This central electrode is housed within a stainless steel tube  with a 27° angle at one end, making it ergonomically suitable for oral applications. NTP generation involves polarizing the reactor electrodes with 13.56 MHz radio frequency signals, using helium gas as a working medium. We introduce plasma-treated water (PTW) as an adjunctive therapy to enhance biofilm eradication within root canals. A synergistic approach combining NTP and PTW is employed and compared to the gold standard , effectively neutralizing Enterococcus faecalis bacteria, even in scenarios involving biofilms. Moreover, applying NTP in both gaseous and liquid environments successfully achieves bacterial inactivation at varying treatment durations, demonstrating the device’s suitability for medical use in treating root canal biofilms. The proposed NTP reactor, characterized by its innovative design, offers a practical and specific approach to plasma treatment in dental applications. It holds promise in combatting bacterial infections in root canals and oral cavities.Non-thermal plasmas (NTPs), known as cold atmospheric plasmas (CAPs), hold great potential for diverse medical applications, including dentistry. However, traditional linear and rigid dielectric barrier discharge reactors used for NTP generation encounter limitations in accessing oral cavities and root canals. To address this issue, we have developed an innovative NTP reactor featuring an angled end for improved accessibility. The central copper electrode, with a 0.59 mm diameter and adjustable length for desired angulation, is coated with zircon powder (ZrSiO Bacteria lodged in the root canal cause pulpitis and apical periodontitis. Once these bacteria organize themselves into a biofilm, dental debridement becomes difficult due to their ability to adhere to and colonize the root canal, where they develop and secrete an exopolysaccharide that acts as an adhesion and protection matrix for the bacteria, making them more resistant to antimicrobial agents than in planktonic bacteria ,2,3. TheThe gold standard for dental disinfection is chemomechanical treatment, which is carried out with manual or assisted rotary instruments and using chemical solutions such as sodium hypochlorite (NaOCl) ,7,8. TheNTP is excellent for wound disinfection, sterilization, and healing in biomedical applications. This is possible because when generating the NTP and depending on the gas used, reactive species, charged particles, and UV photons can be produced, which have a high oxidative potential and, therefore, can attack and disrupt the molecular structures of microorganisms . LikewisThe NTP is generated by providing energy to a gas using an electric field, which achieves its ionization. An NTP is composed of ions, free electrons, and atoms in their elementary or excited state. The electric field causes the dissociation of the gas atoms or molecules into ions and electrons. Subsequently, the atoms or molecules in the excited state lose their energy by colliding with other particles or by the emission of photons . The NTPAnother way to achieve bacterial inactivation is by utilizing water interacting with NTP, a process known as Plasma-Treated Water (PTW). The application of PTW has garnered significant interest due to its potential environmental, medical, and even food technology applications, among others. The primary technique for producing PTW involves generating plasma either outside or within the water to produce ions that result in various highly reactive oxygen species (ROS) and nitrogen species (RNS), which are essential for bacterial inactivation. One advantage of PTW is its ability to be applied rapidly and easily, regardless of the surface’s form. Furthermore, this innovative approach opens new avenues for enhancing disinfection and treatment strategies, contributing to various fields, including public health, agriculture, and industrial processes .Plasma can be generated through various methods, with some requiring high vacuum systems. Examples include glow discharge, microwave discharge, inductively coupled discharge, and capacitively coupled discharge. However, when operating at atmospheric pressure, NTP can be generated using corona discharge and dielectric barrier discharge (DBD). Notably, these methods eliminate the need for high-vacuum systems ,17.In the setup of a DBD-type plasma between two electrodes where the discharge occurs, one or both can be coated with a dielectric material ,19. The An electric field is also established between electrically insulated electrodes, and if the field strength is sufficient, an electric discharge occurs. However, the insulation prevents the generation of significant current flow, preventing the gas from heating. Instead, the electrons migrate toward the positive electrode but cannot escape due to insulation. In a minimal time, the resulting charge compensates for the high-voltage field created, extinguishing the discharge ,20.Various DBD reactor designs have been described . The chaIndeed, some reviews focus on dielectric materials and their influence on the performance of the DBD plasma reactor, considering factors like thickness ,24,25. RCeramic materials serve specific purposes based on their application. Multifunctional ceramics consist of different materials, each contributing a distinct function. Some of the most studied ceramics include aluminium oxide doped with MgO , calciumThe reactors that generate NTP for biomedical applications need specific characteristics, including being manageable and ergonomic. However, conventional NTP reactors are often rigid and straight, making them unsuitable for direct use in oral cavity root canals. A jet-type reactor with a curved terminal has been developed to overcome this limitation ,29. ThesAnother type of reactor used for NTP generation is the Thorns plasma reactor , which cThis study introduces an innovative approach for addressing biofilms within root canals. The primary objective is the efficient elimination of these biofilms by utilizing a novel curved-tip reactor designed for NTP generation. This unique reactor was also employed for PTW production. Both processes were compared to the conventional gold standard in root canal treatments involving sodium hypochlorite. In vitro assessments demonstrated the effectiveness of bacterial and biofilm removal from dentin samples. The curved reactor design enhances manoeuvrability and facilitates application in challenging-to-access regions, potentially offering an improved treatment modality for endodontic infections.The NTP-generating reactor proposed in this study features a coaxial design with a 27° angle at one end. This configuration allows for convenient insertion into the oral cavity and precise positioning to apply NTP from the incisors to the lower and upper molars refer to . ConsequThe reactor has specific characteristics, including an internal electrode length of 99 mm and an external electrode length of 100 mm. Both electrodes have an angle of 27° at one end, enabling easy access to the molars, as depicted in 4) powder  was chosen. This dielectric material exhibits high electrical insulation, thermal conductivity, and anticorrosive properties. When mixed, it forms a chemical setting cement capable of withstanding temperatures above 1400 °C, making it suitable for the plasma conditions and potential temperature gradients and thermal shocks within the reactor.The inner electrode is coated with a dielectric material to create the dielectric barrier discharge. For this purpose, commercial zircon  for 2 min, as shown in After injecting the mixture to cover the copper electrode, it is inserted into a silicone hose with an internal diameter of 2.1 mm and an external diameter of 3 mm. The mixture is carefully injected inside the hose until the entire electrode volume is covered. Before the mixture sets, the silicone hose with the covered electrode is introduced into an 80 mm long carbon fibre tube with an internal diameter of 3.1 mm and an external diameter of 5 mm. The silicone hose and carbon fibre tube are placed in a mould to create the required 27° curvature for the reactor design b.After the zircon mixture has been injected and set around the copper electrode inside the silicone hose and carbon fibre tube, the central electrode, now coated with the zircon material, is carefully retrieved from the hose by making a transverse cut using a scalpel blade. It is then adjusted to the required length to fit inside the reactor c.The schematic diagram of the entire process is depicted in Several publications have discussed using NTP reactors for treating biofilms ,31. VariPeptostreptococcus spp., Streptococcus spp., Enterococcus faecalis, Staphylococcus salivarius, Lactobacillus spp., Actinomyces spp., Candida albicans, Eubacterium spp., Bacillus spp., and Gram-negative bacteria such as Porphyromonas spp., Prevotella spp., Veillonella spp., and Escherichia coli [The literature reports the presence of various bacteria and microorganisms in root canals ,34, inclhia coli , among oEnterococcus faecalis holds significant relevance in the field, given its widespread presence in cases of endodontic failure. It has been detected in necrotic pulps before and after unsuccessful treatments [Enterococcus faecalis ATCC 29212 was selected as the bacterium to be investigated.The enduring presence of microorganisms in the apical region of the root canal can potentially result in endodontic diseases, even following the completion of root canal treatment. eatments . This baThis investigation employed a lower second premolar, which had been extracted for orthodontic purposes. Access to the pulp chamber was established, and a type K file was meticulously inserted until it was visible through the apical foramen. The working length was determined by deducting 0.5 mm, and chemomechanical instrumentation was performed using a solution of 5.25% NaOCl and rotary instruments, ultimately achieving a final diameter of 0.45 mm with a taper of 0.06%.To obtain 3D images of the premolar, high-definition Planmeca OP300 5 × 5 FOV cone beam, computed tomography  produced DICOM format images. Using the inVivo5 3D design program , a three-dimensional recreation of the premolar was generated in STL format. The design included a 3 mm slot above the apex in its vestibular portion, with dimensions of 3 mm in depth and 3.8 mm in height, ensuring it occupied the entire root canal space. Once the final design was completed, the model was 3D printed using standard transparent biocompatible resin, specifically the 3D AnyCubic 8k with LCD technology  see a,b.A 0.45/0.06 rotary file was introduced into the 3D-printed models along their final length to prepare the samples. Additionally, a size 15 K file was passed 0.5 mm through the foramen to ensure the patency of the apical foramen and to check for any resin obstructions in the duct. The samples underwent a 10 min wash with isopropyl alcohol in an ultrasonic bath, and an additional 10 min rinse with distilled water in the same ultrasonic bath.Human mandibular premolars were carefully selected for this study based on specific criteria, including having a fully formed apex, no root curvature, no fractures, and no signs of resorption. Each tooth had a single root canal. Dentin segments were obtained from these premolars using a diamond disc, and their measurements were precisely matched to the groove in the 3D-printed models. To simulate the root canal space, a standardized round groove with dimensions of 1.0 mm in depth and 1.3 mm in width c was creTo verify the structural integrity of the samples, they were scrutinized under an operating microscope at a 20× magnification level . Following this preparation, the dentin samples were treated with 17% ethylenediaminetetraacetic acid for 1 min to remove the smear layer, ensuring a clean and consistent surface. Subsequently, the prepared dentin segments were inserted into the designated spaces in the 3D-printed models d. GingivBefore inoculating and preparing bacterial biofilms, the models shown in Enterococcus faecalis bacterium was cultured in 5 mL of Luria–Bertani (LB) nutrient medium for 20 h at 37 °C. After incubation, the nutrient medium was removed by centrifugation using a Dynamica-brand centrifuge  operating at 5000 rpm for 10 min. This centrifugation process was repeated twice, and, each time, sterile phosphate-buffered saline (PBS) with a pH of 7.2 was used. The supernatants obtained after centrifugation were discarded, and the residues were washed three times with distilled water to eliminate any potential interference from the culture media. Subsequently, the washed residues were resuspended in