons from the ARRONAX accelerator, Nantes were used to produce and investigate the medically interesting Sc radioisotopes. Both natural and isotopically enriched CaCO3 and TiO2 targets were used (42Ca, 43Ca, 44Ca, 48Ca, 48Ti). The production efficiency and isotopic purity were determined and are reported here for the highest commercially available enrichments of the target material. The Thick Target Yield, Activities at the End of Bombardment (EOB) and the relative activities of produced impurities at EOB are reported for 43Sc, 44gSc, 44mSc and 47Sc produced with particle energies below 33 MeV.

Production of medical Sc radioisotopes with an alpha particle beam.

The internal α-particle beam of the Warsaw Heavy Ion Cyclotron was used to produce research quantities of the medically interesting Sc radioisotopes from natural Ca and K and isotopically enriched 42Ca targets. The targets were made of metallic calcium, calcium carbonate and potassium chloride. New data on the production yields and impurities generated during the target irradiations are presented for the positron emitters 43Sc, 44gSc and 44mSc. The different paths for the production of the long lived 44mSc/44gSc in vivo generator, proposed by the ARRONAX team, using proton and deuteron beams as well as alpha-particle beams are discussed. Due to the larger angular momentum transfer in the formation of the compound nucleus in the case of the alpha particle induced reactions, the isomeric ratio of 44mSc/44gSc at a bombarding energy of 29MeV is five times larger than previously determined for a deuteron beam and twenty times larger than for proton induced reactions on enriched CaCO3 targets. Therefore, formation of this generator via the alpha-particle route seems a very attractive way to form these isotopes. The experimental data presented here are compared with theoretical predictions made using the EMPIRE evaporation code. Reasonable agreement is generally observed.

Cyclotron production of (43)Sc for PET imaging.

BACKGROUND: Recently, significant interest in (44)Sc as a tracer for positron emission tomography (PET) imaging has been observed. Unfortunately, the co-emission by (44)Sc of high-energy γ rays (E γ = 1157, 1499 keV) causes a dangerous increase of the radiation dose to the patients and clinical staff. However, it is possible to produce another radionuclide of scandium-(43)Sc-having properties similar to (44)Sc but is characterized by much lower energy of the concurrent gamma emissions. This work presents the production route of (43)Sc by α irradiation of natural calcium, its separation and purification processes, and the labeling of [DOTA,Tyr3] octreotate (DOTATATE) bioconjugate. METHODS: Natural CaCO3 and enriched [(40)Ca]CaCO3 were irradiated with alpha particles for 1 h in an energy range of 14.8-30 MeV at a beam current of 0.5 or 0.25 µA. In order to find the optimum method for the separation of (43)Sc from irradiated calcium targets, three processes previously developed for (44)Sc were tested. Radiolabeling experiments were performed with DOTATATE radiobioconjugate, and the stability of the obtained (43)Sc-DOTATATE was tested in human serum. RESULTS: Studies of (nat)CaCO3 target irradiation by alpha particles show that the optimum alpha particle energies are in the range of 24-27 MeV, giving 102 MBq/µA/h of (43)Sc radioactivity which creates the opportunity to produce several GBq of (43)Sc. The separation experiments performed indicate that, as with (44)Sc, due to the simplicity of the operations and because of the chemical purity of the (43)Sc obtained, the best separation process is when UTEVA resin is used. The DOTATATE conjugate was labeled by the obtained (43)Sc with a yield >98 % at elevated temperature. CONCLUSIONS: Tens of GBq activities of (43)Sc of high radionuclidic purity can be obtainable for clinical applications by irradiation of natural calcium with an alpha beam.

Microfluidic method of pig oocyte quality assessment in relation to different follicular size based on lab-on-chip technology.