a PBS solution.The 6 colony-forming units per mL (CFU/mL) and 1 × 105 CFU/mL for subsequent experiments.Bacterial counting was conducted using a Carl Zeiss phase-contrast microscope  and a Neubauer chamber . To guarantee the precision of the bacterial count, serial dilutions at a 1:10 ratio were meticulously prepared and subsequently incubated for 24 h at 37 °C under aerobic conditions. The bacterial cell count in the suspension was determined through the conventional plate count method, and the bacterial cell density was adjusted to 1 × 10Enterococcus faecalis biofilms to NTP, and it was then diluted in 1 mL of sterile PBS. A total of 20 µL of Enterococcus faecalis culture was injected into each root canal model containing inserted dentin. Then, 15 mL of Luria–Bertani agar was placed into an 80 mL beaker to introduce the root canal models with inserted dentin until the complete coverage of the specimens with the bacterial suspension up to the dentin of the model was achieved. Each group of specimens consisted of 10 individual models. These groups of specimens were carefully placed in a circular plate made of stainless steel schedule 316L. The circular plate had perforations with a diameter of 0.296 mm, which allowed each model to be securely positioned . The SDW container was positioned on a shaker device to ensure a consistent dispersion of the reactive components within the water. The operational parameters of the NTP reactor remained consistent with those detailed in Enterococcus faecalis bacteria in the biofilms was 107 CFU/mL, establishing this as our control condition; in this case, the samples did not undergo any treatment after inoculation, and their initial CFU counts were used as a reference for comparison with the experimental group.Within the scope of this research, we cultivated biofilms on 3D root canal models containing embedded dentin, as depicted in The α condition denoted the application of NTP in the root canal for 5 min, with the reactor’s tip positioned at a 5 mm distance. The γ condition entailed the introduction of PTW into the root canal for 5 min. In another condition, the reactor’s tip was placed directly onto the model, in contact with the dentin, at a distance of 0 mm, designated as the ε condition. Furthermore, we employed NaOCl in 0.6% and 5.25% concentrations. The first concentration was defined as the ζ condition and was applied to the root canal for 5 min, while the second concentration of NaOCl was used for 1 min (β condition) and for 5 min (η condition). All these conditions were considered baseline reference conditions.In the subsequent stage of experimentation, we replicated the baseline procedures, i.e., α + α, γ + γ, and ε + ε. In each instance, the initial condition was administered, the model with the dentin was refrigerated, and the same procedure was repeated one hour later.Moreover, we explored specific combinations of the baseline conditions. For instance, we applied NTP treatment (α), immediately followed by the application of PTW (γ) for an additional 5 min, which was referred to as α + γ. Similarly, the combinations were carried out as γ + ε, and, finally, η + γ.Another phase of the experimentation entailed the combination of the NaOCl at 5.25% (η) and NTP at 0 mm (ε). After applying the NaOCl solution for 5 min, the model with the dentin was refrigerated for 60 min, followed by NTP; we referred to this procedure as η + ε. Lastly, the experiment identified as γ + ε + ε was performed, in which PTW was administered to the model with the dentin for 5 min (γ) without removing the PTW residues. Subsequently, the ε condition was applied, followed by 60 min of refrigeration, and then the ε condition was reapplied for 5 min more.We utilized scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis (EDX) to examine zircon powder during this study. Additionally, SEM was used independently to investigate the ultrastructural and physicochemical characteristics of the formed biofilm, and the morphology of both the biofilms and microorganisms within the dentin treatment areas.Dentin samples were collected from the treated and control root canal models and prepared for SEM analysis using the following described methodology. The samples were harvested, washed, and fixed with glutaraldehyde in solution at 6% in 0.1 M of PBS and placed under refrigeration at 4 °C for 24 h. Samples were then postfixed in osmium tetroxide (solution at 4%) in 0.1 M of PBS and refrigerated at 4 °C for 2 h. Finally, the samples were rinsed with PBS and dehydrated in ascending graded ethanol series . The samples were subsequently affixed to be mounted on a stub and then subjected to an ion sputter process to apply an ultra-thin layer of gold.To perform the SEM analysis, we utilized a scanning electron microscope model JEOL JSM 5900 , with the following radiation parameters: high voltage of 20 kV and a probe current of 25 pA. These settings provided the necessary resolution and sensitivity to examine the surface morphology and composition of the biofilm, enabling us to gain valuable insights into its structure and interactions with the dentin surfaces.The data analysis was conducted using two software tools: a spreadsheet program  and Origin-Pro software ver. 8.0 SR2 . Statistical tests, including the ANOVA with Bonferroni and Tukey’s post hoc tests, were performed at a significance level of 5%. We rigorously analysed and interpreted the experimental results by applying these statistical methods, deriving meaningful conclusions from the data.The analysis of the zircon powder was conducted to evaluate the composition of the dielectric using a scanning electron microscope JEOL JSM 5900, operating at an electron energy of 20 kV. ® CuKa diffractometer  was utilized to determine the crystalline phases in the zircon powder used to coat the central electrode. The diffractometer was operated with an acceleration set at 35 kV and a current of 25 mA. Diffractograms were obtained in the 15–80° (2θ) range using a step size of 0.02°, and each step took approximately 38.9 s.A Siemens D5000The X-ray diffraction (XRD) results were analysed by comparing them with reference standards from the ICDD  database using DIFFRAC.EVA V4.1.1 software . The thermal treatment of the zircon powder during the coating of the central electrode did not lead to substantial changes in peak widths, indicating no significant alterations in the crystal lattice structure.1O2), which give rise to the formation of secondary species, including molecular oxygen (O2), hydrogen peroxide (H2O2), atomic oxygen (O), and ozone (O3). Some of these primary species have a half-life of the order of microseconds [2+) and helium atomic radicals. The presence of the NOγ(A-X) system was also determined in the spectrum, which results from the dissociation of the surrounding air and the subsequent formation of NO molecules from oxygen (O) and nitrogen (N) atoms.The NTP generated by the curved-tip reactor produced a significant concentration of reactive species. In particular, the generated NTP provides an optical emission spectrum (OES) identical to that observed in previous works ,38, wheroseconds . FurtherSecondary reactive species can persist from milliseconds to several days . In summIn biomedicine, the temperature generated in the NTP is critical and should not exceed 40 °C to avoid the thermal damage and destruction of living tissue . In thisSubsequently, at a fixed distance of 5 mm, the plasma application time was varied between 2 and 7 min see . These cEnterococcus faecalis bacterial inactivation. 6 and 105 CFU/mL concentrations, employing a 5 min NTP application while maintaining a 5 mm distance between the reactor tip and the plate. In Experiments were conducted initially in Petri dishes and subsequently on root canal models with embedded dentin to assess the time required for Enterococcus faecalis was exposed to the bacteria at a 106 CFU/mL concentration, leading to complete bacterial inactivation within 5 min.We conducted bacterial inactivation using PTW on the same strain in the subsequent phase. Water distilled with a volume of 5 mL was utilized, and the treatment duration was set to be 5 min. Water characterization revealed a pH of 4.3, ozone concentration of 0.8 ppm, and a hydrogen peroxide concentration of 3 ppm. Employing PTW, The experimental phase aimed at removing biofilms from root canals followed the protocol outlined in Enterococcus faecalis bacteria in the control model was consistent at approximately 1.52 × 107 CFU/mL, as depicted in p < 0.001) and those where significance was not observed .The concentration of Enterococcus faecalis within root canals. Additionally, they provide valuable insights into the effectiveness of different approaches and potential limitations in this specific application. These results contribute to advancing therapeutic strategies in biofilm removal in endodontic procedures.These findings emphasize the importance of treatment combinations and their sequencing to achieve effective inactivation of Enterococcus faecalis biofilm in Enterococcus faecalis cells displaying a deflated and ruptured morphology, accompanied by debris, as indicated by arrows in We utilized SEM to investigate how different treatments affected the morphology of bacteria attached to the dentin surface . The EntEmerging microbial control technologies offer numerous advantages in the scientific field, including the preservation of oral health. Microbial load and the virulence of various oral microorganisms significantly impact microbial contamination assessment, with specific pathogens posing risks in root canals. Over the decades, innovative techniques have been developed to address oral health challenges. However, it is essential to note that some decontamination methods may adversely affect oral properties.Combining multiple treatment methods, especially for root canals, has shown promise by synergistically combating oral health-threatening microorganisms, providing effective alternatives for achieving desired outcomes. Although this multidisciplinary approach creates an inhospitable environment for microorganisms, a definitive solution still needs to be discovered. These approaches aim to optimize therapeutic interventions and enhance patients’ quality of life, contributing to ongoing dental advancements and promoting optimal oral health.This study aimed to develop a non-thermal plasma generator reactor with a curved terminal and assess its performance in the inactivation of biofilms in Petri dishes and root canals. Furthermore, the coating exhibited no detectable porosities, ensuring electrical insulation and the effective operation of the plasma reactor. The absence of porosities is critical to maintaining electrical integrity and preventing any potential plasma leakage, thereby contributing to the overall success of the plasma reactor in its intended applications. Traditional plasma generators typically possess rigid and straight macroscopic characteristics. To initiate the discharge, the polarization of the two electrodes is achieved using a high-voltage source in the order of kilovolts, employing alternating or pulsed current with an operating frequency in the tens of kilohertz range. The gas flow rate during the experimentation was consistently maintained at approximately 10 LPM ,46,47,48The power supply characteristics used in these applications included a voltage range of ±200 V, a frequency of 13.56 MHz, and a gas flow rate of 0.5 LPM .Many research endeavours have been dedicated to unveiling the bacterial species inhabiting root canals ,59,60,61Enterococcus faecalis was chosen as the model organism for our in vitro studies, representing endodontic disinfection investigations. Preclinical and clinical studies have reported that the separate application of NTP and PTW promotes anti-inflammatory and microbicidal functions, facilitating tissue repair and contributing to the restoration of periodontal tissue. The extent of this recovery is contingent on the number of applications, as supported by research such as [Chronic periradicular lesions, both before and after root canal therapy, have garnered significant attention in the literature ,35. Biof such as ,52,63,64Enterococcus faecalis and its treatment with NTP and PTW using an innovative reactor with a curved terminal underscore a remarkable level of success. This achievement can be attributed to our meticulous examination of the distinct phases involved in the inactivation process of Enterococcus faecalis. These findings emphasize the importance of treatment combinations and their sequencing in achieving Enterococcus faecalis inactivation within root canals, offering valuable insights into the effectiveness of different approaches for this specific application.The outcomes of our experiments involving Enterococcus faecalis, a bacterium known for its resistance to conventional sterilization methods, including heat, acid, calcium hydroxide, and UV irradiation [Enterococcus faecalis within root canals. These experiments clearly illustrate the efficacy of treatment combinations involving NTP, PTW, and NaOCl applied under various sequences and conditions. In our study, the microbiological results strongly indicate the susceptibility of Enterococcus faecalis to the NTP and PTW generated by our proposed reactor, with noticeable effects on Enterococcus faecalis biofilms becoming apparent after just 5 min of treatment. Plasma exposure rapidly disrupts the cell wall, as visually depicted in adiation , has posEnterococcus faecalis bacteria was also studied using SEM. These results revealed distinct differences in root canals, with significant effects observed when using NTP and PTW treatments, especially when combined, compared to the individual use of sodium hypochlorite. Notably, the combined treatment of NaOCl and NTP at a 0 mm distance from the nozzle (The colonization of e nozzle i demonst4) powder coating on the central electrode, ensuring stable NTP production suitable for dental applications, particularly in root canal treatments. The curved-terminal reactor offers an ergonomic and efficient solution for accessing root canals, applying NTP, and inactivating biofilms of Enterococcus faecalis. The combined treatments involving NaOCl, NTP, and PTW exposure demonstrated significantly heightened antimicrobial efficacy compared to the control group. The unique antimicrobial efficacy of NTP and PTW is attributed to their ability to disrupt microbial structures, including those within biofilms. This breakthrough can revolutionize root canal disinfection, providing a superior and optimized approach. In summary, our study represents a substantial advancement in dentistry, presenting an innovative method for biofilm inactivation in root canals using NTP generated by a curved-terminal reactor. Implementing this technology can improve the prevention and management of endodontic infections. Further research with this reactor design is essential for complete bacterial inactivation in root canals.In this study, we introduce an innovative reactor design with a curved termination for generating non-thermal plasma (NTP) and plasma-treated water (PTW) to inactivate biofilms in root canals. The reactor utilizes zircon (ZrSiO


Goals: To explore factors associated with inadequate gastric preparation for MCE.Background: Factors associated with inadequate gastric preparation for magnetically controlled capsule endoscopy (MCE) remains unclear.Study: Data of patients who underwent MCE from June 2021 to July 2022 were prospectively collected. The gastric cleanliness score (GCS) of the six stomach regions  was recorded. Patients with GCS score ≥18 were defined as the adequate preparation. Factors related to inadequate gastric preparation were analyzed using a logistic regression model with estimated odds ratios (OR).Results: The mean GCS score of 211 patients was 17.01 ± 2.82. In the multivariable analysis, proton pump inhibitor (PPI) use  and premedication time after administering simethicone <30 min  were independent risk factors for inadequate gastric preparation. Comparing the gastric cleanliness of different locations, the median GCS of the lower stomach  was significantly higher than that of the upper stomach  (p <0.001).Conclusion: PPI use and inadequate premedication time (<30 min) may reduce the quality of gastric preparation for MCE. The type, dose, duration of medication, and discontinuation time of PPIs was well worth further exploration. Appropriate control of the type and time of premedication may be the key to improving overall gastric cleanliness. Magnetically controlled capsule endoscopy (MCE) offers a safe means to screen the gastric mucosa, detect lesions and identify gastric cancer, using a remote magnetic control system to adjust the angle of capsule observation without the need for intubation or sedation . MultiplThe diagnostic performance of MCE relies on clear visualization of the entire gastric mucosa. However, in clinical practice, the presence of chyme, air bubbles, mucus, and bile in the gastric lumen may obscure microscopic lesions, reduce the cleanliness of the gastric mucosa, compromise the integrity of the visual field, and potentially lead to misdiagnosis or omission . Unlike Previous studies have shown that simethicone use can improve gastric image quality by removing air bubbles , and repClinicalTrials.gov (NCT04933643). We included patients aged 18–75 years who required MCE at the endoscopy center of Qilu Hospital, Shandong University, from June 2021 to July 2022. All patients provided written informed consent.This prospective observational study was approved by the Ethics Committee of Qilu Hospital, Shandong University and registered at Patients with the following were excluded: 1) severe physical diseases who were unable to adhere to the examination requirements; 2) dysphagia, known or suspected gastrointestinal fistula, stenosis or obstruction; 3) known active upper gastrointestinal bleeding; 4) altered gastrointestinal anatomy due to previous surgery; 5) exclusion criteria for magnetic resonance imaging examinations, including patients with implanted electronic medical instruments and magnetic metal devices; 6) pregnancy; and 7) claustrophobia or other mental disorders.Patients were instructed to follow a soft diet the day before the examination and fast overnight (>8 h). Colored drinks were not permitted after 8 p.m. To reduce the impact of foam on the visual field, an appropriate bubble-removing agent (10 mL of simethicone dissolved in 50 mL of water) was administered 40 min before the examination. Before undergoing MCE, the patients were instructed to drink 500–1,000 mL of water to fill the stomach and provide the airwater interface for capsule sailing .For the MCE procedure, the patient ingested the capsule with 100 mL of water and was instructed to lay on the examination bed in the supine position. The operator then adjusted the endoscopic capsule using the magnetic control system. In order to observe the cardia, fundus, body, angulus, antrum, and pylorus in sequence, the patient position was adjusted as necessary. The patient position alternated between the left lateral, supine, and right lateral positions. At least two examinations of each gastric area were performed. The patient continuously consumed water when the stomach was underfilled. The gastric examination time for MCE was recorded.H. pylori infection, drug use, diet before examination, fasting time, and premedication time after administering simethicone. Premedication time after administering simethicone was defined as the time from administering simethicone to swallowing capsule.The content of the questionnaire, with the aim to record patient information and evaluate gastric cleanliness, was discussed and formulated jointly by the research team. Considerations included the relevant literature and available information, including patients’ demographic and related clinical data, such as patient sources, history of basic diseases , family history of gastric cancer, The primary outcome was to identify factors associated with inadequate gastric preparation. The gastric preparation quality was expressed as the gastric cleanliness score (GCS). We evaluated the six primary anatomical landmarks of the stomach . A 4-point grading scale was used to objectively assess the cleanliness of each landmark as either excellent (only traces of adherent mucus or foam present: score 4), good , fair (considerable amount of mucus or foam present preventing a completely reliable examination: score 2), or poor  . Since tUsing the scoring criteria, two endoscopists (with at least 3 years of reading experience) independently evaluated the cleanliness of the endoscopic images obtained during MCE. When the grading scores varied between the two endoscopists, the final GCS was determined by a senior endoscopist (with over 5 years of reading experience) who was in charge of quality control.Secondary outcomes include upper and lower GCS and positive lesions. The senior endoscopist described and recorded any positive lesions observed during MCE. In this study, positive lesions were defined as focal lesions of the stomach, including focal erosion, polyps, ulcers, gastric varices, submucosal tumors, etc. Diffuse lesions, including superficial or atrophic gastritis, were regarded as negative. The number of lesions per patient (NLPP) was recorded to evaluate the total detection in the population.p-value of <0.05.Continuous variables are reported as means and standard deviation (SD), or medians and interquartile range (IQR) when not normally distributed, while categorical variables are expressed as percentages. Variables were compared using the chi-square or Kruskal–Wallis tests, when appropriate. Multivariable logistic regression analysis was performed to identify variables independently associated with inadequate gastric preparation quality, using estimated odds ratios (ORs) and 95% confidence intervals (95% CIs). Statistical significance was defined as a two-sided p <0.10, multivariate analysis was performed. All statistical data were analyzed using R Statistics software .The sample size was calculated using events per variable (EPV), which is widely recognized as an effective means of determining sample size when the aim is to develop logistic regression prediction models . AccordiOverall, 214 patients underwent gastric preparation and MCE from June 2021 to July 2022. Three patients were excluded due to upper gastrointestinal bleeding or missing data. Ultimately, 211 patients were enrolled in this study. The patient characteristics, clinical indications for MCE, and comorbidities are shown in p <0.001) and the consistency for assessing the eligibility of gastric preparation between the assessors was ranked as “excellent” .High inter-observer consistency was found between the endoscopists in assessing the quality of gastric preparation. The intra-group correlation coefficient for the GCS was 0.89 (p <0.001), nausea or acid reflux as an indication of MCE , hypertension , and premedication time after administering simethicone <30 min  were associated with inadequate gastric preparation.Inadequate gastric preparation was reported in 54% (114/211) of the patients and the GCS was 17.01 ± 2.82 for the total population. Univariate analysis revealed that proton pump inhibitor (PPI) use  and premedication time after administering simethicone <30 min  were independent risk factors for inadequate gastric preparation in patients requiring MCE  and premedication time after administering simethicone <30 min  were associated with inadequate gastric cleanliness of the upper gastric site  was higher than that of the upper , and the difference was statistically significant (ric site .p = 0.001). The NLPP in the adequate preparation group (0.77 ± 0.86) was significantly higher than that of the inadequate group (0.38 ± 0.83) (p <0.001). The NLPP for the lower stomach in the adequate preparation group (0.35 ± 0.50) was also higher than that of the inadequate group (0.14 ± 0.37) (p = 0.001), while there was no significant difference in the upper stomach comparison (p = 0.084).Positive lesions detected during MCE are shown in Gastric cleanliness greatly affects the quality of MCE, and the presence of gastric mucus and foam reduces clinical observation ability . In ablePPI use as an independent risk factor for reduced gastric mucosal cleanliness may be associated with its impact on gastric emptying . The “acAs for the drugs used for gastric preparation, previous studies have shown that simethicone, an antifoaming substance, can effectively improve the visibility of the gastric mucosa . RegardiSimilar to previous studies , we founH. pylori infection was not included in the final analysis due to the restriction of urea breath test in some patients taking PPIs  after administering simethicone may improve the cleanliness of the gastric mucosa. The type, dose, duration of medication, and discontinuation time of PPIs was well worth further exploration. Improving the type and time of premedication may be the key to improving the overall gastric cleanliness. Better control of these factors in the future will improve gastric cleanliness and a higher lesion detection rate is expected, which will be conducive to promoting the application of MCE in gastric cancer screening.