Since microfollicular environment and the size of the follicle are important markers influencing oocyte quality, the aim of this study is to present the spectral characterization of oocytes isolated from follicles of various sizes using lab-on-chip (LOC) technology and to demonstrate how follicle size may affect oocyte quality. Porcine oocytes (each, n = 100) recovered from follicles of different sizes, for example, from large (>5 mm), medium (3-5 mm), and small (<3 mm), were analyzed after preceding in vitro maturation (IVM). The LOC analysis was performed using a silicon-glass sandwich with two glass optical fibers positioned "face-to-face." Oocytes collected from follicles of different size classes revealed specific and distinguishable spectral characteristics. The absorbance spectra (microspectrometric specificity) for oocytes isolated from large, medium, and small follicles differ significantly (P < 0.05) and the absorbance wavelengths were between 626 and 628 nm, between 618 and 620 nm, and less than 618 nm, respectively. The present study offers a parametric and objective method of porcine oocyte assessment. However, up to now this study has been used to evidence spectral markers associated with follicular size in pigs, only. Further investigations with functional-biological assays and comparing LOC analyses with fertilization and pregnancy success and the outcome of healthy offspring must be performed.

The value of EORTC risk tables in evaluating recurrent non-muscle-invasive bladder cancer in everyday practice.

INTRODUCTION: Due to the risk of recurrence and progression, patients with non-muscle-invasive bladder cancer have to be under observation. The aim of this study is the evaluation of early recurrence at the first control cystoscopy, as a prognostic factor for recurrence and progression based on EORTC risk tables. MATERIAL AND METHODS: This study analyzed 243 patients with non-muscle-invasive bladder cancer, with an average observation time of 46 months. Recurrence was observed in case of 99 patients. Among these patients, we selected 79 who had the first cystoscopy 3 months after the transurethral electroresection of the bladder tumor. Subsequently, 45 patients with early recurrence at the first control cystoscopy were compared with 34 patients whose cancer recurred at later control cystoscopies. The patients were compared with respect to the number of points assigned by EORTC tables. RESULTS: Those patients who had an early recurrence had a significantly higher score in the EORTC table in the progression scale (p = 0.017) but not in the recurrence scale (p = 0.11), as compared with patients who had a late recurrence. CONCLUSIONS: Early recurrence that occurs within 3 months after TURBT indicates a higher risk of progression, as compared with a late recurrence. Patients who had an early recurrence had a significantly higher EORTC risk score for progression. Their EORTC risk score for recurrence was also higher, but the difference was not statistically significant. Every patient with an early recurrence has a worse prognosis and a higher risk of progression.

Supravital fluorometric apoptosis detection in a single mouse embryo using lab-on-a-chip.

Detection of apoptosis is one of the main criteria of preimplantation embryo growth potential assessment. Recent developments in lab-on-a-chip techniques has led to apoptosis detection and monitoring on a single cell or embryo level. However, single embryo apoptosis detection without a change in embryo developmental competence and post-examination "recovery" still remains a challenge. In this paper we present a lab-on-a-chip, co-working with miniaturized optical instrumentation, which allows supravital examination of single embryos for the presence of apoptotic blastomers with full after lab-on-a-chip study "recovery" and maintenance of their further developmental capacity.

The SmartBioPhone, a point of care vision under development through two European projects: OPTOLABCARD and LABONFOIL.

This paper describes how sixteen partners from eight different countries across Europe are working together in two EU projects focused on the development of a point of care system. This system uses disposable Lab on a Chips (LOCs) that carry out the complete assay from sample preparation to result interpretation of raw samples. The LOC is either embedded in a flexible motherboard with the form of a smartcard (Labcard) or in a Skinpatch. The first project, OPTOLABCARD, extended and tested the use of a thick photoresit (SU-8) as a structural material to manufacture LOCs by lamination. This project produced several examples where SU-8 microfluidic circuitry revealed itself as a viable material for several applications, such as the integration on chip of a Polymerase Chain Reaction (PCR) that includes sample concentration, PCR amplification and optical detection of Salmonella spp. using clinical samples. The ongoing project, LABONFOIL, is using two results of OPTOLABCARD: the sample concentration method and the capability to fabricate flexible and ultra thin LOCs based on sheets instead of wafers. This rupture from the limited and expensive wafer surface heritage allows the development of a platform where LOCs are big enough to include all the sample preparation subcomponents at a low price. These LOCs will be used in four point of care applications: environment, food, cancer and drug monitoring. The user will obtain the results of the tests by connecting the Labcard/Skinpatch reader to a very popular interface (a smartphone), creating a new instrument namely "The SmartBioPhone". All standard smartphone capabilities will be at the disposal of the point of care instrument by a simple click. In order to guarantee the future mass production of these LOCs, the project will develop a large dry film equipment where LOCs will be fabricated at a low cost.