Cold stress is among the most important environmental factors reducing the yield of crops. The present study aimed to investigate the impact of increasing cold stress conditions on winter oilseed rape polyamines, proline, and ethylene metabolism in acclimated and non-acclimated winter oilseed rape. This study was carried out under controlled conditions in the laboratory. The winter oilseed rape hybrid ‘Visby’ was used in the experiment. Acclimated and non-acclimated plants were subjected to a two-day-long increasing cold (from −1 °C to −3 °C) treatment. HPTLC, RT-qPCR, spectral analysis, and gas chromatography methods were used to analyse the levels of polyamines, gene expression, proline, and ethylene, respectively. This study showed a decrease in putrescine, spermidine, and spermine content during cold acclimation and a decrease in putrescine and spermidine levels at sub-zero temperatures. There were intensive changes in ADC2 gene expression, proline, and ethylene levels in non-acclimated plants: a substantial increase after exposure to −1 °C temperature and a sharp decrease after exposure to −3 °C temperature. The changes in these parameters were lower or absent in acclimated plants. The phenomena observed in this study add new insights to the knowledge about the plant stress response and suggest questions to be answered in the future. Adverse environmental factors affect the growth, yield potential, and distribution of plants. Thus, research on the plant stress mechanism and its control is one of the most important scientific fields to date. Physiological–biochemical reactions are activated in plants under stressful conditions. These are the defence mechanisms of plants, which help them to survive in unfavourable conditions when the level of an environmental factor exceeds the limit ,2.Brassica napus L.) is among the oldest crops grown in the world and is currently one of the most widely cultivated representatives of the Brassicaceae family. Its distribution mainly depends on climatic conditions [Oilseed rape  are organic polymeric compounds in all living organisms and are implicated in many biological functions. Studies have reported modified levels of PAs in plants under various stress conditions  ,19. MutaProline and ethylene have numerous functions and participate in the stress response of plants. PAs, proline, and ethylene are related through common precursors in their biosynthetic pathways. There is also evidence that catabolism of PAs contributes to proline accumulation. A positive correlation between proline accumulation and putrescine content and a negative correlation with spermidine content has been reported. Exogenous PA treatments were reported to increase the levels of PAs and proline. PAs have also been shown to regulate ethylene biosynthesis by inhibiting the accumulation of 1-aminocyclopropane-1-carboxylic acid (ACC). Nevertheless, there is some evidence of interaction, but the exact relationship between PAs, proline, and ethylene metabolism is still poorly understood. Many studies on the metabolism and interaction of these molecules have been conducted under drought, salinity, and other types of stress conditions. However, studies under cold stress are limited ,24. This study aimed to expand the scientific knowledge on the mechanism underlying the plant response to adverse environmental conditions by investigating the influence of increasing low-temperature stress on the metabolism of closely related plant stress biomarkers  in winter oilseed rape. The insights obtained in this study under the specific environmental conditions may provide a clearer understanding of the plant response to cold and may be useful for future studies aiming to develop means for plant protection against abiotic stress.The PA content analysis showed that the acclimation treatment resulted in lower putrescine content values in oilseed rape plants . A signiThe expression pattern of the PA biosynthetic gene ADC2 was analysed in this study. The acclimation treatment resulted in higher ADC2 expression levels in A than in NA oilseed rape plants . InteresProline content was reduced by 3.72 µmol after the acclimation treatment  and did The levels of emitted ethylene  were 39%Temperature is one of the environmental factors that determine a plant’s geographical distribution. Temperatures above or below a specific range have a negative impact on the yield of agriculturally important crops and lead to economic loss. Some species from temperate regions, including winter oilseed rape, have developed an adaptive process named cold acclimation, which results in tolerance to low-non-freezing temperatures. Current knowledge indicates that plants initiate many physiological and metabolic responses under abiotic stress conditions, which are equipped with post-translational modifications and changes in gene expression. A deeper analysis of these responses may help develop plant protection methods against stress ,25.Many studies have revealed that the overall PA metabolism changes in response to many abiotic stresses. The changes in PA levels in response to stress are important in reducing the effects of stress. PAs were shown to be beneficial for protein homeostasis, detoxification of reactive oxygen species (ROS), activation of the antioxidative machinery, and molecular chaperone activity under stress conditions, etc. ,27. EarlArabidopsis under various environmental stresses. ADC1 was activated mainly by cold, and ADC2 expression was induced with drought, high salinity, mechanical injury, and potassium. Another study showed that the level of both ADC1 and ADC2 transcripts increased during cold stress. Moreover, previous studies have shown that the up-regulation of ADC1 and ADC2 leads to an eventual accumulation of putrescine [The synthesis of putrescine from arginine by the ADC in plants is present under optimal conditions and various stresses . It was shown that ADC1 and ADC2 are among the most significantly cold-induced genes in the PA biosynthesis pathway in plants ,26. Difftrescine ,27,31. In this study, we analysed changes in the ADC2 gene expression pattern under acclimating and subzero conditions. The data showed that the ADC2 gene expression level was increased with the acclimation treatment . The −1 Many studies have shown that plant exposure to suboptimal conditions alters proline metabolism and changes its content in the plant. This amino acid is known to act both as a primary and secondary metabolite. It is a compatible solute and contributes to low-temperature resistance and resistance to other stresses. Proline has been shown to enhance stress tolerance by improving membrane stability, ROS scavenging, and storage of energy and nitrogen ,33,34,35What is more, proline was proposed to act as one of the osmolytes in enhancing plant stress tolerance ,35. HoweMany studies have shown an increase in proline accumulation in various plant species under stress conditions ,35. Our Ethylene is a gaseous plant hormone involved in various physiological and developmental processes as well as in different stress responses. The levels of this secondary metabolite and the expression of its signalling genes are rapidly upregulated in response to multiple abiotic stresses. Ethylene not only inhibits plant growth but also confers stress tolerance that maximises plant survival in adverse conditions by altering physiological and developmental activities in plants, e.g., it balances photosynthesis, controls stomatal aperture, cooperates with the ROS response pathway, promotes adventitious root formation, and regulates stem and petiole growth ,24.The ethylene biosynthesis pathway is related to one of the PA biosynthesis pathways. S-adenosylmethionine (SAM) formed from methionine is a common precursor of PAs and ethylene. Polyamines have been shown to regulate ethylene biosynthesis by inhibiting ACC accumulation. In addition, the common precursors sharing PAs and ethylene have antagonistic effects: PAs inhibit plant cell senescence, while ethylene promotes it ,38,39.The nature of the change in ethylene content varies depending on the species ,40. In tThis study showed a strong increase in the ADC2 gene expression level, proline, and ethylene after exposure to the −1 °C temperature treatment and a sharp decrease after exposure to the −3 °C temperature treatment in N plants. The changes in these parameters were lower or absent in A plants. Also, a reduction in the amount of all PAs during cold acclimation and a decrease in spermidine and putrescine was observed at sub-zero temperatures. The phenomena observed in this study add new insights into the knowledge of the plant stress response and suggest questions to be answered in the future. For example, why did the total PA content decrease when oilseed rape plants were exposed to low temperatures? Do PAs serve as an energy source, or do they serve another purpose under low-temperature stress? How are the changes in ADC2 expression, proline, and ethylene levels related? The answers to these questions would add to the general understanding of the function and action of PAs, proline, and ethylene in plants and provide a prerequisite for the practical use of this knowledge in the future, e.g., to increase plant resistance to various stresses.The specific environmental conditions designed for the present study widened the understanding of how the tested parameters operate. We believe that future studies on plant stress mechanisms in different plant species and under various specific environmental scenarios will be helpful in answering many fundamental questions and will provide new leads for crop protection against stress.v/v) of garden compost and peat moss (pH 5.5–6.5) was used as the substrate. The substrate was irrigated using tap water. No fertilizer was used in this study.This study was carried out under controlled conditions in a laboratory. The winter oilseed rape hybrid ‘Visby’  was used in the experiment. Seeds were supplied by the Lithuanian Seeding Association. The seeds were sown in plastic cube pots of 10 cm edge length. Each pot contained nine plants. A mix  was set. The below-described experimental treatments were performed under the same illumination. Following the design of this study , plants The low-temperature treatments were performed in Friocell  cooling incubators. For the 4-day-long cold acclimation treatment , the pot−1 until −1 °C was reached. On the second day of the treatment, the temperature was reduced by the same rate to −3 °C.Next, for the two-day-long increasing cold (from −1 to −3 °C) treatment , on the Fully expanded leaf blades were collected for assays after the cold acclimation and each day of the increasing cold treatment. Freshly harvested samples were used for ethylene emission analysis. Samples collected for PA content, gene expression, and proline analyses were placed in liquid nitrogen and stored in an ultra-low freezer  at −80 °C.v/v) perchloric acid in chilled mortars (4 °C). The residue was removed with centrifugation at 13,800× g for 15 min. Next, 600 μL of the supernatant was used for the derivatisation reaction. Dansyl chloride (5 mg/mL) was dissolved in acetone. Then, 2× dansyl chloride (1200 μL) and 1× sodium carbonate (600 μL) were added to the samples and then vortexed and incubated in the dark for 16 h. The excess dansyl chloride was removed by adding 300 μL of proline (Roth) solution in deionised water (150 mg/mL) and stored in the dark for a further 30 min. Next, 1500μL of toluene was used to extract dansyl amides from the solution. After vortexing and centrifugation for 10 min at 13,800× g, the dansyl amides in toluene were further analysed with HPTLC using CAMAG (Switzerland) equipment and the visionCATS 2.5 ver. (Switzerland) programme. The automatic TLC Sampler 4 was used to apply standards and samples as 6 mm bands on 10 cm × 20 cm glass silica gel SIL G-25 UV254 chromatography plates. Standard solutions (1 M) of putrescine, spermidine, and spermine  were prepared by dissolving them in deionised water. The elution was carried out in the Automatic Developing Chamber 2. A 25:4 ratio of chloroform:triethylamine mobile phase was used. The dried plate was transferred to the TLC visualiser 2 and illuminated at 365 nm. Finally, the absorption was measured using the TLC Scanner 4. The obtained data were derived from the results of sample peaks, analogous to ascorbic acid in the calibration curve. The content of PAs was determined according to the standard curve of known PA concentrations.The content of PAs was determined according to the method reported by Madhubala  with minTotal RNA from frozen leaf tissues weighing 150 mg was isolated and purified using the commercial RNA isolation kit: Quick-RNA Plant MiniPrep™ Kit . DNase I set  was applied to avoid genomic DNA contamination. The quantity and quality of the isolated RNA were evaluated using a NanoPhotometer® P330 spectrophotometer . RNA purity was assessed using the ratio of optical density (OT) absorbance OT 260 nm/OT 280 nm (range 1.80 to 2.05) and OT 260 nm/OT 230 nm (range 2.00 to 2.30). RNA integrity was assessed using electrophoresis in a 1% agarose gel stained with ethidium bromide. RNA samples were stored at −20 °C until the RT-qPCR analysis.Complementary DNA (cDNA) was synthesised in a PCR thermocycler Lab Cycler  using the commercial Verso cDNA Synthesis Kit . Under the different experimental conditions, two commonly used reference genes (HTB1 and UBC21)  43,44,4,444,45 wThe gene encoding arginine decarboxylase 2 (ADC2) was used as the target gene for this study. Primers for the target gene were designed using the Primer3Plus programme, choosing the optimal length of the nucleotide sequence in the intronic part of the gene .The qRT-PCR analysis of the samples was performed with the Azure Cielo (USA) real-time PCR system using the commercial Maxima SYBR Green qPCR Master Mix (2X) kit  according to the manufacturer’s recommendations in three biological and technical replicates. Data were processed with Quant Studio™ Design and Analysis Software version v1.5.2 . Gene expression levels were calculated using the delta–delta Ct method according to the formula:−∆∆Ct, where ∆∆Ct = ∆Ct (affected sample) − ∆Ct (unaffected sample); ∆Ct = Ct (target gene) − Ct (reference gene) [Gene expression level = 2ce gene) ,46.A colour reaction of acidified ninhydrin  was usedEthylene emission from leaves was evaluated using the method of Child et al.  with slip < 0.05 were considered to be significant. Multiple comparisons for mean values were performed using a post hoc test. The values were presented as mean ± standard deviation (SD). The statistical analyses were performed using the SPSS Statistics 23  program. The research data were subjected to an analysis of variance (ANOVA). Differences at


Detecting authentic and quality banknotes presents a significant challenge, particularly for individuals with low vision or visual impairments. Extensive research has been dedicated to achieving accurate banknote detection. It is crucial for clean banknotes to be readily detectable and accepted in daily transactions. However, existing Indian currency datasets suffer from limitations, including insufficient size, a lack of datasets on damaged/spoiled banknotes, and the unavailability of publicly accessible datasets featuring spoiled, torn, or altered banknotes. Recognizing the vital importance of a spoiled banknote dataset for the benefit of low vision and visually impaired individuals, we introduce a comprehensive dataset of spoiled banknotes comprising 5125 Indian currency notes. This dataset encompasses both old and new denominations of 10, 20, 50, and 100 Rupees, aiming to significantly enhance the accessibility and accuracy of banknote detection systems. By making this dataset openly accessible to the researchers, we aim to promote research and development of solutions for detection of spoiled banknote. Specifications Table1•Comprehensive and Diverse: The dataset comprises 5125 high-quality images, encompassing eight different classes of spoilt Indian banknotes. It includes both old and new denominations, providing a comprehensive representation of spoilt banknotes in Indian currency.•First Open-Access Dataset: To the best of our knowledge, this dataset is the first openly accessible collection of spoilt Indian banknotes. Its availability enables researchers and practitioners to access and utilize the dataset for various applications and research purposes.•Identification and Classification: The dataset serves as a valuable resource for researchers developing algorithms and models to identify and classify spoilt and non-spoilt Indian banknotes accurately. It facilitates advancements in the field of banknote analysis and classification.•Accessibility for Visually Impaired: The dataset can contribute to the development of tailored applications that aid visually impaired individuals in identifying non-spoilt banknotes. This application can enhance their independence and ease of conducting financial transactions.•Sorting Applications: The dataset holds significance for building applications that can effectively sort spoilt and non-spoilt banknotes. This has practical implications for banks, government agencies, and other practitioners involved in monetary transactions, streamlining their processes, and improving efficiency.2The objective of this study is to develop a dataset and associated methodologies for the accurate classification of spoilt and non-spoilt banknotes in the context of day-to-day monetary transactions. It is essential to correctly differentiate between clean and unspoilt banknotes, which are widely accepted by local vendors, and spoilt banknotes, including torn, holed, crumbled, or soiled notes. Such spoilt banknotes can only be exchanged at designated public sector banks, following the guidelines set by the Reserve Bank of India Currency recognition and automated teller machines play a vital role in correctly identifying banknotes While several currency datasets are currently available, there is a scarcity of specific datasets focusing on spoilt banknotes for experimentation by researchers . To addr3The dataset used in this study builds upon the existing dataset of Indian (Rupee) and Thai (Bhatt) banknotes with annotations, created by The spoilt banknotes dataset comprises eight distinct classes, namely: New 10 Rupees, Old 10 Rupees, New 20 Rupees, Old 20 Rupees, New 50 Rupees, Old 50 Rupees, New 10 Rupees, and Old 100 Rupees. These denominations were selected as they are commonly used in day-to-day transactions by the general public.To create this dataset we collected the spoiled banknotes from vegetable vendors, local shopkeepers, and neighbors. We followed the Reserve Bank of India (RBI) guidelines to authenticate Indian banknotes. As per the RBI guidelines we examined each spoiled banknote in the dataset by scrutinizing it for key security features such as watermarks, security threads, and intaglio printing. Additionally, we verify that the year of printing, the language panel, and the theme on the reverse side align with the designated denomination The dataset is organized and stored in specific folders according to the respective denominations. The dataset of spoilt banknotes comprises images that were collected in diverse environments and backgrounds, including illuminated, dark, cluttered, and from both the front and back directions of the banknotes . The ima44.1The spoilt banknote dataset was generated through the acquisition of images using the high-resolution rear cameras of a Samsung A33 5G mobile and an Apple iPhone 6. The mobile cameras were employed to capture images of spoilt banknotes from both the old and new categories.To standardize the dataset, the original images underwent preprocessing, which involved resizing them to a dimension of 1280 × 768 pixels. Following the resizing process, the images were organized and saved in separate folders based on their respective denominations. This organizational structure allows for convenient access and management of the dataset.The steps involved in the spoilt banknote image data acquisition process are outlined in 4.21.•Make and Model: Samsung Galaxy A33 5G Android Mobile.•Rear Primary Camera: It has a 48-megapixel (f/1.8) lens.•Camera Sensor: The camera sensor used is Sony IMX 582 1/2".•Battery: The mobile is equipped with a 5000 mAh battery.For Samsung Galaxy A33 5G Android Mobile:2.•Make and Model: Apple iPhone 6.•Rear Camera: It has an 8-megapixel (f/2.2) lens with rear autofocus capability.•Battery: The iPhone 6 has a 1080 mAh battery.For Apple iPhone 6:The cameras used in the data acquisition process and the specifications of the captured images:In both cases, the captured images were saved in JPG format and resized with a resolution of 1280 × 768 pixels. These specifications provide essential information about the cameras and image properties utilized in the data acquisition process.3.3Samsung Galaxy A33 5G Android Mobile with primary rear camera of 48 megapixel and Apple iphone6 rear camera is used to capture spoilt banknote images. These images are captured in different back grounds. The original images with size 3024 × 4032 and 2448 × 3264 were resized to 1280 × 768 pixel. The proposed data does not involve studies with animals, humans or data collected from social media platform. Therefore, we confirm that our research strictly adheres to the guidelines for authors provided by Data in in terms of ethical considerations.Vidula Meshram: Methodology, Data curation, Writing – original draft. Vishal Meshram: Supervision. Kailas Patil: Conceptualization, Supervision, Writing – review & editing. Yogesh Suryawanshi: Writing – review & editing. Prawit Chumchu: Writing – review & editing.


The study demonstrated that weather conditions and microbial activity induced considerable seasonal variations in PAHs content. Manure increased the content of organic carbon and total nitrogen, the abundance of organotrophic, ammonifying and nitrogen-fixing bacteria, actinobacteria and fungi and enhanced the activity of soil enzymes, including dehydrogenases, catalase, urease, acid phosphatase and alkaline phosphatase.Soil-dwelling microorganisms play an important role in the environment by decomposing organic matter, degrading toxic compounds and participating in the nutrient cycle. The microbiological properties of soil are determined mainly by the soil pH, granulometric composition, temperature and organic carbon content. In agricultural soils, these parameters are modified by agronomic operations, in particular fertilization. Soil enzymes participate in nutrient cycling and they are regarded as sensitive indicators of microbial activity and changes in the soil environment. The aim of the present study was to determine whether PAH content in soil is associated with the microbial activity and biochemical properties of soil during the growing season of spring barley treated with manure and mineral fertilizers. Soil samples for analysis were collected on four dates in 2015 from a long-term field experiment established in 1986 in Bałcyny near Ostróda (Poland). The total content of PAHs was lowest in August (194.8 µg kg Environmental pollution caused by polycyclic aromatic hydrocarbons (PAHs) poses one of the greatest problems in the contemporary world. Polycyclic aromatic hydrocarbons (PAHs) are considered to be especially toxic to humans, likewise to plants, microorganisms and other living organisms. PAH toxicity is a well-known fact, especially its ability to cause cancer ,2,3,4,5.The microbial degradation of PAHs is influenced by various environmental factors, including the availability of nutrients, the abundance and type of soil-dwelling microorganisms, as well as the type and chemical properties of degraded PAHs. Polycyclic aromatic hydrocarbons can be potentially degraded/transformed by a wide range of bacterial and fungal species ,11. MicrLow nutrient availability can also decrease the effectiveness of bioremediation in areas contaminated with PAHs. In addition to easily metabolized sources of carbon, microorganisms also require minerals, including nitrogen, phosphorus, potassium and iron, for metabolic and growth processes. Therefore, contaminated and nutrient-deficient soils should be supplemented to stimulate the growth of autochthonous microorganisms ,10. AccoDissolved organic matter (DOM) is a major source of organic carbon (Corg) in soil and it plays a key role in carbon cycling. The presence of strong bonds between PAHs and soil organic matter (SOM) can significantly decrease the bioavailability and mobility of PAHs. As a result, these pollutants tend to accumulate in carbon-rich organic soils rather than in the deeper strata of mineral soils ,17.Organic matter increases soil moisture content and stimulates microbial growth. At the same time, organic and mineral nutrients enhance the abundance of exogenous microorganisms in the soil microflora, which increases the counts and viability of bacteria and other organisms capable of degrading PAHs .There are several biological remediation techniques  for the treatment of PAH-contaminated soil. Based on the selection of the proper remediation approach, these remediation techniques are carried out by two basic types: (i) in situ  and (ii) ex situ (bioreactors) .The rate of PAH biodegradation is affected by pH, which affects the development of soil microorganisms and enzymes. An increase in soil acidity promotes the accumulation of PAHs in soil ,20. The Environmental contamination with persistent organic pollutants has emerged as a serious threat of pollution. Scientific knowledge upon microbial interactions with individual pollutants over the past decades has helped to abate environmental pollution . For theIn the natural environment, organic compounds are degraded by soil microorganisms and enzymes both under aerobic and anaerobic conditions . IntermeThe research was conducted in order to assess the effect of long-term fertilization with manure and mineral fertilizers on the content of polycyclic aromatic hydrocarbons (PAHs) in soil. Relationships were also explored between the soil content of PAHs and the soil microbiological (counts of bacteria and fungi) and biological activity (enzymatic activity). The combined application of manure and mineral fertilizers has been studied in only a very few research experiments, hence their effect on PAH content in soil is still unexplored. The new insights contribute to a better understanding of PAH biodegradation processes under complex natural conditions. It was assumed that the optimal fertilization both with manure and with mineral fertilizers customized strictly to nutritional requirements to field crops does not exceed the permissible concentrations of the assessed PAHs in the soil.Hordeum vulgare L.): (1) N0P0K0, (2) N1P1K1, (3) N2P1K1, (4) N3P1K1, (5) N2P1K2, (6) N2P1K3, (7) N2P1K2Mg, (8) N2P1K2MgCa   in 1986. The experiment was conducted in three replicates (blocks) on soil developed from sandy loam , accordkg ha−1) .−1 two years before the spring barley cultivation. Before the study, soil composition (per kg) was as follows: 100.0 mg of K, 53.2 mg of Mg, 41.3 mg of P, 7.9 g of organic carbon and 0.79 g of total nitrogen. Soil pH was slightly acidic (6.2 in 1 mol dm−3 KCl). Spring barley was grown in the second year after manure application (at the rate of 40 t ha−1). The content of nutrients, heavy metals and PAHs (LMW and HMW) in the manure was described previously by Krzebietke et al. [The following crops were grown in rotation: sugar beets, spring barley, maize and spring wheat. After the spring wheat harvest, soil was limed with 2.5 t CaO hae et al. .Soil samples for analyses of chemical, biochemical and microbiological properties and PAH levels were collected at a depth of 0–30 cm on four dates during the growing season of spring barley , after harvest and after skimming. Fresh soil samples for microbiological and biochemical analyses were passed through a sieve with a 2 mm mesh size directly after they had been transported to the laboratory.In a study by Smreczak and Maliszewska-Kordybach , spring −3, by the potentiometric method; hydrolytic acidity (Hh), by Kappen’s method; total nitrogen content, by distillation after mineralization in sulfuric (VI) acid with the addition of the selenium reagent mixture; organic carbon content, by the Kurmies method.Selected chemical properties of soil  were analyzed. The following parameters were determined in air-dried soil samples: pH 1 mol KCl∙dmThe content of 16 PAHs was determined with the Trace GC/MS Ultra ITQ900 system with a TRIPlus autosampler  and a flame ionization detector. The total content of 16 PAHs anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, indeno1,2,3-cd)pyrene, dibenzoanthracene and benzoperylene) was determined by the method described by Krzebietke et al. [-cdpyreneThe counts of the following soil-dwelling microorganisms were determined in the soil samples: organotrophic bacteria, on Bunt and Rovira agar ; ammonifMicrobial counts were determined by plating in three replicates. Microbial cultures were incubated at a temperature of 28 °C. The number of colony-forming units (CFU) was determined with a colony counter. The activity of the following soil enzymes was determined in three replicates: dehydrogenases, by the method described by Öhlinger ; urease,i; k; lj; Date is the repeated measures effect; ith rate of NPK fertilization and manure k; ith rate of NPK fertilization and sampling date l; k and sampling date l; l; ith rate of NPK fertilization, manure k and sampling date l; The data  were processed statistically by repeated measures ANOVA, where manure application and varied mineral fertilization were the fixed grouping factors and the sampling date was the repeated measure factor:Before performing statistical analyses, dependent variables in each group were tested for normal distribution. The homogeneity of variance was determined in groups and the sphericity  was evaluated with Mauchly’s test. Data that did not satisfy the sphericity condition were analyzed with the use of Wilk’s lambda test and Pillai’s trace criterion.p < 0.05.The Shapiro–Wilk test revealed that the data did not have normal distribution, therefore they were log transformed. In the next step, data were processed in Tukey’s post hoc HSD test at Microbial counts and enzymatic activity were evaluated with the Kruskal–Wallis non-parametric test for independent samples. The analyses were performed on untransformed data. The relationships between soil microbial activity, biochemical properties, organic carbon and total nitrogen content vs. PAH content (17 parameters) were determined by principal component analysis (PCA). The strength of the correlations in PCA was validated with the use of Bartlett’s test of sphericity. The number of principal components was selected with the Kaiser criterion based on eigenvalues greater than one (λi > 1). The interpretation of individual principal component PCi was simplified by varimax rotation. The results of all chemical, microbiological and biochemical analyses were interpreted by focusing on the main effects. All statistical analyses were performed in the Statistica 13 program .Considerable variations in temperature and precipitation were noted in 2015 . Microor−3) and lower hydrolytic acidity than soil supplied with mineral fertilizers only . Liming decreased soil acidity and increased the availability of nutrients for organotrophic bacteria. Higher N and K rates induced similar effects. In 2015, the abundance of organotrophic bacteria in soil varied widely from 18,108 CFU kg−1 DM to 283,108 CFU kg−1 DM soil (−1 DM soil) and lowest in August (2.5 times lower). In April, the average abundance of organotrophic bacteria reached 69,108 CFU kg−1 DM soil and it was 19% lower than in September. May and September were characterized by the most favorable temperatures for bacterial growth , which could explain the increase in the abundance of organotrophic bacteria in these months. According to Borowik et al. [In soil sown with spring barley, the counts of organotrophic bacteria were 1.7 higher in treatments that were regularly amended with manure than in treatments that were supplied with mineral fertilizers only a. The ab DM soil c. Bacterk et al. , organotManure and mineral fertilizers significantly modified the abundance of ammonifying bacteria in soil a,b. The 2P1K2 had a minor influence on the counts of ammonifying bacteria. As expected, regular liming created the most favorable environment for the growth of ammonifying bacteria.Potassium exerted varied effects on the counts of ammonifying bacteria; a moderate K rate decreased their abundance, whereas the highest K rate stimulated the proliferation of ammonifying bacteria b. Magnes−1 DM soil) which was characterized by highly unfavorable weather conditions during the 7-day monitoring period before sampling . According to Dąbek-Szreniawska et al. [8 CFU kg−1 DM soil) and it was 15% higher in September . The analyzed parameter was highest in May and August and it was considerably lower in April and September. These results could be attributed to optimal temperatures for microbial growth in May and August. Despite low precipitation in these months, soil water content was probably sufficient to promote the growth of ammonifying bacteria. Manure application increased the counts of ammonifying bacteria 1.4-fold relative to treatments supplied with mineral fertilizers only. The decomposition of organic matter supplied to soil with manure increased the content of mineral N and created a favorable environment for the development of soil microorganisms, including N-fixing bacteria. An increase in the content of mineral N as well as higher microbial counts promoted N immobilization in soil.The abundance of ammonifying bacteria varied during the growing season c and it a et al. , a decreManure significantly increased the counts of N-fixing bacteria in soil a. The abIncreasing N rates exerted a minor effect on the abundance of N-fixing bacteria in soil b. Potass8 to 247 × 108 CFU kg−1 DM soil (The counts of N-fixing bacteria in soil varied widely from 18 × 10 DM soil c. Averag−1) did not have a highly stimulating effect on actinobacteria counts. Only the highest N rate (90 kg ha−1) induced a 15% increase in the abundance of actinobacteria relative to the control . According to Vetanovetz and Peterson [−1). The highest K rate induced the greatest (2-fold) increase in actinobacteria counts relative to the lowest K rate. Magnesium did not influence the abundance of the studied bacterial group. Actinobacteria counts clearly increased in regularly limed soil.Long-term manure soil application as well as mineral fertilization significantly modified actinobacteria counts in soil a,b. ActiPeterson , mineralActinobacteria counts varied considerably during the growing season of 2015 c. The me2P1K1. Regular liming also enhanced fungal growth in soil.Fungal abundance was 32% higher in soil amended with manure every other year than in soil supplied with mineral fertilizers only a. Fungal6 to 250 × 106 CFU kg−1 DM soil (6 CFU kg−1 DM soil) and lowest in August. The analyzed parameter was similar in early spring (April) and late summer (September).In the growing season of 2015, fungal counts in soil ranged from 11 × 10 DM soil c. Mean fDehydrogenases (DHA) are regarded as reliable indicators of soil biochemical activity. Dehydrogenase activity is influenced by enzymes secreted by soil-dwelling microorganisms, both aerobic and anaerobic . Dehydro−1) decreased the analyzed parameter by 8% relative to the control treatment (−1). Kucharski [A comparison of the observed changes in DHA activity revealed that the lowest N rate used in the study  and was only somewhat lower in September (5.26 µmol TFF kg−1 DM h−1). In May, DHA activity was 20% higher than in April. The observed variations in the studied parameter could be attributed to changes in the moisture and oxygen content of soil (Dehydrogenase activity in soil was determined in the range of 2.13 to 9.65 µmol TFF kgg season c. This p of soil .−1) had no significant effect on catalase activity in soil (−1) increased catalase activity. Increasing N rates also stimulated catalase activity in the work of Lemanowicz and Koper [2P1K2Mg. Catalase activity in soil varied during the growing season of 2015  and lowest in May (0.04 mmol N-NH4 kg−1 soil h−1). Urease activity was 1.5 times higher in August (0.18 mmol N-NH4 kg−1 soil h−1) than in April.In the growing season of 2015, urease activity ranged from 0.02 to 0.46 mmol N-NHsoil h−1 c. The stAcid phosphatase activity differed significantly between treatments treated by manure and treatments supplied with mineral fertilizers only a,b. In s−1) induced the greatest (20%) increase in acid phosphatase activity relative to the control treatment. In the work of Kucharski [−1) stimulated the activity of acid phosphatase. Lemanowicz and Koper [In turn, mineral fertilizers had no significant influence on the activity of the discussed enzyme. However, higher N rates can stimulate acid phosphatase activity by increasing the concentration of H+ in the soil solution as a result of nitrification and enhancing NH4+ uptake by plants. The highest N rate applied  activity in soil a,b. In s−1) and suppressed by higher N rates (60 and 90 kg ha−1). In the work of Lemanowicz and Koper [−1 also induced a significant (13%) decrease in AlP activity. Higher N rates also inhibited AlP activity in a study by Lemanowicz [−1) decreased the value of this parameter in soil. In the current experiment, regular soil liming increased AlP activity two-fold relative to the treatment fertilized with N2P1K2Mg. Similar results were reported by Kalembasa and Kuziemska [Alkaline phosphatase activity was stimulated by the lowest N rate  and the values noted in May and August were similar. Higher AlP activity in spring could be associated with rapid phosphorus uptake by plant roots and the resulting decrease in the content of available phosphorus in soil. Such conditions support the secretion of phosphatases by plant roots, which catalyze the hydrolysis of organic phosphorus compounds to mineral compounds [−1 h−1).Alkaline phosphatase activity fluctuated in the growing season of 2015 c. This pompounds . Accordiompounds , phosphaStatistical analyses revealed that manure (M), mineral fertilization (Min) and M × Min interactions significantly influenced the total content of 16 PAHs and the content of LMW PAHs  and HMW PAHs (benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzoperylene, indenopyrene and dibenzoanthracene) . In 2015−1) and lowest in August (119.8 µg kg−1). This value was significantly higher in April (259.5 µg kg−1) than in September (210.0 µg kg−1).The content of LMW PAHs in soil differed significantly during the growing season ; it was In the growing season of 2015, the content of LMW PAHs  was highly similar in soil treated with manure and in soil supplied with mineral fertilizers only . The ana−1) and lowest in August (75.0 µg kg−1).The content of HMW PAHs (benzo(a)anthracene, benzo(a)pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzoperylene, indenopyrene and dibenzoanthracene) in soil differed significantly during the growing season . The ana−1) and soil supplied with mineral fertilizers only (117.0 µg kg−1).The content of HMW PAHs was lower in April, May and August, and in September in soil supplied with mineral fertilizers only . In Apri−1) and highest in May (484.6 µg kg−1). The analyzed parameter was significantly lower in April (358.7 µg kg−1) than in September (368.3 µg kg−1). According to the IUNG system [−1).The total content of 16 PAHs in soil varied significantly in the growing season of 2015 . The fluG system , soil caMicrobial abundance and soil enzymatic activity undoubtedly influenced the observed fluctuations in the total content of 16 PAHs. The examined parameter was lowest in August when dehydrogenase activity in soil was much higher . In a stThe presence of correlations between selected properties of soil samples collected on four dates in 2015 was identified by principal component analysis (PCA). In April, the first two principal components explained 65% of total variance in the following variables: abundance of organotrophic bacteria, ammonifying bacteria, nitrogen-fixing bacteria, actinobacteria and fungi; activity of dehydrogenases, catalase, urease, acid phosphatase and alkaline phosphatase; content of total nitrogen and organic carbon; Hh and pH; content of LMW PAHs; content of HMW PAHs; total content of 16 PAHs . The anaThe influence of enzymatic activity on the studied soil parameters increased in May. The strong correlations between the activity of dehydrogenases, catalase, urease, acid phosphatase and alkaline phosphatase, pH, organic carbon content, total nitrogen content and actinobacteria counts explained 41.4% of total variance . An analIn August, PC1 explained 42.1% of total variance in the examined soil parameters. The abundance of organotrophic bacteria and actinobacteria and soil enzymatic activity  were strongly linked with pH and organic carbon and total nitrogen content . SimilarSoil samples collected in September were also characterized by high levels of microbial and enzymatic activity. High microbial abundance can be attributed to a higher content of organic matter that was supplied to soil with harvest residues. An analysis of PC1 demonstrated that strong correlations between all microbial counts , enzymatic activity , pH (in 1 mol KCl) and the content of organic carbon and total nitrogen explained 50.5% of total variance . An analMicrobial abundance increases under supportive conditions for microbial growth . AccordiIn the work of Lemanowicz and Bartkowiak , acid phAccording to Dąbek-Szreniawska et al. , soil pHIn a study by Maliszewska-Kordybach and Smreczak , soil coGałązka et al.  demonstrSoil is a complex matrix whose physical, physicochemical, chemical and biological properties are correlated with microbial activity and the presence of pollutants such as PAHs. Weather fluctuations during the growing season also exert a strong influence on chemical and biochemical processes in soil. The relationships between the examined soil parameters were, at least partly, identified in PCA. The PCA revealed that biological processes in soil are determined mainly by carbon and nitrogen content in soil, soil pH and Hh values. Microbial proliferation rates affect soil enzymatic activity. However, the impact of specific microbial groups on PAH levels in soil could not be determined based on the results of a short-term study. Data covering a longer period of time are also needed to formulate reliable conclusions about the impact of PAHs on soil enzymatic activity. However, the present findings indicate that PCA should be used to evaluate the relationships between diverse soil parameters.The study revealed considerable seasonal variations in PAH levels in soil, depending on weather conditions and the activity of soil-dwelling microorganisms. The total content of 16 PAHs and the content of LMW and HMW PAHs was higher in soil amended with manure than in soil supplied with mineral fertilizers only. Manure application increased organic carbon and total nitrogen content, stimulated the activity of organotrophic, ammonifying and nitrogen-fixing bacteria, actinobacteria and fungi and increased the activity of dehydrogenases, catalase, urease and acid and alkaline phosphatase. Rising N rates increased the abundance of organotrophic bacteria and fungi and enhanced acid phosphatase activity in soil but inhibited the activity of dehydrogenases and alkaline phosphatase. Soil liming was most effective in increasing the counts of ammonifying bacteria, nitrogen-fixing bacteria, organotrophic bacteria and actinobacteria. Liming also enhanced the activity of catalase, urease and alkaline phosphatase and suppressed acid phosphatase activity. The study showed that manure is one of the important sources of polycyclic aromatic hydrocarbons in the soil. Further research, therefore, is still needed to investigate the effects of applied manure and mineral fertilizers under field conditions on the bioremediation of PAH-polluted soils.


Myelodysplastic syndromes (MDS) are incurable diseases characterized by dysplastic hematopoietic cells, cytopenias in the blood and an inherent tendency for transformation to secondary acute myeloid leukemia (AML). Since most therapies fail to prevent rapid clonal evolution and disease resistance, new and non-invasive predictive markers are needed to monitor patients and adapt the therapeutic strategy. By using ISET, a very sensitive approach to isolate cells larger than mature leukocytes from peripheral blood samples, we looked for cellular markers in 99 patients (158 samples) with MDS and 66 healthy individuals (76 samples) used as controls. We found a total of 680 Giant Cells, defined as cells having a size of 40 microns or larger in 46 MDS patients (80 samples) and 28 Giant Cells in 11 healthy individuals (11 samples). In order to understand if we had enriched from peripheral blood atypical cells of the megakaryocyte line, we studied the Giant Cells using immunolabeling with megakaryocytes and tumor-specific markers. We report that the Giant Cells we found in the peripheral blood of MDS patients primarily express tumor markers. Our results show that Polyploid Giant Cancer Cells (PGCC), similar to those described in solid tumors, are found in the peripheral blood of patients with MDS and suggest the working hypothesis that they could play a role in hematological malignancies. Myelodysplastic syndromes (MDS) are a heterogeneous group of stem cell disorders characterized by cytopenia with an inherent tendency for transformation to secondary acute myeloid leukemia (AML). MDS are incurable diseases except for < 10% individuals who can be successfully treated with allogeneic transplantation. Most therapies fail to obtain sustained responses because of rapid clonal evolution and appearance of disease resistance. Therefore, it would be important to find new predictive markers of clonal evolution and disease recurrence detectable in the peripheral blood that could be used as non-invasive approach to monitor patients and keep their disease under constant surveillance. To this aim, we applied ISET, a very sensitive approach to isolate cells larger than mature leukocytes, to the peripheral blood of patients with MDS. Through the enrichment of large cells, we found a relevant number of giant cells with one or multiple nuclei.In solid cancers, polyploid giant cancer cells (PGCC) have been suggested to initiate tumorigenesis and tumor recurrence after therapy . AccordiHowever, a high rate of cells called “dysplastic hypogranular megakaryocytes” and somehow morphologically similar to PGCC have been described in 80.3% of bone marrow samples from patients with MDS . These aInterestingly, the majority of the Giant Cells we have isolated from peripheral blood of MDS patients express tumor markers, raising the intriguing question of their nature and similarity with the PGCC described in solid tumors.Although these data need further investigations, they open the way to efforts aimed at understanding the potential clinical impact of these newly described Giant Cells in patients with liquid cancers.Blood samples were obtained from patients and healthy individuals who consented to donate their sample to an institutional review board (IRB) approved tissue repository at New York Presbyterian Hospital/Columbia University Medical Center. Informed consent was obtained from all the subjects who participated in the study. This study is approved by the IRB under protocol AAAR7591 of Columbia University in accordance with the Declaration of Helsinki.Complete blood count, percentage of peripheral blasts, serum levels of erythropoietin, ferritin, lactate dehydrogenase, and vitamin B12 were recorded for each patient. On a subset of patients, data from bone marrow measurements including the percentage of myeloblast, ring sideroblasts, and cellularity were recorded from the bone marrow aspiration performed on the same day as blood collection.Trypsin-treated cultured cells were filtered using ISET® technology with or without blood according to the manufacturer’s instructions . Generated filters were then stained for 5 min with cytopathological staining using MERK’s Giemsa’s azur-eosin-methylene blue solution .We have used the ISET platform , which is currently applied to study circulating tumor cells in patients with solid cancers, in order to eliminate by size the majority of mature blood cells. ISET concentrates cells which are larger than lymphocytes and neutrophils from a much larger volume of blood (5 to 10 mL) than the one used for peripheral blood smears.The CE-IVD ISET® platform and its consumables were used as previously reported  to isolate and analyze large cells in blood. Peripheral blood samples were drawn into EDTA tubes  with immediate gentle agitation . They weThe filtration membrane has 10 spots, making it possible to process blood samples of 10 mL (1 mL per spot). Cells were stained with Giemsa for visualization of nuclei, cytoplasm and cell morphology and images were digitalized using an Olympus scanner BX-61VS.Subsequently, the Giemsa stain was washed 5 min with PBS1X. Antigen retrieval was then performed for 20 min at 50 °C using EnVision™ FLEX Target Retrieval Solution High pH (50x) (Dako Omnis) (Agilent). Permeabilization was performed in Triton 0.1% PBS1X solution. The immono-cytochemistry was performed using EnVision™ G|2 Doublestain System, Rabbit/Mouse (DAB + /Permanent Red) (Agilent technologies). The spots were incubated in an ISET® ICC Staining Box, one hour at room temperature with specific antibodies diluted with DAKO Antibody Diluent (Agilent technologies). After immunostaining, cells were counterstained with hematoxylin 3 min and the spots were scanned using an Olympus scanner BX-61VS. The double digitalization allowed to visualize both cytomorphological details and immunolabeling on the same cell.Giant Cells were arbitrarily defined as cells having a size of 40 microns (maximum diameter) or more. They were identified with Giemsa staining and counted. After immunocytochemistry, all these cells were individually analyzed by visualizing side by side the cytomorphological (Giemsa staining) and the immunostaining details.For positive controls, we used the following cells/ cell lines: HeLa cells, MEG01, primary culture of monocyte-derived macrophages, alone or spiked in blood from healthy subjects, before filtration by ISET. A549 epithelial cell line from patient with lung carcinoma was used as a negative control for all the antibodies used. We also performed tests omitting the primary antibody, to ensure the absence of cross-reactivity in parallel with tests including the primary antibody, to guarantee the specificity of the antibody.Megakaryoblastic cell line  was purchased from The European Collection of Authenticated Cell Cultures (ECACC) . MEG-01 cells were grown in standardized conditions in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 2 mM Glutamine, and 1% penicillin–streptomycin solution at 37 ℃ in an incubator with 5% CO2 atmosphere. This cell line was used as positive control for CD61 and CD41 staining.HeLa cell line , product code ATCC-CCL-2) was purchased from American Type Culture Collection . Hela cells were grown in standardized conditions in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), and 1% penicillin–streptomycin solution at 37 ℃ in an incubator with 5% CO2 atmosphere. Cell culture mediums and FBS were purchased from Thermo Fisher Scientific, Les Ulis, France. Penicillin–streptomycin solution and Glutamine were purchased from Sigma-Aldrich, Darmstadt, Germany. This cell line was used as positive control for Syncytin-1, Runx2, and telomerase staining.THP-1 cells were differentiated into macrophage-like cells by incubation in the presence of 200 ng/ml of Phorbol 12-myristate 13-acetate (PMA) for 24 h, which leads to a macrophage-like phenotype characterized by changes in morphology and increased cell surface adherence. PMA was purchased from Sigma-Aldrich, Darmstadt, Germany. This cell line was used as positive control for CD163, CD68, and CD11b staining.C-terminal , CD61 (Integrin beta 3) Recombinant Rabbit Monoclonal Antibody , CD41 mouse Monoclonal Antibody , Rabbit Polyclonal Anti-Syncytin-1 antibody (Clinisciences), Anticorps mouse monoclonal RUNX2 , and Anti-Telomerase reverse transcriptase antibody .Antibodies used in this study are as follows: Recombinant Anti-CD163 antibody , recombinant Anti-CD68 antibody , Recombinant Anti-CD11b antibody [EP1345Y]—We have analyzed a total of 234 peripheral blood samples including 158 samples from 99 patients with MDS and 76 samples from 66 healthy individuals used as controls  as cells having a size (maximum diameter) equal to or larger than 40 microns. Using this definition, we identified 680 GC in 80 out of 158 samples from patients with MDS and 28 GC in 11 out of 76 samples from healthy individuals. Of the 158 samples from patients with MDS, 80 (50.6%) showed 680 cells having a size of 40 microns or larger and 50 (32%) showed 271 cells with a size equal or larger than 50 microns . In the 46 MDS patients, almost a third of the patients have GC equal or greater than 10 Fig. D.Morphological analysis of GC was performed after Giemsa staining. Representative images of GC identified in MDS patients are shown in Fig. Using immunocytochemistry, we studied the expression of several proteins including CD61, CD41, Telomerase, Runx2, Syncytin-1, CD11b, CD68, and CD163  of patients with MDS. These cells were detected by eliminating the vast majority of mature blood cells using ISET to concentrate the larger cells and analyze them. Although the cytomorphological aspects of some of these GC are similar to those of atypical megakaryocytes and megakaryoblasts, the GC we describe express tumor markers: telomerase, syncytin and Runx2, and macrophage markers which are not expressed by cells of the megakaryocyte series. Thus, given their tumor-like characteristics, these new GC found in the peripheral blood of MDS patients should be further investigated to explore the fascinating hypothesis that they could represent in liquid tumors counterpart of PGCC described in solid cancers.We thought that the giant cells we found could be morphologically assessed as atypical megakaryocytes. In the literature, the observation of megakaryocytes in peripheral blood is extremely rare. Ku et al. reported a single megakaryocyte in a patient with Myelofibrosis (MF) and leukoerythroblastosis . The imaIn order to understand if the GC we found are atypical megakaryocytes or not, we have used macrophage and tumor-specific markers and have applied a specific approach allowing to examine, side by side, the cell morphology and its immunolabeling by ICC (immunocytochemistry). In fact, immunofluorescence does not allow to explore in detail the morphological characteristics nor ICC which, used alone, covers the cytomorphological details.The vast majority of the Giant Cells that we have identified were found positive for the CD61 marker. CD61, or integrin beta 3, also known as GPIIIa, is a glycoprotein of 105 kD found on platelets, monocytes, endothelial cells, smooth muscle cells, B cells, macrophages, mast cells and fibroblasts. Only the coexpression of CD61 with CD41 is considered specific of megakaryocytes. However, the GC we tested for both markers and which were positive for CD61, were negative for CD41, making it impossible to confirm their megakaryocyte origin.Nearly 70% of GC with a size of 40 microns or larger were positive to a cocktail of macrophage markers . This is suprising as megakaryocytes are not known to express macrophage markers and we did not find any report on the expression of macrophage markers in megakaryocytes. Furthermore, Syncytin expression was found in approximately 70% of the GC detected in MDS patients. Syncytin-1 is a cell–cell fusion protein whose function is best characterized in placental development , 21. SynRUNX2 gene encodes a transcription factor that acts as a “master switch,” regulating genes involved in the development of several tissues including bones, teeth, and cartilage. However, it has also been demonstrated that RUNX2 is involved in tumor cells invasion, especially in bone metastases and in the development of malignant tumors. Thus, RUNX2 expression in the GC we found in the blood of MDS patients could be linked to the tumor characteristics of the GC, increasing the probability for the GC to be, in fact, PGCC.We also found that RunX2 is expressed in 54% of the GC. The Finally, 77.6% of the GC strongly expressed telomerase. Telomeres maintain genomic integrity in normal cells, and their progressive shortening during successive cell divisions induces chromosomal instability. In the large majority of cancer cells, telomere length is maintained by telomerase. Thus, telomere length and telomerase activity are crucial for cancer initiation and the survival of tumors . In factLongitudinal analysis of samples from healthy patients indicated a consistent pattern of GC. In general, patients or healthy subjects who did not have any GC in initial collection did not show them in subsequent collection when followed for up to 200 weeks. Patients who showed GC on initial collection consistently showed GC on subsequent collections, with some patients showing an increase in GC overtime. The pattern we observed in our study of repetitive samples is consistent with observations in the field of circulating rare cells. In fact, even with a highly sensitive approach, the probability to find rare cells depends on their frequency in blood. The lower limit of detection (LLOD) of ISET is one tumor cell in 10 ml of blood . If the In conclusion, we have described novel circulating giant cells with unique characteristics in patients with MDS. These GC are not megakaryocytes and express tumor markers and macrophage markers, thus suggesting the stimulating hypothesis that they could be analogous to the PGCC found in patients with solid cancers and thought to be at the origin of tumor formation and tumor recurrence. Further in vitro studies are needed to explore their proliferative capacity, their resistance to treatment and their possible ability to generate blasts cells by budding. Animal studies could evaluate their tumorigenic/leukemic potential. In patients, their presence and phenotype in the bone marrow should be assessed, as well as the correlation of their presence in peripheral blood with clinical data and disease evolution.Ultimately, our findings provide more questions than answers but open a new field of investigations aimed at understanding if the GC we detected in MDS patients can be considered the equivalent of PGCC described in solid cancers.